19 International Picornavirus Meeting FINAL PROGRAMME

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1 19th International Picornavirus Meeting FINAL PROGRAMME & ABSTRACTS

2 R-Biopharm AG RIDAGENE Multiplex real-time PCR More than a PCR test Gastrointestinalinfections Respiratoryinfections Hospital-acquiredinfections Sexuallytransmittedinfections Bacterialimbalance FlexibleuseinroutinediagnosticsRIDAGENEkits runoncommonlyusedPCRinstruments Distributed in Switzerland by: RUWAG Handels AG Bielstrasse 52 2544 Bettlach Tel. 032 644 27 27 Fax 032 644 27 37 [email protected] www.ruwag.ch R-Biopharm AG An der neuen Bergstrae 17, 64297 Darmstadt, Germany E-mail: [email protected] www.r-biopharm.com

3 TABLE OF CONTENT Welcome Words 5 Committee 6 Programme 7 Programme at a glance 8 Monday 12 Tuesday 70 Wednesday 114 Thursday 154 Index of authors 191 List of participants 210 Sponsors 215 General information 218 Congress Venue 218 Registration desk opening hours 218 WIFI 218 Excursions 219 About Les Diablerets 221 Free Access Card 222 Train schedule 222 Tourist board office 223 Europic 2016 3

4 Epithelix in vitro Solutions for Respiratory Diseases and Chemical Testing Provider of long shelf-life human 3D in vitro lung tissues & testing services MUCILAIR MUCILAIR -HF SMALLAIR SMALLAIR -HF ONCOCILAIR CULTURE PRIMARY CELL & TISSUES MEDIA HUMAN CELLS EXTRACTS

5 WELCOME WORDS Welcome to EUROPIC 2016 We are very pleased to welcome you all to the 19th European Study Group on the Molecular Biology of Picornaviruses in the beautiful village in the Swiss mountains: Les Diablerets. When we were printing the programme you were 195 participants registered from 20 countries! We hope that this year meeting will reach all your expectations! We are most grateful to those who helped us prepare this meeting, thanks to the members of the Scientific Committee for their valuable assistance in organizing the Scientific Programme and all the Session Chairs who kindly accepted to help us in the abstracts selection. Thank you to the invited speakers giving Keynote Lectures: Nihal Altan Bonnet, James Gern, Lo James, Karla Kirkegaard, Frank Kuppeveld, Andrew Macadam, Johan Neyts, Ann Palmemberg, Shin-Ru Shih, Marco Vignuzzi and Eckard Wim- mer. Also a special thank you to Symporg and the secretaries who helped us with the organisation. We hope that the fresh swiss alp air of Les Diablerets (1200 meters high) will be a real benefit for all of you. At the end we would like to thanks our sponsors who accepted to support our meeting! We wish you a wonderful meeting and look forward to meet you! Bienvenue! Welcome! The EUROPIC 2016 Organising Committee Caroline Tapparel Vu, Laurent Kaiser and Urs Greber Europic 2016 5

6 COMMITTEE Organizing Committee Urs Greber Laurent Kaiser Caroline Tapparel Vu Scientific and Reviewing Committee Raoul Andino Laurent Kaiser Bert Semler Urs Greber Thomas Michiels Caroline Tapparel Vu Reviewing Committee Nihal Altan-Bonnet Alexander Gorbalenya Hiroyuki Shimizu George Belov Lo James Peter Simmonds Kimberley Benschop Satoshi Koike Toby Tuthill Dieter Blaas Mark Pallansch Frank van Kuppeveld Francis Delpeyroux Rosa Pint Eckard Wimmer David Evans David Rowlands Katja Wolthers Faculty Keynote Lecturer Nihal Altan-Bonnet Andrew Macadam Frank van Kuppeveld James Gern Johan Neyts Marco Vignuzzi Lo James Ann Palmenberg Eckard Wimmer Karla Kirkegaard Shin-Ru Shih 6 Europic 2016

7 Programme Keynote Lectures List Programme by day with abstracts

8 8 16:00 18:30 Arrival and Registration 18:30 21:00 Welcome address and dinner at Les Diablerets SUNDAY 04/09/2016 MONDAY 05/09/2016 8:45 9:00 INTRODUCTION AND ANNOUNCEMENTS with Caroline Tapparel, Laurent Kaiser and Urs Greber 14:00 Session 2 PICORNAVIRUS-CELL INTERACTIONS 9:00 Session 1 PICORNAVIRUS STRUCTURE AND LIFE CYCLE Chairs: 1st part, Dieter Blass & George Belov 18:10 2nd part, Thomas Michiels & Rosa Pint Chairs: 1st part, Bert Semler & Nihal Altan-Bonnet 12:35 Host responses to viral infection: innate immunity, membrane 2nd part, Toby Tuthill & Eckard Wimmer 14:00 Karla Kirkegaard biology and viral transmission OLD AND NEW THOUGHTS ABOUT THE REPLICATION AND ASSEMBLY 09:00 Eckard Wimmer SINGLE-CELL VIROLOGY: ON-CHIP INVESTIGATION OF VIRAL OF POLIOVIRUS 14:35 Craig Cameron INFECTION DYNAMICS ATOMIC STRUCTURE OF A RHINOVIRUS C, A VIRUS SPECIES LINKED 09:35 Yue Liu ILLUMINATING THE SITES OF ENTEROVIRUS REPLICATION IN LIVING TO CHILDHOOD ASTHMA EXACERBATIONS 14:50 CELLS USING A SPLIT-GFP-TAGGED VIRAL PROTEIN Hilde van der Schaar GENOMIC RNA FOLDING MEDIATES ASSEMBLY OF HUMAN 09:50 Peter Stockley UBIQUITYLATION OF VP1PX IN THE BIOGENESIS OF QUASI- PARECHOVIRUS-1. 15:05 ENVELOPED HEPATITIS A VIRIONS Kevin L. McKnight VIRUS QUASISPECIES REVEALS RNA STRUCTURES REQUIRED FOR 10:05 Grace Logan 15:20 INFECTIOUS ENTRY PATHWAY OF COXSACKIEVIRUS B1 Varpu Marjomki GENOME PACKAGING. 10:11 INVESTIGATING THE ASSEMBLY MECHANISM OF ENTEROVIRUSES Rebecca Chandler-Bostock IDENTIFICATION AND CHARACTERIZATION OF NOVEL VIRUS-HOST 15:26 INTERACTIONS THROUGH ANALYSIS OF NUCLEAR PROTEIN EFFLUX Dylan Flather FOOT-AND-MOUTH DISEASE VIRUS REPLICATES INDEPENDENTLY DURING RHINOVIRUS INFECTION 10:17 OF PHOSPHATIDYLINOSITOL 4-PHOSPHATE AND TYPE III Stephen Berryman PHOSPHATIDYLINOSITOL-4-KINASES COFFEE BREAK COFFEE BREAK FAT(AL) ATTRACTION: PICORNAVIRUSES HIJACK LIPID TRANSFER 16:05 Frank van Kuppeveld MACHINERY TO CREATE REPLICATION ORGANELLES PROGRAMME AT A GLANCE PROTEIN COMPOSITION OF THE QUASI-ENVELOPE OF HEPATITIS A 11:00 Kevin L. McKnight ACTIVATION OF PHOSPHOLIPID SYNTHESIS IS REQUIRED FOR THE VIRUS 16:40 DEVELOPMENT OF PICORNAVIRUS REPLICATION ORGANELLES AND George Belov 11:15 RHINOVIRUS SUPERINFECTION EXCLUSION IS INDEPENDENT OF Robert Witte PROTECTION OF THE REPLICATION COMPLEXES VIRUS ENTRY AND REQUIRES REPLICATION STUDIES OF A PICORNAVIRAL PHOSPHOINOSITIDE-BINDING CRYO-EM STRUCTURES OF HONEY BEE DEFORMED WING VIRUS 16:55 Djoshkun Shengjuler 11:30 REVEAL CONFORMATIONAL CHANGES LINKED TO GENOME RELEASE Lindsey Organtini PROTEIN IDENTIFICATION OF NOVEL SUBSTRATES OF POLIOVIRUS AND A CYSTEINE RESIDUE IN THE RHINOVIRUS-A16 CAPSID HAS KEY 11:45 Urs Greber 17:10 COXSACKIEVIRUS 3C PROTEINASES USING TERMINAL AMINE Julienne Jagdeo FUNCTIONS IN VIRUS ENTRY ISOTOPIC LABELING OF SUBSTRATES EFFECTS OF A LARGE DELETION IN NON-STRUCTURAL PROTEIN 3A 11:51 Emma Howes DIFFERENTIAL PATHOGENESIS OF RHINOVIRUSES FROM SPECIES A, ON FMDV REPLICATION 17:25 Manel Essaidi-Laziosi B AND C AND OF ENTEROVIRUS D68 IN HUMAN AIRWAY EPITHELIA REDUNDANCY WITHIN THE TANDEM 3B REPEATS IN FOOT AND 11:57 Morgan Herod EXPLORING THE REPERTOIRE OF ENTEROVIRUS D68 GLYCAN MOUTH DISEASE VIRUS REPLICATION 17:40 Hendrik Jan Thibaut RECEPTORS EXTRACELLULAR VESICLES ARE THE TROJAN HORSES OF VIRAL THE SYNERGISTIC ROLES OF ICAM-1 AND SIALIC ACIDS IN 12:00 Nihal Altan-Bonnet 17:55 Jim Baggen INFECTION COXSACKIEVIRUS A24 INFECTION LUNCH BREAK POTENTIAL MODELS TO SEPARATE TWO DIFFERENT HEPATITIS A 18:01 Tiing Tiing Chua VIRUS POPULATIONS 18:15 19:15 1st POSTER SESSION ( posters of sessions 1,2,3,4) 19:30 21:30 DINNER AT THE HOTEL EUROTEL VICTORIA Keynote Lecture Communication (12 + 3 min) Shotgun presentation (5 + 1 min) 6 septembre 2016 Europic 2016

9 14:00 Session 4 VIRUS GENETICS, GENOME PLASTICITY, EVOLUTION, CLASSIFICATION Chairs: 1st part, Raul Andino & David Evans 18:05 2nd part, Peter Simmons & Alexander Gorbaleyna Europic 2016 VIRUS EVOLUTION: MATHEMATICAL REDUCTION OF SEQUENCE 14:00 Marco Vignuzzi TUESDAY 06/09/2016 SPACE AND EMPIRICAL FITNESS LANDSCAPES UNCOVERING RNA VIRAL POPULATION DYNAMICS: SEQUENCE 14:35 Rasmus Henningsson SPACE AND EMPIRICAL FITNESS LANDSCAPES 8:45 Session 3 IMMUNE MODULATION OF PICORNAVIRAL INFECTIONS SEQUENCE SPACE AND TRANSLATIONAL FITNESS LANDSCAPE IN 14:50 Rosa Pint Chairs: Urs Greber, Frank van Kuppeveld & Lo James HEPATITIS A VIRUS 11:20 ADAPTATION OF RV-C15 TO CDHR3-EXPRESSING HELA CELLS 15:05 Kelly Watters INTRACELLULAR NEUTRALIZATION AND INNATE IMMUNE ACTIVATION ALLOWS HIGH-TITER VIRUS PRODUCTION IN TISSUE CULTURE 08:45 Lo James THE INFLUENCE OF SEQUENCE AND RNA STRUCTURE ON BY THE CYTOSOLIC ANTIBODY RECEPTOR TRIM21 15:20 Kirsten Bentley MAVS SIGNALING DEFINES THE HOST SPECIES RANGE AND RECOMBINATION IN ENTEROVIRUSES 09:20 Stanley M. Lemon THIRTY YEARS OF POLIOVIRUS REPLICATION IN AN PATHOGENICITY OF HUMAN HEPATITIS A VIRUS 15:35 IMMUNODEFICIENT INDIVIDUAL: MOLECULAR EVOLUTION AND Dimitra Klapsa 09:35 CONVERGENT MECHANISMS USED BY CARDIOVIRUSES, KSHV AND Michael Peeters EFFECT OF ANTI-VIRAL TREATMENTS YERSINIA TO ACTIVATE RSK KINASES THEILER'S VIRUS L* PROTEIN INHIBITS RNASE L ACTIVATION 15:50 PICORNAVIRUS RNA RECOMBINATION David Barton 09:50 Thomas Michiels THROUGH COMPETITION WITH 2-5A BINDING COFFEE BREAK COFFEE BREAK 16:25 PICORNAVIRIDAE: THE EVER-GROWING VIRUS FAMILY Roland Zell HUMAN RHINOVIRUS 3C PROTEASE CLEAVES RIPK1, AN IMPORTANT 10:35 Sarah Croft INTERMEDIATE IN EXTRINSIC APOPTOSIS COMPLETE REVERSION OF A MULTIPLY MUTATED COXSACKIEVIRUS RECOGNITION OF ENTEROVIRUS 71 BY TOLL-LIKE-RECEPTOR 8 AND 16:40 B3 (CVB3) CRE(2C) DURING REPLICATION OF 5 TERMINALLY Nora Chapman 10:50 RIG-I ACTIVATE THE INFLAMMASOME IN HUMAN MYELOID CELLS Hsing-I Huang DELETED VIRUS RSK MAY CONTROL STRESS GRANULES ASSEMBLY THROUGH PKR NEXT GENERATION SEQUENCING REVEALS NEW SAT GENOTYPES 11:05 Yohei Hayashi INHIBITION 16:50 BRINGING US CLOSER TO UNDERSTANDING THE HISTORY OF FOOT- Nick J. Knowles AND-MOUTH DISEASE VIRUS IN AFRICA nd 11:20 12:30 2 POSTER SESSION ( posters of sessions 1,2,3,4) PREDICTION OF CONSERVED RNA STRUCTURES WITHIN THE FOOT- LUNCH BREAK 17:00 AND-MOUTH DISEASE VIRUS GENOME REVEALS FUNCTIONAL CIS- Fiona Tulloch ACTING ELEMENTS LOCALISED IN THE 3D CODING REGION EXCHANGES OF GENOMIC DOMAINS BETWEEN POLIOVIRUS TYPE 17:10 2 AND A PANEL OF CO-CIRCULATING SPECIES C ENTEROVIRUSES Mal Bessaud REVEAL A HIGH DEGREE OF GENOMIC PLASTICITY IMPORTANCE OF 5 TERMINAL SEQUENCE IN COXSACKIEVIRUS B3 17:20 RNA IN REGULATING VIRAL RNA REPLICATION AND A SWITCH TO James Flanegan VPG-PRIMED POSITIVE-STRAND INITIATION MISINCORPORATION OF NUCLEOTIDES OR DRUGS DURING 17:30 POLIOVIRUS RNA-DEPENDENT RNA POLYMERASE ELONGATION Nynke Dekker LEAVES FINGERPRINTS IN THE PAUSING KINETICS RNA VIRUS DIVERSITY IS IMPORTANT TO COUNTERACT INNATE 17:40 Raul Andino IMMUNITY AND SPREAD WITHIN THE HOST CONNECTING METAGENOMICS EXPLORATION AND EXPERIMENTAL 17:50 RESEARCH OF PICORNAVIRUSES WITH THE EVOLUTIONARY BASED Alexander Gorbalenya PARTITIONING OF GENOMIC DIVERSITY BY DEMARC 18:05 19:00 3rd POSTER SESSION ( posters of sessions 5,6,7) FREE FOR DINNER Keynote Lecture Communication (12 + 3 min) Short Communication (8 + 2 min) 6 septembre 2016 9 PROGRAMME AT A GLANCE

10 10 WEDNESDAY 07/09/2016 8:45 Session 5 EMERGING/REEMERGING PATHOGENS 13:30 Session 6 ANTIVIRAL STRATEGIES st Chairs: 1 part, Satoshi Koike & Marc Pallansch Chairs: Kimberley Benschop & Katja Wolthers 12:00 15:45 2nd part, Hiroyuki Shimizu & Francis Delpeyroux VIRAL AND HOST FACTORS INVOLVED IN ENTEROVIRUS 71 TOWARDS THE DEVELOPMENT OF HIGHLY POTENT RHINO- AND 08:45 INFECTION POTENTIAL APPLICATIONS IN THERAPY AND Shin-Ru Shih 13:30 Johan Neyts ENTEROVIRUS INHIBITORS PROPHYLAXIS AN ENTEROVIRUS INFECTION MOUSE MODELS TO STUDY ANTIVIRAL ENTEROVIRUS D68 AND ACUTE FLACCID MYELITIS: ASSOCIATION OR 14:05 Els Scheers 09:20 INCIDENTAL FINDING? Steve Oberste THERAPY AND THE POTENTIAL DEVELOPMENT OF RESISTANCE THE TALE OF HOW AN IN SILICO DESIGNED COXSACKIEVIRUS B3 ENTEROVIRUS 71 INFECTION STUDIES IN HUMAN LUNG AND 14:20 PROTEASE INHIBITOR TURNED OUT TO BE A CAPSID BINDER WITH A Rana Abdelnabi 09:35 GUT ORGANOIDS REVEAL A POTENTIAL VIRAL DETERMINANT OF Sabine van der Sanden NOVEL MECHANISM INSTEAD INCREASED SUSCEPTIBILITY A REVERSE GENETIC SYSTEM FOR DEFORMED WING VIRUS, THE 14:35 PROTEIN KINASE D INHIBITORS BLOCK PICORNAVIRUS REPLICATION Anabel Guedan 09:50 MOST IMPORTANT VIRAL PATHOGEN OF HONEY BEES. David Evans CROSS-NEUTRALIZATION OF MULTIPLE POLIOVIRUS TYPES BY 14:50 Scott Dessain COFFEE BREAK HUMAN MONOCLONAL ANTIBODIES COXSACKIEVIRUS B1 INFECTIONS ARE ASSOCIATED WITH INSULIN- EXPLORING THE PERMISSIVITY OF GENETIC EXCHANGES IN THE 2C 10:35 DRIVEN AUTOIMMUNE PROCESS IN CHILDREN WITH INCREASED Amirbabak Sioofy Khojine 15:05 REGION OF RHINOVIRUS TO ESTABLISH A MODEL OF INFECTION FOR Carmen Mirabelli RISK OF TYPE 1 DIABETES IN FINLAND (DIPP STUDY) RV-C ARE ENTEROVIRUSES ASSOCIATED WITH TYPE 1 DIABETES? TARGETING THE HOST: ROLE OF N-MYRISTOYLTRANSFERASES IN 10:50 Richard Lloyd 15:15 Irena Corbic Ramljak REPORTS FROM THE NPOD-V CONSORTIUM AND THE TEDDY STUDY THE INFECTIVITY OF COXSACKIEVIRUS B3 (CVB3) PROGRAMME AT A GLANCE OUTBREAK OF TYPE 1 VACCINE-DERIVED POLIOVIRUS IN LAO THERMOSTABLE, IMMUNOGENIC POLIOVIRUS VLPS OF ALL THREE 11:05 Hiroyuki Shimizu 15:25 Helen Fox PEOPLE'S DEMOCRATIC REPUBLIC IN 2015-2016 SEROTYPES DETECTION OF ENTEROVIRUS D68 BASED ON THE NATIONAL HUMAN MONOCLONAL ANTBODIES AS POTENTIAL THERAPEUTIC 15:35 Konstantin Chumakov 11:15 EPIDEMIOLOGICAL SURVEILLANCE OF INFECTIOUS DISEASES Hitomi Kinoshita AGENTS AGAINST POLIOMYELITIS SYSTEM IN JAPAN FROM 2005 TO 2015 COFFEE BREAK AMBULATORY PAEDIATRIC SURVEILLANCE OF HAND, FOOT AND 11:25 MOUTH DISEASE: OUTBREAK OF CV-A6 INFECTIONS IN FRANCE, Audrey Mirand 15:40 17:00 4th POSTER SESSION ( posters of sessions 5,6,7) 2014-2015 19:00 APERITIF AND GALA DINNER WITH SPECIAL SWISS ATHMOSPHERE COXSACKIEVIRUS B1 IS ASSOCIATED WITH TYPE 1 DIABETES 11:35 RESULTS FROM VIRUS ANTIBODY SURVEY IN DIFFERENT EUROPEAN Amirbabak Sioofy Khojine POPULATIONS LUNCH BREAK Keynote Lecture Communication (12 + 3 min) Short Communication (8 + 2 min) 6 septembre 2016 Europic 2016

11 Europic 2016 THURSDAY 08/09/2016 8:45 Session 7 VACCINES, DISEASE PREVENTION AND CONTROL, ERADICATION Chairs: David Rowland & Andrew Macadam 12:30 CAN NEW VACCINES HELP MAINTAIN A POLIO-FREE WORLD AFTER 08:45 Andrew Macadam ERADICATION? THE VP1 AMINO ACID RESIDUE 145 OF EV71 IS A VIRULENCE 09:10 Ken Fujii DETERMINANT IN SCARB2-DEPENDENT INFECTION MOLECULAR BASIS OF POLIOVIRUS VACCINE ATTENUATION IN NON- 09:20 HUMAN PRIMATES AND POLIOVIRUS-SUSCEPTIBLE TRANSGENIC Begona Valdazo-Gonzalez MICE ASSESSED BY DEEP SEQUENCING ANALYSIS 09:30 VECOTRED VACCINE AGAINST POLIOMYELITIS Ekaterina Viktorova PLANT-MADE POLIOVIRUS-LIKE PARTICLES: DEVELOPING A 09:40 Johanna Marsian SYNTHETIC POLIO VACCINE PHENOTYPE OF CONGO-2010 POLIOVIRUS THAT IS POORLY 09:50 NEUTRALIZABLE BY SERA FROM VACCINE RECIPIENTS SUGGESTS Alexander Lukashev THE REASONS FOR ITS EMERGENCE AND EXTINCTION NOVEL HIS-TAG MARKER FOOT-AND-MOUTH DISEASE VIRUS 10:00 VACCINE BOUND TO NANOLIPOPROTEIN ADJUVANT VIA METAL IONS: Elizabeth Rieder UTILITY ON VACCINE AND DIAGNOSTIC DEVELOPMENTS EUROPEAN NON-POLIO ENTEROVIRUS SURVEILLANCE AND 10:10 LABORATORY DETECTION ARE WE PREPARED TO DETECT AN Heli Harvala ENTEROVIRUS OUTBREAK? ENVIRONMENTAL ENTEROVIRUS SURVEILLANCE IN THE 10:20 Kimberley Benschop NETHERLANDS: POLIO AND BEYOND COFFEE BREAK 11:00 MEETING REPRISE Ann Palmenberg ALBERT SABIN LECTURE 11:45 James Gern DETERMINANTS OF RHINOVIRUS ILLNESS SEVERITY SELECTED VISITS OR DEPARTURE WITH LUNCH BOXES Keynote Lecture Sabin Lecture Short Communication (8 + 2 min) 6 septembre 2016 11 PROGRAMME AT A GLANCE

12 MONDAY 05/09/2016 9:00 Session 1 PICORNAVIRUS STRUCTURE AND LIFE CYCLE Chairs: 1st part, Bert Semler & Nihal Altan-Bonnet 12:35 2nd part, Toby Tuthill & Eckard Wimmer K01: OLD AND NEW THOUGHTS ABOUT THE REPLICATION AND ASSEMBLY OF Eckard Wimmer (Stony Brook 09:00 POLIOVIRUS university, USA) A01: ATOMIC STRUCTURE OF A RHINOVIRUS C, A VIRUS SPECIES LINKED TO Yue Liu (Purdue University, 09:35 CHILDHOOD ASTHMA EXACERBATIONS United States of America) Peter Stockley (University of 09:50 A02: GENOMIC RNA FOLDING MEDIATES ASSEMBLY OF HUMAN PARECHOVIRUS-1. Leeds, United Kingdom) A03: VIRUS QUASISPECIES REVEALS RNA STRUCTURES REQUIRED FOR GENOME Toby Tuthill (The Pirbright 10:05 PACKAGING. Instituten United Kingdom) Rebecca Chandler-Bostock 10:11 A04: INVESTIGATING THE ASSEMBLY MECHANISM OF ENTEROVIRUSES (University of Leeds, United Kingdom) A05: FOOT-AND-MOUTH DISEASE VIRUS REPLICATES INDEPENDENTLY OF Stephen Berryman (Pirbright 10:17 PHOSPHATIDYLINOSITOL 4-PHOSPHATE AND TYPE III PHOSPHATIDYLINOSITOL-4- Institute, United Kingdom) KINASES COFFEE BREAK Kevin L. McKnight (University of 11:00 A07: PROTEIN COMPOSITION OF THE QUASI-ENVELOPE OF HEPATITIS A VIRUS North Carolina, United States of America) A08: RHINOVIRUS SUPERINFECTION EXCLUSION IS INDEPENDENT OF VIRUS Robert Witte (University of 11:15 ENTRY AND REQUIRES REPLICATION Zurich, Switzerland) Lindsey Organtini (Penn State A09: CRYO-EM STRUCTURES OF HONEY BEE DEFORMED WING VIRUS REVEAL 11:30 CONFORMATIONAL CHANGES LINKED TO GENOME RELEASE Hershey College of Medicine, United States of America) A10: A CYSTEINE RESIDUE IN THE RHINOVIRUS-A16 CAPSID HAS KEY FUNCTIONS Urs Greber (University of Zurich, 11:45 IN VIRUS ENTRY Switzerland) A11: EFFECTS OF A LARGE DELETION IN NON-STRUCTURAL PROTEIN 3A ON Emma Howes (The Pirbright 11:51 FMDV REPLICATION Institute, United Kingdom) A12: REDUNDANCY WITHIN THE TANDEM 3B REPEATS IN FOOT AND MOUTH Morgan Herod (University of 11:57 DISEASE VIRUS REPLICATION Leeds, United Kingdom) Nihal Altan-Bonnet (NHLBI, K02: INTERCELLULAR TRANSMISSION OF RNA VIRUS POPULATIONS AND ITS 12:00 National Institutes of Health, IMPACT ON VIRAL FITNESS United States of America) LUNCH BREAK 12 Keynote Lecture Communication (12 + 3 min) Shotgun presentation (5 + 1 min) Europic 2016

13 MONDAY 05/09/2016 POSTER SESSION 1 - PICORNAVIRUS STRUCTURE ANDLIFE CYCLE A13: THE NOVEL ASYMMETRIC ENTRY INTERMEDIATE OF A PICORNAVIRUS CAPTURED WITH NANODISCS Susan Hafenstein (Penn State College of Medicine, United States of America) A14: CONSERVED ELEMENTS WITHIN THE GENOME OF FOOT-AND MOUTH DISEASE VIRUS; THEIR INFLUENCE ON VIRUS REPLICATION Jonas Kjr (DTU Vet, Denmark) A15: A SINGLE AMINO ACID SUBSTITUTION IN POLIOVIRUS NONSTRUCTURAL PROTEIN 2CATPASE CAUSES CONDITIONAL DEFECTS IN ENCAPSIDATION AND UNCOATING Emmanuel Asare (Stony Brook University, United States of America) A17: DEFINING THE ROLE OF FOOT-AND-MOUTH DISEASE VIRUS 2B PROTEIN IN THE VIRAL GENOME REPLICATION COMPLEX Eleni-Anna Loundras (University of Leeds, United Kingdom) A18: CHARACTERISATION OF MONOCLONAL ANTIBODY RESISTANT POLIOVIRUS MUTANTS BY DEEP SEQUENCING ANALYSIS Bethany Charlton (National Institute of Biological Standards and Control, United Kingdom) A19: INSIDE OUT FOOT-AND-MOUTH DISEASE VIRUS (FMDV) PARTICLES FROM DISSOCIATED PENTAMERS Toby Tuthill (University of Oxford, United Kingdom) A20: PICORNAVIRUS CAPSID PROTEIN VP4: ROLE IN CELL ENTRY AND TARGET FOR STRATEGIES TO PREVENT INFECTION Jessica Swanson (The Pirbright Institute, United Kingdom) A22: DISSECTING HUMAN PARECHOVIRUS STRUCTURE AT NEAR-ATOMIC LEVEL Shabih Shakeel (University of Helsinki, Finland) A23: MODES OF INTERACTION BETWEEN FOOT-AND-MOUTH DISEASE VIRUS AND INTEGRIN RECEPTOR VISUALISED AT HIGH RESOLUTION BY CRYOEM Abhay Kotecha (University of Oxford, United Kingdom) A24: IN VITRO CONDITIONS AFFECTING TO UNCOATING OF ECHOVIRUS 1 Visa Ruokolainen (University of Jyvskyl, Finland) A25: CHARACTERIZATION OF NOVEL PAN-COXSACKIEVIRUS 2A AND 3C ANTIBODIES Emma Svedin (The Center for Infectious Medicine, Department of Medicine HS, Karolinska Institutet, Sweden) A26: STRUCTURE AND GENOME RELEASE OF HONEYBEE VIRUSES Pavel Plevka (CEITEC MU, Czech Republic) A27: STRUCTURE OF HUMAN AICHI VIRUS AND IMPLICATIONS FOR RECEPTOR BINDING David Stuart (University of Oxford) A28: INVESTIGATING THE ASSEMBLY MECHANISM OF ENTEROVIRUSES Rebecca Chandler-Bostock A29: FOOT-AND-MOUTH DISEASE VIRUS REPLICATES INDEPENDENTLY OF PHOSPHATIDYLINOSITOL 4-PHOSPHATE AND TYPE III PHOSPHATIDYLINOSITOL-4-KINASES Stephen Berryman A30: EFFECTS OF A LARGE DELETION IN NON-STRUCTURAL PROTEIN 3A ON FMDV REPLICATION Emma Howes A31: REDUNDANCY WITHIN THE TANDEM 3B REPEATS IN FOOT AND MOUTH DISEASE VIRUS REPLICATION Morgan Herod A32: THE VIRAL CAPSID ACCOUNTS FOR THE DIFFERENCES IN TROPISM AND ACID SENSITIVITY OBSERVED BETWEEN RESPIRATORY AND NON RESPIRATORY ENTEROVIRUSES Lna Royston Europic 2016 13

14 MONDAY 05/09/2016 K01 OLD AND NEW THOUGHTS ABOUT THE REPLICATION AND ASSEMBLY OF POLIOVIRUS Yutong Song, Alexander Gorbatsevych, Ying Liu, JoAnn Mugavero, Sam Shen, Emmanuel Asare, Ping Jiang, Aniko V. Paul, Steffen Mueller, and Eckard Wimmer Stony Brook university, USA Using a combination of computer design and chemical synthesis we have generated variants of poliovirus type 1 (Mahoney) (PV1) whose ORF (6,189 nt) carried in Max variants (excess of over-represented favored - synonymous codon pairs) up to 1,304 and in SD variants (randomly scrambled synonymous codons) up to 2,104 synonymous mutations without loss of viability. With one exception (P2Min), Min variants (excess of under-represented unfavored - synonymous codon pairs) are non-viable. P2Min (471/2154 silent mutations in the P2 domain of the ORF) expressed reduced protein and RNA synthesis in HeLa cells at 37 C and was non-viable at 33C and 37.5C. Compared with wt PV1, P2Min displayed a vastly reduced specific infectivity, a phenotype with little comprehension in virology, which we will discuss. We have searched in vain by computer analyses in genomes of wt polio-1, -2, -3, CAV20 and our vastly mutagenized variants (all genomes packaged into PV capsids) for the occurrence of dozens of conserved degenerate short RNA structures (called by us dRSs) that recently have been forcefully proposed as the only important genome signals for +RNA virus assembly. This hypothesis of dozens of degenerative short RNA structures has largely ignored the crucial function of unique packaging signals (PSs) studied for decades for +strand virus and retrovirus assembly. We find that no phase of PV proliferation accommodates the dRS hypothesis: 1. No significant number of any conserved degenerate structures (dRSs) has been found in PV1, -2, -3, CAV20 genomes or in the hugely modified variants, which could solely function as sole triggers or cause of specificity in packaging. 2. Specific packaging is triggered through the recognition between a replication protein and capsid precursors. 3. Assembly depends stringently on genome replication, an observation suggesting that only newly synthesized genomes are packaged. 4. No free genomic RNA in infected cells is packaged. In fact free genomic RNA (eventually engaged in translation) is always being modified through its loss of its VPg. However, although genomes lacking VPg are still infectious, they have never been found in virions. We suggest a two-step mechanism in all +RNA virus assembly: I. Unique RNA/protein or protein/protein signaling essential for the recognition of cognate genome/capsid precursors; II. Co-condensation of genome/capsid- proteins, perhaps involving dRSs. 14 Europic 2016

15 MONDAY 05/09/2016 A01 ATOMIC STRUCTURE OF A RHINOVIRUS C, A VIRUS SPECIES LINKED TO CHILDHOOD ASTHMA EXACERBATIONS Yue Liu1, Marchel Hill2, Thomas Klose1, Zhenguo Chen1, Kelly Watters2, Yury Bochkov2, Ann Palmenberg2, Michael Rossmann1 1 Purdue University, 2University of Wisconsin-Madison Isolates of rhinovirus C (RV-C), a recently identified Enterovirus (EV) species, are the causative agents of mild to severe respiratory infections among children and are linked to childhood asthma exacerbations. The RV-C have been refractory to structure determination because they are difficult to propagate in vitro. In the present work, adaptation of a recombinant RV-C15 virus for tissue culture via serial passage in a transduced HeLa cell line leads to new protocols for improved virus yields. Using cryo-electron microscopy, the atomic structures have been determined of the full virion and native empty particle (NEP) of the derivative RV-C15a. The virus has 60 fingers on the virus outer surface that probably function as dominant immunogens. Since the NEPs also display these fingers, they may have utility as vaccine candidates. A sequence-conserved surface depression adjacent to each finger forms a likely binding site for the sialic acid on the RV-C receptor cadherin related family member 3. The RV-C, unlike other EV, are resistant to capsid-binding antiviral compounds because the hydrophobic pocket in VP1 is filled with multiple bulky residues. These results define potential molecular determinants for designing anti-viral therapeutics and vaccines. Europic 2016 15

16 MONDAY 05/09/2016 A02 GENOMIC RNA FOLDING MEDIATES ASSEMBLY OF HUMAN PARECHOVIRUS-1. Peter Stockley1, Shabih Shakeel2, Simon White3, Eric Dykeman4, David Rowlands3, Joseph Cockburn3, Sarah Butcher2, Reidun Twarock4 1 University of Leeds, 2Institute of Biotechnology and Department of Biosciences, University of Helsinki, Helsinki, Finland., 3Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK., 4Departments of Mathematics and Biology and York Centre for Complex Systems Analysis, University of York, York, UK. Recent EM and X-ray structures of picornaviruses (PVs) that fail to cleave their VP0 polyproteins show that the infectious virion assembles to make 60 RNA-coat protein contacts that surround each five-fold vertex. Recently, we introduced the concept of Packaging Signal (PS) Mediated virion assembly of ssRNA virions that supersedes the previous paradigm of a unique assembly initiation site within the RNA. In PS-mediated reactions multiple sites across the viral genome, that can be sequence degenerate and thus difficult to spot by sequence gazing, act co-operatively to ensure faithful, rapid and complete assembly at low concentrations. Mathematical modeling of this process suggest that virions assembling via this mechanism have distinct selective advantages, and conversely that it may be a widespread phenomenon. We have established the basic features of PS-mediated assembly in model virions from phage to plant viruses. This mechanism makes multiple predictions about the three-dimensional layout of virions that have subsequently been confirmed either by us or others, leaving little doubt that it is an important aspect of assembly processes. The structures of PVs showing multiple RNA-CP contacts suggest that they have assembled via this route but PVs, and especially enteroviruses such as polio, have been intensively studied and no RNA packaging signals have been detected. In order to examine whether the RNA-CP contacts in Human Parechovirus-1 correspond to PSs, we used RNA SELEX and bioinformatics to identify CP binding RNA motifs, comparing these sequences with the genome. This reveals matches at multiple sites across the genome, putative PSs, that appear conserved in aspects of sequence, secondary structure (stem-loop) and genomic location across known HPEV-1 strains. The loop consensus sequence fits the RNA density of the X-ray structure perfectly making sequence-specific hydrogen bonds with CP residues. RNA oligos encompassing single PS sites trigger sequence/structure specific self-association of HPEV-1 CP pentamers in vitro but do not yield virus-like particles (VLPs). Nor are VLPs produced when the entire genome is titrated with pentamer despite containing the sequences of all the PSs. We conclude that RNA PSs do exist in this class of PVs but assembly must involve the correctly folded RNA genome, perhaps as it emerges from the replicase and in concert with the virally encoded 2C system. I will report our latest experiments aimed at testing these ideas. References: Dykeman EC, Stockley PG, Twarock R. (2014) Solving a Levinthals paradox for virus assembly identifies a unique antiviral strategy. PNAS 111 5361-5366. Patel et al. (2015) Revea the density of encoded functions in a viral RNA. PNAS 112 2227-2232. 16 Europic 2016

17 MONDAY 05/09/2016 A03 VIRUS QUASISPECIES REVEALS RNA STRUCTURES REQUIRED FOR GENOME PACKAGING. Grace Logan1, Joseph Newman1, Caroline F. Wright1, Lidia Lasecka1, Don P. King1, Dan T. Haydon2, Eleanor M. Cottam3, Toby J. Tuthill1 1 The Pirbright Institute, 2The University of Glasgow, 3General Bioinformatics Many viruses protect their genomes by packaging them into an outer capsid or shell of virus-encoded proteins. Folding and packaging of viral genomic material into the assembling capsid has been demonstrated as a target for antiviral strategies for significant pathogens from diverse viral families. Packaging and capsid assembly in RNA viruses may involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and humans. In the picornavirus family of mammalian RNA viruses, requirements for packaging remain controversial. A packaging signal comprising an RNA stem loop has been previously identified for one virus in the family, Aichi virus and direct interactions between capsid and RNA are suggested in the structures of several other picornaviruses. In contrast, no packaging signals have been found in poliovirus and interaction between genome and capsid has instead been shown to be mediated via the non structural protein 2C. Here we show a novel and simple approach to identify RNA packaging signals in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data of viral quasispecies from both packaged and unpackaged RNA we have determined twelve regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study improves understanding of RNA virus packaging and confirms a new target for antivirals. These approaches offer a readily transferable methodology for identifying packaging requirements in many other viruses. Europic 2016 17

18 MONDAY 05/09/2016 A04 & POSTER A28 INVESTIGATING THE ASSEMBLY MECHANISM OF ENTEROVIRUSES Rebecca Chandler-Bostock1, Bo Meng2, German Leonov3, Simon White1, Oluwapelumi Adeyemi1, Toby Tuthill4, Reidun Twarock3, David Rowlands1, Peter Stockley1 1 University of Leeds, 2University of Cambridge, 3University of York, 4Pirbright Institute There is now extensive evidence that picornaviruses (PVs) that do not cleave their VP0 polyprotein, such as human parechovirus (HPeV), Ljungan virus and Aichi virus, assemble infectious virions in which sixty RNA-coat protein (CP) contacts are made (Sasaki et al, 2003; Seitsonen et al, 2010; Zhu et al, 2015). For HPeV, SELEX and bioinformatic analysis suggest that these RNA-CP contacts are sequence-specific, involving short sequence motifs that can vary across the sixty copies. This situation is entirely consistent with predictions based on assembly via multiple, degenerate RNA packaging signals (PSs) which we have shown to occur in many viruses, and replaces the more classical, single PS assembly initiation idea (Dykeman et al, 2014; Patel et al, 2015). This contrasts with extensive evidence against any role for RNA in assembly of enteroviruses that do mature their VP0 proteins. In order to explore whether these two groups of viruses use distinct assembly pathways or whether the roles of RNA PSs are obscured in enteroviruses, we have applied similar techniques to poliovirus and enterovirus E (EV-E). We have shown that SELEX against poliovirus uncleaved CP protomer yields sequences that match multiple sites within the poliovirus genome, i.e. putative PS sites. Some of these genomic sites trigger partial self-association of poliovirus capsomers, behaviour we have shown also occurs in HPeV. Neither virus assembles virus-like particles in the presence of full-length genomic RNA, highlighting the need for RNA chaperones to mediate the RNA folding for efficient assembly. Many of these viruses naturally produce empty particles with uncleaved VP0. In vitro reassembly of EV-E yields extremely stable dodecahedral empty paticles (Li et al, 2012). However we have shown that empty particles assembled in vivo are unstable, can be recycled into mature virions and therefore act as intermediates in virion formation. 18 Europic 2016

19 MONDAY 05/09/2016 A05 & POSTER A29 FOOT-AND-MOUTH DISEASE VIRUS REPLICATES INDEPENDENTLY OF PHOSPHATIDYLINOSITOL 4-PHOSPHATE AND TYPE III PHOSPHATIDYLINOSITOL-4- KINASES Stephen Berryman1, Katy Moffat1, Christian Harak2, Volker Lohmann2, Terry Jackson1 1 Pirbright Institute, 2University of Heidelberg Picornaviruses form replication complexes in association with membranes in structures called replication organelles (RO). Common themes to emerge from studies of picornavirus replication are the need for cholesterol and phosphatidylinositol 4-phosphate (PI4P). In infected cells, type III phosphatidylinositol-4-kinases (PI4KIII) generate elevated levels of PI4P, which is then exchanged for cholesterol at replication organelles by oxysterol- binding protein (OSBP). For the enteroviruses, replication organelles form at Golgi membranes in a process that utilises PI4KIIIb. Other picornaviruses, for example the cardioviruses, are believed to initiate replication at the ER and subvert PI4KIIIa to generate PI4P. Here we have used a variety of techniques including chemical inhibitors, PI4KIII knockout cell lines, and quantification and visualisation of PI4P by confocal microscopy, to investigate the role of type III PI4K and PI4P in FMDV replication. Our results show that, in contrast to the enteroviruses and cardioviruses, FMDV replication is independent of PI4KIII (PI4KIIIa and PI4KIIIb), and PI4P levels do not increase in FMDV-infected cells and PI4P is not seen at RO. Furthermore, preliminary experiments using inhibitors of cholesterol synthesis and transport suggest that, consistent with PI4P-independent replication, FMDV infection does not require OSBP and may be less dependent on cholesterol than other picornaviruses, pointing to a unique requirement towards lipids at the FMDV replication membranes. Europic 2016 19

20 MONDAY 05/09/2016 A07 PROTEIN COMPOSITION OF THE QUASI-ENVELOPE OF HEPATITIS A VIRUS Kevin L. McKnight1, Ling Xie1, Olga Gonzalez-Lopez1, Zongdi Feng2, Xian Chen1, Stanley M. Lemon1 1 University of North Carolina, 2Research Institute at Nationwide Childrens Hospital OBJECTIVES: Human hepatitis A virus (HAV) is released in a noncytolytic fashion from infected cells both in vitro and in vivo as membrane-wrapped, quasi-enveloped virions (eHAV). These virions are completely cloaked in host-derived membranes that protect the capsid from neutralizing antibody, but nonetheless have specific infectivity similar to naked, nonenveloped virus particles. Several lines of evidence suggest that the biogenesis of eHAV is dependent upon components of the endosomal sorting complex required for transport (ESCRT), but the protein composition of the membranes surrounding the quasi-enveloped capsid is unknown. Our goal in this study was to define the host protein composition of the eHAV particle. METHODS: We adopted a quantitative proteomics approach incorporating amino acid-coded tagging with stable isotopes (AACT/SILAC) to distinguish eHAV-associated proteins from proteins associated with exosomes similar in size and density to eHAV. Low-passage, noncytopathic HM175/p16 virus released into supernatant fluids from infected Huh-7.5 cells maintained in exosome-free 13C6-Lys/13C6-Arg heavy media was concentrated by ultracentrifugation and mixed with a similar concentrate from mock-infected cells cultured in parallel in 12C6- Lys/12C6-Arg light media (or vice versa) prior to purification by isopycnic ultracentrifugation in an iodixanol gradient. eHAV-containing gradient fractions were identified by RT-PCR for HAV RNA and either subjected to SDS- PAGE and in gel digestion or directly proteolytically digested in solution prior to identification of peptide fragments by mass spectrometry. RESULTS: Between 231 and 439 host proteins were identified by the presence of at least one unique peptide in 4 independent labeling experiments. When peptides were ranked according to intensity of heavy vs. light isotopes, substantial skewing was evident in all 4 samples based on the cell culture of origin (heavy vs. light media), consistent with significant enrichment of the samples for eHAV vs. nonviral exosomes. Peptide coverage for structural HAV proteins was 49.8% whereas no peptides were identified from nonstructural proteins. Peptides spanning VP4-VP2 and the VP1pX cleavage were present, consistent with the presence of some empty capsids and previous studies demonstrating that VP1pX is uncleaved in eHAV particles (Feng et al. Nature 496:367-71, 2013). Based on relative heavy/light isotype peptide intensities, 30 host proteins were predicted to be eHAV- associated in each of the 4 labeling experiments; 83 proteins were so identified in at least 3 of 4 experiments. Eighty of these 83 proteins (96%) are among the 2911 previously reported exosome proteins in the Vesiclepedia database (www.microvesicles.org); 63% are components of lysosomes, 34% of mitochondria, 72% of cytoplasm, whereas 9% are cell surface, 10% endosome, and 18 % Golgi apparatus components (p

21 MONDAY 05/09/2016 A08 RHINOVIRUS SUPERINFECTION EXCLUSION IS INDEPENDENT OF VIRUS ENTRY AND REQUIRES REPLICATION Robert Witte, Artur Yakimovich, Fanny Georgi, Vardan Andriasyan, Urs Greber University of Zurich Rhinoviruses (RVs) of the species enterovirus comprise more than 150 known genotypes. RV infections occur frequently, and lead to significant economic loss and potentially lethal exacerbations in patients with chronic obstructive pulmonary disease or asthmatic conditions. The large number of different genotypes and infections makes sporadic co-infection events possible. We investigated whether simultaneous infection of cell cultures with multiple RV genotypes affects virus propagation and spreading. We infected cell cultures with one or two genotypes at very low multiplicity of infection, and analyzed viral spreading by measurement of plaques, defined as a group of cells infected from a single founder cell. We found that different genotypes showed very strong competition which resulted in almost perfect exclusion of propagating plaques, a process so far undescribed for picornaviruses. This phenotype is different to other viruses, such as Adenovirus, where co-infections show only mild exclusion or even synergistic spreading effects within the first 12 hours of infection. The exclusion of RV infections occurred for genotypes which utilize the same receptor, as well as genotypes that bind and enter cells through different receptors. Further studies indicated that exclusion did not occur at the level of entry. We investigated the exclusion phenotype by fluorescence microscopy at different time points, and showed that exclusion occurs between 2 and 4 hours post infection. Using compounds that block the replication of only one genotype we could show that initiation of replication is necessary to establish the exclusion phenotype. We conclude that the exclusion effect is virus mediated and not dependent on a cellular response to incoming virus. Furthermore exclusion occurs prior to the exponential replication phase, making it unlikely that exhaustion of essential factors for genome amplification is involved in exclusion. Previous work has shown that the formation of a replication compartment involves components from several different cell organelles, and is sensitive to interference with drugs targeting lipid metabolism or cellular trafficking which reduces the levels of lipid species that are normally upregulated in RV replication phase. We propose that RV infection exclusion is a side effect of competition for essential resources for the formation of a replication compartment, and are currently investigating the nature of one or several limiting cellular components. Our data suggest that replication compartments differ between genotypes in a non-redundant manner, or that their composition is functionally linked to genotype- specific replication steps. We will test the hypothesis that one RV genotype exerts metabolic effects on the host cells and thereby precludes the proper formation of the replication compartment for a competing genotype. We plan to carry out genetic and metabolic supplementation studies in order to override the blocking effects of one virus on the competing virus. Europic 2016 21

22 MONDAY 05/09/2016 A09 CRYO-EM STRUCTURES OF HONEY BEE DEFORMED WING VIRUS REVEAL CONFORMATIONAL CHANGES LINKED TO GENOME RELEASE Lindsey Organtini1, Kristin Shingler1, Robert Ashley1, Elizabeth Capaldi2, Kulsoom Durrani1, Kelly Dryden3, Alexander Mahkov4, James Conway4, Marie Pizzorno2, Susan Hafenstein1 1 Penn State Hershey College of Medicine, 2Bucknell University, 3University of Virginia, 4University of Pittsburg School of Medicine Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported since 2006. The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the life cycle of DWV or its molecular and structural details. Here we report the first structures of DWV capsids isolated from infected adult worker bees and solved by cryo-EM, including the immature procapsid, infectious virion capsid, and the empty capsid after genome release. The capsids are decorated by large spikes around the five-fold vertices. For the procapsid and genome filled capsids the five-fold spikes had an open- flower like conformation whereas the empty particles that have released genome had a close-flower like spike conformation. Between the two conformations the spikes undergo a significant hinge-like movement that we predict using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, must change conformation to release the genome, and that genome escapes from a five-fold vertex to initiate infection. Finally the structures illustrate that DWV has a lifecycle similar to picornaviruses. 22 Europic 2016

23 MONDAY 05/09/2016 A10 A CYSTEINE RESIDUE IN THE RHINOVIRUS-A16 CAPSID HAS KEY FUNCTIONS IN VIRUS ENTRY Urs Greber1, Daria Seiler1, Simon Gndinger1, Mark Ltzerich2, Pascal Roulin1, Andreas Jurgeit3, Peer Mittl1, Oleg Georgiev1, Peer Mittl1 1 University of Zurich, 2University of Zurich / The Hussman Institute, 3Merck Darmstadt Rhinoviruses (RV) cause the common cold, asthma exacerbations and severe respiratory disease, and are a major health concern. Virus entry into cells and uncoating are essential for infection and disease. Uncoating of non-enveloped viruses, such as RV, depends on cues from the host, which act in a time and space-controlled manner during cell entry (1). Specifically, uncoating of RV depends on virus binding to a cell surface receptor, such as intercellular adhesion molecule-1 (ICAM-1), low density lipoprotein receptor (LDLR) and related proteins, or cadherin-related family member 3 (CDHR3). Downstream steps in endocytosis expose the virion to chemical cues, which may complement effects from receptor binding and result in conformational changes of the virion and genome uncoating. For example, exposure of RV-A2 to low pH (or high temperatures of 50-56C) in vitro leads to extrusion of the RNA genome and yields empty capsids (B-particles), which have an expanded structure compared to native virions (2). B-particles of RV-A2 also contain a disulfide bond between cysteine C298 and C450 of the viral polyprotein, corresponding to C296 and C450 of RV-A16, respectively. This disulfide links the viral protein (VP) 2 and VP3. Here we explored the function of cysteine residues in RV-A16 and RV-A1A in virus entry. Treating isolated A16 or A1A virions with thiol targeting reagents, such as N-ethylmaleimide (NEM) or para- chloromercury benzoate (PCMB) severely reduced infectivity. PCMB inhibited virus uncoating, as indicated by the neutral red / white light infection assay, where virions grown in presence of the RNA binding dye neutral red are inactivated by white light, unless their RNA has dissociated from the capsid and hence, the dye separated from the RNA (3, 4). PCMB did not impair RV-A16 binding to cells, as indicated by 35S-methionine labeled virions. About half of the cysteine residues were found to be modified by alkylating agents,targeting C450 and three highly conserved Cys, as indicated by the results obtained from high performance liquid chromatography combined with tandem mass spectrometry (LC/MS/MS). Mutagenesis of the highly conserved Cys residues gave no infectious virions, but the C450V mutant was viable, and it was resistant to inactivation by PCMB, albeit at a fitness cost. We conclude that the free thiol group of C450 in VP3 is key for RV-A entry. C450, which is conserved in RV-A16 and RV-A1A may form a disulfide bond with C296, and thereby promote the release of the viral RNA from the capsid. Literature 1. Yamauchi Y, Greber UF. 2016. Traffic 17: 569-92 2. Garriga D, et al. 2012. PLoS Pathog 8: e1002473 3. Mandel B. 1967. Virology 31: 702-12 4. Conti C, et al. 1992. Antimicrob Agents Chemother 36: 95-9 Europic 2016 23

24 MONDAY 05/09/2016 A11 & POSTER A30 EFFECTS OF A LARGE DELETION IN NON-STRUCTURAL PROTEIN 3A ON FMDV REPLICATION Emma Howes1, Caroline Wright1, Begoa Valdazo-Gonzlez1, Kris De Clercq2, Annebel De Vleeschauwer2, Sarah Gold1, Toby Tuthill1, Donald King1, Martin Ryan3, Terry Jackson1 1 The Pirbright Institute, 2CODA-CERVA, Unit Vesicular Exotic diseases, 3University of St Andrews OBJECTIVES: The non-structural protein 3A of Foot-and-Mouth Disease Virus (FMDV) is much longer than in other picornaviruses, 153 amino acids compared to just 87 amino acids in poliovirus. Naturally occurring deletions in 3A (of approximately 10 amino acids) have previously been discovered and appear to attenuate the viruss ability to infect cattle and grow in bovine cells in vitro, while remaining infectious to pigs and capable of growing in porcine cell lines, therefore implying FMDV 3A may have a role in determining viral host range. This work investigates effects of a much larger naturally occurring deletion in the C terminal region of 3A of 57 amino acids. The deletion was identified as part of a mixed viral population, and its effects on replication and ability to survive independently of virus containing full length 3A investigated in different host cell lines. METHODS: The 57 amino acid deletion found in 3A (a.a 81-137) was introduced in an FMDV sub-genomic replicon with GFP reporter gene. Replicon RNA was transfected into BHK (hamster), MDBK (bovine) and SK-RST (porcine) cells and replication measured based on GFP expression. The virus with this deletion was also plaque purified from the original mixed population. BHK, MDBK and IBRS2 cells were infected to establish if the variation with a deletion in 3A was able to survive alone and measure rate of virus growth compared to that of viruses with full length 3A. RESULTS: Replicon assays showed that replication in BHK and porcine cells was unaffected with GFP intensity matching that produced by a replicon with wild type 3A. However in bovine cells replication was completely attenuated. Assays measuring CPE with the plaque purified 3A deletion virus showed a similar pattern, in that CPE appeared sooner in BHK and porcine cell lines but was slower and had a smaller effect in bovine cells. Similar assays to look at rate of CPE appearance are also being carried out using a form of 3A deleted virus recovered from replacing the reporter gene of the replicon with the FMDV capsid proteins. CONCLUSIONS: These results suggest that full length 3A is required for FMDV to replicate efficiently in bovine cells, but that it remains able to replicate efficiently in porcine cells when missing a large amount of the C terminal domain, supporting the idea of a possible role for FMDV 3A in viral host range. 24 Europic 2016

25 MONDAY 05/09/2016 A12 & POSTER A31 REDUNDANCY WITHIN THE TANDEM 3B REPEATS IN FOOT AND MOUTH DISEASE VIRUS REPLICATION Morgan Herod1, Sarah Gold2, Lidia Lasecka2, Caroline Wright2, Tobias Tuthill2, David Rowlands1, Nicola Stonehouse1 1 University of Leeds, 2The Pirbright Institute OBJECTIVES: The short 3B peptide of the Picornaviridae undergoes 3D-mediated uridylation, generating VPg- pUpU, which acts as the protein primer for viral RNA replication. Uniquely among the Picornaviridae, foot-and- mouth disease virus (FMDV) encodes three non-identical copies of the 3B peptide in tandem (3B1, 3B2 and 3B3, respectively). Limited studies previously suggested the importance of 3B3 in viral replication, however, the functional roles of the multiple copies of 3B have not been elucidated. We therefore investigated the roles of the multiple copies of 3B in FMDV viral replication. METHODS: We employed both a FMDV replicon system and full-length virus to investigate the effects of 3B deletion and functional mutations on viral replication in a variety of host cell lines. Subsequent complementation studies were utilised to probe the function of 3B-containing polyprotein precursors in viral RNA replication. Finally, the effect of 3B and 3BC boundary mutations on polyprotein processing were investigated by in vitro pulse-chase analysis. RESULTS: Studies with mutant replicons suggest that the 3B repeats are largely redundant for viral RNA replication. However, the presence of all three functional copies of 3B permits a maximal rate of replication. There appears to be no additional effects of 3B in determining host species specificity or the production of virus particles. Furthermore, we identified residues at the C-terminus of 3B3, specifically at the 3BC boundary, which are important for viral RNA replication. Our data suggest that these residues are not required for 3B to act as a primer for replication, but that conservation of the 3BC boundary sequence is essential for providing an active 3Dpol molecule in trans for viral replication. Subsequently, 3B molecules are provided by a precursor which does not contain functional 3Dpol, suggesting intermolecular 3B uridylation. Work is in progress to investigate the effects of 3BC boundary mutations on the processing of the P3 polyprotein precursor. CONCLUSION: Our observations suggest that the sequence of the 3BC boundary, and therefore the relative rate of 3BC cleavage, is essential for supplying functional 3Dpol to the viral replication complex. These data support a model where separate molecules of the P3 precursor subsequently supply 3B in trans for replication so involving intermolecular 3B uridylation. These results, in combination with our previously published studies, start to build an understanding of how multiple, separate, non-structural protein precursors contribute to FMDV RNA replication. Europic 2016 25

26 MONDAY 05/09/2016 K02 INTERCELLULAR TRANSMISSION OF RNA VIRUS POPULATIONS AND ITS IMPACT ON VIRAL FITNESS Nihal Altan-Bonnet1, Marianita Santiana1, Yael Mutsafi1, Ying-Han Chen1 1 Laboratory of Host pathogen-Dynamics, Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Intercellular transmission of RNA virus populations and its impact on viral fitness. We recently discovered an entirely novel type of viral transmission whereby enteroviruses are transmitted as populations, instead of as single viral particles, among cells (Chen YH et al., Cell 160(4):619-30 2015). We found this type of infectivity to be mediated by phosphatidylserine-enriched vesicles derived from host membranes. This mode of transmission, due to its essentially quantal nature, delivers large quantities of viral genomes simultaneously into the cytoplasm of susceptible host cells. We find that this results in very high multiplicities of infection that both enhance viral replication and modulate the host immune system. We discuss the impact of these findings on viral pathogenesis. 26 Europic 2016

27 MONDAY 05/09/2016 A13 THE NOVEL ASYMMETRIC ENTRY INTERMEDIATE OF A PICORNAVIRUS CAPTURED WITH NANODISCS Susan Hafenstein1, Hyunwook Lee2, Kristin Shingler2, Lindsey Organtini2, Robert Ashley2, Alexander Makhov3, James Conway3 1 Penn State College of Medicine, 2Penn State COM, 3University of Pittsburgh Many non-enveloped viruses engage host receptors that initiate capsid conformational changes necessary for genome release. Structural studies on mechanisms of picornavirus entry have relied on in vitro approaches of virus incubated at high temperatures or with excess receptor molecules in order to trigger the entry intermediate, or A-particle. We have induced the coxsackievirus B3 entry intermediate by triggering the virus with full-length receptors embedded in lipid bilayer nanodiscs. These asymmetrically formed A-particles were reconstructed using cryo-electron microscopy and a direct electron detector. We present the first high-resolution structures of a picornavirus entry intermediate captured at a membrane with and without imposing icosahedral symmetry (3.9 and 7.8 , respectively). The asymmetric receptor binding triggers global capsid expansion and local conformational changes at the site of interaction. Viral proteins extrude from the capsid at the site of extensive protein remodeling adjacent to the nanodisc. Thus, the binding of receptor triggers formation of a unique site in preparation for genome release. Europic 2016 27

28 MONDAY 05/09/2016 A14 CONSERVED ELEMENTS WITHIN THE GENOME OF FOOT-AND MOUTH DISEASE VIRUS; THEIR INFLUENCE ON VIRUS REPLICATION Jonas Kjr1, Line Dahl Poulsen2, Jeppe Vinther2, Thomas Bruun Rasmussen1, Graham John Belsham1 1 DTU Vet, 2University of Copenhagen OBJECTIVES: Several conserved elements within the genome of foot-and-mouth disease virus (FMDV) have been identified, e.g. the IRES. Such elements can be crucial for the efficient replication of the genomic RNA. Previously, SHAPE analysis of the entire FMDV genome (Poulsen et al., 2016 submitted) has identified a conserved RNA structure within the 3Dpol coding region (the RNA-dependent RNA polymerase) which might have an important role in virus replication. The FMDV 2A peptide, another conserved element, is responsible for the primary cleavage at its own C-terminus (2A/2B junction). It is believed that this cleavage is achieved by ribosomal skipping, in which the 2A peptide prevents the ribosome from linking the next amino acid (aa) to the growing polypeptide. The nature of this cleavage has so far not been investigated in the context of the full-length FMDV RNA within cells. Through reverse genetics, this study aims to identify how these distinct conserved elements influence the replication of FMDV RNA. METHODS: Changes were made within the predicted 3Dpol RNA structure and the 2A peptide coding sequence which were expected to be detrimental for their function. These were: Silent mutations, to disrupt the 3Dpol RNA secondary structure, were generated in a FMDV replicon containing Gaussia Sequence changes encoding selected modifications of the 2A peptide (as described by Donnelly et al., 2001) were introduced into a full-length FMDV cDNA and in a FMDV replicon cDNA containing Gaussia RNA transcripts were generated in vitro from the plasmids, and introduced into BHK cells by electroporation. The replication efficiency was assessed by measurement of luciferase activity or by rescue of mutant viruses. The rescued viruses derived from the 2A mutant cDNAs were passaged 3 times and the rescued RNAs were sequenced. RESULTS: Initial results indicate that 3 different replicon mutants, with the disrupted 3Dpol RNA structure, had very similar RNA replication efficiencies as the wt FMDV replicon. Furthermore, the replicon system showed that the 2A mutants were also able to undergo replication, although at a lower rate than for the wt FMDV replicon. One mutant which previously (Donnelly et al., 2001) was found not to undergo cleavage was still replication competent. Analysis of rescued viruses by sequencing of the third passage revealed that the 2A mutants with the lowest cleavage activity had reverted to the wt but some mutants with defective cleavage activity were viable. CONCLUSIONS: Initial results confirm that efficient cleavage at the 2A/2B junction is required for optimal replication. Rescue of viable mutant viruses with mutants previously characterized as non-cleaving indicates a discrepancy between in vitro and cell-based experiments. Detrimental changes to the 3Dpol RNA structure did not change the replication efficiency in a replicon system. However these results do not eliminate a possible effect of this structure on virus replication; such analyses are in progress. Further study of these two conserved elements should provide additional insights into mechanisms underlying FMDV virus replication. 28 Europic 2016

29 MONDAY 05/09/2016 A15 A SINGLE AMINO ACID SUBSTITUTION IN POLIOVIRUS NONSTRUCTURAL PROTEIN 2CATPASE CAUSES CONDITIONAL DEFECTS IN ENCAPSIDATION AND UNCOATING Emmanuel Asare, Eckard Wimmer, Aniko Paul, Ping Jiang, Joann Mugavero Stony Brook University The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252 of poliovirus 2CATPase interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPase has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near the N252 residue, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive growth phenotypes or had a lethal growth phenotype due to defective RNA replication. A K259A mutation, mapping to an helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33C relative to the wt. In conclusion, we have for the first time linked a cold- sensitive encapsidation defect in 2CATPase (K259A) to a subsequent delay in uncoating of the virus particle at 33C during the next cycle of infection. Europic 2016 29

30 MONDAY 05/09/2016 A17 DEFINING THE ROLE OF FOOT-AND-MOUTH DISEASE VIRUS 2B PROTEIN IN THE VIRAL GENOME REPLICATION COMPLEX Eleni-Anna Loundras, Morgan R. Herod, Joseph C. Ward, Mark Harris, Nicola J. Stonehouse University of Leeds OBJECTIVES: Due to the highly contagious nature of FMDV, research with infectious virus is restricted to a limited number of key facilities worldwide. As a result, the biochemistry of viral genome replication is poorly understood. In particular, although it is implicit that synthesis of both positive and negative sense RNA genomes is undertaken by the 3Dpol enzyme (located within the P3 region of the genome), the roles of the other non-structural proteins remain to be determined. In this study, we focus on the non-structural proteins encoded by the P2 region, particularly 2B. Previous studies have assigned putative roles for 2B as a helicase, as a viroporin, or as being involved in membrane rearrangements. Understanding the details of these functions will enable the development of novel vaccines and antiviral compounds. METHODS: The development of FMDV sub-genomic replicons containing a reporter transgene has allowed for the study of viral genome replication in a non-infectious system and helps elucidate the initial events in replication complex formation, together with the molecular interactions with the complex. Using the sub-genomic replicon system, complementation studies have been undertaken to ascertain the minimum requirements for the recovery of replicons with replication-defective mutations introduced into 3Dpol. The use of helper transcripts containing a series of deletions within the P2 region have been used to probe the role of P2 proteins in genomic replication. RESULTS: We have shown that there is a significant 10x increase above background levels of reporter expression if the lethal 3Dpol mutant is complemented by a replicon containing an active 3Dpol together with the P2 region. In contrast, no complementation is observed with helper transcripts expressing just the P3 precursor or part thereof. Deletion analysis has revealed a critical requirement for the 2B coding sequence. CONCLUSIONS: The results suggest that there are essential interactions between the non-structural proteins in the P2 and P3 regions of the FMDV genome. These interactions appear to be necessary for replication in the context of a sub-genomic replicon. It appears that 2B must be expressed in cis with a functional 3Dpol to be able to recover replication of 3Dpol mutations. 30 Europic 2016

31 MONDAY 05/09/2016 A18 CHARACTERISATION OF MONOCLONAL ANTIBODY RESISTANT POLIOVIRUS MUTANTS BY DEEP SEQUENCING ANALYSIS Bethany Charlton, Javier Martin National Institute of Biological Standards and Control Poliovirus is a human enterovirus responsible for paralytic poliomyelitis and exists as three distinct serotypes. The antigenic regions recognised by neutralising antibodies on the surface of poliovirus have been extensively studied by the characterisation of plaque-purified monoclonal antibody (mAb) resistant viruses. However, the antigenic profile of poliovirus type-2 (PV2) is not fully established, as there are some conflicting results on the location of independent antigenic sites. It is important to fully characterise the antigenic structure of PV2 in order to better understand the impact of poliovirus evolution on antigenic changes in humans and the relevance of such mutations on vaccine immunogenicity/efficacy. This would help the design and characterisation of new vaccines that might still be required for the endgame of poliovirus eradication. OBJECTIVES: This study aimed to use deep sequencing (DS) analysis of mAb-resistant poliovirus mutants as a means to fully refine the antigenic profile of PV2. We hypothesise that this technique will provide a more comprehensive structural profile of PV2, compared to conventional plaque purification techniques. METHODS: Two independent populations of Sabin strain PV2 were generated from purified vaccine seed virus preparations by passage in poliovirus sensitive HEp-2c cells at a multiplicity of infection (MOI) of 0.01 infectious virus per cell. A low MOI avoids phenotypic hiding of mutant genomes. These PV2 populations were challenged with a panel of type-2 specific mAbs. Serial viral dilutions were used to infect HEp-2c cells in the presence of antibody in successive passages. The virus RNA was then extracted and the whole genome amplified using a One-Step RT-PCR system. The viral genomes were purified and processed by DS on the Illumina MiSeq. Single nucleotide polymorphism (SNP) variations from the consensus Sabin type-2 sequence (GenBank AY184220) were analysed for mutations which likely confer antibody resistance. RESULTS: We found that DS techniques can much more sensitively detect mutations in the poliovirus genome: detecting variants present in less than 1% of the sample. This is in comparison to plaque purification techniques which would require the characterisation of numerous plaque-purified viruses before detection of mutations at this level. Results revealed that mutant PV2 populations generated with a type-2 specific antibody contained 14 structural mutations which were likely selected for by antibody pressure, as visualised on a 3D protein model. The mutations mapped to amino acid residues previously located at antigenic sites 3a and 3b, confirming that they are part of a unique antigenic site. The number and proportion of mutants found in antibody-resistant populations varied depending on the MOI in the first passage, which was directly related to the proportion of mutants present in the original population. This was presumably generated by the high viral polymerase error rate. CONCLUSION: This repeatable method provides evidence which is promising to more comprehensively define the antigenic sites of PV2 with our larger panel of mAbs. This would be useful in the post-eradication era of PV2, particularly in characterising circulating vaccine-derived PV2 strains which may reappear in the population and the environment. Europic 2016 31

32 MONDAY 05/09/2016 A19 INSIDE OUT FOOT-AND-MOUTH DISEASE VIRUS (FMDV) PARTICLES FROM DISSOCIATED PENTAMERS Nayab Malik1, Abhay Kotecha1, Sarah Gold2, Amin Asfor2, Jingshan Ren1, Juha Huiskonen1, Tobias Tuthill2, Elizabeth Fry1, David Stuart1 1 University of Oxford, 2The Pirbright Institute, UK FMDV is a highly infectious pathogen, and is one of the biggest hindrances to the international trade of animals and animal products in the world. The virus capsids are unstable at a pH lower than 7 and readily dissociate to release the genome inside the host cell. While there have been several studies into how the enteroviruses undergo uncoating by forming an intermediate particle, this process is more poorly understood for Aphthoviruses and there are no published structures elucidating how FMDV undergoes this transformation to infect cells. To the best of our knowledge, there has been only one paper claiming to show, at low resolution, a disassembly intermediate for another Aphthovirus, Equine Rhinitis A Virus (ERAV)1, and no structures for a dissociated pentamer. Here, we present the medium resolution (5.2 ) structure of an FMDV pentameric subunit assembly formed from acid-dissociated pentamers in the absence of VP4, using cryo-electron microscopy. In this inside-out assembly, the pentamers have associated with each other such that the hydrophobic edges that usually lead to the self assembly of a native capsid, are shielded or stabilised by interactions between the HI loop of VP2 with its symmetry related partners at the 3-fold axis, and between the BC and HI loops of VP3 with the VP2 sheet at the two-fold axis. Overall there are significant large-scale conformational changes in the pentamer. We also present a lower resolution structure of the isolated pentamer (8.2 ), which is extremely similar to the pentamers comprising the inside-out particle and less like those in the native particle. These structural analyses help us understand why the pentamers are unable to re-assemble properly. Taking these data into consideration, we suggest that the ERAV structure mooted to be a disassembly intermediate1 may also have formed from re-assembled dissociated pentamers. Thus there is still no hard structural evidence that Aphthoviruses form an intermediate particle during disassembly. References: 1. S.E.Bakker et al., The limits of structural plasticity in a picornavirus capsid revealed by a massively expanded equine rhinitis A virus particle. J. Virol. 2014 Jun;88(11):6093-9 32 Europic 2016

33 MONDAY 05/09/2016 A20 PICORNAVIRUS CAPSID PROTEIN VP4: ROLE IN CELL ENTRY AND TARGET FOR STRATEGIES TO PREVENT INFECTION Anusha Panjwani1, Jessica Swanson2, Amin Asfor3, David J Rowlands4, Nicola J Stonehouse4, Tobias J Tuthill3 1 The Pirbright Institute, 2The Pirbright Institute, Ash Road, Pirbright, Woking GU24 0NF and School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, 3The Pirbright Institute, Ash Road, Pirbright, Woking GU24 0NF, 4School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT VP4 is a small, myristoylated and conserved internal capsid protein, which is released from the virus during entry to cells. Studies with the enteroviruses poliovirus and human rhinovirus (HRV) by our group and others have demonstrated that VP4 is required for cell entry and interacts with membranes to form size-selective pores, consistent with a role in facilitating genome delivery through the membrane. The aims of the current work are to understand which parts of VP4 are required for membrane activity, to test the ability of antibodies or other molecules to disrupt VP4 function, and to investigate the role of VP4 in more distantly related picornaviruses such as foot-and-mouth disease virus (FMDV). The ability of purified viruses or recombinant VP4 to permeabilise membranes was characterised using liposome dye release assays. Studies with truncated forms of HRV VP4, showed that pore-forming activity is contained in the N-terminus of the protein. Pores formed by this N-terminal membrane-active region of HRV VP4 retained similar properties to pores formed by full length recombinant protein, consistent with a size required for the release of the viral genome. Antibodies to the N-terminus of HRV VP4, and not the C-terminus, had virus-neutralising activity and inhibited virus-induced permeability in model membranes. With FMDV VP4, both N- and C- terminal regions of the protein induced membrane permeability. However, antibodies against the N-terminus of FMDV VP4 also contained virus-neutralising activity and inhibited virus- induced permeability in model membranes, suggesting a generally conserved mechanism for VP4 activity during cell entry of picornaviruses. This study suggests that antibodies against the N-terminus of VP4 can neutralise virus infectivity by interfering with the ability of VP4 to interact with membranes thereby blocking cell entry. VP4 may therefore present a useful antiviral or vaccine target for picornaviruses. Europic 2016 33

34 MONDAY 05/09/2016 A22 DISSECTING HUMAN PARECHOVIRUS STRUCTURE AT NEAR-ATOMIC LEVEL Shabih Shakeel1, Brenda Westerhuis2, Ausra Domanska1, Roman Koning3, Rishi Matadeen4, Abraham Koster3, Arjen Bakker5, Tim Beaumont5, Katja Wolthers2, Sarah Butcher1 1 University of Helsinki, 2Amsterdam Medical Center, 3Leiden University Medical Center, 4Netherlands Centre for Electron Nanoscopy, 5 AIMM Therapeutics Human parechoviruses are emerging pathogens of the Picornaviridae family. Here, we present a 4 resolution structure of human parechovirus 3 (HPeV3) using electron cryo-microscopy and single particle analysis. HPeV3 is one of the most medically relevant virus among the parechoviruses and is known to cause neonatal sepsis in infants. Currently, there are no therapies available to combat its infection, but insight into its structure could help design of antiviral therapies. Structural analysis shows that the virus shares common features with other picornaviruses such as a canyon on the capsid surface and an annulus composed of VP3 around the 5-fold vertices. However, HPeV3 possess many distinguishing features compared to other picornviruses such as: 1) the N-terminus of VP0 stabilises the inter-pentamer interactions, in contrast to other picornaviruses where it primes the capsid for RNA release; 2) the VP1 hydrophobic pocket is blocked and cannot be used as a drug target as has been done for other picornaviruses; 3) HPeV3 RNA is highly ordered around the vertices and interact with conserved regions of the capsid proteins VP1 and VP3. These RNA-capsid protein interactions could serve as potential new drug targets. 34 Europic 2016

35 MONDAY 05/09/2016 A23 MODES OF INTERACTION BETWEEN FOOT-AND-MOUTH DISEASE VIRUS AND INTEGRIN RECEPTOR VISUALISED AT HIGH RESOLUTION BY CRYOEM Abhay Kotecha1, Quan Wang2, Xiangchi Dong3, Serban Ilca4, Julian Seago5, Bryan Charleston5, Elizabeth Fry1, Nicola Abrescia6, Timothy Springer3, Juha Huiskonen1, David Stuart1 1 University of Oxford, 2Chinese Academy of Science, China, 3Harvard Medical School, Boston, 4University of Oxford, Oxford, 5The Pirbright Institute, 6CIC bioGUNE, Bilbao Foot-and-mouth disease virus (FMDV) is a global scourge of domesticated animals. The conserved arginine- glycine-aspartic acid (RGD) motif present in the highly mobile, exposed and antigenic GH loop of the viral capsid protein VP1 mediates cell entry by attachment to an integrin receptor, generally v6. As the particle is unstable below pH 6.8, acidic conditions are thought to assist entry by particle uncoating. FMDV adaptation to tissue culture occurs often by basic mutations (e.g. H56R in VP3) conferring affinity for sulphated sugars such as heparin sulphate (HS) but attenuating the virus in natural infections. HS interaction has been mapped structurally to a conserved site in two serotypes and it has been suggested that this common mode of interaction reflects an underlying propensity for sugar binding. Here we determined the detailed interactions between v6 and two tissue culture adapted FMDV strains by cryo-electron microscopy (cryo-EM). In the preferred mode of engagement, the fully open form of the integrin attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role. Europic 2016 35

36 MONDAY 05/09/2016 A24 IN VITRO CONDITIONS AFFECTING TO UNCOATING OF ECHOVIRUS 1 Visa Ruokolainen, Marjo Haapakoski, Mira Myllynen, Varpu Marjomki University of Jyvskyl One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on earlier studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N-terminus of VP1 and to have reduced infectivity. Very recently, our group identified another type of infectious Echovirus 1 (E1) particle population during infection, a novel open infectious form of E1 (Myllynen et al., 2016, in press, JVI spotlight). This novel particle is denser in CsCl gradient, permeable to SYBR Green II, and in contrast to for example poliovirus, it still contains most of its VP4. Furthermore, in contrast to information around poliovirus it is able to bind to its receptor 21 integrin and show high infectivity. Heating of E1 particles to super-physiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results thus suggest that among enteroviruses there may exist different mechanisms on virus uncoating and led us to explore more details of enterovirus uncoating. Here we focused on the effect of key ions (Na+, Cl-, Mg2+, Ca2+ and K+) and explored their direct effect on the virus particle. The formation of porous, SYBR Green II permeable virus particle and RNA release was studied by using real-time spectroscopy and radioative gradients. We hypothesized, that since the uncoating of E1 is not induced by low pH, and virus is not accumulating in acidic endosomes, other ions might contribute to opening of the virus capsid. We also wanted to see if the in vitro formed porous virus particle was as infective as was previously shown for the dense virion formed in vivo, and using various infectivity and viability assays we could determine the infection potency of the treated virus. The preliminary results show that E1 is a very stable virus and can withstand lengthy periods at room temperature under various ion combinations. However, at 37 degrees, some ionic conditions contributed to formation of the porous but still infectious virion. Several ion concentrations e.g. high Mg2+, Ca2+ and low K+ seemed to have a protective role in virus opening. In contrast, high NaCl concentration as such and low NaCl with high K+ seemed to open the structure. Even if some of the tested ionic conditions were promoting the formation of the porous intermediate E1 particle, the virions seemed to retain rather high infectivity suggesting that further cues in cells may be participating in efficient genome release. These results were further supported with radioactive gradients, where protective conditions maintained the virus mostly in intact form and opening conditions led to notable formation of dense E1, but neither showed increased amounts of empty capsids. This study provides further knowledge about the uncoating and genome release of E1. New information on the uncoating of enterovirus B group is needed since several details leading to the uncoating of E1 differs remarkably from the model enterovirus species poliovirus. 36 Europic 2016

37 MONDAY 05/09/2016 A25 CHARACTERIZATION OF NOVEL PAN-COXSACKIEVIRUS 2A AND 3C ANTIBODIES Emma Svedin1, Olli Laitinen1, Pr Larsson1, Sebastian Kapell1, Erna Domsgen1, Virginia Stone1, Heikki Hyty2, Vesa Hytnen3, Malin Flodstrm-Tullberg1 1 The Center for Infectious Medicine, Department of Medicine HS, Karolinska Institutet, 2BioMediTech, University of Tampere, 3 Department of Virology, University of Tampere BACKGROUND: Coxsackie B viruses (CVBs) are common viruses usually causing mild infections, but which in some cases can give rise to more severe infections such as meningitis, myocarditis and neonatal sepsis. In some of these diseases, the viral proteases 2Apro and 3Cpro are directly involved in causing tissue damage. Antibodies (Abs) directed to the viral encoded proteases could allow studies on protein-protein interactions, cellular localization and be used as a tool to identify new targets for 2Apro and 3Cpro that might contribute to CVB- induced pathology. OBJECTIVES: Our goal was to develop new antibodies to the CVB-encoded proteases 2Apro and 3Cpro that would detect the proteases using different methodologies such as Western Blot, histochemistry, flow cytometry and confocal microscopy. METHODS: Recombinant His-tagged 2Apro and 3Cpro proteases cloned from CVB3 (Nancy) were produced in E. coli. Rats were immunized with purified proteases and hybridomas generated (GenScript, Hong Kong). Monoclonal Abs were selected based on negativity for binding to His and positivity in ELISA. Cell lines (HeLa, GMK, Vero, RD and A549) where infected with selected viruses belonging to the species enterovirus A, B and C. Mice were infected with CVB3 or CVB4. Western Blot, immunocyto- and immunohistochemistry and flow cytometry were used to evaluate the specificity of the 2A and 3C Abs, and to compare their specificities with the commonly used Ab against a viral capsid protein, virus protein-1 (VP-1)(Dako). RESULTS: In Western Blot, the 2A and VP-1 Abs could detect all CVB strains (1-6) and Echovirus 6. The 3C Ab could detect all CVB strains but was not able to detect Echovirus 6. In immunocytochemistry the 3C and VP-1 Abs could detect all viruses tested that belonged to the EV B genus. The 2A Ab could detect all viruses tested within the EV B genus except for Echovirus 9. The VP-1 Ab detected viruses belonging to the EV A or EV C genus, which the 2A and 3C Abs were unable to do. All three Abs stained CVB3 and CVB4-infected mouse pancreas. Preliminary experiments using intracellular staining and analysis by flow cytometry on CVB3 infected cells showed that 2A, 3C and VP1 Ab staining overlapped. CONCLUSION: Both 2A and 3C antibodies recognized all CVB viruses using Western blot and immunocytochemistry. Viruses belonging to the EV B genus were detected using 2A and 3C to varying extent. However, both 2A and 3C seemed to be more specific, detecting viruses within the EV B genus, compared to the VP-1 antibody that recognized viruses from other EV genus as well. In summary, the 2A and 3C Abs generated can be used in different applications to detect the proteases in infected cells. Future studies will be conducted to establish protocols for the use of 2A and 3C in other applications such as confocal microscopy. Europic 2016 37

38 MONDAY 05/09/2016 A26 STRUCTURE AND GENOME RELEASE OF HONEYBEE VIRUSES Pavel Plevka1, Sergei Kalynych1, Karel kubnk1, Edukondalu Mullapudi1, Radovan Spurn1, Lenka Plkov1, Michaela Veselkov1, Antonn Pidal2, Joachim Rodrigues De Miranda3, Robert Paxton4 1 CEITEC MU, 2Mendel University, 3SLU, 4Martin Luther University Halle-Wittenberg Honeybee (Apis mellifera) is the most important insect pollinator. However, the worldwide population of honeybee is under pressure from environmental stress and pathogens, including viruses that cause collapses of honeybee colonies. The resulting limited pollination has negative impact on agricultural productivity and on biodiversity of wild flowering plants. Majority of honeybee viruses belong to the order Picornavirales specifically to Dicistroviridae and Iflaviridae families. Although honeybee viruses cause substantial economic and environmental damage, structures of their virions are unknown. Here we present structures of several Iflaviruses and Dicistroviruses. Capsid proteins VP3 of several iflaviruses have a large C-terminal domain. Five of these domains form crowns on virion surface located around icosahedral fivefold symmetry axes. Although the crown domains are conserved among iflaviruses, they have not been observed in any other virus from the order Picornavirales. The domain contains a putative conserved active site that is likely to play a role in iflavirus cell entry. Honeybee dicistroviruses have non-canonical major capsid proteins VP1 and VP3 containing jellyroll -barrels composed of only seven instead of eight -strands, as is the rule for jellyroll proteins of other viruses. Unlike many picornaviruses the honeybee viruses do not contain a hydrophobic pocket in capsid protein VP1 that could be targeted with capsid- binding antiviral compounds. 38 Europic 2016

39 MONDAY 05/09/2016 A27 STRUCTURE OF HUMAN AICHI VIRUS AND IMPLICATIONS FOR RECEPTOR BINDING Elizabeth Fry1, Ling Zhu2, Xiangxi Wang3, Jingshan Ren2, Abhay Kotecha2, Thomas Walter2, Shuai Yuan3, Teruo Yamashita4, Tobias Tuthill5, Zihe Rao3, David Stuart2 1 Oxford University, 2University of Oxford, 3Chinese Academy of Science, 4Aichi Prefectural Institute of Public Health, 5The Pirbright Institute Aichi virus is a rather unusual but poorly characterized picornavirus. It can cause severe gastroenteritis in children. We have determined its three dimensional structure at 3.7 resolution, revealing a structure intermediate between the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops, replacing the canyon characteristic of enteroviruses. VP0 is not cleaved to form VP2 and VP4 so that the VP2 -barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface a polyproline helix structure, not seen previously in picornaviruses is present at the C-terminus of VP1, corresponding to the position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity suggesting a role in attachment to a cellular receptor. Europic 2016 39

40 MONDAY 05/09/2016 A32 THE VIRAL CAPSID ACCOUNTS FOR THE DIFFERENCES IN TROPISM AND ACID SENSITIVITY OBSERVED BETWEEN RESPIRATORY AND NON RESPIRATORY ENTEROVIRUSES Lna Royston1, Johan Geiser2, Manel Essaidi-Laziosi2, Isabelle Piuz2, Karl-Heinz Krause2, Caroline Tapparel2 1 University of Geneva, 2University of Geneva, Faculty of Medicine BACKGROUND: Rhinoviruses (RVs) and enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide. Classified in the Enterovirus genus within the Picornaviridae family, these viruses have a single positive-stranded RNA genome. Although closely related at a genetic level, respiratory EVs (including RVs) and non respiratory EVs have remarkably different in vivo and in vitro phenotypic characteristics. Whereas respiratory EVs infect upper respiratory airways cells and cause mostly benign, self-limited cold-like illnesses, non respiratory EVs in contrast have a wide tropism and are associated to a broad range of clinical syndromes. In vitro properties of respiratory EVs and non respiratory EVs vary considerably as well, whether it is in terms of optimal growth temperature, acid sensitivity or cell tropism. The purpose of our work is to define the genetic determinants underlying these different phenotypic characteristics. METHODS: We artificially generated chimeric viruses by exchanging genomic regions of two different EV types from the same species, one displaying a typical EV phenotype (EV-D94) and the other exhibiting a respiratory phenotype (EV-D68). By propagating the chimeric viruses in cell cultures and in 3D human reconstituted tissues, we assessed which part of the genome is related to each phenotype of interest. RESULTS: The exchange of the capsid-encoding P1 region resulted in a change of tropism of the chimeric virus, whether in cell lines or 3D reconstituted tissues. The acid sensitivity phenotype was also affected by the P1 exchange. In contrast, the optimal growth temperature phenotype seemed not strictly associated to the capsid encoding region. CONCLUSION: Capsid proteins are the only determinants of the acid sensitivity and tissue tropism of enteroviruses, but not of the optimal growth temperature. 40 Europic 2016

41 MONDAY 05/09/2016 14:00 Session 2 PICORNAVIRUS-CELL INTERACTIONS Chairs: 1st part, Dieter Blass & George Belov 18:10 2nd part, Thomas Michiels & Rosa Pint Karla Kirkegaard (Stanford 14:00 K03: VIRAL EXCURSIONS AND DIVERSIONS ALONG THE AUTOPHAGY PATHWAY University School of Medicine, United States of America) Craig Cameron (The B01: SINGLE-CELL VIROLOGY: ON-CHIP INVESTIGATION OF VIRAL INFECTION 14:35 DYNAMICS Pennsylvania State University, United States of America) B02: ILLUMINATING THE SITES OF ENTEROVIRUS REPLICATION IN LIVING CELLS Hilde van der Schaar (Utrecht 14:50 USING A SPLIT-GFP-TAGGED VIRAL PROTEIN University, Netherlands) Kevin L. McKnight (University of B03: UBIQUITYLATION OF VP1PX IN THE BIOGENESIS OF QUASI-ENVELOPED 15:05 HEPATITIS A VIRIONS North Carolina, United States of America) Varpu Marjomki (University of 15:20 B04: INFECTIOUS ENTRY PATHWAY OF COXSACKIEVIRUS B1 Jyvaskyla / Nanoscience center, Finland) B05: IDENTIFICATION AND CHARACTERIZATION OF NOVEL VIRUS-HOST Dylan Flather (University of 15:26 INTERACTIONS THROUGH ANALYSIS OF NUCLEAR PROTEIN EFFLUX DURING California, Irvine USA, United RHINOVIRUS INFECTION States of America) COFFEE BREAK K04: FAT(AL) ATTRACTION: PICORNAVIRUSES HIJACK LIPID TRANSFER MACHINERY Frank van Kuppeveld (Utrecht 16:05 TO CREATE REPLICATION ORGANELLES University, Netherlands) George Belov (University of B06: ACTIVATION OF PHOSPHOLIPID SYNTHESIS IS REQUIRED FOR THE Maryland, and Virginia-Maryland 16:40 DEVELOPMENT OF PICORNAVIRUS REPLICATION ORGANELLES AND PROTECTION Regional College of Veterinary OF THE REPLICATION COMPLEXES Medicine, United States of America) Djoshkun Shengjuler (The 16:55 B07: STUDIES OF A PICORNAVIRAL PHOSPHOINOSITIDE-BINDING PROTEIN Pennsylvania State University, United States of America) B08: IDENTIFICATION OF NOVEL SUBSTRATES OF POLIOVIRUS AND Julienne Jagdeo (University of 17:10 COXSACKIEVIRUS 3C PROTEINASES USING TERMINAL AMINE ISOTOPIC LABELING British Columbia, Canada) OF SUBSTRATES B09: DIFFERENTIAL PATHOGENESIS OF RHINOVIRUSES FROM SPECIES A, B AND C Manel Essaidi-Laziosi (CMU, 17:25 AND OF ENTEROVIRUS D68 IN HUMAN AIRWAY EPITHELIA Switzerland) Hendrik Jan Thibaut (Utrecht 17:40 B10: EXPLORING THE REPERTOIRE OF ENTEROVIRUS D68 GLYCAN RECEPTORS University, Netherlands) B11: THE SYNERGISTIC ROLES OF ICAM-1 AND SIALIC ACIDS IN COXSACKIEVIRUS Jim Baggen (Utrecht University, 17:55 A24 INFECTION Netherlands) B12: POTENTIAL MODELS TO SEPARATE TWO DIFFERENT HEPATITIS A VIRUS Tiing Tiing Chua (University of 18:01 POPULATIONS Alberta, Canada) Europic 2016 Keynote Lecture Communication (12 + 3 min) Shotgun presentation (5 + 1 min) 41

42 MONDAY 05/09/2016 POSTER SESSION 2 - PICORNAVIRUS-CELL INTERACTIONS B13: PHOSPHATIDYL-INOSITOL-4 PHOSPHATE (PI4P) / CHOLESTEROL COUNTERCURRENT DEPENDENT RHINOVIRUS REPLICATION IN ABSENCE OF PI4K3B ACTIVITY Urs Greber (University of Zurich, Switzerland) B14: ICAM-1 BINDING RHINOVIRUSES A89 AND B14 UNCOAT IN DIFFERENT ENDOSOMAL COMPARTMENTS Dieter Blaas (Dept. Med. Biochem., Med. Univ. Vienna, Austria) B15: INTERACTION OF FMDV LPRO WITH THE TRANSCRIPTION FACTOR ADNP IS REQUIRED FOR EFFICIENT VIRAL REPLICATION Gisselle N Medina (ORISE/PIADC/ARS, United States of America) B16: AN ENTEROVIRUS MUTANT THAT CAN REPLICATE IN THE ABSENCE OF REPLICATION ORGANELLES WHEN PI4KB IS INHIBITED Charlotte Melia (Leiden University Medical Center, Netherlands) B17: ENTEROVIRUS MUTANTS THAT OVERCOME PI4KB INHIBITION BY MODULATING POLYPROTEIN PROCESSING Heyrhyoung Lyoo (Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Netherlands) B18: SECRETORY CARRIER MEMBRANE PROTEIN 3 INTERACTS WITH 3A PROTEIN OF ENTEROVIRUS 71 AND REGULATES VIRAL REPLICATION Jing-Yi Lin (School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taiwan) B19: AUGMENTING ENTEROVIRUS REPLICATION THROUGH SPECIFIC MODULATION OF HOST RESTRICTION FACTORS ENHANCES VACCINE STRAIN PRODUCTION Jessica Ciomperlik (CDC/NCIRD/DVD, United States of America) B20: FOOT-AND-MOUTH DISEASE VIRUS AND JUMONJI C-DOMAIN CONTAINING PROTEIN 6 (JMJD6) INTERACTIONS Elizabeth Rieder (USDA, ARS, United States of America) B21: THE UNRAVELLING OF PROTEASE-SPECIFIC CELLULAR TARGETS AS A WAY TO DISCOVER NOVEL MECHANISMS CONTRIBUTING TO ENTEROVIRAL DISEASE Sebastian Kapell (Karolinska Institutet, Sweden) B22: VIRULENCE DETERMINANTS OF TWO FOOT-AND-MOUTH DISEASE VIRUSES OF SEROTYPE A EXHIBITING DIFFERENT PATHOGENICITY IN ADULT MICE Mara Ins Gismondi (Biotechnology Institute, INTA, Hurlingham, BA, Argentina) B23: POLIOVIRUS RNA IS PROTECTED FROM CLEAVAGE BY RNASE DURING VIRION UNCOATING AND TRANSFER ACROSS CELLULAR AND MODEL MEMBRANES Elisabetta Groppelli (University of Leeds, United Kingdom) B24: THE STRUCTURAL INTEGRIN PERSPECTIVE FOR FMDV SPECIFICITY, AN IN SILICO STUDY Mara Ins Gismondi (Biotechnology Institute, INTA, Hurlingham, BA, Argentina) B25: AUTOPHAGY IN ENTEROVIRUS 71 -INFECTED NEURON Jhao-Yin Lin (Chang Gung University, Taiwan) B26: SINGLE-CELL VIROLOGY: ON-CHIP INVESTIGATION OF VIRAL INFECTION DYNAMICS Craig Cameron B27: IDENTIFICATION AND CHARACTERIZATION OF NOVEL VIRUS-HOST INTERACTIONS THROUGH ANALYSIS OF NUCLEAR PROTEIN EFFLUX DURING RHINOVIRUS INFECTION Dylan Flather B28: STUDIES OF A PICORNAVIRAL PHOSPHOINOSITIDE-BINDING PROTEIN Djoshkun Shengjuler B29: IDENTIFICATION OF NOVEL SUBSTRATES OF POLIOVIRUS AND COXSACKIEVIRUS 3C PROTEINASES USING TERMINAL AMINE ISOTOPIC LABELING OF SUBSTRATES Julienne Jagdeo B30: POTENTIAL MODELS TO SEPARATE TWO DIFFERENT HEPATITIS A VIRUS POPULATIONS Tiing Tiing Chua 42 Europic 2016

43 MONDAY 05/09/2016 K03 VIRAL EXCURSIONS AND DIVERSIONS ALONG THE AUTOPHAGY PATHWAY Karla Kirkegaard, Roberto Mateo, Emma Abernathy, Matthew Taylor and Sara Bird Stanford University School of Medicine, Stanford CA, USA 94305 Autophagy is an ancient and highly conserved cellular process. Once thought to be only a starvation response, it is now appreciated to be an important aspect of many signaling, trafficking and degradation pathways within eukaryotic cells. Like a long and exciting hiking trail through diverse terrain, the autophagy pathway can be entered and exited at many points. The canonical autophagy pathway is stimulated by nutrient deprivation via mTOR inhibition and culminates in the degradation of cytoplasmic contents. However, cellular processes such as growth factor signaling, antigen presentation and unconventional secretion are known to utilize segments and components of this pathway for processes that neither begin with mTOR nor end with autolysosomal degradation of cytoplasmic contents. Numerous microbes, including poliovirus and Dengue virus, have evolved to subvert segments of the cellular autophagy pathway or its individual constituents to promote their infections cycles. In the case of poliovirus infection, the autophagy pathway, its components or both decorate membranous compartments on which RNA replication occurs and faciliate non-lytic viral exit. In the case of dengue virus, destruction of the beclin1/Vps34/ Atg14 complex by a small-molecule inhibitor results in the formation of incompletely processed, uninfectious particles. Here, the effect of CRISPR/Cas9-mediated oblation of individual constituents of the cellular autophagy pathway on the infectious cycles of both poliovirus and dengue virus are presented. Interestingly, these two viruses utilize different segments and components of the autophagy pathway. However, one common component is Atg9, a protein that scavenges lipids and contributes to the growth of nascent autophagosomes. Experiments are in progress to determine whether the absence of Atg9 similarly decreases lipid recruitment to the membranous structures induced during poliovirus and dengue infections. Although every step of the autophagy pathway is not utilized by these viral infections, upstream stimulation of the canonical autophagy pathway can exacerbate infections, presumably through upregulation of those steps that are utilized. For example, in mouse models of dengue virus infection, short periods of murine fasting increase viral growth and pathology. Should this be shown to be dependent on stimulation of the autophagy pathway, this finding could elucidate one reason for the increased severity of infections in developing countries. Europic 2016 43

44 MONDAY 05/09/2016 B01 & POSTER B26 SINGLE-CELL VIROLOGY: ON-CHIP INVESTIGATION OF VIRAL INFECTION DYNAMICS Craig Cameron1, Feng Guo1, Sixing Li1, Mehmet Caglar2, Zhangming Mao1, Claus Wilke2, Tony Huang1 1 The Pennsylvania State University, 2The University of Texas at Austin OBJECTIVE: Studies of the virus-host interaction are typically performed on the population level, which completely ignores the stochastic nature of cellular gene expression. Expression levels of receptors for entry, host-encoded antagonists of infection and the myriad host proteins and nucleic acids hijacked by the virus to facilitate multiplication are different in each cell. This circumstance leads to the very likely possibility that only a few cells in the population provide an ideal environment for virus replication. At the population level, this situation manifests as intermediate responses to reduced or ablated host factor gene expression, to mutations in viral genes or even drug treatment. To remove the ambiguities inherent in studies performed at the population level, it is critical to be able to interrogate the virus-host interaction, viral mutants or antiviral therapeutics at the single- cell level. METHODS: We employ a novel, high-throughput analysis platform that allows us to carefully manipulate the multiplicity of infection, compare mutant to wt populations, and study the effect of an antiviral agent, all at the single-cell level. Our analysis platform consists of a multi-layer, pneumatically-controlled, microfluidic cell-culture system with live-cell imaging. This system can (i) separate single, virus-infected cells into individual wells in a high-throughput manner; (ii) eliminate cross infection by sealing each well using pneumatic valves; (iii) monitor the kinetics of viral replication in single cells by using an automatic, time-lapse, live-cell-imaging approach; (iv) extract biologically meaningful parameters describing viral growth from the imaging data; and (v) compare these parameters across experimental conditions and/or viral mutants. RESULTS: Our studies reveal substantial variation among individual infections at the cellular level; changes in this variation due to genetic mutation can explain in vivo post-infection outcomes. Our studies also show that an antiviral agent targets a specific subset of infected cells, rather than targeting the entire population in a uniform manner. CONCLUSION: Observations made using this next-generation-virology platform will likely be as transformational to virology as single-molecule approaches have been for enzymology and cell biology. 44 Europic 2016

45 MONDAY 05/09/2016 B02 ILLUMINATING THE SITES OF ENTEROVIRUS REPLICATION IN LIVING CELLS USING A SPLIT-GFP-TAGGED VIRAL PROTEIN Hilde van der Schaar1, Charlotte Melia2, Armando van Bruggen3, Jeroen Strating3, Maartje van Geenen3, Bram Koster2, Montserrat Brcena2, Frank van Kuppeveld3 1 Utrecht University, 2Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands, 3Department of Infectious Diseases & Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands OBJECTIVES: Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles, ROs) with a unique protein and lipid composition on which they multiply their viral genome. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would therefore be preferred, suitable tools are currently unavailable, as recombinant enteroviruses that encode RO-anchored viral proteins tagged with fluorescent reporters have to date not been reported. To overcome this limitation, we in this study used a split-GFP system to visualize enterovirus ROs in living cells. METHODS: The split-GFP system used comprises a large fragment (S1-10) and a small fragment (S11) of only 16 residues. The GFP(S11) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. Using correlative light electron microscopy (CLEM), we studied whether GFP fluorescence was emitted from ROs. Live-cell imaging was conducted to monitor the development of ROs in real time. RESULTS: The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. With CLEM we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. Although the GFP fluorescence was dimmer compared to CVB3 with GFP(S11)-tagged 3A in BGM cells that stably expressed GFP(S1-10), the virus encoding both GFP fragments generated GFP fluorescent foci that colocalized with 3A, indicating that this virus is also suitable for live-cell imaging. CONCLUSION: This new tool bridges a methodological gap by circumventing the need for immunolabeling of fixed cells. It allows the study of the dynamics and formation of enterovirus ROs in living cells and will have multiple applications in future studies on their origin, location, and function. Europic 2016 45

46 MONDAY 05/09/2016 B03 UBIQUITYLATION OF VP1PX IN THE BIOGENESIS OF QUASI-ENVELOPED HEPATITIS A VIRIONS Kevin L. McKnight, Fengyu Hu, Jonathan K. Mitchell, Stanley M. Lemon University of North Carolina OBJECTIVES: Hepatitis A virus (HAV) is released non-cytolytically from infected cells in vitro and in vivo as infectious quasi-enveloped virions (eHAV), cloaked in host-derived membranes and resistant to neutralizing anti- capsid antibodies (Feng et al., Nature 496:367-71, 2013). Several lines of evidence indicate the biogenesis of these exosome-like virions is dependent upon components of the endosomal sorting complex required for transport (ESCRT) and involves release of virions via multivesicular bodies (MVBs). K-63 ubiquitylation is a common signal in the selection and sorting of cargo destined for interactions with ESCRT components and vesicular trafficking via the MVB pathway. An in silico search for potential ubiquitylation sites in the HAV polyprotein (ubipred.org) identified with medium-to-high confidence 3 of 7 Lys residues in the 8 kDa pX domain of VP1pX. Only one other Lys residue in the entire polyprotein was similarly predicted to be ubiquitylated. Since VP1pX is unprocessed and present in eHAV, these results led us to hypothesize that ubiquitylation of pX may be key in the recruitment of assembled HAV capsids to MVBs. METHODS: To assess the importance of the 3 pX Lys residues to eHAV biogenesis, we mutated each to Ala, individually or in combination, in a newly created molecular clone of low passage, non-cytopathic HM175/p16 virus. Huh-7.5 hepatoma cells were infected by RNA transfection and intracellular and extracellular virus was quantified by RT-qPCR and analyzed in isopycnic gradients. Direct evidence of ubiquitylation was sought by immunoprecipitation of intracellular virus with anti-ubiquitin antibodies and by immunoblotting and pull-down assays of 293T cells expressing Flag-VP1pX and HA-ubiquitin-K0. RESULTS: Each of 3 mutant viruses containing various combinations of pX Lys to Ala substitutions demonstrated significant reductions in eHAV released into supernatant fluids by infected cells. A 3K mutant in which all three Lys residues were replaced with Ala replicated well as RNA, but failed to release eHAV. Cells infected with this mutant contained a high intracellular abundance of a unique, membrane-associated form of HAV considered to be a possible assembly or transport intermediate of eHAV in addition to naked virions normally present in infected cells. FACS analysis confirmed a defect in the capacity of the 3K mutant to spread to uninfected cells. Immunoblotting provided no direct evidence of pX ubiquitylation, consistent with a requirement for ubiquitylation of a minor population of pX molecules or rapid deubiquitylation as is known to occur among proteins interacting with ESCRT components. In contrast, anti-ubiquitin antibody was capable of precipitating readily detectable amounts of parental but not 3K mutant virus from infected cell lysates. CONCLUSIONS: Guided by bioinformatics analyses showing a strong likelihood of pX ubiquitylation, we have demonstrated that 3 Lys residues within the pX domain are important for eHAV production. Virus in which these residues were mutated was unable to efficiently exit the infected cell and instead remained stalled within the cell in a membrane-associated form. We conclude that ubiquitylation of pX is an important signal that facilitates the selection of HAV capsids as cargo for trafficking out of infected cells via the MVB pathway. 46 Europic 2016

47 MONDAY 05/09/2016 B04 INFECTIOUS ENTRY PATHWAY OF COXSACKIEVIRUS B1 Varpu Marjomki1, Mari Martikainen1, Amirbabak Khojine2, Heikki Hyty2 1 University of Jyvaskyla / Nanoscience center, 2University of Tampere Coxsackievirus B1 (CVB1) is a member of the species B Enteroviruses (EV-B) belonging to the family of Picornaviridae. CVB1 has been pinpointed recently as a risk factor for Type I diabetes. Therefore, we set out to explore the infectious entry pathway of CVB1 in order to increase our understanding of the disease processes in the cellular and tissue levels. We have recently shown that Echovirus 1 (E1) and Coxsackievirus A9 (CVA9), internalize via macropinocytic uptake into cytoplasmic endosomes that mature into nonacidic multivesicular bodies (MVBs). In these MVBs viruses uncoat leading to the release of their genome finally to the cytoplasm, and to replication and production of new viral particles. In this work we examined the infectious entry pathway of CVB1 into cells including receptor binding, regulators of the entry and the involvement of the multivesicular structures in the pathway. DAF distribution was changed upon CVB1 infection but it did not colocalize with the virus. In contrast, CAR colocalized to some extent with CVB1 during the early time points of entry but was not particularly accumulating in late time points with the virus. We found out that the CVB1 entry resembles the infectious entry pathway of the EV-B species EV1 and CVA9 for the time table of uncoating, replication and the nature of the endosomal structures involved in the entry. CVB1 entered cytoplasmic endosomes that were not colocalizing with EEA1 or LAMP1. In addition, Baf1 had no effect and nocodazole had a marginal effet on infection suggesting that CVB1 entered non-acidic endosomes. The drug experiments pinpointed Rac1 and Na+/H+ exchanger to be involved in the pathway but not dynamin, suggesting that CVB1 may be following macripinocytic-like pathway as its close relatives. Over-expression of the ESCRT proteins Hrs and Vps4 had a strong inhibitory phenotype in CVB1 infection suggesting further that CVB1 entered non-acidic MVBs as E1 and CVA9. Despite relying on different receptors for their primary entry, the results strongly suggest that these closely related EV-B species share the similar entry pathway pathway to non-acidic multivesicular bodies and collectively suggest that there exists an enterovirus B group entry pathway. Europic 2016 47

48 MONDAY 05/09/2016 B05 & POSTER B27 IDENTIFICATION AND CHARACTERIZATION OF NOVEL VIRUS-HOST INTERACTIONS THROUGH ANALYSIS OF NUCLEAR PROTEIN EFFLUX DURING RHINOVIRUS INFECTION Dylan Flather, Paul D. Gershon, Bert L. Semler University of California, Irvine USA Despite enteroviruses being canonical cytoplasmic viruses, a growing body of evidence points to the importance of nuclear-resident and nuclear-shuttling proteins in the replication of these viruses. Through virus-induced modifications to the host cell nucleo-cytoplasmic trafficking machinery, enterovirus infections lead to the cytoplasmic accumulation of nuclear proteins, expanding the repertoire of protein functions available where viral replication is carried out. To better understand the contribution of nuclear proteins in the life cycle of human rhinovirus 16 (HRV16), we have characterized protein redistribution during HRV16 infection by quantitative proteomics. This resulted in the identification of a number of proteins that increased in abundance in the cytoplasm, while simultaneously decreasing in abundance in the nucleus, throughout the course of infection. The relocalization of nuclear proteins to the cytoplasm (where viral replication takes place) is suggestive of the possible pro- or anti- viral roles of these proteins during HRV16 infection. Proteins known to redistribute to the cytoplasm of enterovirus- infected cells and perform functions associated with viral replication, such as polypyrimidine tract-binding protein 1 (PTB1) and heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C1/C2), were identified in our analysis. Additionally, we identified heterogeneous nuclear ribonucleoprotein M (hnRNP M), which was recently shown to redistribute to the cytoplasm of poliovirus-infected cells and facilitate viral replication, and which served as a relocalization control in subsequent studies. We have selected several candidate proteins to investigate for their possible novel roles in the infectious cycle of HRV16. Studies focused on the validation of relocalization events and the functional characterization of novel roles in HRV16 replication have initially focused on two RNA-binding, nuclear-resident proteins: Splicing factor proline/glutamine-rich (SFPQ) and DEAH box helicase 9 (DHX9). SFPQ is a known internal ribosome entry site (IRES) trans-acting factor for some cellular mRNAs, while DHX9 has been shown to participate in the replication of a related picornavirus, namely foot-and-mouth disease virus. We have confirmed that HRV16 infection causes the nuclear efflux of these proteins by Western blot analysis and confocal immunofluorescence microscopy. Through siRNA-mediated knockdown of these proteins, we are attempting to better understand their roles in translation, genome replication, and overall amplification of HRV16 during infection. In ongoing experiments, nuclear-cytoplasmic fractionations of infected cells of respiratory lineage, which more closely represent a natural infection target of HRV16, are being subjected to a comparable analysis. We will explore the differences in the infection-induced nuclear efflux proteome between the standard (HeLa) and respiratory-derived cells, with a particular focus on those proteins that are unique to a respiratory virus within a relevant infection model. By analyzing the impact of HRV16 infection on the sub-cellular distribution dynamics of nuclear proteins, our studies have exposed novel HRV16-host interactions, revealed mechanistic insights into HRV16 replication, and highlighted the important role nuclear proteins play during enterovirus infections. 48 Europic 2016

49 MONDAY 05/09/2016 K04 FAT(AL) ATTRACTION: PICORNAVIRUSES HIJACK LIPID TRANSFER MACHINERY TO CREATE REPLICATION ORGANELLES Frank van Kuppeveld Utrecht University Picornaviruses, like all other (+)RNA viruses, extensively rearrange intracellular membranes to create replication organelles (ROs), which are generally thought to provide a scaffold for genome replication and lend protection against sensors that can initiate antiviral host responses. First insights in the 3D structure of the picornavirus replication organelles have been obtained, but little is known about the identity of the membrane-modifying host cell factors and pathways that are hijacked by viral proteins to create these unique structures. Our current knowledge on the identity of these factors, which may be targets for broad-spectrum antiviral therapy, will be summarized, with particular emphasis on how enteroviruses and cardioviruses usurp lipid transfer machinery (e.g. PI4 kinases and OSBP) at membrane contact sites to create a lipid environment that drives viral RNA replication. Furthermore, new data will be presented on the membrane alterations induced by escape mutants that are largely insensitive to PI4K and OSBP inhibitors, which challenge the dogma that accumulation of cytosolic ROs is indispensable for viral RNA replication and evasion of innate antiviral host responses. Europic 2016 49

50 MONDAY 05/09/2016 B06 ACTIVATION OF PHOSPHOLIPID SYNTHESIS IS REQUIRED FOR THE DEVELOPMENT OF PICORNAVIRUS REPLICATION ORGANELLES AND PROTECTION OF THE REPLICATION COMPLEXES George Belov1, Jules Nchoutmboube1, Ekaterina Viktorova1, Alison Scott2, Courtney Chandler2, Lauren Ford-Siltz1, Shane Ellis3, Robert Ernst2 1 Department of Veterinary Medicine, University of Maryland, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, MD 20742, 2Department of Microbiology, University of Maryland, School of Dentistry, Baltimore, MD, 21201, 3M4I, The Maastricht Multimodal Molecular Imaging Institute, University of Maastricht, Maastricht, The Netherlands, 6229 ER In poliovirus-infected cells the membrane architecture is totally reorganized by the end of ~six hour long infectious cycle. Early in infection, loose clusters of single membrane replication compartments are detected in the vicinity of the Golgi membranes and ER tubules. As infection progresses, these clusters grow in size and complexity replacing normal cellular organelles. The 3D reconstruction revealed that picornavirus-induced membranes represent complex agglomerates of branching tubular structures with no homology in non-infected cells. Finally, these tubular structures collapse into double membrane vesicles. The molecular mechanisms that underlie such dramatic remodeling of membranes in infected cells and the significance of their intricate shapes in the viral replication cycle remains largely unknown. Here we investigated the phospholipid synthesis in poliovirus-infected cells and its role in the development of the replication organelles. We implemented a novel, ultrafast lipidomic technique that allows identification of lipids in situ, without prior extraction and purification. We found that lipid profile of infected cells is very different from that of control cells. Statistical comparison yielded at least 316 mass intervals discriminating infected and control cells. Among other changes, the synthesis of phosphatidylcholines with mixed acyl configurations (largely based on C16 incorporation) was significantly increased. Phosphatidylcholine is the major structural component of the cellular membranes, thus the accumulation of molecules with shorter acyl chains (including C16) indicates that membranes in infected cell become more fluid. We found that the rate limiting enzyme of the phosphatidylcholine synthesis pathway, CCT-a, undergoes massive translocation from the nucleus where it normally resides in a non-active conformation to the cytoplasm of infected cells where it associates with the replication membranes. We identified lipid droplets as the source of fatty acids for infection-specific phospholipid synthesis, which is in accordance with decreasing triglyceride content of infected cells as evidenced from the lipidomic data. Surprisingly, poliovirus replication under conditions of high multiplicity of infection did not require active phosphatidylcholine synthesis. However, the electron microscopy revealed that the development of the replication organelles was severely compromised. Instead of tight clusters of convoluted replication membranes developing in permissive conditions, the replication organelles were sparse and isolated, often resembling highly enlarged ER tubules. Membrane permeabilization assay showed that the viral replication complexes formed in cells with inhibited phospholipid synthesis become highly accessible. Accordingly, under conditions of low multiplicity of infection and multiple rounds of viral propagation, the virus yield strongly depended on phosphatidylcholine synthesis, suggesting that its inhibition allows mounting of anti-viral response. Altogether, our data support a model that infection-specific activation of phospholipid synthesis results in massive extrusion of fluid membranes which assemble in characteristic convoluted clusters of the replication organelles. Such elaborate membranous structures are dispensable for the viral RNA replication but are necessary to protect the replication complexes from the cellular anti-viral mechanisms. Targeting the cellular factors involved in infection-specific activation of phospholipid synthesis provides a novel anti-viral approach based not on inhibition of the viral replication, but on increasing visibility of the replication complexes to the cellular sensors of infection. 50 Europic 2016

51 MONDAY 05/09/2016 B07 & POSTER B28 STUDIES OF A PICORNAVIRAL PHOSPHOINOSITIDE-BINDING PROTEIN Djoshkun Shengjuler1, Yan Chan1, Simou Sun1, Ibrahim Moustafa1, Joseph Moran1, Akira Uchida1, Jamie Arnold1, Nai-Yun Hsu2, Nihal Altan-Bonnet3, Paul Cremer1, David Boehr1, Craig Cameron1 1 The Pennsylvania State University, 2Rutgers University, 3National Heart, Lung, and Blood Institute OBJECTIVES: One exciting advance in picornavirology over the past few years has been the realization that phosphatidylinositol 4-phosphate (PI4P) is an essential component of the enterovirus replication organelle. In the cell, phosphoinositides (PIs) are used as markers of specific membranes that proteins and enzymes use to ensure appropriate localization and/or activation. Enteroviruses do not appear to encode conventional PI-binding domains, although early studies observed such activity in the RNA-dependent RNA polymerase, 3Dpol. Our objective was to discover other PI-binding determinants in enteroviruses and characterize them biochemically and biophysically. METHODS: Hints of the location of a second putative PI-binding site(s) on 3C were obtained by molecular docking experiments and confirmed by nuclear magnetic resonance (NMR) spectroscopy experiments. To assess specificity, fluorescence polarization (FP)-based PI-binding experiments were conducted. However, this assay was unable to reveal the impact of overall lipid composition on PI specificity or affinity. To this end, we have established a supported lipid bilayer (SLB) in the context of a microfluidic platform to study 3C-PI interaction using a label-free, fluorescence method. RESULTS: Docking studies suggested a single, predominant PI-binding site towards the N-terminus of 3C and NMR experiments confirmed these observations. Our initial FP-based PI-binding studies suggested a broad specificity towards PIs. However, SLB-binding experiments revealed a clear impact of the bilayer setting and its composition on affinity, which is in the low micromolar range. CONCLUSIONS: Using a variety of approaches, we show that 3C binds to PIs with broad specificity. Compared to cellular PI-binding domains, 3C seems to have a novel structural scaffold for PI-binding. SLB-binding experiments suggest that association of 3C molecules and/or formation of PI microdomains may be a consequence of 3C- PI interaction in the fluid bilayer. Finally, PI-binding may be a unifying theme for many, if not all, viruses, thus representing a new, tractable target for the design of antiviral agents. Europic 2016 51

52 MONDAY 05/09/2016 B08 & POSTER B29 IDENTIFICATION OF NOVEL SUBSTRATES OF POLIOVIRUS AND COXSACKIEVIRUS 3C PROTEINASES USING TERMINAL AMINE ISOTOPIC LABELING OF SUBSTRATES Julienne Jagdeo1, Antoine Dufour2, Christopher Overall2, Eric Jan1 1 Department of Biochemistry and Molecular Biology, University of British Columbia, 2Department of Oral Biological and Medical Sciences, University of British Columbia Picornaviruses utilize a strategy whereby the viral polyprotein is processed by virally-encoded proteinases. In addition, viral proteinases cleave host proteins to manipulate cellular processes to direct synthesis of new virions and subvert host antiviral responses. Several host proteins have already been identified as targets of picornaviral proteinases, however, the full repertoire of targets is not known. Here, we used a novel proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of protease-generated N-terminal peptides by mass spectrometry and identify novel substrates of the poliovirus and coxsackievirus B3 (CVB3) 3C proteinase (3Cpro). TAILS provides a unique advantage over other methods as it enriches for N-terminal peptides, allowing for greater coverage of the proteome and simultaneously revealing the cleavage site through identification of protease-generated peptides. TAILS was performed on HeLa cell and mouse HL1 cardiomyocyte extracts subjected to purified poliovirus and CVB3 3Cpro, respectively. We identified 90 and 40 high confidence candidate substrates of poliovirus and CVB3 3Cpro, respectively, including the known poliovirus 3Cpro substrate polypyrimidine tract binding protein at a known cleavage site, thus validating this approach. Three identical peptides were identified in both TAILS screens, encoding for the heterogeneous nuclear ribonucleoprotein (hnRNP) M, hnRNP K and phosphoribosylformylglycinamidine synthase (PFAS). We showed that these common host proteins are targets of poliovirus and CVB3 3Cpro in vitro and are cleaved in poliovirus- and CVB3-infected cells. Moreover, mutations in the TAILS-identified cleavage sites for several candidates blocked cleavage in vitro and under infection. Depletion of these proteins by siRNAs modulated virus infection, suggesting that cleavage either promoted or inhibited virus infection. Loss of hnRNP K decreased poliovirus production in infected HeLa cells and led to a reduction in poliovirus IRES-mediated translation, suggesting a role for hnRNP K in facilitating viral translation. In contrast, loss of PFAS enhanced poliovirus production and modulated expression of NFB signalling proteins, indicative of an antiviral role for PFAS under poliovirus infection. In summary, identification of common cleaved substrates of poliovirus and CVB3 3Cpro may contribute to a general strategy of enterovirus infections. Importantly, identification of distinct host substrates may be key to understanding the specific tropism and virus host interactions of each picornavirus infection. 52 Europic 2016

53 MONDAY 05/09/2016 B09 DIFFERENTIAL PATHOGENESIS OF RHINOVIRUSES FROM SPECIES A, B AND C AND OF ENTEROVIRUS D68 IN HUMAN AIRWAY EPITHELIA Manel Essaidi-Laziosi1, Manel Essaidi-Laziosi2, Sacha Benaoudia3, Melanie Fernandes-Rocha4, Isabelle Piuz4, Samuel Constant5, Laurent Kaiser4, Caroline Tapparel Vu2 1 CMU, 2Faculty of Medicine Geneva, 3Ecole Normale Suprieure de Lyon, 4HUG, 5Epithelix OBJECTIVES: Viral respiratory infections are the most frequent causes of acute illnesses worldwide and are associated with mild to severe diseases. Respiratory enteroviruses (EV), particularly rhinoviruses (RV), are the most common causative agents. The human airway epithelium is the first line of defence against respiratory infections. Mucociliary clearance (MCC) serves as a mechanical barrier while epithelial cells recognize and fight the infection via cytokine secretion. This study aimed to compare the in vitro pathogenesis of clinically relevant RV and EV strains using standardized in vitro 3D human airway epithelial models. METHODS: Respiratory tissues were infected with RV from species A (RV-A55 and RV-A49), B (RV-B48) and C (RV-C8 and RV-C15) and EV-D68. Viruses were isolated directly from clinical specimen without prior amplification in immortalized cells. After infection with comparable viral loads, the replication kinetics, cell tropism, cytokine induction and effects on tissue integrity and mucociliary clearance were analyzed for each viral strain. RESULTS: The six viral strains tested infected only a small proportion of ciliated cells but all affected cilia beating and mucociliary clearance except RV-B48 which appeared less pathogenic. In contrast, EV-D68 showed a significantly higher replication rate and caused massive loss of ciliated cells, along with reversible impairment of tissue integrity. Interestingly, all viral stains persisted for more than 28 days in the tissue culture system, suggesting that infection cannot be resolved in the absence of immune cells. CONCLUSION: Altogether this work provides new insights on the differential pathogenesis of the most frequent causes of viral respiratory infections and confirms that 3D airway epithelia are valid surrogate models to study the initial phase of contained respiratory viral infections, prior to the action of immune cells. Europic 2016 53

54 MONDAY 05/09/2016 B10 EXPLORING THE REPERTOIRE OF ENTEROVIRUS D68 GLYCAN RECEPTORS Hendrik Jan Thibaut1, Jim Baggen2, Jacqueline Staring3, Yue Liu4, Richard Roberts2, Jost W. de Bruin2, Arno L. W. van Vliet2, Lucas T. Jae3, Vincent A. Blomen3, Michael G. Rossmann4, Thijn R. Brummelkamp3, Frank J. M. van Kuppeveld2 1 Department of Infectious Diseases and Immunology, Utrecht University, 2Department of Infectious Diseases and Immunology, Utrecht University, the Netherlands, 3Netherlands Cancer Institute, Amsterdam, The Netherlands, 4Department of Biological Sciences, Purdue University, USA INTRODUCTION & OBJECTIVES: In the past decade, the frequency of detecting EV-D68 during outbreaks of respiratory disease has increased worldwide. In 2014, EV-D68 caused a nationwide outbreak of respiratory tract disease in the United States with clustered cases of central nervous system disease. Despite some evidence for a role of sialic acid (Sia), many unresolved puzzles existed concerning EV-D68 host factor requirements. METHODS & RESULTS: Genome-wide genetic screening in human haploid cells is a powerful tool to uncover factors implicated in the entry of pathogens, including viruses. We recently performed such a genome-wide haploid screen with the prototype EV-D68 strain Fermon and identified several genes involved in sialic acid (Sia) biology to be essential for infection1. CRISPR-/Cas9-mediated knock-out and gene reconstitution demonstrated that both 2,3- and 2,6-linked Sia can be used as functional cellular EV-D68 receptors. Upon comparing Sia- preferences of EV-D68 clinical isolates, we identified several isolates that can infect cells in a Sia-independent manner, indicating that these strains can use an alternative non-sialylated receptor. To identify this alternative receptor, we performed a second haploid screen in the background of a SLC35A1 knockout cell line (lacking cell surface Sia) to ensure exclusive usage of the non-sialylated receptor(s). We found that in the absence of Sia, glycosaminoglycans (GAGs) can serve as alternative receptor for infection of cells. By employing GAG-deficient knock-out cells and sodium chlorate treatment (to prevent sulfation of cell surface GAGs), we showed that Sia- independent strains can engage both Sia and sulfated GAGs as functional receptor. Competition between both glycan receptors for virus binding suggested overlapping binding sites. We constructed infectious cDNA clones of a Sia-dependent isolate and a Sia-independent isolate, and performed loss-of-function and gain-of-function mutagenesis studies to either disrupt or introduce the Sia-independent phenotype. Through this mutational analysis, we identified two residues adjacent to the Sia-binding site that allow dual receptor usage. CONCLUSION: We identified several EV-D68 isolates that can employ multiple receptors where both Sia and sulfated GAGs can be engaged to productively infect cells. The plasticity by which Sia-independent EV-D68 strains can switch between different glycan receptors, might reflect an in vivo situation where a complex virus glycan interplay might influence tropism and pathogenesis. 1. Baggen et al. Enterovirus D68 receptor requirements unveiled by haploid genetics. Proc Natl Acad Sci U S A, 113(5):1399-404 (2016) 54 Europic 2016

55 MONDAY 05/09/2016 B11 THE SYNERGISTIC ROLES OF ICAM-1 AND SIALIC ACIDS IN COXSACKIEVIRUS A24 INFECTION Jim Baggen1, Hendrik Jan Thibaut1, Richard Roberts1, Jasper Slager1, Arno van Vliet1, Georg Zocher2, Nitesh Mistry3, Kimberley Benschop4, Erwin Duizer4, Niklas Arnberg3, Thilo Stehle2, Frank van Kuppeveld1 1 Department of Infectious Diseases and Immunology, Utrecht University, 2Interfaculty Institute of Biochemistry, University Tbingen, 3 Division of Virology, Department of Clinical Microbiology, Ume University, 4Division of Virology, Department of Clinical Microbiology, National Institute for Public Health and the Environment INTRODUCTION & OBJECTIVES: Coxsackievirus A24 (CV-A24) belongs to the species Enterovirus C and was first isolated in South Africa in 1952. In 1970, a highly contagious genetic variant of CV-A24 (CV-A24 variant) emerged that causes acute hemorrhagic conjunctivitis (AHC), respiratory disease and occasionally acute flaccid paralysis. Since its emergence, CV-A24 variant has been implicated in numerous AHC outbreaks and two pandemics. CV- A24 strains that do not belong to the variant cluster (non-variant strains) still circulate in the human population, but have not been associated with severe disease. Previous studies showed that CV-A24 variant can use sialic acids (Sia) for infection and revealed a Sia-binding site in the capsid protein VP1 (1, 2). However, we observed that cells lacking cell surface Sia are still susceptible to CVA24 variant infection, suggesting that this virus can engage other receptor(s) besides Sia. This study was conducted to identify other receptor(s) and to define the role of Sia in infection with CV-A24 variant and non-variant strains. METHODS & RESULTS: Treatment of cells with various receptor-specific antisera showed that CV-A24 variant infection was specifically inhibited by ICAM-1-blocking antiserum. Moreover, CRISPR/Cas9-mediated ICAM-1 knockout and gene reconstitution in several cellular backgrounds showed that ICAM-1 is the major CV-A24 receptor for both CV-A24 variant and non-variant strains. Bio-layer interferometry confirmed the direct interaction between CV-A24 and ICAM-1. Depletion of Sia expression showed that, in addition to ICAM-1, CV-A24 variant can use Sia as a secondary attachment receptor on specific cell types. Because only the variant strains are associated with ocular disease, we hypothesized that the acquisition of a Sia-binding site might be an important determinant for ocular tropism. Consistently, comparison of Sia-dependencies between several variant and non-variant strains showed that most non-variant strains cannot employ Sia for infection. CONCLUSION: We conclude that ICAM-1 is the major receptor for both CV-A24 variant and non-variant strains. In addition, CV-A24 variant strains have a binding site for Sia, which can facilitate infection of specific cell types. Differences in Sia-dependency between CV-A24 variant and non-variant strains may reflect a beneficial role of a Sia-binding site in infection of ocular tissues. 1. E. C. Nilsson et al., Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential. J. Virol. 82, 30618 (2008). 2. G. Zocher et al., A Sialic Acid Binding Site in a Human Picornavirus. PLoS Pathog. 10, e1004401 (2014). Europic 2016 55

56 MONDAY 05/09/2016 B12 & POSTER B30 POTENTIAL MODELS TO SEPARATE TWO DIFFERENT HEPATITIS A VIRUS POPULATIONS Tiing Tiing Chua1, Daniel Pang2, Justin Shields1, Michael A. Joyce3, Rineke H.G. Steenbergern3, Lorne D. Tyrrell3 1 University of Alberta, Canada, 2University of Alberta, Canada (graduated), 3University of Alberta/Li Ka Shing Institute of Virology, Canada Hepatitis A Virus (HAV) is grouped under the family of Picornaviridae, a group of viruses that has in common that they are non-enveloped, single stranded, positive RNA viruses. In 2013, two different forms of HAV were discovered in vivo: enveloped (eHAV) and non-enveloped. eHAV was found exclusively in serum samples while non-enveloped HAV was found in feces samples only. However, how these two varaints are formed, and the reasons for the existence of two different HAV variants remain unknown. To answer these questions, a cell culture system representative of hepatocytes is needed.Thus, this study focuses on the development of in vitro system mimicking the polarized hepatocyte. Huh7 (a hepatoma cell line) was cultured on dual-chamber systems with DMEM supplemented with 2% human serum (HS cells), instead of the typical 10% fetal bovine serum (FBS). HS cultured Huh7 cells undergo growth arrest, and become differentiated, and we predicated they also become polarized. The proper differentiation and polarization of HS cells was confirmed by dextran diffusion studies. The columnar character of HS cells was also shown under the electron microscope (EM). We then characterized the viruses that were released from both sides of the polarized cells. HAV variants were separated by isopycnic gradient ultracentrifugation and identified by buoyant density. Further characterizations were performed by Western blot analysis, EM, TCID50 assays and neutralization assays. Our results show that in polarized cells, higher proportion of one of the HAV populations is secreted, which is enveloped form. This supports the hypothesis that in vivo the membranes are stripped off in the bile, possibly by the action of bile acids. Meanwhile, we also developed an in vivo model to study HAV infection, using a non-primate model with chimeric human livers (SCID-beige/Alb-uPA mice). This model was generated by transplanting normal human hepatocyte into SCID mice carrying a plasminogen activator transgene (Alb-uPA). A persistent HAV infection was observed in this model through intraperitoneal (IP), tail intravenous (IV) as well as by placing uninfected mice in a HAV contaminated environment (bedding and cohousing). Surprisingly, HAV infection was also detected in the mice infected through nasal installation route. By performing isopycnic gradient ultracentrifugation, eHAV was identified only in mice sera while non-enveloped HAV was identified only in both mice bile and mice feces. In short, both HS cells and chimeric mice model were shown to be a suitable in vitro and in vivo model, respectively, to further the study the formation of two different HAV populations. 56 Europic 2016

57 MONDAY 05/09/2016 B13 PHOSPHATIDYL-INOSITOL-4 PHOSPHATE (PI4P) / CHOLESTEROL COUNTERCURRENT DEPENDENT RHINOVIRUS REPLICATION IN ABSENCE OF PI4K3B ACTIVITY Urs Greber, Pascal Roulin University of Zurich Rhinoviruses (RVs) replicate their positive-sense RNA genome on cytoplasmic membranes derived from the Golgi apparatus. The viral genome encodes a poly-protein with membrane-targeted proteins 2B, 2C and 3A, which control host membrane traffic and lipid composition of the replication membranes (Belov, 2016). The virus recruits host factors for replication, such as the phosphatidylinositol 4 (PI4)-kinase 3 beta (PI4K3b), which enhances PI4- phosphate (PI4P) levels on the replication membranes. This establishes a PI4P lipid gradient, and drives counter- current exchange of PI4P and cholesterol lipids at contact sites with the endoplasmic reticulum and the lipid shuttling protein oxysterol binding protein 1 (OSBP1) (Roulin et al., 2014). Here we aimed to better understand the essence of PI4K3b for RV replication. From a pool of RV-A16 mutants selected for resistance against small chemical inhibitors of PI4K3b, we identified a single point mutation in the highly conserved viral 2B membrane protein, isoleucine 92 to threonine [I92T] located near the cytosolic carboxy-terminus. Unlike earlier enterovirus 3A mutants found to be resistant to PI4K inhibitors (Thibaut et al., 2014), our 2B [I92T] mutant disrupted the Golgi morphology, enhanced intracellular PI4P levels, recruited PI4K3b to replication membranes and was sensitive to OSBP1 inhibitors, either in presence or absence of PI4K3b inhibitors. While 3A alone recruited PI4K3b to perinuclear membranes resembling replication membranes, the 2B [I92T] mutant virus recruited PI4K2a to replication membranes. It was more sensitive to RNA interference against PI4K2a, PI4K2b and PI4K3a, but less sensitive against PI4K3b depletion, indicating that it takes advantage of numerous PI4-kinases to establish a PI4P lipid gradient on the replication membranes. The data highlight the importance of the PI4P/cholesterol counter- current lipid flux for establishing Golgi-derived RV replication membranes, even in presence of potent inhibitors of the Golgi-resident PI4K3b. Literature: Belov, G.A. 2016. Dynamic lipid landscape of picornavirus replication organelles. Current opinion in virology. 19:1-6. Roulin, P.S., M. Lotzerich, F. Torta, L.B. Tanner, F.J. van Kuppeveld, M.R. Wenk, and U.F. Greber. 2014. Rhinovirus Uses a Phosphatidylinositol 4-Phosphate/Cholesterol Counter-Current for the Formation of Replication Compartments at the ER-Golgi Interface. Cell Host Microbe. 16:677-690. Thibaut, H.J., H.M. van der Schaar, K.H. Lanke, E. Verbeken, M. Andrews, P. Leyssen, J. Neyts, and F.J. van Kuppeveld. 2014. Fitness and virulence of a coxsackievirus mutant that can circumnavigate the need for phosphatidylinositol 4-kinase class III beta. J Virol. 88:3048- 3051. Europic 2016 57

58 MONDAY 05/09/2016 B14 ICAM-1 BINDING RHINOVIRUSES A89 AND B14 UNCOAT IN DIFFERENT ENDOSOMAL COMPARTMENTS Rick Conzemius1, Haleh Ganjian2, Renate Fuchs2, Dieter Blaas1 1 Dept. Med. Biochem., Med. Univ. Vienna, 2Dept. Pathophys. & Allergy Research, Med. Univ. Vienna OBJECTIVES: The more than 150 currently known common cold virus serotypes are phylogenetically classified as species A, B, and C. Most RV-A and all RV-B use ICAM-1 for cell attachment and entry (the major group); only twelve RV-A bind members of the LDLR family (the minor group). RV-A89 and RV-B14 belong to the major group. The RNA genome of RV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. RV-A89 is similarly internalized into early endosomes but was not detected in late endosomes. We thus comparatively investigated endocytic pathways and location of productive uncoating of these two serotypes. METHODS: By using infectivity assays and quantitative microscopy (TissueFAXS) we compared the kinetics of internalization, generation of subviral particles and RNA release for RV-B14 and RV-A89. Subcellular co-localization with late endosomes and transferrin-recycling endosomes was assessed in the presence and absence of drugs interfering with the lysosomal (EGA) and the recycling (nocodazole, ciliobrevin A) route by confocal microscopy. We also studied uncoating and replication in cells transfected to either express constitutively active or dominant negative mutants of Rab11-GTPase. Rab11-GTPase is involved in transferrin transport from sorting endosomes to the perinuclear recycling compartment and from sorting endosomes back to the plasma membrane. RESULTS: Our experiments revealed much slower uncoating of HRV-A89 while HRV-B14 uncoating was comparatively quick suggesting differences in endocytic pathways. Immunofluorescence microscopy showed that RV-A89, but not RV-B14, colocalizes with transferrin in the endocytic recycling compartment (ERC). RV-B14 colocalized with LAMP2-positive late endosomes. Applying the above drugs we confirm that RV-B14 productively uncoats in compartments of the lysosomal pathway and demonstrate that RV-A89 uncoats in the perinuclear recycling compartment. Uncoating of RV-A89 depends on functional Rab11. CONCLUSIONS: Two major group rhinovirus serotypes, belonging to species A and B respectively, exploit different endocytic pathways for infection, despite binding the same receptor. It is possible that the big difference in affinity for ICAM-1 by RV-A89 (IC50 = 70 nM) and RV-B14 (IC50 = 3272 nM) accounts for the different pathways exploited, directing RV-A89 into the recycling pathway whereas RV-B14 dissociates from ICAM-1 already in an earlier compartment. 58 Europic 2016

59 MONDAY 05/09/2016 B15 INTERACTION OF FMDV LPRO WITH THE TRANSCRIPTION FACTOR ADNP IS REQUIRED FOR EFFICIENT VIRAL REPLICATION Gisselle N Medina1, Giselle Knudsen2, Alexander Greninger3, Anna Kloc4, Fayna Diaz-San Segundo5, Elizabeth Rieder6, Marvin Grubman6, Joe Derisi2, Teresa De los Santos6 1 ORISE/PIADC/ARS, 2University of California, San Francisco, 3University of California at San Francisco, 4PIADC/ORISE, 5University of Connecticut, 6United States Department of Agriculture The foot-and-mouth disease virus (FMDV) leader protease (Lpro) inhibits cap-dependent host translation and host transcription affecting several factors involved in innate immunity. While specific cleavage of the translation initiation factor eIF-4G1 mediates the shutoff of cap-dependent translation, the mechanisms involved in controlling the cellular transcriptional machinery remain elusive. In this study, we have identified the host transcription factor ADNP (activity dependent neuroprotective protein) as an Lpro interacting protein by affinity purification and mass spectrometry. We show that Lpro can bind to ADNP in vitro and in cell culture. During infection, endogenous ADNP levels decreased over time and discrete cleavage products were detected. Infection with a chimeric cardiovirus (TMEV-L) containing FMDV Lpro showed that the cleavage is specific for FMDV Lpro and that its proteolytic activity is required. Interestingly depletion of endogenous ADNP by RNAi negatively affected virus replication and higher levels of interferon (IFN) and IFN inducible gene (ISG) mRNAs were detected. Furthermore, we found that Lpro and ADNP are in the same complex with the chromatin remodeling protein Brg-1, a member of the basal host transcriptional machinery. Interestingly, endogenous levels of Brg-1 were also affected by FMDV infection. Our results uncover a novel role of FMDV Lpro in modulating the host innate immune response by targeting ADNP and the general transcriptional machinery. Europic 2016 59

60 MONDAY 05/09/2016 B16 AN ENTEROVIRUS MUTANT THAT CAN REPLICATE IN THE ABSENCE OF REPLICATION ORGANELLES WHEN PI4KB IS INHIBITED Charlotte Melia1, Hilde M. van der Schaar2, Ronald W. A. L. Limpens1, Abraham J. Koster1, Frank J. M. van Kuppeveld2, Montserrat Brcena1 1 Leiden University Medical Center, 2Faculty of Veterinary Medicine, Utrecht University OBJECTIVES: One of the defining features of positive-sense RNA virus replication is the generation of cytoplasmic replication organelles (ROs) that provide a platform for efficient viral RNA synthesis, and may confer additional benefits with regard to innate immune evasion and spatial organization of the virus life cycle. For the enteroviruses, RO formation is concomitant with viral recruitment of phosphatidylinositol 4-kinase III beta (PI4KB) and enterovirus replication is dependent upon its phosphorylated lipid product, PI4P. Interestingly, a single point mutation in the 3A protein can be sufficient to overcome this requirement for active PI4KB for instance the coxsackievirus 3B (CVB3) 3A-H57Y mutation. Using this CVB3 mutant we explore the relationship between PI4P and RO development by utilizing a range of light and electron microscopy techniques. METHODS: Buffalo Green Monkey (BGM) cells infected under PI4KB inhibition were assessed by electron microscopy to determine whether limiting levels of PI4P altered RO morphology or development. To monitor the dynamics of infection under these conditions, we used a split-GFP system to track the CVB3 3A-H57Y protein during infection. Furthermore, the cellular morphology underlying 3A-H57Y-GFP signal was revealed using correlative light and electron microscopy (CLEM). The subcellular location of CVB3 3A-H57Y viral RNA synthesis in the presence or absence of PI4KB inhibitors was determined through metabolic labelling ([3H]uridine) and subsequent autoradiography of infected cells. RESULTS: In both wt and 3A-H57Y CVB3 infections, ROs develop in an identical manner in the absence of PI4KB inhibitor. Remarkably, when PI4KB is inhibited, the CVB3 3A-H57Y mutant did not generate ROs during the exponential phase of RNA synthesis. Studies using the split-GPF system revealed that in the absence of ROs the 3A protein instead localizes to the Golgi apparatus, suggesting that the Golgi provides an alternative site for RNA replication under PI4KB inhibition. This was corroborated through the use of autoradiography, which revealed the presence of newly synthesized RNA at Golgi membranes. Interestingly, at very late time points in infection, the Golgi apparatus did disassemble and structures resembling wt ROs could be visualized by electron microscopy. CONCLUSIONS: Here we show that ROs are not inherently required for enterovirus RNA synthesis. Instead, an intact Golgi apparatus is sufficient to host viral RNA replication for the majority of the CVB3 3A-H57Y viral infection cycle. The appearance of ROs only very late in PI4KB-inhibited CVB3 3A-H57Y infection suggests that high levels of PI4P may not be strictly required for enterovirus RO generation, but do accelerate the process. 60 Europic 2016

61 MONDAY 05/09/2016 B17 ENTEROVIRUS MUTANTS THAT OVERCOME PI4KB INHIBITION BY MODULATING POLYPROTEIN PROCESSING Heyrhyoung Lyoo, Cristina Dorobantu, Hilde van der Schaar, Frank van Kuppeveld Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands OBJECTIVES: All positive-strand RNA viruses modulate host cell membranes to build new organelles, so- called replication organelles, to replicate their genome. Enteroviruses require several host factors such as phosphatidylinositol-4-kinase III (PI4KB) and oxysterol binding protein (OSBP) to form their replication organelles. Inhibitors of PI4KB and OSBP have been studied as putative antivirals against enteroviruses. Enterovirus mutants resistant to PI4KB and OSBP inhibitors were isolated that carried multiple substitutions in the non-structural proteins. However, the single substitutions in the 3A protein (e.g. 3A-H57Y in coxsackievirus B3, CVB3) were found to be sufficient to confer resistance. In addition to 3A-H57Y, mutation N2D in 2C was identified in each of the PI4KB-inhibitor resistant CVB3 pools, but the possible benefit of this substitution for the CVB3 3A-H57Y mutant has not been investigated yet. The aim of this study was to define the effects of the 2C substitution on the resistance to inhibitors of PI4KB and OSBP. METHODS: The 2C-N2D mutation was inserted by reverse genetics techniques into wild-type and mutant (3A-H57Y) CVB3 that also encodes Renilla luciferase upstream of the capsid coding region. To assess viral replication rates, virus-infected HeLa cells were incubated in the presence of inhibitors of PI4KB or OSBP, and lysed at different time points post infection to determine the intracellular luciferase activity. To analyze viral polyprotein processing, cell lysates were subjected to Western blot analysis with antibodies directed against 2C, 3A, and 3D. RESULTS: As expected, the 2C-N2D substitution by itself did not confer any resistance to the inhibitors of PI4KB and OSBP. The double mutant (i.e. 2C-N2D/3A-H57Y) on the other hand showed a better resistance than the single 3A-H57Y mutant, indicating that 2C-N2D provided additional benefits only in combination with 3A-H57Y. Since the 2C substitution is located near the cleavage site between 2B and 2C, we hypothesized that the polyprotein processing may play a role in the acquisition of resistance. To test this, we compared the polyprotein processing profiles of wild-type and mutant virus by Western blot analysis. The double mutant and 3A-H57Y single mutant showed a better cleavage efficiency of 3AB into 3A in the absence of PI4KB inhibitor. In the presence of the inhibitor, both mutants maintained this ability, while wild-type virus accumulated more of the 3AB precursor. Importantly, we consistently observed that the 2C-N2D/3A-H57Y double mutant showed better processing of 3AB and 2BC than the 3A-H57Y single mutant at increasing concentrations of the inhibitor. Processing of 3CD was not affected by the substitutions nor by the inhibitor. CONCLUSIONS: This study suggests that alterations in lipid homeostasis can affect enterovirus polyprotein processing. Yet, enteroviruses can acquire mutations that can modulate polyprotein processing in order to overcome these effects. Besides the 3A-H57Y mutation, which is key to overcome PI4KB inhibition, also mutations at cleavage junctions of other proteins (e.g. 2C-N2D) can contribute to this adjustment. This modulation of processing efficiency might be the consequence of topological changes in the polyprotein induced by the mutations which makes the cleavage sites more accessible to the 3C(D) protease. Europic 2016 61

62 MONDAY 05/09/2016 B18 SECRETORY CARRIER MEMBRANE PROTEIN 3 INTERACTS WITH 3A PROTEIN OF ENTEROVIRUS 71 AND REGULATES VIRAL REPLICATION Jing-Yi Lin1, Kai-Zhe Lin2, Jia-Ying Lu1, Shin-Ru Shih2 1 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taiwan, 2Research Centre for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan Picornaviruses are a diverse and major cause of human disease, and their genomes replicate with intracellular membranes. The functionality of these replication organelles depends on the activities of both the viral nonstructural proteins and the co-opted host proteins. The mechanism by which viral-host interactions generate viral replication organelles and regulate viral RNA synthesis is unclear. To elucidate this mechanism, enterovirus 71 (EV71) is used herein as a virus model to investigate how these replication organelles are formed and to identify the cellular components that are critical in this process. Immunoprecipitation assay is combined with LC- MS/MS analysis to identify 172 cellular proteins and four viral proteins associating with viral 3A protein. Secretory carrier membrane protein 3 (SCAMP3) is one of the host proteins that selected for further investigation. Here, we demonstrate that SCAMP3 associates with 3A protein by immunoprecipitation assay and colocalizes with 3A protein during virus infection. SCAMP3 knockdown in infected cells decreases synthesis of EV71 viral RNA, viral proteins, and viral growth. Taken together, the results suggest that SCAMP3 interacts with 3A protein of EV71 and positively regulates viral replication. 62 Europic 2016

63 MONDAY 05/09/2016 B19 AUGMENTING ENTEROVIRUS REPLICATION THROUGH SPECIFIC MODULATION OF HOST RESTRICTION FACTORS ENHANCES VACCINE STRAIN PRODUCTION Jessica Ciomperlik1, William C. Weldon2, M. Steven Oberste2, Jennifer L. Anstadt2 1 CDC/NCIRD/DVD, 2Centers for Disease Control and Prevention A challenge commonly associated with vaccine production is decreased yield due to attenuation of virus strains, particularly given that only a few cell lines are approved for vaccine production. Such challenges can lead to reduced vaccine supply and increased production costs, both of which restrict the availability of crucial vaccine resources in developing country settings. This includes the inactivated polio vaccine (IPV), which must replace the oral polio vaccine (OPV) worldwide to support the final stages of polio eradication. A recent study by our group used a whole-genome siRNA library screen to identify cellular target genes that act as restriction factors during poliovirus (PV) replication, including for PV vaccine strains. The cellular transcription factor p300 was identified as a one such restriction factor, with knockdown of p300 resulting in a significant increase in PV production. Using CRISPR technology, a stable cell line (DEP300) was engineered with p300 function abolished, to potentially serve as a high-performing poliovirus vaccine production cell line. Since transcription factors modulate expression of complex networks of genes, we sought to understand the precise mechanism by which p300 restricts PV replication. As a histone acetyltransferase, p300 regulates cellular transcription via chromatin remodeling. It also mediates cAMP-gene regulation by binding specifically to phosphorylated CREB, a host factor known to be degraded by PV protease 3Cpro. Interestingly, we found that unlike CREB1, p300 is not degraded during PV infection. To further explore the impact of cellular transcription factors on PV replication, we utilized siRNAs to target the p300, CREB1, and pCAF pathways in infected Vero cells. Transcription factor-associated restriction of PV replication is not universal, as knockdown of p300 and CREB1 enhanced viral replication while knockdown of pCAF did not, further suggesting a novel viral restriction mechanism mediated by the p300 pathway. To assess the stability of PV vaccine strains in cells lacking p300, virus was passaged five times in the DEP300 cell line and viral genomes analyzed for mutations. The development of an enhanced vaccine production cell line that produces increased yield of safe, genetically stable PV vaccine strains would dramatically lower cost and greatly increase vaccine supply, both of which are critically necessary to achieve full PV eradication and to maintain elimination post-eradication. Europic 2016 63

64 MONDAY 05/09/2016 B20 FOOT-AND-MOUTH DISEASE VIRUS AND JUMONJI C-DOMAIN CONTAINING PROTEIN 6 (JMJD6) INTERACTIONS Elizabeth Rieder1, Paul Lawrence2, Devendra Rai2, Carolina Stenfeldt2, Juan Pacheco2, Jonathan Arzt2 1 USDA, ARS, 2Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA Foot-and-mouth disease virus (FMDV) utilizes four integrins (v1, v3, v6, and v8) as its primary cell receptor. In cell culture, FMDV has been shown to adapt to use heparan sulfate (HS) for entry, and rarely utilizes an unidentified third receptor. Capsid mutations acquired by a soluble integrin resistant FMDV cause (i) adaptation to CHO-677 cells (ii) increased affinity to membrane-bound Jumonji C-domain containing protein 6 (JMJD6) (iii) induced JMJD6 re-localization from the cell surface and cytoplasm to the nucleus. Interestingly, pre-treatment of cells with N- and C-terminal JMJD6 antibodies or by simultaneous incubation of mutant virus with soluble JMJD6 (but not by treatment with HS or v6) impaired virus infectivity in cultured cells. JMJD6 and mutant virus co-purified by reciprocal co-immunoprecipitation. Molecular docking predictions suggested JMJD6 C-terminus interacts with mutated VP1 capsid protein. Interestingly, JMJD6-FMDV cattle pathogenesis was equivalent to parental A24-WT administered by intraepithelial lingual inoculation route. In contrast, JMJD6-FMDV aerosol- infected cattle did not manifest signs of FMD and animals showed no detectable viremia. Immunofluorescent microscopy of post-mortem tissue revealed JMJD6-FMDV exclusively co-localized with JMJD6+ cells while A24- WT was occasionally found in JMJD6+ cells. 64 Europic 2016

65 MONDAY 05/09/2016 B21 THE UNRAVELLING OF PROTEASE-SPECIFIC CELLULAR TARGETS AS A WAY TO DISCOVER NOVEL MECHANISMS CONTRIBUTING TO ENTEROVIRAL DISEASE Sebastian Kapell1, Anssi Nurminen2, Emma Svedin3, Vesa Hytnen2, Olli Laitinen2, Malin Flodstrm-Tullberg3 1 Karolinska Institutet, 2BioMediTech, University of Tampere, 3The center of infectious medicine, Karolinska Institute OBJECTIVE: The two enterovirus encoded proteases, 2Apro and 3Cpro, perform the posttranslational proteolytic processing of the viral polyprotein. They also cleave several host cell proteins in order to promote the production of new virus particles, and to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, and improved tools to identify targets for these proteases may reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we set out to summarize possibilities for the identification of protease-specific cellular targets including novel tools such as bioinformatics and in silico analyses. METHODS: We used bioinformatics, and performed in silico and wet lab analyses. Results We provide an overview of a multidisciplinary approach to identify novel protease targets. We also present the methodology to produce updated substrate sequence LOGOs for enteroviral proteases, sequence alignments and primary amino acid sequence percentage identity matrix for the enteroviral proteases (2Apro and 3Cpro). CONCLUSIONS: The approaches outlined here should provide a better understanding of how the proteases, in concert with other viral proteins, contribute to the pathogenesis of different enterovirus associated diseases. Such information will also be of immediate importance for the development of novel drugs to prevent and treat diseases caused by enteroviruses. New prediction methods and proteome-wide approaches are critical for the successful completion of this goal. Europic 2016 65

66 MONDAY 05/09/2016 B22 VIRULENCE DETERMINANTS OF TWO FOOT-AND-MOUTH DISEASE VIRUSES OF SEROTYPE A EXHIBITING DIFFERENT PATHOGENICITY IN ADULT MICE Marco Cacciabue1, Mara Soledad Garca Nez1, Rubn Marrero1, Elizabeth Rieder2, Elisa Carrillo3, Mara Ins Gismondi1 1 Biotechnology Institute, INTA, Hurlingham, BA, Argentina, 2Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA, 3 CICVyA, INTA, Hurlingham, BA, Argentina Foot-and-mouth disease virus (FMDV) causes an acute disease of cloven-hoofed animals with devastating consequences. Different models including adult C57 Bl/6J mice have been used to study FMDV biology and host susceptibility to viral infection. In this context, we identified two FMDV viruses of serotype A/Arg/01 (derived from field isolates that circulated in Argentina in 2001) exhibiting differential virulence in adult C57 Bl/6J mice. The non-lethal (NL) variant caused mild signs of disease whereas the lethal (L) variant caused death within 24-48 h, independently of the dose used (102-106 pfu/mice). In this work, we evaluated pathological changes induced by NL and L variants in mice inoculated with 105 pfu virus inoculum and examined serum and various organs at 22 hpi. Severe lesions in the exocrine pancreas were observed (60% and 80% necrosis of acinar cells in NL and L inoculated mice, respectively); multifocal apoptosis was evident in thymus and spleen of inoculated mice. Viral load in serum, pancreas, liver and spleen was significantly higher in FMDV-L inoculated mice (p

67 MONDAY 05/09/2016 B23 POLIOVIRUS RNA IS PROTECTED FROM CLEAVAGE BY RNASE DURING VIRION UNCOATING AND TRANSFER ACROSS CELLULAR AND MODEL MEMBRANES Elisabetta Groppelli1, Hazel Levy2, Eileen Sun3, Mike Strauss2, Clare Nicol1, Sarah Gold4, Xiaowei Zhuang2, Toby Tuthill4, James Hogle2, David Rowlands1 1 University of Leeds, 2Harvard University, 3Harvard university, 4The Pirbright Institute As non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis, picornaviruses face the challenge of delivering the RNA genome across the membrane of the endocytic vesicle into the cytoplasm, where it is translated. Given the hydrophobic nature of the membrane and the charged backbone of the RNA, this process has to be mediated by viral and/or cellular components. Recent structural studies using an in-vitro virus-receptor- liposome system captured poliovirus particles in the act of RNA translocation. Most particles were found to be at a distance from the lipid membrane but were connected to it by one or two stems (umbilici) of polypeptide and perhaps lipid. Raw images from this data set clearly showed RNA crossing intact membranes. Analysis of the relative densities of the capsid and umbilical connections are consistent with the presence of translocating RNA within the umbilici and, importantly, the RNA of a number of particles has been captured specifically in the process of crossing the liposome membrane. These data suggest a coordinated mechanism in which the genomic RNA is directed from the inside of the viral particle across the membrane and into the cytoplasm without exposing the RNA to the lumen of the endocytic vesicle. Here we show that in the same in-vitro system, large amounts of RNase added outside the liposomes do not affect RNA translocation. In parallel, we show that poliovirus replication in HeLa cells is not decreased by co-internalisation of excess RNase. Importantly, full length genomic RNA can be recovered after RNase co-internalisation suggesting that the viral RNA is protected from RNase degradation at all stages of cell entry during infection. Our results also would support that the integrity of the endocytic vesicle is maintained during entry as disruption of the lipid membrane would remove viral RNA/RNAse segregation and therefore reduce, at least in part, the integrity of the bulk viral RNA. The uncoating and cell entry process for aphthoviruses is less well understood than for enteroviruses such as poliovirus. Aphthovirus particles dissociate into pentamer subunits and release the RNA at pH values encountered during endocytosis. This would be expected to expose the RNA to the contents of the endosome lumen. However, transiently formed RNA free empty particles have been described for equine rhinitis A virus (ERAV) which may represent RNA delivery structures able to protect the RNA during the infection process. To test this possibility we compared the resistance of the poliovirus infectious process to the presence of high concentrations of RNase with that of ERAV. We found that ERAV infectivity was unaffected by the presence of high concentrations of RNase, as was the case with poliovirus. The observation that RNA release is insensitive to RNase in members of two distantly related genera of picornaviruses (the enterovirus genus (poliovirus) and the aphthovirus genus (ERAV)) that were once thought to release their genomes by very different processes, suggests that RNase resistant RNA translocation across intact membranes may be a general property of the entire family. Europic 2016 67

68 MONDAY 05/09/2016 B24 THE STRUCTURAL INTEGRIN PERSPECTIVE FOR FMDV SPECIFICITY, AN IN SILICO STUDY Rubn Marrero1, Mara Ins Gismondi2, Guido Knig2 1 Biotechnology Institute, INTA, Hurlingham, BA, Argentina, 2Biotechnology Institute, INTA, Hurlingham, BA, Argentina; CONICET, Argentina FMDV is an agro-economic relevant picornavirus which infects wild and domestic cloven-hoofed animals. At a molecular level, the viral entrance takes place due to host-pathogen interactions between cell adhesion molecules (integrins) and FMDV capsid motif (aminoacidic triplet RGD). The general picture is more complex: the virus not only displays the simplest integrin recognition motif in its capsid surface; but has also evolved the RGD aminoacidic surroundings for a high affinity to certain integrin highly expressed in nasopharynx tissues, the primary infection site. The recent resolution of the tridimensional structure of the human, alfa V beta 6 integrin, in complex with an RGD peptide, has paved an avenue for diverse structural studies. Alfa V beta 6 integrin is the preferred molecule for FMDV entrance in their natural host (bovine). Based on the published human integrin structure, we have computationally modelled (by homology) several bovine integrins interacting with an FMDV peptide (interaction complexes), and fully studied (building single mutants), the preferred amino acidic substitutions at the interaction interface. In other words, we have obtained the integrin recognition profile or the preferred sequence space of the bovine integrins for any peptide. The emergent data shows that the bovine integrin beta 6 subunit preferentially presents a unique hydrophobic interface (amino acids usage) which supports their specificity for the alfa helix presented at the end of the RGD peptide. Other integrin beta subunits (beta 3, 5 ) at the same positions of the interaction interface present more polar or bulky amino acids which result in a less compatible interaction. As a whole, our work makes usage of new structures to recapitulate the bovine model and brings new information concerning the specificity determinants of the recognition between FMDV and bovine integrins. 68 Europic 2016

69 MONDAY 05/09/2016 B25 AUTOPHAGY IN ENTEROVIRUS 71 -INFECTED NEURON Jhao-Yin Lin, Hsing-I Huang Chang Gung University Autophagy is an important cellular process that can deliver the cytoplasmic macromolecules, long-lived protein and dysfunctional organelles to lysosomes for degradation. Autophagy usually occurs in basal level for maintain cellular homeostasis especially in differentiated cells, like neuron. Because differentiated cells cannot proliferate, basal autophagy can protect cells from apoptosis. When cells suffered stressful stimulants, like starvation or virus infection, autophagy could also be induced. Several RNA viruses have been identified that can induce autophagy and autophagy played a role in virus replication. Enterovirus 71 belongs to enterovirus strain of piconarvirus. In clinic reports, EV71 especially infects the children below 5-year-old and may cause serious CNS complications. EV71 could induce autophagy in some cell lines and found autophagosomes was as replication complex for EV71. But the detail mechanism of EV71-induced autophagy in neuron-like cells is still unclear. We used human neural stem cell and neuroblastoma IMR-32 to differentiate neuron-like cells. We used EV71 to infect these neuron-like cells and found they could be infected and induced autophagy following EV71 infection. According to the turn over assay results, autophagy flux induced by EV71 was complete in neuron-like cells. To infect neuron-like cells by different MOI of EV71, we could find that autophagy was increased following more viral protein expression. And autophagy was decreased when inhibiting the newly synthesis of EV71 viral protein. And then we transfected separate EV71 viral protein plasmids to neuron-like cells, we found EV71 2B and 2BC could induce autophagy compare to vector only control. Next, we want to know how these viral protein to induce autophagy and effect of autophagy for virus growth and neuron survival when EV71 infection. Europic 2016 69

70 TUESDAY 06/09/2016 8:45 Session 3 IMMUNE MODULATION OF PICORNAVIRAL INFECTIONS Chairs: Urs Greber, Frank van Kuppeveld & Lo James 11:20 Lo James (Medical Research K05: INTRACELLULAR NEUTRALIZATION AND INNATE IMMUNE ACTIVATION BY THE 09:00 Council Laboratory of Molecular CYTOSOLIC ANTIBODY RECEPTOR TRIM21 Biology, United Kingdom) Stanley M. Lemon (University of C01: MAVS SIGNALING DEFINES THE HOST SPECIES RANGE AND PATHOGENICITY 09:20 OF HUMAN HEPATITIS A VIRUS North Carolina, United States of America) Michael Peeters (Universit C02: CONVERGENT MECHANISMS USED BY CARDIOVIRUSES, KSHV AND YERSINIA 09:35 TO ACTIVATE RSK KINASES catholique de Louvain, de Duve Institute, Belgium) C03: THEILER'S VIRUS L* PROTEIN INHIBITS RNASE L ACTIVATION THROUGH Thomas Michiels (Universite 09:50 COMPETITION WITH 2-5A BINDING catholique de Louvain, Belgium) COFFEE BREAK C04: HUMAN RHINOVIRUS 3C PROTEASE CLEAVES RIPK1, AN IMPORTANT Sarah Croft (University of 10:35 INTERMEDIATE IN EXTRINSIC APOPTOSIS Canberra, Australia) C05: RECOGNITION OF ENTEROVIRUS 71 BY TOLL-LIKE-RECEPTOR 8 AND RIG-I Hsing-I Huang (Chang Gung 10:50 ACTIVATE THE INFLAMMASOME IN HUMAN MYELOID CELLS University, Taiwan) C06: RSK MAY CONTROL STRESS GRANULES ASSEMBLY THROUGH PKR Yohei Hayashi (Universit 11:05 INHIBITION catholique de Louvain, Belgium) LUNCH BREAK 70 Keynote Lecture Communication (12 + 3 min) Europic 2016

71 TUESDAY 06/09/2016 POSTER SESSION 3 - IMMUNE MODULATION OF PICORNAVIRAL INFECTIONS C07: POTENT NEUTRALIZATION OF HEPATITIS A VIRUS SUGGESTS A RECEPTOR MIMIC MECHANISM Elizabeth Fry (Oxford University) C08: DIFFERENTIAL INNATE IMMUNE ACTIVATION AS AN ATTENUATION MECHANISM UNDERLYING CODON-DEOPTIMIZED POLIOVIRUS STRAINS Shane Smithee (CDC/NCIRD/DVD, United States of America) C09: APHTHOVIRUS LEADER PROTEINASE CLEAVES G3BP1 AND G3BP2 AND AFFECTS STRESS GRANULE FORMATION Linda Visser (Utrecht University, Netherlands) C10: IMMUNODOMINANT IGM AND IGG EPITOPES RECOGNIZED BY ANTIBODIES INDUCED IN ENTEROVIRUS A71-ASSOCIATED HAND, FOOT AND MOUTH DISEASE PATIENTS Yoke Fun Chan (University Malaya, Malaysia) C11: ANTIGENIC CHARACTERIZATION OF HUMAN PARECHOVIRUSES USING MIMOTOPE VARIATION ANALYSIS Maria Anastasina (Institute of Biotechnology, University of Helsinki, Finland) Europic 2016 71

72 TUESDAY 06/09/2016 K05 INTRACELLULAR NEUTRALIZATION AND INNATE IMMUNE ACTIVATION BY THE CYTOLSOLIC ANTIBODY RECEPTOR TRIM21 Lo James Medical Research Council Laboratory of Molecular Biology, United Kingdom TRIM21 is a recently discovered mammalian Fc receptor and E3 ubiquitin ligase expressed in the cytosol of most cells1. It is the highest affinity IgG receptor in man and has the widest known antibody specificity, being capable of also binding IgM and IgA. TRIM21 mediates an intracellular humoral response that protects against antibody-opsonized pathogens that invade the cytosol during infection. Upon detection, TRIM21 activates key immune transcription pathways to induce a potent antiviral state2. Simultaneous with and independent of immune activation, TRIM21 recruits cellular degradation machinery, which catalyse the disassembly and destruction of the virion to prevent its replication. Initially shown to protect against adenovirus infection, recent data suggests that this antiviral mechanism may also be effective against certain picornaviruses such as rhinovirus 14. In my talk I will summarize key aspects of TRIM21 biology, including recent work showing how sequential ubiquitination and deubiquitination control sensing and effector functions3 and how TRIM21 integrates with other cytosolic sensors such as RIG-I and cGAS to promote a rapid antiviral response4. 1. Mallery, D. L. et al. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proc Natl Acad Sci U S A 107, 19985-19990, doi:10.1073/pnas.1014074107 (2010). 2. McEwan, W. A. et al. Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nat Immunol 14, 327-336, doi:10.1038/ni.2548 (2013). 3. Fletcher, A. J., Mallery, D. L., Watkinson, R. E., Dickson, C. F. & James, L. C. Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21. Proc Natl Acad Sci U S A, doi:10.1073/pnas.1507534112 (2015). 4. Watkinson, R. E., McEwan, W. A., Tam, J. C., Vaysburd, M. & James, L. C. TRIM21 Promotes cGAS and RIG-I Sensing of Viral Genomes during Infection by Antibody-Opsonized Virus. PLoS Pathog 11, e1005253, doi:10.1371/journal.ppat.1005253 (2015). 72 Europic 2016

73 TUESDAY 06/09/2016 C01 MAVS SIGNALING DEFINES THE HOST SPECIES RANGE AND PATHOGENICITY OF HUMAN HEPATITIS A VIRUS Asuka Hirai-Yuki1, Lucinda Hensley1, John M. Cullen2, Jason K. Whitmire1, Stanley M. Lemon1 1 University of North Carolina, 2North Carolina State University OBJECTIVES: Host recognition of viral RNAs by RIG-I-like helicases and Toll-like receptors activates signaling pathways that induce interferons (IFNs) and proinflammatory cytokines, resulting in inflammation and limiting virus replication. Human hepatitis A virus (HAV) is a primitive hepatotropic picornavirus with a capsid structure intermediate between that of mammalian picornaviruses and insect dicistroviruses. It has a unique lifestyle, circulating during acute infection cloaked in a quasi-envelope derived from host cell membranes and resistant to neutralizing antibodies, but shed in feces as a stable non-enveloped virion. Infection in humans typically results in acute inflammatory liver injury following several weeks of subclinical intrahepatic infection. Viral RNA is abundant and increasing in the liver throughout this prodromal period, but evokes little type I IFN-stimulated gene expression, due in part to cleavage of the innate immune adaptor proteins, MAVS and TRIF (TICAM-1), by HAV-encoded proteases, 3ABC and 3CD. Like other human hepatitis viruses, the host range of HAV has been considered for many years to be limited to higher primates. Our goal in this study was to determine whether mice defective in innate or adaptive immunity could serve as effective small animal models of HAV infection and disease. METHODS AND RESULTS: Wild-type BL6 mice demonstrated no detectable virus replication or liver injury following intravenous challenge with wild-type HAV recovered from chimpanzee feces. However, type I IFN receptor knockout (Ifnar1-/-) mice supported robust intrahepatic replication of HAV, resulting in fecal shedding of virus, viremia, marked elevation of serum alanine aminotransferase (ALT), and histologic evidence of inflammatory hepatitis with numerous apoptotic hepatocytes and high levels of circulating IFN-beta. Virus was passaged 6x mouse-to-mouse using either feces or liver as inoculum, with only a single nonsynonymous mutation (3D-Arg468 to Lys) evident within the polyprotein by the 4th passage. Infection was highly hepatotropic, with viral genome copy numbers in the liver 500-fold greater than spleen, and 10,000-fold more than intestinal tissue. Mavs-/- and Irf3-/- Irf7-/- mice were equally permissive for HAV infection but demonstrated neither inflammation nor hepatocellular apoptosis despite high intrahepatic HAV genome copy numbers, whereas Trif-/-, Ifngr1-/-, Rag1-/- and NRG mice were non-permissive for HAV infection. Hepatocellular apoptosis and hepatic inflammation in infected Ifnar1-/- mice was associated with transcriptional activation of IFIT2 and Noxa. CONCLUSIONS: This remarkable new murine model of HAV infection recapitulates many aspects of the human disease, reveals that the capacity of HAV to evade type I IFN responses effectively defines its host species range, and identifies a key role for MAVS in inducing an IFN-independent pathway leading to hepatocellular apoptosis following infection with an hepatotropic human picornavirus. Europic 2016 73

74 TUESDAY 06/09/2016 C02 CONVERGENT MECHANISMS USED BY CARDIOVIRUSES, KSHV AND YERSINIA TO ACTIVATE RSK KINASES Michael Peeters1, Frederic Sorgeloos2, Didier Vertommen3, Felix Mller-Planitz4, Thomas Michiels3 1 Universit catholique de Louvain, de Duve Institute, 2University of Cambridge, Cambridge, UK, 3Universit catholique de Louvain, de Duve Institute, Brussels, Belgium, 4Ludwig-Maximillians University, Munich, Germany BACKGROUND & AIM: Theilers virus (TMEV) belongs to the Cardiovirus genus. As other Cardioviruses, it expresses a Leader (L) protein, which is implicated in the escape of the host immune response. Amongst its functions, L protein interacts with and maintains RSK kinases activated by inhibiting their dephosphorylation by phosphatases. Strikingly, two other very different pathogens (Kaposis Sarcoma-associated Herpes Virus [KSHV] and bacteria Yersinia) express proteins (respectively ORF45 and YopM) that interact with and activate RSK exactly as the L protein of TMEV does (1,2). This work aimed at defining the interface between L and RSK in order to understand the basis of RSK activation by pathogens. RESULTS: By mutagenesis and co-immunoprecipitation, we identified a DDVF motif of the L protein implicated in the interaction with RSK2, where the Phenylalanine is critical for the interaction. Very interestingly, ORF45 and YopM possess the same DDVF motif. In the case of KSHV ORF45, the Phe residue of this motif was also shown to contribute to RSK binding (3). We showed that the YopM Phe residue was also involved. By competition experiments, we showed that L and YopM interact with the same region of RSK. Thus, the RSK activation mechanism used by the different pathogens is likely identical. Cross-linking experiments identified a putative binding site of pathogens protein on RSK2. This suggests a model for activation of the kinase. The DDVF motif could tilt an alpha helix which is close to the activation loop of RSK2 and thereby maintain the kinase in an active conformation. CONCLUSIONS: To conclude, we highlighted an evolutionary convergence of three very different pathogens (RNA virus, DNA virus and bacteria). They activate RSK kinases via a common DDVF motif, probably via the same mechanism. Current efforts are now exerted to confirm the RSK binding site, the RSK activation mechanisms, and RSK targets that are involved in host-pathogen interaction. References: 1. Kuang et al., 2008, J Virol 82:1838-50; 2. Hentschke et al., 2010, PLoS One 5:10; Kuang et al., 2011, J Biol Chem 286:41171-82. 74 Europic 2016

75 TUESDAY 06/09/2016 C03 THEILERS VIRUS L* PROTEIN INHIBITS RNASE L ACTIVATION THROUGH COMPETITION WITH 2-5A BINDING Thomas Michiels1, Melissa Drappier1, Babal K. Jha2, Robert H. Silverman2, Susan R. Weiss3 1 Universit catholique de Louvain, de Duve Inst., Brussels, Belgium, 2Lerner Research Institute, Cleveland, USA, 3Perelman school of Medicine at the Univ. of Pennsylvania, Philadelphia, USA We showed previously that Theilers virus L* inhibits RNase L through direct protein-protein interaction. RNase L is one of the best-studied effector of the interferon (IFN) response. Interestingly, RNase L inhibition by L* exhibits species-specificity as L* inhibits mouse but not human RNase L (1). The aim of this work was to define the mechanism of RNase L inhibition by L*. We took advantage of this species specificity to map the L* binding site on RNase L, by testing L*-mediated inhibition of a series of human-mouse or rat-mouse RNase L chimeras. Our results suggest that L* interacts at two sites within RNase L ankyrin repeats 1 and 2. According to the recently solved structure of dimeric RNase L (2, 3), these target sites are compatible with RNase L inhibition through antagonism of both dimerization and 2-5A-binding. We therefore established a crosslinking-based RNase L dimerization assay and showed that L* inhibits RNase L dimerization. Next, we analyzed whether L* could interfere with 2-5A binding. Competitive binding studies by surface plasmon resonance demonstated that L* indeed competes with 2-5A for binding to mouse but not to human RNase L. Significance of RNase L inhibition by L* was confirmed in vivo, using recombinant Mouse hepatitis virus (MHV) where the RNase L antagonist protein NS2 was substituted for L*. In conclusion, protein L* of Theilers virus inhibits the OAS/RNase L pathway by binding to RNase L ankyrin repeats 1 and 2, thereby preventing 2-5A-mediated dimerization and activation of RNase L. This inhibition significantly supports virus replication in primary macrophages and in vivo. 1. Sorgeloos et al. 2013, PLoS pathog. 9, e1003474; 2. Huang et al., 2014, Mol Cell 53: 221-34; 3. Han et al., 2014, Science 343: 1244-8; 4. Zhao et al., 2012, Cell Host Microbe 11: 607-16. Europic 2016 75

76 TUESDAY 06/09/2016 C04 HUMAN RHINOVIRUS 3C PROTEASE CLEAVES RIPK1, AN IMPORTANT INTERMEDIATE IN EXTRINSIC APOPTOSIS Sarah Croft, Erin Walker, Reena Ghildyal University of Canberra Human Rhinovirus (HRV) is a human pathogen of significant medical importance, being a major cause of upper respiratory tract infections and causing the majority of the virus-induced asthma exacerbations. One cellular response to viral infection is the initiation of apoptosis; apoptotic signals are propagated via caspase cascades that lead to cell death, thereby reducing viral replication which relies on cellular machinery. OBJECTIVES: In this study, we investigated whether HRV could modulate apoptosis, a key antiviral innate immune response, and induce a cellular environment conducive to viral replication. We investigated the HRV mediated cleavage of RIPK1, an extrinsic apoptosis adaptor protein and aimed to determine the effects of this cleavage on viral replication. RESULTS: We have used HRV16 infected cells, cells treated with chemical inducers and inhibitors of extrinsic apoptosis, and in vitro protease cleavage assays to show that HRV16 3C protease cleaves a key intermediate in extrinsic apoptosis. Receptor interacting protein kinase 1 (RIPK1) was cleaved by caspase 8, as expected, during chemical induction of extrinsic apoptosis. RIPK1 was also cleaved in HRV infection albeit at a different cleavage site. Interestingly, caspase 8 activation, which is associated with extrinsic apoptosis, was required for optimal HRV 3C protease mediated cleavage of RIPK1. This was potentially achieved by increasing the accessibility of the HRV 3C cleavage site within RIPK1. Additionally, preliminary data suggests that the chemical inhibition of RIPK1 facilitates viral replication, even with the induction of apoptosis. CONCLUSION: The caspase 8 mediated RIPK1 cleavage product has a pro-apoptotic function, and further cleavage of this pro-apoptotic product by HRV 3C may provide a mechanism by which HRV regulates apoptosis. 76 Europic 2016

77 TUESDAY 06/09/2016 C05 RECOGNITION OF ENTEROVIRUS 71 BY TOLL-LIKE-RECEPTOR 8 AND RIG-I ACTIVATE THE INFLAMMASOME IN HUMAN MYELOID CELLS Hsing-I Huang, Chia-Jung Chou, Chi-Chong Chio Chang Gung University Objectives: EV71 is a pathogen that causes outbreaks in many countries in past decades, which is associated with wide-range pathological conditions including hand-foot-and mouth disease, herpagina, and even neurological manifestations. Clinical observations suggest cytokine production is correlated to the outcome of EV71 infection. Especially the production of IL-1b in the cerebrospinal fluid is correlated with the occurrence of pulmonary edema. It has been known that IL-1b secretion is through the activation of inflammasome, a multiprotein complex expressed in myeloid cells. However, how EV71 infection triggers the IL-1b production and the associated mechanisms are not clear. In this study, we showed that NLRP3 inflammasome is activated in myeloid cells upon EV71 infection. METHODS: PMA primed THP-1 cells were used as cell model in this study, since these cells are able to produce huge amounts of IL-1beta upon stimulation. Knockdown of TLR in monocytes by RNA interference was used to evaluate their roles in mediating inflammasome activation. Furthermore, the obtained results were further confirmed by using human peripheral blood mononuclear cells-derived macrophages. RESULTS: Our data showed that not only live viruses but also UV inactivated viral particles can evoke the IL- 1b production. Various clinical isolates are able to activate inflammasome formation in monocytic cells. Stable knockdown of NLRP3 ameliorates EV71-induced IL-1b expression. Furthermore, Treatment with Ac-YVAD-cmk or CA-074-me abrogated the EV71 induced IL-1b secretion, which indicates the acidification of endosome plays an essential role. Our results revealed that the expression of TLR8 and RIG-Iaffected inflammasome activation by EV71 in myeloid cells, while TLR3 was not. CONCLUSION: In summary, EV71 infection activates the NLRP3 inflammasome formation via the TLR8 and RIG-I in myeloid cells. Our results suggest innate immune sensing plays important roles in mediating EV71-induced inflammasome activation. Europic 2016 77

78 TUESDAY 06/09/2016 C06 RSK MAY CONTROL STRESS GRANULES ASSEMBLY THROUGH PKR INHIBITION Yohei Hayashi, Fabian Borghese, Thomas Michiels Universit catholique de Louvain OBJECTIVES: Theilers Murine encephalomyelitis virus (TMEV) belongs to the Theilovirus species within the Cardiovirus genus. The leader (L) protein encoded at the N terminus of viral polyprotein is multifunctional and was shown to antagonize the innate immune response. We reported that wild type but not mutant L protein inhibits stress granules (SG) assembly in infected cells. Recent results suggested that this inhibition was consequent to L-mediated inhibition of the double-stranded RNA (dsRNA)-activated protein kinase R (PKR). However, we found no evidence for interaction between L protein and PKR or viral dsRNA suggesting that PKR inhibition by L might be indirect. Recently, we identified cellular kinases of the RSK family as binding partners of the L protein. In this study, we evaluated the influence of RSK in inhibition of PKR activation and SG assembly by TMEV L protein. RESULTS: L-Mutant (LZn) but not wild type TMEV induces SG formation in HeLa cells from 8 hours post-infection. PKR-KO HeLa M cells were generated using the CRISPR/Cas9 technology. Unlike wild type HeLa M, these cells failed to produce SG after infection with LZn TMEV although they readily formed SG after sodium arsenite treatment. To analyze the involvement of RSK kinases on SG assembly, eIF3 (SG marker) immunolabeling was performed on cells treated with the RSK inhibitor: BID1870. BI-D1870 treatment, however, influenced neither SG assembly nor TMEV infection. In contrast, knocking down RSK1 and/or RSK2 using shRNAs led to the induction of small SG-like eIF3 dots in the cytoplasm, even after infection with the wild type virus. Western blot analysis revealed that PKR was activated by Lwt TMEV infection in the RSK knocked down cells. CONCLUSIONS: In this study, we confirmed that PKR has an important role in SG assembly after TMEV infection. Interestingly, knock down of RSK kinases (that are activated by L) led to the emergence of SG in cells infected with the wild type virus. Thus TMEV may utilize RSK kinases to inhibit SG assembly through PKR inhibition. 78 Europic 2016

79 TUESDAY 06/09/2016 C07 POTENT NEUTRALIZATION OF HEPATITIS A VIRUS SUGGESTS A RECEPTOR MIMIC MECHANISM Elizabeth Fry1, Xiangxi Wang2, Ling Zhu1, Minghao Dang2, Zhongyu Hu3, Qiang Gao2, Shuai Yuan2, Yao Sun2, Bo Zhang4, Jingshan Ren5, Thomas Walter5, Junzhi Wang3, David Stuart1, Zihe Rao2 1 Oxford University, 2Chinese Academy of Science, 3National Institutes for Food and Drug Control, 4Sinovac Biotech, 5University of Oxford Hepatitis A virus (HAV) infects ~1.4 million people annually and whilst there is a vaccine, there are no licensed therapeutic drugs. Hepatoviruses are unusual among picornaviruses, both in targeting the liver and also in their structure and life cycle. Cryo-EM structures of HAV full particles and empty particles at 3.4 and 3.8 resolution reveal a layered RNA genome and altered inner capsid layer distinguishing the full and empty particles. A potent HAV-specific monoclonal antibody R10 neutralizes virus infection at nM concentrations. The cryo-EM structure of full particles complexed with R10 Fab at 4.2 resolution, together with the crystal structure of the R10 Fab, allowed us to build a reliable model of the complex. R10 binds across the pentamer interface interfering with receptor attachment and viral uncoating and the R10 Fab destabilizes the virus at temperatures above 60C, shedding light on the hitherto unknown mechanism by which this extraordinarily stable virus releases its RNA and demonstrating new opportunities for therapeutic intervention. Europic 2016 79

80 TUESDAY 06/09/2016 C08 DIFFERENTIAL INNATE IMMUNE ACTIVATION AS AN ATTENUATION MECHANISM UNDERLYING CODON-DEOPTIMIZED POLIOVIRUS STRAINS Shane Smithee1, Nhien T. Wynn2, Cara C. Burns2, M. Steven Oberste2, Jennifer L. Anstadt2 1 CDC/NCIRD/DVD, 2Centers for Disease Control and Prevention Codon usage bias, or frequency of synonymous codon usage, is a cellular mechanism that regulates the function and expression of genes. Codon usage can determine the efficiency of translation based on availability of intracellular pools of transfer RNAs. Due to codon bias, interchange of synonymous codons can have profound effects on the expression of genes. Human viruses, in general, have a low codon usage bias when compared to their host. Further altering viral genomes via codon-deoptimization (e.g., changing the codon usage within viral genes) has become an increasingly utilized method to create attenuated viral strains to serve as potential vaccine candidates. Previous work in our lab to characterize codon-deoptimized polioviruses demonstrated that viral protein expression remained relatively unchanged, suggesting that the mechanism of attenuation was not related to an effect on overall translation efficiency of viral proteins. We hypothesized that differential activation of host innate immune responses contributes to the attenuation mediated by codon-deoptimization. While codon- deoptimized poliovirus strains are not overtly more sensitive to interferon pretreatment in a HeLa cell model, clear differences in the expression of innate immune genes such as IRF-7, RNaseL, and MDA-5 were observed when infection with unaltered wildtype strains was compared. Taken together, these data suggest that codon- deoptimized polioviruses do indeed interact differently with host cells, likely due to altered innate immune sensing and activation. Investigating these differences in cells of neural origin may reveal core components of the innate immune response that contribute to the attenuation of codon-deoptimized poliovirus in the biological context of neuronal infection. Identifying the precise mechanisms underlying pathogen attenuation as mediated by codon deoptimization is paramount to fully understanding the strategy as a platform for vaccine development. 80 Europic 2016

81 TUESDAY 06/09/2016 C09 APHTHOVIRUS LEADER PROTEINASE CLEAVES G3BP1 AND G3BP2 AND AFFECTS STRESS GRANULE FORMATION Linda Visser, Martijn Langereis, Huib Rabouw, Raoul De Groot, Frank Van Kuppeveld Utrecht University INTRODUCTION AND OBJECTIVES: Innate antiviral responses are the first line of defense against viral infections. Next to the well known type I interferon (IFN-/) response pathway, the stress response pathway is increasingly recognized as an important innate antiviral pathway. Activation of this pathway leads to an inhibitory phosphorylation of eIF2, thus inhibiting viral and host translation. The resulting inactive pre-initiation mRNA complexes aggregate to so-called stress granules (SG). These SGs may also contribute to the antiviral state within the host cell by sequestering translation factors or, alternatively, by functioning as antiviral signal transduction platforms. Picornaviruses are known to evade innate antiviral responses. Cardioviruses suppress both the IFN-/ and stress response pathways via their leader protein (L), although the mechanism remains elusive. In contrast, aphthoviruses produce two viral proteinases (Lpro and 3Cpro), which are known to limit IFN-/ pathway activation. Whether aphthoviruses also repress the stress response pathway remains to be elucidated. In this study, we investigated the role of the aphthovirus proteinases (Lpro and 3Cpro) in the suppression of the stress response pathway. METHODS: In order to study the role of the aphthovirus proteinases in repressing innate antiviral responses, in the context of viral infection, we generated recombinant cardioviruses. Utilizing the infectious clone of encephalomyocarditis virus (EMCV), in which the leader protein was inactivated (EMCV-LZn), we inserted the gene of an aphthoviral proteinase (Lpro or 3Cpro). Subsequently, these recombinant viruses were used to study whether the aphthovirus proteinases can suppress the stress response pathway. Stress response pathway activation was determined using flow cytometry analysis of PKR and eIF2 phosphorylation. The formation of SGs was analysed using immune fluorescence microscopy. RESULTS: Stress response pathway activation (i.e. PKR and eIF2 phosphorylation) is unaffected upon infection with the recombinant EMCV-LZn viruses expressing either the aphthovirus Lpro or the 3Cpro. However, stress granule formation is reduced upon infection with EMCV-LZn expressing aphthovirus Lpro, but not 3Cpro. The number of stress granules per cell is unaffected, but the observed granules are significantly smaller (reduced by more than 50% compared to EMCV-LZn). A common mechanism by which viruses inhibit SG formation is by cleavage of scaffolding proteins that are essential in SG formation. Indeed, we observe that infection with recombinant EMCV- LZn virus expressing the aphthovirus Lpro results in cleavage of G3BP1 and G3BP2, two known scaffold proteins. CONCLUSION: In conclusion, we show that aphthovirus proteinases do not affect activation of the stress response pathway. Nevertheless, the formation of SGs was altered. We identify Lpro as the viral proteinase responsible for SG repression. Lpro cleaves both G3BP1 and G3BP2, which most likely is responsible for the reduced SG size. These results put aphthoviruses in a long list of viruses that impair SG formation. Why numerous viruses benefit from repressed SG formation, without affecting pathway activation, remains to be further investigated. Europic 2016 81

82 TUESDAY 06/09/2016 C10 IMMUNODOMINANT IGM AND IGG EPITOPES RECOGNIZED BY ANTIBODIES INDUCED IN ENTEROVIRUS A71-ASSOCIATED HAND, FOOT AND MOUTH DISEASE PATIENTS Yoke Fun Chan1, Kam Leng Aw Yong2, Jamal I-Ching Sam2, Mia Tuang Koh2, Yoke Fun Chan2 1 University Malaya, 2University Malaya, Malaysia Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and 4 IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142-156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope; and PEP23 mapped at VP1 41-55 was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates. 82 Europic 2016

83 TUESDAY 06/09/2016 C11 ANTIGENIC CHARACTERIZATION OF HUMAN PARECHOVIRUSES USING MIMOTOPE VARIATION ANALYSIS Maria Anastasina1, Arno Pihlak2, Anri Kivil2, Katja Wolthers3, Kaia Palm2, Sarah Butcher1 Institute of Biotechnology, University of Helsinki, 2Protobios LLC, 3Academic Medical Centre, AMC 1 Considerable antigenic diversity within species of picornaviruses complicates development of surveillance and control measures for relevant human infections. Our knowledge of viral antigenicity is still limited and is based primarily on investigations of lab-adapted strains, rather than clinical isolates. Here we show how picornavirus antigenic determinants can be studied in human sera in a high throughput manner using Mimotope Variation Analysis (MVA) that combines phage display with massive parallel sequencing. We utilized the MVA for antigenic characterization of parechovirus types 1 and 3 in a collection of human sera and for identification of individual and common antibody responses to these viruses. We demonstrate how comprehensive sera immunoprofiling using MVA could improve our understanding of viral antigenicity and facilitate identification of common and strain-specific epitopes. Furthermore, such an approach aids discovery of infection biomarkers and supports development of diagnostic and surveillance tools for these viruses. Europic 2016 83

84 TUESDAY 06/09/2016 VIRUS GENETICS, GENOME PLASTICITY, EVOLUTION, 14:00 Session 4 CLASSIFICATION 18:05 Chairs: 1st part, Raul Andino & David Evans 2nd part, Peter Simmons & Alexander Gorbaleyna K06: VIRUS EVOLUTION: MATHEMATICAL REDUCTION OF SEQUENCE SPACE AND Marco Vignuzzi (Pasteur Institute, 14:00 EMPIRICAL FITNESS LANDSCAPES France) Rasmus Henningsson (Lund D01: UNCOVERING RNA VIRAL POPULATION DYNAMICS: SEQUENCE SPACE AND 14:35 EMPIRICAL FITNESS LANDSCAPES University / Institut Pasteur, Paris, Sweden) D02: SEQUENCE SPACE AND TRANSLATIONAL FITNESS LANDSCAPE IN HEPATITIS Rosa Pint (University of Barcelona, 14:50 A VIRUS Spain) Kelly Watters (University of D03: ADAPTATION OF RV-C15 TO CDHR3-EXPRESSING HELA CELLS ALLOWS 15:05 HIGH-TITER VIRUS PRODUCTION IN TISSUE CULTURE Wisconsin-Madison, United States of America) D04: THE INFLUENCE OF SEQUENCE AND RNA STRUCTURE ON RECOMBINATION Kirsten Bentley (University of St 15:20 IN ENTEROVIRUSES Andrews, United Kingdom) D05: THIRTY YEARS OF POLIOVIRUS REPLICATION IN AN IMMUNODEFICIENT Dimitra Klapsa (NIBSC, United 15:35 INDIVIDUAL: MOLECULAR EVOLUTION AND EFFECT OF ANTI-VIRAL TREATMENTS Kingdom) David Barton (University of 15:50 D07: PICORNAVIRUS RNA RECOMBINATION Colorado, United States of America) COFFEE BREAK Nick Knowles (The Pirbright 16:25 D08: PICORNAVIRIDAE: THE EVER-GROWING VIRUS FAMILY Institute, United Kingdom) Nora Chapman (University of D09: COMPLETE REVERSION OF A MULTIPLY MUTATED COXSACKIEVIRUS B3 16:40 (CVB3) CRE(2C) DURING REPLICATION OF 5 TERMINALLY DELETED VIRUS Nebraska Medical Center, United States of America) D10: NEXT GENERATION SEQUENCING REVEALS NEW SAT GENOTYPES BRINGING Nick J. Knowles (The Pirbright 16:50 US CLOSER TO UNDERSTANDING THE HISTORY OF FOOT-AND-MOUTH DISEASE Institute, United Kingdom) VIRUS IN AFRICA D11: PREDICTION OF CONSERVED RNA STRUCTURES WITHIN THE FOOT-AND- Fiona Tulloch (University of St 17:00 MOUTH DISEASE VIRUS GENOME REVEALS FUNCTIONAL CIS-ACTING ELEMENTS Andrews, United Kingdom) LOCALISED IN THE 3D CODING REGION D12: EXCHANGES OF GENOMIC DOMAINS BETWEEN POLIOVIRUS TYPE 2 AND A Mal Bessaud (INSTITUT PASTEUR, 17:10 PANEL OF CO-CIRCULATING SPECIES C ENTEROVIRUSES REVEAL A HIGH DEGREE France) OF GENOMIC PLASTICITY D13: IMPORTANCE OF 5 TERMINAL SEQUENCE IN COXSACKIEVIRUS B3 RNA IN James Flanegan (University of 17:20 REGULATING VIRAL RNA REPLICATION AND A SWITCH TO VPG-PRIMED POSITIVE- Florida, United States of America) STRAND INITIATION D14: MISINCORPORATION OF NUCLEOTIDES OR DRUGS DURING POLIOVIRUS Nynke Dekker (TU Delft, 17:30 RNA-DEPENDENT RNA POLYMERASE ELONGATION LEAVES FINGERPRINTS IN THE Netherlands) PAUSING KINETICS Raul Andino (University of D15: RNA VIRUS DIVERSITY IS IMPORTANT TO COUNTERACT INNATE IMMUNITY 17:40 AND SPREAD WITHIN THE HOST California, San Francisco, United States of America) D16: CONNECTING METAGENOMICS EXPLORATION AND EXPERIMENTAL Alexander Gorbalenya (Leiden 17:50 RESEARCH OF PICORNAVIRUSES WITH THE EVOLUTIONARY BASED PARTITIONING University Medical Center, OF GENOMIC DIVERSITY BY DEMARC Netherlands) FREE FOR DINNER 84 Keynote Lecture Communication (12 + 3 min) Short Communication (8 + 2 min) Europic 2016

85 TUESDAY 06/09/2016 POSTER SESSION 4 - VIRUS GENETICS, GENOME PLASTICITY, EVOLUTION, CLASSIFICATION D17: MECHANISM OF RNA VIRUS RECOMBINATION Jamie Arnold (Penn State, United States of America) D18: 5 TERMINAL CHARACTERIZATION OF PERSISTENT ENTEROVIRUS IN CARDIAC TISSUE OF PATIENT SUFFERING FROM DILATED CARDIOMYOPATHY Alexis Bouin (University of California, Irvine, United States of America) D19: ESTABLISHING A SYSTEM TO STUDY RECOMBINATION IN ENTEROVIRUS 71 Andrew Woodman (Penn State University, United States of America) D20: PATTERNS OF INTERTYPIC RECOMBINATION BETWEEN VACCINE-DERIVED POLIOVIRUSES REVEAL GENOMIC REGIONS WITH REDUCED FITNESS Konstantin Chumakov (US Food and Drug Administration, United States of America) D21: UNTANGLING THE ROLE OF THE PSEUDOKNOTS IN FMDV REPLICATION Joseph Ward (University of Leeds, United Kingdom) D22: CORNERING RNA VIRUSES BY CONSTRAINING THEIR SEQUENCE SPACE Gonzalo Moratorio (Institut Pasteur, France) D23: IMPACTS OF GENETIC BOTTLENECKS ON THE FOOT-AND-MOUTH DISEASE VIRUS CONSENSUS SEQUENCE: IMPLICATIONS FOR VIRUS FITNESS AND EVOLUTION Caroline Wright (The Pirbright Institute, United Kingdom) D24: MOSAIC EVOLUTION OF ENTEROVIRUS A71 GENOGROUP F THROUGH FREQUENT INTRASPECIES GENETIC RECOMBINATION Romain Volle (INSTITUT PASTEUR, France) D25: SCREENING OF ENTEROVIRUS 71 TRANSCRIPTION DYNAMICS: ESTABLISHING HIGH-THROUGHPUT ASSAY FOR SINGLE-MOLECULE INVESTIGATION VIA MAGNETIC TWEEZERS Richard Janissen (Kavli Institute of Nanoscience, Delft University of Technology, Netherlands) D26: DELETION OF DOMAIN I IN THE 5' NTR OF COXSACKIEVIRUS B3 (CVB3) GENOMIC RNA DOES NOT PREVENT REPLICATION Nora Chapman (University of Nebraska Medical Center, United States of America) D27: GENOME SEQUENCE OF AVIAN ENTERO-LIKE VIRUS 2 SHOWS IT TO BE A CHICKEN MEGRIVIRUS Nick Knowles (The Pirbright Institute, United Kingdom) D28: INTERTYPIC RECOMBINATION OF HUMAN PARECHOVIRUS 4 ISOLATED FROM INFANTS WITH SEPSIS-LIKE DISEASE Sisko Tauriainen (University of Turku, Finland) D30: PICORNAVIRUS RNA RECOMBINATION David Barton Europic 2016 85

86 TUESDAY 06/09/2016 K06 VISUALIZING, MONITORING AND TARGETING RNA VIRUS EVOLUTION IN SEQUENCE SPACE AND FITNESS LANDSCAPES Marco Vignuzzi, Institut Pasteur, Paris, France RNA viruses comprise the majority of emerging pathogens of clinical and agricultural relevance, and their characteristically elevated mutation rates and frequencies are the basis for their rapid evolution, and the emergence of adaptive mutations resulting in increased fitness, virulence, resistance and tropism. A replicating RNA virus population generates thousands of variants from which only a few provide considerable adaptive benefits. But how can we probe this mutant spectrum to identify the variants of interest? How can we track the evolution of these viruses taking into account all of the minority mutations? Can our knowledge on how viruses fill and move in sequence space be used to predict, control and target them? I will present our recent efforts to better monitor and visualize virus population dynamics in terms of sequence space and fitness landscapes using combined experimental and mathematical approaches. I will highlight how we have used Coxsackie virus as a tool to track evolution and target RNA viruses using evolutionary principles. 86 Europic 2016

87 TUESDAY 06/09/2016 D01 UNCOVERING RNA VIRAL POPULATION DYNAMICS: SEQUENCE SPACE AND EMPIRICAL FITNESS LANDSCAPES Rasmus Henningsson1, Gonzalo Moratorio2, Antonio Borderia2, Marco Vignuzzi2, Magnus Fontes2 1 Lund University / Institut Pasteur, Paris, 2Institut Pasteur, Paris Due to their rapid mutation rates, RNA viruses circulates as a complex networks of closely related genetic variants, a mutant spectrum linked through mutation. Indeed, it has been show how the composition of the mutant swarm greatly affects pathogenesis and viral fitness, in a way that could not have been explained at the consensus level. This leads to the view that the mutant swarm is not an unwanted side-effect of imperfect viral replication, but rather an important feature of viral infections. A plethora of approaches have been used to try to understand impact of the mutant swarm composition on viral fitness. Previous work have however imposed severe restrictions on how the data is analyzed, either by focusing analysis on a few selected loci, a short region of interest, or a few dominant haplotypes, often combined with theoretical models for fitness and/or viral populations behavior. We present a bottom-up framework for analyzing mutant swarms, based on empirical whole-genome deep sequencing data. Briefly, we represent mutant swarms by the variant frequencies across all loci, allowing for analysis of how each mutant swarm explores sequence space. Great care is taken to avoid the impact of sequencing errors that are otherwise easily mistaken for minority variants. Our methods were applied to a large dataset of sequencing data coming from mutant swarms passaged under different mutagenic conditions. Applying dimension reduction techniques, we find a new representation that captures the informative part of the data set. Visualization of this representation uncover distinct patterns relating to lineage and mutagen used in the experiments, showing differences in how the different mutant swarms spread out in sequence space. Coupling with hundreds of empirical fitness values from competitive fitness assays, we show that fitness can be predicted with good accuracy, greatly outperforming consensus-level analysis. Finally, a fitness landscape, based entirely on empirical data, is produced by a further step of nonlinear dimension reduction, giving an easily accessible overview of the impact of the mutant swarm composition on viral fitness. Europic 2016 87

88 TUESDAY 06/09/2016 D02 SEQUENCE SPACE AND TRANSLATIONAL FITNESS LANDSCAPE IN HEPATITIS A VIRUS Rosa Pint, Francisco-Javier Prez-Rodrguez, Luca DAndrea, Albert Bosch University of Barcelona Hepatitis A virus (HAV) has a very unique genomic composition showing the highest codon deoptimization among picornaviruses, particularly in the capsid region. Additionally, it possesses a very inefficient IRES and is unable to shutdown the cellular protein synthesis. These three features are interrelated and adaptation of the virus to conditions of artificially induced cellular shutoff is anticipated to be associated to changes in the sequence space which in turn may induce modifications in the translational landscape. Deep-sequencing of two capsid fragments (in the VP3 and VP1 proteins) belonging to regions of low amino acid volatitlity (codons that after mutation show low tendency to induce nonsynonymous replacements) and high codon volatility (codons that after mutation show high tendency to change of frequency class), revealed replacements of the original haplotypes by new emerging ones in the sequence space of two populations adapted to different degrees of cellular shutoff. The translation efficiency of the different VP3- and VP1-derived haplotypes was tested in vitro and the landscape of each population in different conditions of shutoff determined. These analyses revealed that HAV, in terms of codon volatility, is less robust than other picornaviruses and this lack of robustness allows for its evolvability to conditions of cellular shutoff. 88 Europic 2016

89 TUESDAY 06/09/2016 D03 ADAPTATION OF RV-C15 TO CDHR3-EXPRESSING HELA CELLS ALLOWS HIGH-TITER VIRUS PRODUCTION IN TISSUE CULTURE Kelly Watters, Yury Bochkov, Shakher Sijapati, Sarmila Basnet, Marchel Hill, James Gern, Ann Palmenberg University of Wisconsin-Madison Rhinovirus C species (RV-C) can cause severe wheezing illnesses and asthma exacerbations in children. We discovered that expression of human cadherin-related family member 3 (CDHR3) with the single nucleotide polymorphism (rs6967330, C529Y) that is linked to the development of early childhood asthma with severe exacerbations (1) enables cells normally unsusceptible to RV-C infection to support RV-C attachment and propagation. We developed HeLa cell lines stably expressing CDHR3-C529 or CDHR3-Y529 variants. Cells transduced with the Y529 variant expressed more CDHR3 on their surfaces and increased RV-C binding and replication (10- fold) compared to control cells or cells transduced with the C529 variant. Although RV-C clinical isolates, including C15, can replicate in transfected HeLa cell lines expressing CDHR3-Y529, they induce only mild CPE in these cells. By serial passage of C15 in the cultured CDHR3-Y529 stable HeLa-E8 cell line (HeLa-E8), we were able to adapt the virus to increase virus yields with strong CPE. After 10 passages, the binding and replication of the adapted virus (C15a) increased (10-fold) compared to wild-type C15. Surprisingly, C15a bound both control and CDHR3- transduced HeLa cells to almost similar levels; but replication in transduced cells was about 2 logs higher than in control cells. Complete genome sequencing of C15a identified several missense mutations in capsid (VP3 and VP1) and nonstructural (3A) proteins. These were then introduced individually or in combination into the C15 cDNA. The recombinant C15 viruses indicated that adaptation was acquired by two key mutations: T125K in VP1, which increased virus attachment, and E41K in 3A, which improved virus replication. Results of virus binding assays suggest that the T125K VP1 mutation allows C15a to use heparin as a receptor/co-receptor for attachment and entry, in addition to CDHR3. The generation of stable CDHR3-expressing cell lines and an adapted C15a virus now allow for high titer RV-C production and provide useful tools for the investigation of RV-C. 1. Bnnelykke K, Sleiman P, Nielsen K, Kreiner-Mller E, Mercader JM, Belgrave D, den Dekker HT, Husby A, Sevelsted A, Faura-Tellez G, Mortensen LJ, Paternoster L, Flaaten R, Mlgaard A, Smart DE, Thomsen PF, Rasmussen MA, Bons-Guarch S, Holst C, Nohr EA, Yadav R, March ME, Blicher T, Lackie PM, Jaddoe VW, Simpson A, Holloway JW, Duijts L, Custovic A, Davies DE, Torrents D, Gupta R, Hollegaard MV, Hougaard DM, Hakonarson H, Bisgaard H (2014) A genome-wide association study identifies CDHR3 as susceptibility locus for early childhood asthma with severe exacerbations. Nat Genet. 46(1):51-55. Europic 2016 89

90 TUESDAY 06/09/2016 D04 THE INFLUENCE OF SEQUENCE AND RNA STRUCTURE ON RECOMBINATION IN ENTEROVIRUSES Kirsten Bentley1, Ashley Pearson1, Andrew Woodman2, Sin Jones1, David Evans1 1 University of St Andrews, 2Penn State The extensive genetic diversity in positive strand RNA viruses can be attributed to the error prone polymerase and is compounded by the process of recombination, which can occur during replication if a single cell is infected with more than one virus. Via what is believed to be a copy-choice mechanism viral RNA-dependent RNA polymerases can undergo a template switching event that leads to the generation of hybrid genomes containing genetic information derived from both parental viruses. It is currently proposed that this template switching event is promoted by sequence motifs and/or secondary structure in the RNA genome. Using a poliovirus-based in vitro replication assay (CRE-REP assay) we have isolated viable, early recombination products following dual transfection of independently replication-compromised PV1 (donor) and PV3 (acceptor) genomes. Strikingly, recovered primary recombinants exhibited junctions straddling either the VP1/2A or 2A/2B proteolytic polyprotein cleavage sites and the majority, termed imprecise recombinants, contain in-frame duplications of up to 400nt. Subsequently, in a process termed resolution, passage of these imprecise recombinant viruses resulted in the loss of sequence duplications and a return to wildtype-length genomes. To investigate the effect of sequence and RNA secondary structure on both the template switching event, i.e. recombination event, and the subsequent resolution event we have modified sequences around the 2A/2B junction of both the donor and acceptor partners of the CRE-REP assay. Here, we demonstrate the effects of maximising sequence identity, or maximising/minimising the degree of RNA secondary structure within a defined region of the 2A/2B junction, on the generation of recombinant viruses in a replicative system, and the subsequent resolution of differing imprecise recombinant viruses. Our data strongly supports a model in which recombination is a random event, independent of RNA sequence, but that structure may have limited influence on the location of the crossover junction. In contrast, RNA sequence demonstrates a potential influence on the process of resolution of imprecise recombinant genomes that is currently being investigated further. 90 Europic 2016

91 TUESDAY 06/09/2016 D05 THIRTY YEARS OF POLIOVIRUS REPLICATION IN AN IMMUNODEFICIENT INDIVIDUAL: MOLECULAR EVOLUTION AND EFFECT OF ANTI-VIRAL TREATMENTS Dimitra Klapsa, Philip Minor, Glynis Dunn, Javier Martin NIBSC Poliovirus strains in live-attenuated vaccines can replicate for very long periods of time in humans with antibody deficiencies leading to chronic infection. Here, we describe an individual who has been excreting type 2 vaccine- derived poliovirus for almost thirty years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we showed that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme, particularly after the recent withdrawal of the type 2 component from the oral polio vaccine. As it is typical of poliovirus isolates from chronic excreters, virus samples in stools from this individual consisted of mixed virus populations resulting in heterogeneous bases in Sanger nucleotide sequence analysis. However, little is known about how and why these mixed viral populations occur and in what manner they behave and evolve. We have performed extensive plaque-purification and deep sequencing analyses of poliovirus isolates from sequential stool extracts obtained during a 20-year period. The results revealed an intricate natural history with viruses from divergent genetic clusters co-evolving for long periods of time, consistent with independent virus replication in distant parts of the gut, but also with sporadic inter-cluster recombination events and recurrent elimination and re-emergence of specific genetic clusters. The presence of diverse viral populations contributed to an increased overall genetic plasticity. This complex replication strategy might provide poliovirus with better potential to respond to changes in the environment or any other challenges that the virus encounters during replication such as the different forms of anti-viral therapy employed with this patient. Although none of the treatments cleared the infection from the patient, at least some of the medical interventions appear to have had an effect on the virus population dynamics. Determining the antigenic and virulence properties of vaccine-derived viruses from chronic excretes could help assessing the potential for these strains to circulate and cause disease in different vaccination contexts. Understanding the population dynamics of poliovirus evolution in immunodeficient individuals could help designing effective anti-viral treatments, not only it would help estimating the efficacy of any given compound to inhibit replication of any of the different virus strain components of the viral population, but it could also allow determining a suitable schedule for treatment. Europic 2016 91

92 TUESDAY 06/09/2016 D07 & POSTER D30 PICORNAVIRUS RNA RECOMBINATION David Barton1, Brian Kempf1, Olve Peersen2 1 University of Colorado, 2Colorado State University OBJECTIVES: RNA recombination is important in the formation and ongoing evolution of picornavirus species groups. In this study we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to replicative RNA recombination. We hypothesized that some features of 3Dpol exist to facilitate RNA recombination. METHODS: We engineered alanine substitution mutations into 3Dpol and measured their impact on the frequency of RNA recombination, the fidelity of RNA synthesis and the sensitivity of virus to inhibition by ribavirin. Recombination was evident when infectious virus was recovered from murine cells co-transfected with donor and recipient RNAs containing lethal mutations, a method described by Lowry et al. (PLoS Pathog. 2014). Alanine substitution mutations were engineered into the polymerase gene of the donor replicon to disrupt protein-RNA interactions within polymerase-RNA elongation complexes. We predicted that such mutations might increase or decrease magnitudes of recombination. RESULTS: Some 3Dpol mutations inhibited the frequency of replicative RNA recombination whereas others increased the frequency of replicative RNA recombination. Within the 3Dpol thumb domain we observed a dramatic difference between a L420A mutation that inhibited recombination by more than 10-fold and an adjacent L419A mutation that increased the frequency of recombination by 2-fold. Neither mutation had major effects on the magnitudes of virus replication in tissue culture cells. In addition to inhibiting RNA recombination, the L420A mutation rendered virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin- induced mutations in viral RNA. The atomic structure of 3Dpol-RNA elongation complexes shows how leucines 419 and 420 form hydrophobic contacts with ribose groups in the RNA template and RNA product, respectively. CONCLUSIONS: Our experimental evidence shows that 3Dpol Leu420, a residue conserved in viruses throughout the Picornaviridae family, is required for replicative RNA recombination. Furthermore, our results suggest that replicative RNA recombination contributes to ribavirin resistance. Structural and functional insights into replicative RNA recombination are evident by visualizing the 3Dpol Leu420 interaction with the nascent RNA product three bases from the polymerase active site. This protein-RNA interaction is important in the ongoing evolution of picornaviruses. 92 Europic 2016

93 TUESDAY 06/09/2016 D08 PICORNAVIRIDAE: THE EVER-GROWING VIRUS FAMILY Roland Zell1, Eric Delwart2, Alexander E. Gorbalenya3, Tapani Hovi4, Andrew M.Q. King5, Nick J. Knowles5, A. Michael Lindberg6, Mark A. Pallansch7, Ann C. Palmenberg8, Gabor Reuter9, Peter Simmonds10, Tim Skern11, Glyn Stanway12, Teruo Yamashita13 1 Jena University Hospital, 2Blood Systems Res. Inst., University of California San Francisco, 3Dept. Medical Microbiology, Leiden University Medical Center, 4National Institute for Health and Welfare, 5The Pirbright Institute, 6Dept. Chemistry and Biomedical Science, Linnaeus University Kalmar, 7Division of Viral Diseases, Centers for Disease Control and Prevention, 8Institute for Molecular Virology, University of Wisconsin, 9National Reference Laboratory of Gastroenteric Viruses, ANTSZ Reg. Inst. of State Public Health Service, 10 Nuffield Dept. Medicine, University of Oxford, 11Max F. Perutz Laboratories, Medical University of Vienna, 12Dept. Biological Science, University of Essex, 13Dept. Microbiology, Aichi Prefecture Institute of Public Health The family Picornaviridae presently comprises 31 genera and 54 species. New species proposals include Avisivirus B and C (chicken picornaviruses), Cosavirus B, D, E, F, Enterovirus K (dromedary camel enterovirus), Kobuvirus D to F (kagovirus from cattle, rabbit picornavirus, bat kobuvirus), Mischivirus B and C (bat picornaviruses) and Parechovirus C and D (Sebokele virus and ferret parechovirus, respectively) as well as eight Hepatovirus species (hepatoviruses from harbour seals, rodents, hedgehogs, shrews, treeshrews, bats, marmots). In addition, three species/genus proposals will be submitted to the ICTV in 2016 (Rabovirus A/Rabovirus, Ampivirus A/Ampivirus, Falcopivirus A/Falcopivirus). Nevertheless, more than 30 new picornaviruses from cattle, pigs, cats, dogs, sea lions, tortoises, various bird species and many bats await classification. The discovery of more than 20 divergent sapelo-like picornaviruses challenges genus and species demarcation rules of this genus and its species. Further, the renaming of the species of the Aphthovirus genus is discussed with the aim of removing host species and disease-specific names. The recent expansion in the number of classified picornavirus genera and ongoing continued characterization of future candidate genera suggests that picornaviruses might usefully be further grouped at a higher taxonomic level (subfamily). However, the diverse genome layouts, host ranges and host interactions of members of such groupings make their definition problematic, and would lack general, overarching inclusion criteria other than phylogenetic clustering. www.picornastudygroup.com Europic 2016 93

94 TUESDAY 06/09/2016 D09 COMPLETE REVERSION OF A MULTIPLY MUTATED COXSACKIEVIRUS B3 (CVB3) CRE(2C) DURING REPLICATION OF 5 TERMINALLY DELETED VIRUS Nora Chapman1, Shane Smithee2, Steven Tracy1 1 University of Nebraska Medical Center, 2Centers for Disease Control and Prevention CVB can delete 5 genomic termini in culture, in experimentally inoculated mice and in naturally infected humans, resulting in populations with 5 terminally deleted genomes (CVB-TD) which are not constrained to 5 di-uridine termini which do have covalently attached VPg. OBJECTIVE: Determine whether the cis-acting replication element, CRE(2C), was required for replication of a CVB population lacking wildtype 5 genomic termini. METHODS: The CRE(2C) in an infectious cDNA copy of a CVB3 genome was altered with 16 mutations (termed CRE knockout or CKO) as in van Ooij et al. (J. Virol., 2006). Transcripts (CVB3-CKO) and two others of CVB3 genomic clones with a deletion of the 5 terminal 49 nucleotides with or without the CKO (CVB3-TD50 or CVB3- TD50-CKO) generated infectious virus in HeLa cell culture or in mice. Purified virion RNA was used to confirm replication extents by RT-PCR and sequence of progeny virus. Progeny virus infectivity was shown by passaging in cell cultures, a process prevented by CVB3-neutralizing antibody. RESULTS: CVB3-CKO replicates 100,000 times less efficiently than wildtype CVB3 but produced infectious virions. CVB3-TD50-CKO and CVB3-TD50 also replicated at the same level, indicating that CRE(2C)-mediated VPg uridylylation was not required for genomes lacking wildtype 5 genomic termini. When CVB3-CKO was passaged in culture, the virus population had developed 5 terminal deletions within 3 days, producing a CVB3-TD-CKO population. By 8 days of passage in culture, sequence analysis indicated all 16 mutations in the CKO sequence had reverted to wildtype sequence. When the CVB3-TD50-CKO strain was similarly passaged, it too reverted completely to a wildtype CRE(2C) but retained the terminal deletion. CONCLUSIONS: CKO viruses in both CVB3 wildtype and in CVB3-TD backgrounds replicate in HeLa cell culture at a level not significantly different from replication extents observed with non-CKO CVB3-TD. As replication of the CVB3-CKO (wildtype 5 end) resulted in CVB3-TD progeny, loss of the CRE(2C) altered specificity of the positive strand RNA initiation. In addition, passage of CKO viruses resulted in reversion of the 16 CRE(2C) mutations. As these viruses are replicating with 5 terminal deletions and to a similar extent as CRE-intact CVB3-TD50, uridylylated VPg is not necessary for positive strand RNA initiation. CVB3-TD viruses replicate with a positive to negative strand ratio approaching unity. If the initiation of negative strand RNA is more efficient with a functional CRE(2C), the dependence of the very poor positive strand RNA replication of CVB3-TD upon the amount of negative strand template can create a selective pressure for reversion of this structure. This reversion demonstrates a role of CRE(2C)-generated uridylylated primer in negative strand RNA initiation in the context of poor positive strand RNA initiation. The generation of TD viruses is likely to occur naturally with the loss of essential elements for correct positive strand RNA initiation but negative strand RNA replication levels can dictate the level of TD virus replication. 94 Europic 2016

95 TUESDAY 06/09/2016 D10 NEXT GENERATION SEQUENCING REVEALS NEW SAT GENOTYPES BRINGING US CLOSER TO UNDERSTANDING THE HISTORY OF FOOT-AND-MOUTH DISEASE VIRUS IN AFRICA Lidia Lasecka-Dykes, Nick J. Knowles, Caroline F. Wright, Grace Logan, Toby Tuthill, Terry Jackson, Donald P. King The Pirbright Institute Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals caused by FMD virus (FMDV), a positive-sense RNA virus of the family Picornaviridae. The disease can lead to huge economical loses and is present in Africa, Asia and sporadically in South America posing a constant threat to the livestock industry. Despite increased number of publicly available whole genome sequences, current FMDV genomic data are biased by the opportunistic nature of sampling, which makes it difficult to evaluate the real variability of FMDV. Using next generation sequencing we have generated full genome sequences of FMDV field isolates from a variety of hosts and geographical locations which will help to characterise the true sequence space of the FMDV genome. Since whole genome sequences of Southern African Territories (SAT) genotypes are underrepresented, 50 SAT isolates collected from various locations in East Africa were sequenced using PCR-free protocol pioneered at The Pirbright Institute. Complete genome sequences were reconstructed de novo using an optimised bioinformatics pipeline. Phylogenetic analysis such as maximum likelihood, detection of phylogeny violation and Bayesian analysis were applied to characterise these SAT isolates. Sequencing of FMDV SAT 1-3 serotypes from East Africa revealed new distinct genotypes of SAT viruses. Phylogenetic analysis suggests that while the newly characterised SAT isolates are capable of recombination with both SAT and non-SAT viruses, these new genotypes did not arise from recombination but probably from geographical isolation of FMDV within Africa caused by the Great African Rinderpest Pandemic (1887-1897). While it is generally considered that FMDV originated in Africa, the Great African Rinderpest Pandemic led to a massive die-off of FMDV hosts (both domestic and wildlife) leading to the eradication of a number of FMDV genotypes in Africa. It is thought that many currently circulating FMDV O and A genotypes were re-introduced into Africa from Europe and Asia after 1900; however, SAT isolates probably re-emerged from remaining African buffalo herds. The newly identified SAT genotypes may represent virus lineages which existed before the 1890s and survived the Great African Rinderpest Pandemic. Sequencing of African FMDV isolates allows us to better understand history of this virus and its natural variability. Europic 2016 95

96 TUESDAY 06/09/2016 D11 PREDICTION OF CONSERVED RNA STRUCTURES WITHIN THE FOOT-AND-MOUTH DISEASE VIRUS GENOME REVEALS FUNCTIONAL CIS-ACTING ELEMENTS LOCALISED IN THE 3D CODING REGION Lidia Lasecka-Dykes1, Fiona Tulloch2, Sarah Gold1, Garry Luke2, Caroline Wright1, Nick Knowles1, Grace Logan1, Toby Tuthill1, Terry Jackson1, Peter Simmonds3, Martin Ryan2, Donald King1 1 The Pirbright Institute, 2University of St Andrews, 3University of Oxford OBJECTIVES: RNA structure plays a crucial role in the replication of positive-sense RNA viruses forming cis- acting elements present in both untranslated regions (UTRs) and within the open reading frame (ORF). While RNA structures of several viruses from the Picornaviridae - including foot-and-mouth disease (FMDV) - have been characterised biochemically, structural prediction analyses of FMDV suggest structures exist within the non- structural region of the genome, but remain undefined. FMDV is the causative agent of foot-and-mouth disease (FMD) - a highly contagious disease of cloven-hoofed animals. While FMD is present in Africa, Asia and sporadically in South America, it can lead to huge economic loses posing a constant threat to the livestock industry. Alongside a wider investigation of FMDV sequence variability, we examined FMDV functional RNA secondary structures and, since cis-acting elements of FMDV UTRs are well studied, we focused our analyses within the coding region of the FMDV genome. METHODS & RESULTS: Extensive comparative RNA structure prediction carried out on a large number of isolates (including sequences available on GenBank and Southern African Territories (SAT) isolates sequenced during this study), representing genome variability across all seven FMDV serotypes, indicated numerous conserved RNA structures present in the non-structural region of the FMDV genome. These bioinformatic analyses predicted structures both previously described, and, novel structures not yet reported. Therefore, we sought to investigate these findings further by designing sequences computationally via randomised codon shuffling to deliberately destroy these potential RNA structures - whilst maintaining dinucleotide frequencies and codon usage. These synthetic sequences have been inserted into a fluorescent FMDV replicon system to examine the effect such changes have on replicative fitness in comparison to published, control, attenuated replicons generated previously. This fluorescent replicon system allows for detailed, facile analyses of replication kinetics directly comparable to data obtained from qRT-PCR and viral growth curves. Our results suggest that several RNA structures located at the end of 3Dpol (the viral RNA-dependent RNA polymerase) are crucial for RNA replication/ translation, as disruption of these RNA structures abolished FMDV RNA replication. Furthermore, we have shown such disruptions cannot be complemented in trans. CONCLUSIONS: RNA secondary structural predictions of FMDV in comparison with other viruses from the Picornaviridae showed that FMDV appears to contain extensive RNA structures distributed throughout the non- structural region of the genome. It has been suggested that some of these RNA structures may be important for the establishment of persistent infections by FMDV in its natural host. Our data show, however, that only a small sub-set of such structures are directly involved in RNA replication/translation within tissue-cultured cells. We have shown that bioinformatic predictions supported by functional studies can aid in the identification of critical FMDV RNA structures for genome replication. Furthermore, these data are facilitating genome modifications in the process of attenuating FMDV. 96 Europic 2016

97 TUESDAY 06/09/2016 D12 EXCHANGES OF GENOMIC DOMAINS BETWEEN POLIOVIRUS TYPE 2 AND A PANEL OF CO- CIRCULATING SPECIES C ENTEROVIRUSES REVEAL A HIGH DEGREE OF GENOMIC PLASTICITY Mal Bessaud, Marie-Line Joffret, Bruno Blondel, Francis Delpeyroux Institut Pasteur OBJECTIVES: Vaccination against poliomyelitis has mostly been based on the oral polio vaccine, which contains live attenuated Sabin strains of the three poliovirus (PV) serotypes. These strains are genetically unstable, and their circulation in non-immunised populations can lead to genetic drift and the emergence of pathogenic circulating vaccine-derived PVs (cVDPVs). In recent decades, several countries, including Madagascar, have experienced poliomyelitis outbreaks due to such viruses. Most cVDPVs have recombinant genomes including non-structural sequences from non-polio enterovirus species C (EV- C) strains and mutations within the Sabin genomic sequences. Furthermore, most cVDPV genomes also include a 5-UTR from a non-polio enterovirus. The recombinant nature of most cVDPVs raises questions about the permissiveness of intertypic genetic exchanges and their possible impact on phenotype. In particular, the respective contributions of recombination events in the 5-UTR and the non-structural part of the genome to viral phenotype remain to be deciphered. METHODS: We constructed 33 chimeric genomes with the same Sabin 2 capsid-encoding sequence. The other sequences (5-UTR and/or non-structural-3-UTR sequences) were from Sabin 2 or from non-polio EV-C collected in Madagascar from healthy children living in the area of a poliomyelitis outbreak. The functionality of the recombinant genomes was checked in permissive cells. We characterised various phenotypic features of the viruses recovered, including their ability to cause paralysis in transgenic mice. RESULTS: Only two of the 33 genomes constructed were not functional. The recombination of Sabin 2 with other EV-C had no deleterious effect on replication in vitro, regardless of the sequences that recombined, because all viable recombinant viruses replicated similarly in cell cultures. However, recombination modified plaque size and temperature sensitivity. Thus, the Sabin 2 capsid was functionally compatible with most 5-UTRs and other non-structural regions from the non- polio EV-C tested, with recombination modifying phenotypic features. This suggests that the genomic regions of most EV-C viruses remain functionally compatible for intertypic recombination during the evolution and differentiation of these species. None of the viruses with an unmodified Sabin 2 5-UTR was neurovirulent, whereas almost all the viruses with a non-polio 5-UTR caused disease in mice, confirming the crucial role of the 5-UTR in the loss of attenuation in Sabin 2-derived recombinants. Furthermore, attenuation levels differed between some viruses with the same 5-UTR. The 5-UTR sequence therefore appears to play a major role in the neurovirulence of recombinants, whereas the 3 half of the genome may modulate pathogenicity. Unlike the type 2 cVDPVs isolated from poliomyelitis patients, these recombinant viruses had a low degree of neurovirulence. This observation suggests that, in addition to recombination events, mutations within Sabin 2 sequences are required for field cVDPVs to acquire a high level of pathogenicity. We found that Sabin 2 sequences were highly compatible with most homologous sequences from other EV-C strains. This high degree of genomic plasticity facilitates intertypic recombination between EV-C strains, leading to phenotypic changes that may, together with mutations, favour the emergence of viral strains with particular phenotypes. Europic 2016 97

98 TUESDAY 06/09/2016 D13 IMPORTANCE OF 5 TERMINAL SEQUENCE IN COXSACKIEVIRUS B3 RNA IN REGULATING VIRAL RNA REPLICATION AND A SWITCH TO VPG-PRIMED POSITIVE-STRAND INITIATION James Flanegan1, Sushma Ogram1, Joan Morasco1, Benjamin Looney1, Michael Clare-Salzler1, Nora Chapman2 1 University of Florida, 2University of Nebraska Medical Center Coxsackievirus B3 (CVB3) is a member of the Picornaviridae family of small positive-strand RNA viruses. CVB3 infections have been associated with inflammatory heart diseases such as acute and chronic myocarditis and have been shown to be present in pancreatic tissue from patients with type 1 diabetes. The 5 terminal sequence in picornavirus RNA is a critical cis-active element that regulates all stages of viral RNA replication. Noncytolytic CVB RNAs containing 5 terminal deletions (5TDs) have been isolated from persistently infected hearts of mice, human myocarditic heart tissue and from the pancreas of diabetic mice. These noncytolytic viruses support very low levels of virus replication and contain 5TDs that range in size up to about 49 nt long (49). We investigated how small 5TDs (3 to 11) affected viral RNA replication and VPgpUpU synthesis in cell-free reactions. In addition, we have investigated the effect of 5TDs on CVB3 RNA replication in the adult -cell derived cell line, Lox5. Similar results were observed in both systems. Although the 5 TD RNAs all translated at levels similar to wild-type (WT) RNA, the overall level of RNA replication varied significantly and ranged from about 17%-0.5% of WT levels. In contrast, the synthesis of VPgpUpU with the 5 TD RNAs (3, 5 and 7) was similar to WT levels and then gradually decreased to about 5% of WT levels with 11 RNA. Negative-strand synthesis also remained at high levels with the 3, 5 and 7 TD RNAs and then gradually decreased to lower levels with 11 RNA. In contrast, the level of positive-strand strand synthesis was very low, even in reactions containing 3 RNA where high levels of VPgpUpU and negative-strand synthesis were observed. These results led us to ask if the low level of positive-strand synthesis observed with the 5TD RNAs was primarily due to the absence of the 3 terminal AA sequence normally present at the 3 end of negative-strand WT RNA or due to the deletions in the highly conserved 5 terminal sequence of positive-strand RNA, or more importantly the 3 terminal sequence of negative-strand RNA. The results indicated that restoration of the 3 terminal AA sequence in these 5 TD RNAs had little effect on positive-strand synthesis. This suggested that in addition to the 3 terminal AA sequence, that the highly conserved sequence at the 3 end of the negative-strand RNA is required for VPgpUpU-primed positive-strand initiation. Even small deletions in the 3 terminal sequence in negative-strand RNA resulted in a switch from the VPgpUpU priming mechanism used with WT RNA to a less efficient VPg-priming mechanism with the 5 TD RNAs. 98 Europic 2016

99 TUESDAY 06/09/2016 D14 MISINCORPORATION OF NUCLEOTIDES OR DRUGS DURING POLIOVIRUS RNA- DEPENDENT RNA POLYMERASE ELONGATION LEAVES FINGERPRINTS IN THE PAUSING KINETICS Nynke Dekker1, David Dulin1, Hyung-Suk Oh2, Cheri Lee2, Theo van Laar1, Jamie Arnold2, Craig Cameron2, Martin Depken1 1 TU Delft, 2Pennsylvania State University Positive (+) RNA viruses represent a major family of pathogens. The development of antiviral therapeutics is therefore a global and intense field of research, constantly in demand of new approaches. Here, we utilize our high-throughput magnetic tweezers assay to characterize the elongation dynamics of the (+) RNA virus model RdRp from poliovirus (3Dpol) on RNA templates of kb-lengths, with or without antiviral nucleotide analogues. Together with in vivo and in vitro bulk assays, we characterize a novel nucleotide analogue, T-1106, from the pyrazinecarboxamide family. Our experiments demonstrate that the fidelity of 3Dpol and its incorporation of nucleotide analogues are governed by low fidelity, catalytically competent pauses. They also lay bare a new mechanism of action for nucleotide analogue drugs, by showing, first, that T-1106 presents a strong antiviral activity on polio virus-infected cells without the cellular toxicity of ribavirin, and second that its incorporation of T-1106 opposite an adenosine results in pauses originating in polymerase backtracks. These findings demonstrate the potential of high-throughput single-molecule techniques for drug characterization. Europic 2016 99

100 TUESDAY 06/09/2016 D15 RNA VIRUS DIVERSITY IS IMPORTANT TO COUNTERACT INNATE IMMUNITY AND SPREAD WITHIN THE HOST Yinghong Xiao, Raul Andino University of California, San Francisco The immune system represents a major barrier to viral infection and a virus ability to overcome these responses enables virus replication and spread within the host. Given that innate immunity is the first line of defense, we hypothesized that during the acute phase of virus infection, rapid adaptation to tissue specific innate responses is critical to establishing a successful infection. Furthermore, interferon appears to elicit tissue specific responses, thus viral intrahost dissemination may require adaptation to such diverse antiviral defense mechanisms. We show that the interferon (IFN) response is indeed a major barrier to intrahost viral spread. However, virus diversity allows rapid adaptation to tissue-specific interferon responses. Our study suggests that diversity enables spread of virus between host tissues through constant optimization of the proportion of variants with distinct functions to circumvent tissue specific challenges. The genetic composition of an RNA virus population is a virulence determinant allowing dynamic regulation of viral functions required to overcome innate barriers to infection. We have developed a technology to rapidly identify determinants of adaptation, including virus functions that suppress IFN responses. The significance of this application is that may provide information to engineering a safe and effective vaccine, as adaptation appear to be a general determinant of RNA virus virulence, our finding may yield a general strategy for the development of RNA virus vaccines. 100 Europic 2016

101 TUESDAY 06/09/2016 D16 CONNECTING METAGENOMICS EXPLORATION AND EXPERIMENTAL RESEARCH OF PICORNAVIRUSES WITH THE EVOLUTIONARY BASED PARTITIONING OF GENOMIC DIVERSITY BY DEMARC Alexander Gorbalenya1, Anastasia Gulyaeva1, Chris Lauber1, Nikolai Kondratiev1, Matvey Zakharov1, Andrey Leontovich2, Dmitry Samborskiy2, Igor Sidorov1 1 Leiden University Medical Center, 2Belozersky Inst. of Physico-Chemical Biology, Lomonosov Moscow State University Until recently our knowledge about picornaviruses was largely derived through characterization of a dozen or so species which have been studied due to their high impact on human population. Researchers active in the characterization of picornaviruses also developed the taxonomy of picornaviruses. With this approach, taxonomy served as a structural framework for accumulated knowledge about picornaviruses that ensured its relevance to the field. This established link has been under increasing strain over the last years due to the ongoing flood of many new picornaviruses discovered by metagenomics, which are not characterized beyond genome sequencing. Their number may have already exceeded the number of the characterized picornaviruses by ten-fold or so, thus making genomes a dominant component of the knowledge base. Even relying on phylogenetic analysis of two sets of genes, VP2-VP3-VP1 and 2C-3C-3D, respectively, of the newly sequenced genomes, the Picornavirus Study Group of the International Committee on Taxonomy of Viruses found it increasingly difficult to establish new taxa unequivocally. With the goal of improving the practice of taxonomy and its link to fundamental and applied research, we have previously introduced a measure for quantifying the quality of virus taxa. It forms the core of a computational approach (DEmARC, DivErsity pArtitioning by hieRarchical Clustering) for classifying viruses of a large monophyletic group (family or order) using pairwise evolutionary distances (1). The inferred genetics-based classification of some 1000 sequences was demonstrated to approximate closely the ICTV taxonomy of picornaviruses of 2010 with few notable deviations (2). In this presentation, we will describe the current state of the genetics-based classification of picornaviruses and compare results obtained by analyzing different protein domain combinations and more than 3000 genome sequences. To this end, we have employed an advanced version of DEmARC that can analyse patristic distances and account for biased species sampling, while quality of the resulting classification is assessed using several measures. We show that the most conserved non-structural proteins retain the strongest phylogenetic signal about a wave-like generation of the genetic diversity of picornaviruses, possibly shaped through constraints imposed by recombination and mass extinction-birth of host species (2). This pattern could be captured in a multi-level hierarchical classification, whose biological implications will be discussed. 1. Lauber, C. & Gorbalenya, A.E. (2012). J. Virol. 86: 3890-3904. 2. Lauber, C. & Gorbalenya, A.E. (2012). J. Virol. 86: 3905-3915. Europic 2016 101

102 TUESDAY 06/09/2016 D17 MECHANISM OF RNA VIRUS RECOMBINATION Jamie Arnold1, Andrew Woodman2, Ashley Acevedo3, Raul Andino3, David Evans4, Craig Cameron2 1 Penn State, 2Penn State University, 3University of California San Francisco, 4University of St. Andrews OBJECTIVES: RNA virus evolution occurs on two levels: genetic drift and genetic shift. Genetic drift is caused in large part by the low incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp), and it is now known that polymerase fidelity is a determinant of viral fitness and virulence. Genetic shift is caused by recombination and is a major contributor to the expansion of the host range of viruses and restoration of virulence to vaccine strains. Very little is known about the mechanistic basis of RNA virus recombination, although it is clear from studies of the picornavirus poliovirus (PV) that the viral RdRp is at the heart of this process. Homologous recombination in RNA viruses is thought to occur by a template-switching mechanism. The elongating viral RdRp will move from one template (donor) to a second template (acceptor). What triggers the jump from donor to acceptor is not known but could include the following: rate of polymerase elongation (speed), nucleotide misincorporation by polymerase (fidelity), thermodynamic stability of the duplex, and the presence of stable/ complex secondary structure. The objectives of our study are to begin to elucidate the impact these factors have on template switching/strand transfer by the viral RdRp and establish its relevance to RNA virus recombination. METHODS: We have developed a robust, biochemical assay for template switching/strand transfer by PV RdRp in vitro using heteropolymeric templates. We also employ a recently developed cell-based recombination assay. This assay utilizes two parental RNAs that are independently unable to generate viable progeny, however following co- transfection viable progeny is produced if a recombination strand transfer event occurs during RNA replication. RESULTS: Using our defined in vitro biochemical assay, we find that in the presence of a ssRNA acceptor template complementary to the 3-end of the nascent elongated RNA, additional synthesis is observed that produces a specific transfer product. The transfer product is not observed in the absence of acceptor RNA or in the presence of a non-complementary, random, acceptor RNA. Sequencing of the transfer product revealed that either a misincorporation or non-templated nucleotide addition event occurred prior to the template switch. In addition, by using a panel of RdRp fidelity mutants, we find that altered fidelity of the RdRp greatly impacts the efficiency of strand-transfer. This result is further supported by using the cell-based recombination assay. Higher and lower fidelity RdRp derivatives yield a reduced and increased number of viable recombinants, respectively. CONCLUSIONS: We conclude that RdRp fidelity contributes to the efficiency of strand transfer. Misincorporation may also be a trigger for template switching followed by a forced-copy-choice mechanism for recombination. Overall, we have uncovered several unexpected features of PV recombination complexes that are beginning to assist us in our understanding of this fundamental process of viral evolution. 102 Europic 2016

103 TUESDAY 06/09/2016 D18 5 TERMINAL CHARACTERIZATION OF PERSISTENT ENTEROVIRUS IN CARDIAC TISSUE OF PATIENT SUFFERING FROM DILATED CARDIOMYOPATHY Alexis Bouin1, Fanny Renois2, Yohan Nguyen2, Michel Wehbe2, Nicolas Lvque2, Paul Fornes2, Laurent Androletti2 1 University of California, Irvine, 2Universit de Reims Champagne-Ardenne OBJECTIVE: Coxsackievirus B3 (CVB3) is a ubiquitous pathogen involved in chronic myocarditis and dilated cardiomyopathy, which is the second leading cause of heart transplantation worldwide. The mechanisms of enteroviral persistence in cardiomyocytes are still unknown but could be linked to 5 terminal deletions of viral genomic RNAs. These deleted forms have been previously identified in fulminant myocarditis and more recently in chronic dilated cardiomyopathy. METHODS: In our study, endomyocardial biopsies from 8 patients suffering from end stage dilated cardiomyopathy and displaying an enterovirus infection were selected. Viral persistence was assessed by two quantitative RT- PCR assays targeting both total and negative strand RNA. Viral populations harboring terminal deletions were identified and their proportions estimated using a deep sequencing strategy targeting specifically the 5 terminal extremity of enteroviruses genome. Pathological involvement of the virus was observed by detection of dystrophin disruption in infected cardiomyocyte using immunofluorescence and confocal microscopy. RESULTS: Our quantitative RT-PCR data on total RNA revealed low viral loads (5.4x104 copies/g RNA extract, range: 3.4x103-3.7x106), consistent with persistent infection. The ratio of positive-strand to negative-strand RNA levels had a mean of 2.65 and ranged from 1.06 to 5.28. Interestingly, there was a strong correlation between viral load and the positive-strand to negative-strand RNA ratio (R2 = 0.78). These results confirmed the persistence of an enterovirus in the 8 patients. The 5 terminal deletions of viral genome were identified for 6 patients by deep sequencing. Deletions ranged from 19 to 50 nucleotides, and 5 out of 6 patients (83%) also harbored complete genomic RNA sequences. The viral forms were separated in 4 different groups: complete, 5 deletions of 19 to 31 nucleotides, 32 to 36 nucleotides, or 37 to 50 nucleotides. The majority of deleted forms belonged to the 37 to 50 nucleotides group (79.5%). The complete, 19 to 31 nucleotides, and 32 to 36 nucleotide groups accounted for 1.3, 7.1, and 12.1% respectively. The deletions all impacted the predicted structure of the 5 cloverleaf RNA element and induced a disruption of the PCBP binding site in the cloverleaf. A reverse correlation was found between the proportion of deleted genomes and the viral load (R2= 0.53), highlighting the association between terminal deletions and persistence. Finally, immunofluorescence staining demonstrated the association of viral RNA and proteins with the disruption of cardiac dystrophin. CONCLUSIONS: This is the first identification of deleted enterovirus genomes in a cohort of patients suffering from dilated cardiomyopathy. Our deep sequencing strategy allowed us to identify both complete and deleted viral forms and to correlate the deleted population proportion with viral persistence. Even when viral RNA accumulation levels were low, the pathophysiological effects of the infection on dystrophin disruption were still observed in cardiac tissues. Taken together these data strongly support the hypothesis that persistent cardiac enterovirus infections are composed of complete and deleted genomes involved in dilated cardiomyopathy pathogenesis. A further understanding of these mechanisms is important to design new treatments to prevent the progression of viral myocarditis to dilated cardiomyopathy. Europic 2016 103

104 TUESDAY 06/09/2016 D19 ESTABLISHING A SYSTEM TO STUDY RECOMBINATION IN ENTEROVIRUS 71 Andrew Woodman, Jamie J. Arnold, Craig E. Cameron Penn State University OBJECTIVES: We have recently shown that the frequency of recombination in poliovirus (species C) tracks with the fidelity of nucleotide incorporation by the RNA-dependent RNA polymerase (RdRp) (Woodman et al., 2016, In submission). These results suggest that a universal approach to stabilise live attenuated vaccines may be possible. We are nearing the end game with poliovirus, which will result in the need for alternate model systems. EV71 (species A) is the etiological agent of hand foot and mouth disease (HFMD) and has been associated with severe and sometimes fatal neurological illness. The founding events and emergence of EV71 subgenogroups and lineages have been associated with recombination (McWilliam-Leitch et al., 2011). These observations suggested that EV71 might provide a suitable new model system to study recombination. We are currently involved in a cross-disciplinary approach that aims to dissect and characterise the triggers and mechanism(s) of RdRp mediated recombination. One of our objectives was to develop an optimised, reproducible approach based upon the previously reported CRE-REP assay (Lowry et al., 2014). METHODS: Briefly, two replicative EV71 parental genomes and two non-replicative genomes that are unable to produce viable virus have been constructed. Only a recombination event between the two parental genomes can produce infectious virions following transfection. Four cell lines considered suitable for the assay (HeLa, L929, Vero and RD) were used during this study along with various doses of parental genome. RESULTS: We have developed and optimised the EV71 CRE-REP assay and show reproducible recombination yield. We observe recombination only from partners able to replicate and do not observe infectious virions from the non-replicative partners. CONCLUSIONS: Firstly, recombination in enteroviruses is primarily a replicative process mediated by the RdRp. Our results support the current observations of recombination based upon the poliovirus model. Secondly, we are close to entering a post-poliovirus world, which will inhibit the use of this virus in a laboratory environment. The ability to establish an alternative EV71 recombination model system is therefore of vital importance. Our system will allow the analysis of a panel of known RdRp variants. In addition, conserved non-recombinogenic alleles identified in polio (Acevedo et al., unpublished) can be analyzed. 104 Europic 2016

105 TUESDAY 06/09/2016 D20 PATTERNS OF INTERTYPIC RECOMBINATION BETWEEN VACCINE-DERIVED POLIOVIRUSES REVEAL GENOMIC REGIONS WITH REDUCED FITNESS Konstantin Chumakov1, Majid Laassri2, Elena Cherkasova3, Elvira Rodionova2, Tatiana Zagorodnyaya2, Svetlana Petrovskaya2, Ekaterina Korotkova4, Olga Ivanova5, Anatoly Gmyl5, Vadim I. Agol5 1 US Food and Drug Administration, 2Food and Drug Administration, 3Food and Drug Administration and National Insiztiutes of Health, 4 Lomonosov Moscow State University, 5Chumakov Institute of Poliomyelitis and Viral Encephalitides Oral Poliovirus Vaccine (OPV) is made from live attenuated poliovirus strains of three serotypes developed by Albert Sabin. The vaccine is the main tool used in the worldwide campaign to eradicate the disease. The attenuated strains can replicate in vaccine recipients and transmit to their contacts, and therefore have the advantage of inducing herd immunity. Over the years, this advantage began to be viewed as a shortcoming, since the vaccine viruses can uncontrollably replicate and accumulate mutations thus reverting to virulence. Vaccine-derived polioviruses (VDPV) that regained virulence can cause outbreaks of poliomyelitis similar to those caused by wild virus. VDPV can also persistently infect some individuals with primary immunodeficiencies. The major ways by which VDPVs evolve and regain virulence is accumulation of point mutations leading to phenotypic reversion. The evolution of VDPV is also known to be often accompanied with recombination of the primary derivatives of OPV with either other strains of attenuated poliovirus and their descendants or with wild poliovirus strains or even non- polio enteroviruses belonging to species C Enterovirus. The biological relevance of VDPV recombination has not been strictly documented so far, but is hypothesized to play a role in phenotypic reversion. Genetic recombination is a general biological phenomenon common to higher organisms and microorganisms, both eukaryotes and prokaryotes, as well as both DNA and RNA viruses. It provides a mechanism for generating diversity helping to adapt to new conditions and to purge deleterious mutations from the genomes. The latter aspect may be especially important for evolution of RNA viruses with error-prone replicases. To understand the evolutionary force driving recombination of attenuated polioviruses we have determined complete nucleotide sequences of over 300 genomes of intertypic poliovirus recombinants. The viruses included both highly evolved VDPV isolated from paralytic cases, immunodeficient chronic carriers, and environment. Comparison of these sequences with published sequences of other recombinant polioviruses revealed the preferred recombination patterns. Genomic region coding for 2A-2B proteins of Sabin 1 poliovirus, 3A-3B region of Sabin 3, and the distal part of 3D of Sabin 2 strain appeared to have lower fitness and were frequently replaced by homologous parts of other Sabin serotypes. Crossover points were located in different parts of P2 and P3 region, while no recombination events found in the capsid-coding region except in the immediate vicinity of P1-P2 boundary. The result suggests that each Sabin strain contains genomic regions that impair their fitness. Selection of recombinant genomes may be driven by removal of the regions and restoration of fitness lost during attenuation process. These findings could be used for rational vaccine design. For instance, genetically stable vaccine strains could be constructed by combining high-fitness P2-P3 regions with the capsid region containing all attenuating mutations. Such viruses would be under weaker pressure to recombine, and thus possess a more stable attenuated phenotype. Europic 2016 105

106 TUESDAY 06/09/2016 D21 UNTANGLING THE ROLE OF THE PSEUDOKNOTS IN FMDV REPLICATION Joseph Ward1, Emma Warner2, Sarah Gold3, Lidia Lasecka3, Caroline Wright3, Morgan Herod2, Tobias Tuthill3, David Rowlands2, Nicola Stonehouse2 1 Univeristy of Leeds, 2University of Leeds, 3Pirbright Institute OBJECTIVES: Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, affecting cloven hooved animals. Outbreaks can have a severe economic impact due to loss of stock and cessation of trade. Within the 5 untranslated region (UTR) of FMDV there is a series of 2-4 tandemly repeated pseudoknots. While the presence of pseudoknots in the UTR of picornaviruses is not unusual, their location in the FMDV UTR is unique. The role of the pseudoknots and their essentiality in FMDV replication are currently unknown. The aim of these studies is to uncover the role of the pseudoknots in the replication of FMDV. METHODS: Mutations were initially introduced into a sub-genomic replicon, which encodes a green fluorescent reporter protein. Mutated replicons where transfected into a variety of cell lines including BHK-21 (hamster), SK- RST (porcine) and MDBK (bovine) cells, this allowed any potential cell-dependent differences to be investigated. Replication was monitored by expression of the reporter protein. Mutations were then introduced into virus and infectivity of BHK-21 cells was assayed. Passaging experiments are being undertaken and virus deep-sequenced to identify any potential mutations. RESULTS: Our current data shows that sequential deletion of up to 3 pseudoknots results in no change in replication in replicon or virus. In comparison, upon removing all 4 pseudoknots, replication is significantly reduced in replicon and is not observed in virus in up to 4 blind passages, signifying a strong requirement for at least one pseudoknot. However, there appears to be no preference for which pseudoknot is retained, suggesting there is some redundancy between pseudoknot repeats in viral replication. Transfection into different cell lines also suggests the pseudoknots do not play a role in host species specificity. To investigate whether the pseudoknots provide a functional role or solely act as a spacer region they were substituted with an equal length of random nucleotides, this yielded a phenotype similar to that of deleting all 4 pseudoknots. Investigation into this is continuing by disrupting the nucleotide interactions key in forming the pseudoknots structure. Work is also in progress to investigate the potential role of the pseudoknots in preventing exonuclease digestion. CONCLUSION: Our results show that while the pseudoknots are not essential for replicon replication, at least one is required for wildtype levels to be reached. However, current evidence suggests that at least one pseudoknot is required for viral replication. This is interesting as no field isolates have been found with fewer than 2 pseudoknots present. These results start to build an idea of the importance of the pseudoknots in FMDV replication. 106 Europic 2016

107 TUESDAY 06/09/2016 D22 CORNERING RNA VIRUSES BY CONSTRAINING THEIR SEQUENCE SPACE Gonzalo Moratorio1, Rasmus Henningsson2, Cyril Barbezange1, Lucia Carrau1, Antonio Bordera3, Herve Blanc1, Enzo Poirier1, Stephanie Beaucourt1, Bryan Mounce1, Magnus Fontes2, Marco Vignuzzi1 1 Institut Pasteur, 2Centre for Mathematical Sciences, Lund University, Lund, Sweden. International Group for Data Analysis, Center for Bioinformatics, Biostatistics and Integrative Biology, Institut Pasteur, Paris, France, 3International Group for Data Analysis, Center for Bioinformatics, Biostatistics and Integrative Biology, Institut Pasteur, Paris, France The tolerance of a virus to mutation, also known as genetic robustness or mutational buffering, determines the extent of genetic diversity that can be maintained in the population. Thus, the observed viral population diversity results from both the generation of, and the tolerance to, mutations. Robustness is modulated by codon usage, as synonymous codons differ in their propensity to mutate non-synonymously and non-conservatively. Due to their error-prone polymerase, RNA viruses have elevated mutation rates and exist as a swarm of closely related sequences. At each replication cycle, many of the newly synthesized genomes differ from the consensus sequence, with possible deleterious effects on viral population fitness. Viral populations can thus occupy different regions of the genotypic sequence space, and their dynamics within this space also depend on mutational robustness. We chose a novel approach to address mutational robustness by genetically engineering Coxsackie (CVB3) and influenza A (IVA) virus populations to occupy different regions of sequence space. Based on the potential effect of a point mutation, a 1 to Stop group was defined, among the six synonymous codons for Leucine and for Serine, as codons with nonsense mutation targets (NSMTs), which are sites that can generate a stop codon after a single nucleotide substitution. For both amino acids, the codons within the P1 region of CVB3 (structural proteins) and PA and HA of IVA (polymerase and surface glycoprotein), were all replaced by this group of codons. The replicative fitness and evolutionary trajectories of these synonymous mutants were evaluated by deep sequencing analysis following tissue culture passage under mutagenic conditions and in a mouse model. Here, we show how cornering viruses in risky areas of sequence space could lower a virus natural robustness. Finally by rewiring robustness and fidelity together, we drastically decreased viral fitness in vivo. Europic 2016 107

108 TUESDAY 06/09/2016 D23 IMPACTS OF GENETIC BOTTLENECKS ON THE FOOT-AND-MOUTH DISEASE VIRUS CONSENSUS SEQUENCE: IMPLICATIONS FOR VIRUS FITNESS AND EVOLUTION Caroline Wright1, Richard Orton2, Marco Morelli3, David Paton1, Dan Haydon2, Don King1 1 The Pirbright Institute, 2University of Glasgow, 3Center for Genomic Science of [email protected] OBJECTIVES: RNA viruses exist as diverse populations of closely related genomes due to errors made by the viral polymerase during genome replication. Transfer of more or less of this genetic diversity, from one environment to another, is generally accompanied by fitness increase or decrease of the virus population, due to competitive optimization or loss of adaptability respectively. This study investigated the impacts of bottleneck severity on the fine scale transfer of genetic diversity and the process by which mutations become established into the foot-and- mouth disease virus (FMDV) consensus sequence. METHODS: Large (diverse) and small (restricted) populations of rescued FMDV, generated from a full-length FMDV cDNA clone, were subjected to three consecutive in vitro passages, simulating relaxed and severe bottleneck transmission respectively. This cell culture adapted FMDV utilizes the sulfated glycan, heparan sulfate (HS), as a cellular receptor during infection in culture. However, in vitro passage of virus was carried out in a fetal goat tongue cell line (ZZ-R 127), which predominantly expresses the integrin receptor v6, one of the main cellular receptors used by FMDV during infection in a natural host. This cell line was specifically chosen to exert a selective pressure on this cell culture adapted virus, in the form of reversion from HS to integrin receptor binding at known genomic positions. Subsequent viral populations were characterized by Illumina deep sequencing. RESULTS: Deep sequence coverage (16,000 to 65,000X) was achieved for virus populations subjected to both relaxed and severe bottleneck passage. After an initial increase in both populations, subsequent analysis revealed that the genetic complexity, measured by Shannon Entropy, remained relatively constant within the relaxed but decreased within the severe bottleneck passaged cultures. Additionally, an increase in mutation frequency at FMDV amino acid positions associated with reversion in cell receptor usage (HS to integrin), was observed with each passage and reached a maximum of 9.8% by passage 3 within the relaxed bottleneck passage. In contrast, mutation frequency was more erratic within the severe bottleneck passage and no mutations were observed at FMDV cell receptor binding residues. However, no individual mutation reached above 1% until passage two in the relaxed bottleneck, whereas six mutations were observed above 1% by passage one in the severe bottleneck passage, one of which reached and remained around 10% through subsequent passages. CONCLUSIONS: Observations made here indicate that mutations are more quickly established within viral populations during severe compared to relaxed bottleneck passage. However, as well as being fewer in number, these mutations lacked the selective advantage of those observed during relaxed bottleneck passage, pointing towards a loss of adaptability. This study provides proof of concept for an experimental design capable of dissecting early impacts of a fundamental process to the evolution of RNA viruses at an unprecedented level of resolution. 108 Europic 2016

109 TUESDAY 06/09/2016 D24 MOSAIC EVOLUTION OF ENTEROVIRUS A71 GENOGROUP F THROUGH FREQUENT INTRASPECIES GENETIC RECOMBINATION Romain Volle1, Richter Razafindratsimandresy2, Marie-Line Joffret3, Serge Sadeuh-Mba4, Ionela Gouandjika5, Mal Bessaud3, Richard Njouom4, Jean-Michel Heraud2, Bruno Blondel3, Jean-Luc Bailly6, Francis Delpeyroux3 1 INSTITUT PASTEUR, 2Institut Pasteur de Madagascar, Antananarivo, Madagascar, 3Biology of Enteric Viruses (BVE), INSERM U994, Institut Pasteur Paris, France, 4Centre Pasteur du Cameroun, Yaound, Cameroun, 5Institut Pasteur de Rpublique Centrafricaine, Bangui, RCA, 6Universit dAuvergne, Facult de Mdecine, Clermont-Ferrand, France INTRODUCTION: Enterovirus A71 (EV-A71) is a member of the Enterovirus A species (EV-A). EV-A71 infections range from mild hand-foot-and-mouth disease (HFMD) to severe neurological manifestations. Based on comparisons of the gene encoding the VP1 capsid protein, EV-A71 strains can be classified into seven genogroups: A to G. Genogroup A includes the historical prototype strain; genogroups B and C have been reported worldwide and were implicated in the principal outbreaks in recent years. Little is known about genogroups D to G, which were more recently identified in India, Africa and Madagascar. In particular, genogroup F has been found only in Madagascar and, as no HFMD outbreak due to this genogroup has yet been reported, the circulation of EV-A71 on this island is probably recent, underestimated or silent. OBJECTIVES: We studied the molecular epidemiology of EV-A71 genogroup F, focusing on certain aspects of its evolution and its time of emergence, together with its possible interaction (recombination) with other EV-A types circulating in Madagascar and elsewhere. METHODS: Whole-genome sequences were determined for 23 strains of seven EV-A types reported in Madagascar (2004-2011). These genomes were added to sequence datasets representative of the EV-A strains circulating worldwide. Phylogenies were reconstructed by maximum likelihood methods, and recombination patterns were analysed by determining pairwise similarity between sequences. Coalescence times were estimated for the EV-A71 genealogy, with Bayesian MCMC methods (BEAST). RESULTS: Capsid coding sequences (the P1 region of the genome) clustered unambiguously, defining serotype and (sub)genogroup monophyletic clades. Phylogenetic analysis of the VP1 gene dated the origin of all EV-A71 lineages to 1901 (18701931). Genogroups D, E, and G had a common ancestor dating back to 1950, but genogroup F arose in 1995 (19902000) and shared a common ancestor with genogroup B in 1946 (19371955). The phylogenies obtained with the sequences of the P2 and P3 genomic regions were not congruent with that obtained with P1 sequences. The sequences of most EV-A71 strains remained essentially associated with the corresponding (sub)genogroup, but those of genogroup F strains were clustered in a clade grouping together most of the other EV-A sequences from Madagascar. Pairwise similarity analysis revealed the existence of mosaic EV-A71 genome associated with recent recombination with Madagascan CV-A5, CV-A7, and CV-A10 strains, in particular. The phylogeny reconstructed with the largest recombinant region common to EV-A71, CV-A7 and CV-A10 from Madagascar included a common ancestor dating back to 2007.1 (2005.42008.5). CONCLUSIONS: Phylogenetic and molecular dating analyses provided evidence for the recent emergence of EV-A71 genogroup F in Madagascar. The capsid coding sequences of genogroups F and B were consistently related, whereas the non-structural coding sequences originated from multiple recent genomic exchanges with co-circulating EV-A strains. As no particular outbreak due to genogroup F strains has been reported in Madagascar to date, we hypothesise that genogroup F is currently undergoing an evolutionary process involving mutations and frequent recombination with other co-circulating EV-A types. This process may or may not lead to the selection and spread of epidemic pathogenic strains in the future. Europic 2016 109

110 TUESDAY 06/09/2016 D25 SCREENING OF ENTEROVIRUS 71 TRANSCRIPTION DYNAMICS: ESTABLISHING HIGH-THROUGHPUT ASSAY FOR SINGLE-MOLECULE INVESTIGATION VIA MAGNETIC TWEEZERS Richard Janissen1, Aysen Norte2, Andrew Woodman3, Jamie Arnold3, Craig Cameron3, Nynke H. Dekker4 1 Kavli Institute of Nanoscience, Delft University of Technology, 2Kavli Institute of Nanoscience Delft, 32Department of Biochemistry and Molecular Biology, Pennsylvania State University, 4Kavli Institute of Nanoscience Picornaviruses remain a threat to public health: for example, polio virus (PV) infections have not been fully eradicated, and in Asia outbreaks of enterovirus (EV) 71 have been on the rise for the past 15 years. Genetic recombination of these pathogens represents the major contributor to the evolution of viruses. This process not only challenges medicine and pharmacology in keeping up with the rate of viral evolution, but it is also difficult to target with classical treatment methods such as vaccines and anti-viral medication. Its molecular mechanism involved has received little attention so far, and how recombination is triggered remains to be elucidated. Previous studies of PV have shown that recombination relies on the viral RNA-dependent RNA polymerase (RdRp). In vitro experiments based on Single-molecule Magnetic Tweezers are able to monitor the RdRp transcription dynamics in real time and at resolutions as high as few base-pairs, all on a massively parallel scale. Together with an unbiased analysis procedure to extract the associated kinetic parameters, we have developed a platform that allows for study of the events that may occur when a polymerase encounters triggers that lead to recombination. To study EV71 RdRp in our Magnetic Tweezers platform, we first designed and tested different RNA substrates to determine the optimal template sequence for EV71 RdRp transcription initiation. Single-nucleotide biochemical incorporation assays demonstrate substantial slower initiation rates for EV71 RdRp compared to PV RdRp. We determined an RNA template sequence that significantly increased EV71 initiation and nucleotide incorporation efficiency and further optimized the protein purification process. Based on these developments, the successful implementation of the single-molecule EV71 RdRp assay will allow us to probe how the dynamics and specific properties this RdRp influence recombination frequency. Parameters measured will include nucleotide incorporation rate, sequence dependence, processivity and fidelity. Ultimately, such high-resolution characterization of the molecular transcription process of EV71 RdRP may thus facilitate the development of strategies to suppress recombination and/or design recombinant-deficient vaccine strains. 110 Europic 2016

111 TUESDAY 06/09/2016 D26 DELETION OF DOMAIN I IN THE 5 NTR OF COXSACKIEVIRUS B3 (CVB3) GENOMIC RNA DOES NOT PREVENT REPLICATION Nora Chapman1, Shane Smithee2, Lee Jaramillo1, Steven Tracy1 1 University of Nebraska Medical Center, 2Centers for Disease Control and Prevention Previous work demonstrated that the group B coxsackieviruses (CVB) can delete 5 terminal genomic sequence without loss of viability in human and murine heart and in primary cells in culture. The largest TD observed was 49 nucleotides, which encompassed the loss of stem b and c and part of d in the secondary structure termed domain I. OBJECTIVE: Having not observed TDs greater than 49 nucleotides, we hypothesized that complete loss of the rest of domain I would be lethal for the virus. We tested this hypothesis by engineering the deletion of domain I in an infectious cDNA clone of a CVB3 genome. METHODS: Deletions of all or part of the secondary structure of domain I (altering or deleting the stemloop d structure with 5 terminal deletions of 56 and 77 nt) were engineered into an infectious cDNA genome of CVB3. Transcripts of these genomes were transfected into HeLa cells in culture to produce virion RNA. Purified virion RNA was used to detect the extent of replication of the CVB3-TD virus by RT-PCR. Infectivity of progeny virus was shown by passaging to fresh cell cultures. RESULTS: CVB3-TD57 (5 deletion of 56 nt) and CVB3-TD78 (5 deletion of 77 nt) replicated without apparent cytopathic effect in HeLa cell culture but did generate virion RNA detectable by RT-PCR. The level of replication of CVB3-TD78 was 100,000 fold less than wildtype parental CVB3 but the infectivity by passage in HeLa cell culture was detectable and could be prevented by CVB3-neutralizing antibody. CONCLUSIONS: Stemloop d can be partially or completely deleted in CVB3-TD viruses without preventing replication of the CVB3-TD virus in HeLa cells. As the level of replication of CVB3-TD78 (preventing all normal domain I secondary structure) is similar to the level of HeLa cell replication seen with CVB3-TD50 (a deletion that arises naturally in persisting CVB3 in hearts of infected mice), why havent we found significant levels of larger deletions in passage of CVB3-TD viruses in primary cell cultures and mice? We know the heart and pancreas or primary cell cultures from these tissues are more resistant to replication of CVB3 than HeLa cells. Stemloop d binding by 3CD in the ternary complex is important for circularization of the genome leading to negative strand initiation. CVB3-TD viruses replicate with nearly equal levels of positive and negative strand RNA, suggesting the importance of negative strand RNA replication for CVB3-TD virus replication. Selection to retain stemloop d in CVB3-TD viruses in animals and pancreatic cells suggests that it increases the fitness of the replicating CVB3-TDs in persistence in natural infections of cardiac and pancreatic tissues but is not essential in the more permissive environment of HeLa cells. Europic 2016 111

112 TUESDAY 06/09/2016 D27 GENOME SEQUENCE OF AVIAN ENTERO-LIKE VIRUS 2 SHOWS IT TO BE A CHICKEN MEGRIVIRUS Nick Knowles1, Daniel Todd2, Katarzyna Bachanek-Bankowska1 1 The Pirbright Institute, 2Agri-Food & Biosciences Institute, Stoney Road, Stormont, Belfast, BT4 3SD, United Kingdom. A picorna-like virus, named EF84/700, was isolated from the faeces of a 27-day-old broiler chicken in Northern Ireland in 1984 (McNulty et al., 1987, Avian Pathology 16: 331-337). It was shown to be antigenically distinguishable from other picorna-like viruses from chickens, ducks and turkeys and designated entero-like virus 2 (ELV-2; McNulty et al., 1990, Avian Pathology 19: 75-87). Subsequently, many avian putative picornaviruses were either confirmed as picornaviruses [e.g. avian encephalomyelitis virus and duck hepatitis virus (DHV) 1] or shown to be astroviruses (e.g. ELV-1, ELV-3, ELV-4, DHV-2 and DHV-3). Avian ELV-2 (AELV-2) was passaged by growth in the chorioallantoic membrane (CAM) of specific-pathogen-free chick embryos. Using Illumina MiSeq technology we have determined the genome sequence of AELV-2 EF84/700. The sequence was 9,571 nt in length, excluding the poly(A) tail. It had a long ORF of 8,253 nt encoding a polyprotein of 2,750 aa. The 5 untranslated region (UTR) was 641 nt long and the 3 UTR was 677 nt. The genome sequence of AELV-2 was most closely related (81% nt id) to the chicken megrivirus CHK-IV-CHV/2013/HUN (KF961187). The genome layout of AELV-2 was predicted to be identical to chicken megrivirus, i.e. VPg+5UTR[1AB-1C-1D/2A1-2A2-2A3 -2B-2C/3A-3B-3C-3D]3UTR-poly(A). Phylogenetic analysis of the P1 capsid region showed that megriviruses fall into three major lineages, i) turkey hepatitis virus (THV), chicken picornavirus (ChPV) 4 and duck megrivirus (DuMV); ii) pigeon mesivirus (MeV) 1 and 2; and iii) chicken megrivirus (ChMV), chicken proventriculitis virus (ChPrV), CPV-5 and AELV-2. In the non- structural protein region (e.g. 3Dpol), megriviruses again fall into three genetic lineages, however, they displayed a different grouping pattern: i) THV, CPV-4, CPV-5, ChPrV, ChMV and AELV-2; ii) MeV-1 and 2; and iii) DuMV. This suggests significant recombination events in their history and complicates their classification into species within the genus Megrivirus. Within the 3 UTR a putative short open reading frame (ORF-2; 98-114 aa) has been described in all megriviruses except for the pigeon mesiviruses. This putative ORF-2 was also present in AELV-2 and would code for a polypeptide 114 aa long. 112 Europic 2016

113 TUESDAY 06/09/2016 D28 INTERTYPIC RECOMBINATION OF HUMAN PARECHOVIRUS 4 ISOLATED FROM INFANTS WITH SEPSIS-LIKE DISEASE Sisko Tauriainen1, Pekka Kolehmainen2, Anu Siponen1, Teemu Smura3, Anni Kauppinen1, Hannimari Kallio-Kokko4, Olli Vapalahti4, Anne Jskelinen4 1 University of Turku, 2University of Turku, Helsinki University, 3Helsinki University, 4Helsinki University and Helsinki University Hospital OBJECTIVES: Human parechoviruses (HPeVs) can cause severe infections of the central nervous system in infants, most of these are caused by HPeV type 3. In 2012 we detected an outbreak in Helsinki, Finland, caused by HPeV4, most babies had sepsis-like symptoms. Because of the unusual symptoms for HPeV4 infections, we sequenced the whole genome of the three rescued viruses to detect any differences in the genome compared to earlier sequenced ones. Despite of HPeV disease associations and frequency in population, research on their genetic identity, diversity and evolution has remained scarce. Deciphering HPeVs on the molecular level is not only vital for understanding their virulence characteristics, but also for getting a complete picture of their epidemiology. METHODS: Complete coding sequences of three previously reported HPeV4 isolates were generated using traditional as well as next generation sequencing techniques. Phylogenetic analysis was conducted with ClustalW and MEGA 6.0 software, whereas recombination analysis was carried out using Simplot and Bootscan. RESULTS: The complete coding sequences of three Finnish HPeV4 isolates were highly similar to each other with 99.1% overall nucleotide sequence similarity. According to their structural capsid-encoding sequence, they were closely related to other HPeV4 strains, whereas based on their non-structural region, they associated only to a single HPeV4, namely the reference strain K251176-02. Interestingly, the 3D-encoding sequence of the Finnish isolates did not cluster with the HPeV4 reference strain anymore, but were instead located in a group comprised of HPeV1 and HPeV3 strains. Analysis with Simplot and Bootscan further indicated intertypic recombination events in the evolution of the Finnish HPeV4 isolates, one of them being the cause for the lost similarity between the Finnish isolates and the reference strain K251176-02 in the 3D-gene. CONCLUSIONS: An intertypic recombination event has led to the evolution of an HPeV mosaic virus, which based on overall sequence similarity, is still classified as HPeV type 4. In the 3D-gene-encoding region, however, the virus appears to have sequence elements similar to those of HPeV1 and HPeV3 in this area. HPeV3 are known to include the most virulent strains out of all HPeVs isolated thus far. Also the Finnish HPeV4 isolates were linked to a disease of higher severity, sepsis-like disease in infants. Complete sequence analysis provides better understanding on the genetic background of a specific strain, however, such analysis is depended on the reference material available. The data from this study adds three further HPeV4 complete coding sequences to the four preexisting ones in GenBank. Europic 2016 113

114 WEDNESDAY 07/09/2016 8:45 Session 5 EMERGING/REEMERGING PATHOGENS Chairs: 1st part, Satoshi Koike & Marc Pallansch 12:00 2nd part, Hiroyuki Shimizu & Francis Delpeyroux K07: VIRAL AND HOST FACTORS INVOLVED IN ENTEROVIRUS 71 INFECTION Shin-Ru Shih (Chang Gung 09:00 POTENTIAL APPLICATIONS IN THERAPY AND PROPHYLAXIS University, Taiwan) E01: ENTEROVIRUS D68 AND ACUTE FLACCID MYELITIS: ASSOCIATION OR Mark Pallansch (CDC/NCIRD/ 09:20 INCIDENTAL FINDING? DVD, United States of America) E02: ENTEROVIRUS 71 INFECTION STUDIES IN HUMAN LUNG AND GUT Sabine van der Sanden (Medical 09:35 ORGANOIDS REVEAL A POTENTIAL VIRAL DETERMINANT OF INCREASED Microbiology, Academic Medical SUSCEPTIBILITY Center, Netherlands) E03: A REVERSE GENETIC SYSTEM FOR DEFORMED WING VIRUS, THE MOST David Evans (University of St. 09:50 IMPORTANT VIRAL PATHOGEN OF HONEY BEES. Andrews, United Kingdom) COFFEE BREAK E04: COXSACKIEVIRUS B1 INFECTIONS ARE ASSOCIATED WITH INSULIN-DRIVEN Amirbabak Sioofy Khojine 10:35 AUTOIMMUNE PROCESS IN CHILDREN WITH INCREASED RISK OF TYPE 1 DIABETES (University of Tampere, Finland) IN FINLAND (DIPP STUDY) Richard Lloyd (Baylor College E05: ARE ENTEROVIRUSES ASSOCIATED WITH TYPE 1 DIABETES? REPORTS FROM 10:50 THE NPOD-V CONSORTIUM AND THE TEDDY STUDY of Medicine, United States of America) Hiroyuki Shimizu (National E06: OUTBREAK OF TYPE 1 VACCINE-DERIVED POLIOVIRUS IN LAO PEOPLE'S 11:05 DEMOCRATIC REPUBLIC IN 2015-2016 Institute of Infectious Diseases, Japan) E07: DETECTION OF ENTEROVIRUS D68 BASED ON THE NATIONAL Hitomi Kinoshita (National 11:15 EPIDEMIOLOGICAL SURVEILLANCE OF INFECTIOUS DISEASES SYSTEM IN JAPAN Institute of Infectious Diseases, FROM 2005 TO 2015 Japan) E08: AMBULATORY PAEDIATRIC SURVEILLANCE OF HAND, FOOT AND MOUTH Audrey Mirand (CHU Clermont- 11:25 DISEASE: OUTBREAK OF CV-A6 INFECTIONS IN FRANCE, 2014-2015 Ferrand, France) E09: COXSACKIEVIRUS B1 IS ASSOCIATED WITH TYPE 1 DIABETES RESULTS Amirbabak Sioofy Khojine 11:35 FROM VIRUS ANTIBODY SURVEY IN DIFFERENT EUROPEAN POPULATIONS (University of Tampere, Finland) LUNCH BREAK 114 Keynote Lecture Communication (12 + 3 min) Short Communication (8 + 2 min) Europic 2016

115 WEDNESDAY 07/09/2016 POSTER SESSION 5 - EMERGING/REEMERGING PATHOGENS E10: THE CROSS-NEUTRALIZING CAPACITY OF SERA FROM DUTCH DONORS PREDICTS PROTECTION AGAINST ASIAN ENTEROVIRUS 71 OUTBREAK STRAINS Sabine van der Sanden (Medical Microbiology, Academic Medical Center, Amsterdam, Netherlands) E11: ENTEROVIRUS D68 INFECTIONS IN SPAIN, 2014-2016 Maria Cabrerizo (Institute of Health Carlos III, Spain) E12: THE SUCKLING MOUSE AS A MODEL FOR STUDYING ENTEROVIRUS D68 PATHOGENESIS Jennifer L. Anstadt (CDC/NCIRD/DVD, United States of America) E13: FURTHER STUDIES ON THE EVOLUTION OF SENECA VALLEY VIRUS Nick Knowles (The Pirbright Institute, United Kingdom) E14: SAFFOLD VIRUS IN VIVO PASSAGES DRIVE THE STRUCTURAL EVOLUTION OF THE CAPSID PROTEIN FOR ENHANCING REPLICATION FITNESS IN NEURAL CELLS. Osamu Kotani (National Institute of Infectious Diseases,Pathogen Genomics Center, Japan) E15: EMERGING RECOMBINANT NEW GENOGROUP OF COXSACKIEVIRUS A16 HAS RAPIDLY DISPERSED IN FRANCE, 20112014 Jean-Luc Bailly (Universit dAuvergne, France) E16: SCREENING AND ANALYSIS OF SAFFOLD VIRUS IN SWEDEN Helena Vandesande (Linnaeus University, Sweden) E17: CHRONIC ENTEROVIRUS INFECTION IN A PATIENT WITH MYALGIC ENCEPHALOMYELITIS/CHRONIC FATIGUE SYNDROME (ME/CFS) CLINICAL, VIROLOGIC AND PATHOLOGICAL ANALYSIS John Chia (United States of America) E18: PHENOTYPIC ANALYSIS OF THE 2014 EPIDEMIC ENTEROVIRUS D68 STRAIN FROM NORTH AMERICA IN RESPIRATORY EPITHELIAL CELLS Debra Lugo (UCLA Department of Pediatric, Infectious Diseases, United States of America) E19: VIRULENCE OF RECENT COXSACKIEVIRUS B2 ISOLATES IN A NEONATAL MOUSE MODEL Noriyo Nagata (National Institute of Infectious Diseases, Japan) Europic 2016 115

116 WEDNESDAY 07/09/2016 K07 VIRAL AND HOST FACTORS INVOLVED IN ENTEROVIRUS 71 INFECTION POTENTIAL APPLICATIONS IN THERAPY AND PROPHYLAXIS Shin-Ru Shih Research Center for Emerging Viral Infections, Chang Gung University, Taiwan Although enterovirus replication occurs in cytoplasm, host nucleus is tightly associated with viral infection. Several host nuclear-resident proteins redistribute into cytoplasm and interact with viral internal ribosomal entry site (IRES) that is responsible for viral translation. Using RNA-protein pull down assay, we identified a novel protein, FBP2, which negatively regulated viral IRES activity. We further found a small RNA, vsRNA1, generated from viral genome, associated with FBP2 and augmented its inhibition of IRES activity. Therefore, vsRNA1 has a great potential to be developed as an antiviral agent. On the other hand, viral 3D polymerase, enters nucleus and interacts with splicing factor Prp8, and such interaction impairs host cell splicing machinery. By doing so, the maturation of host mRNA are strongly reduced. We speculated a mutant virus without nuclear localization signal (NLS) in its 3CD would be attenuated because of less interference of host nuclear functions. Indeed, an NLS mutant virus showed a reduced pathogenicity and mortality rate in mice challenged with EV-A71. Therefore, NLS mutation can be introduced into a vaccine virus candidate to generate a safer attenuated live vaccine. In the final part of this talk, a new strategy to prevent severe EV-A71 infection will be discussed. Pretreatment of heat- killed enterococcus can modulate a host factor, monocyte chemoattractant protein-1 (MCP-1,) and reduce the pathogenicity of EV-A71 infection. 116 Europic 2016

117 WEDNESDAY 07/09/2016 E01 ENTEROVIRUS D68 AND ACUTE FLACCID MYELITIS: ASSOCIATION OR INCIDENTAL FINDING? Steve Oberste1, Terry Ng1, Anna Montmayeur2, Shannon Rogers1, Kaija Maher1, Christina Castro1, Shur-wern Chern1, Allan Nix1, James Sejvar1, Mark Pallansch1 1 CDC, 2CSRA Enterovirus D68 can cause severe respiratory illness, with clinical manifestations that include bronchiolitis and pneumonia, especially in children. In the summer-fall of 2014, enterovirus D68 (EV-D68) was associated with a large outbreak of severe respiratory disease among children throughout the United States, with over 1100 laboratory-confirmed cases, making it the largest ever EV-D68 outbreak. During the same period, sporadic cases and small clusters of pediatric acute flaccid myelitis (AFM) cases were identified in a number of states, leading to speculation of a causal link between EV-D68 infection and AFM. However, EV-D68 has not been detected in cerebrospinal fluid for any AFM case, except for one which was heavily contaminated by blood. To understand the molecular epidemiology of EV-D68 and further examine the possible association with AFM, we sequenced 100 complete genomes of EV-D68 in respiratory cases, as well as 10 EV-D68 genomes from respiratory samples associated with AFM cases, using an amplicon-based NGS protocol that is efficient in sequencing ~100 genomes in parallel. Using all publicly available EV-D68 sequences, we identified conserved sites in the 5UTR, VP1, 2C, and 3UTR regions, allowing amplification of the entire genome in three overlapping amplicons with minimally-degenerate primers for sequencing on the Illumina MiSeq platform. The 110 EV-D68 genome sequences shared genome-wide nucleotide identities of 94-100%. In concordance with recent reports, we observed several sub-clades based on genetic distance and phylogenetic reconstruction. One previous study reported that AFM-associated EV-D68 were grouped into a specific subclade B1, based on a smaller, geographically limited sample set. However, our findings with a larger sample size and more complete geographic representation suggested AFM cases are not specifically associated with any particular sub-clade. The possible association of AFM with EV-D68 infection remains an open question that must be addressed by additional epidemiologic and laboratory investigations. Europic 2016 117

118 WEDNESDAY 07/09/2016 E02 ENTEROVIRUS 71 INFECTION STUDIES IN HUMAN LUNG AND GUT ORGANOIDS REVEAL A POTENTIAL VIRAL DETERMINANT OF INCREASED SUSCEPTIBILITY Sabine van der Sanden1, Norman Sachs2, Evelyn Fessler3, Janneke F. Linnekamp3, Jarno Drost2, Gerrit Koen1, Sylvie M. Koekkoek1, Christianne Buskens4, Pieter J. Tanis4, Jan Paul Medema5, Hans Clevers2, Katja Wolthers1 1 Medical Microbiology, Academic Medical Center, Amsterdam, 2Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), UMC Utrecht and Cancer Genomics Netherlands, 3Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Academic Medical Center, Amsterdam and Cancer Genomics Center, 4Department of Surgery, Academic Medical Center, Amsterdam, 5Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine Academic Medical Center, Amsterdam and Cancer Genomics Center While poliovirus is getting close to extinction, human enterovirus (EV) 71 has been causing massive outbreaks of Hand Foot and Mouth Disease and neurologic disease among children in Asia since 1997. The increased incidence is associated with the emergence of new genotypes. However, no correlation has been found between severity of disease outcome and genotype. Knowledge on EV71 pathogenesis in general is limited due to the lack of suitable animal models mimicking human disease development. Recent advances in human 3D tissue culturing have created the unique opportunity to study virus-host interactions in a human setting. Using recently established human lung and gut organoid culture models, representing the primary enterovirus entry sites, we identified EV71 strain specific replication kinetics in the airway and gut epithelia by RT-qPCR and virus titration assays. Comparative analysis of the capsid amino acid sequences revealed a potential determinant for increased susceptibility in the VP1 capsid protein. Infection of organoids with additional clinical isolates harboring mutations of this residue confirmed our observations. Detailed studies on the role of this residue in determining susceptibility, using site directed mutagenesis, and the underlying mechanism are currently ongoing. The results from this study show that human lung and gut organoids are susceptible for EV71 infection and thereby form relevant models for studying EV71-host interactions. We identified a potential viral marker determining increased susceptibility of the human airway and intestinal epithelia. This strain dependent susceptibility potentially explains differences in severity of disease outcome in EV71 infected children. 118 Europic 2016

119 WEDNESDAY 07/09/2016 E03 A REVERSE GENETIC SYSTEM FOR DEFORMED WING VIRUS, THE MOST IMPORTANT VIRAL PATHOGEN OF HONEY BEES. David Evans1, Olesya Gusachenko1, Christopher Moffat1, Jess Fannon2, Eugene Ryabov2, Adite Kibe2, Katharin Balbirnie1 1 University of St. Andrews, 2University of Warwick Deformed wing virus (DWV) is the most important viral pathogen of honey bees (Apis mellifera), which are a globally-important managed pollinator of high-value crops. The virus is a picorna-like single-stranded, positive- sense RNA virus and belongs to the Iflaviridae, a family that contains other economically-important insect pathogens. When transmitted horizontally in the colony during larval feeding, or vertically via eggs, the virus persists at low levels and causes no overt disease. In contrast, in colonies infested with the ectoparasitic mite Varroa destructor levels of the virus are amplified 1,000 - 10,000 times in mite-exposed pupae and cause overt pathogenesis, including developmental defects in wing and abdomen formation. In addition, high levels of virus are associated with reduced longevity of winter bees. Since the latter are responsible for maintaining the colony overwinter in temperate climates, elevated Varroa and DWV levels lead to increased winter mortality which approaches 50% in long or severe winters. In previous studies we have demonstrated that direct injection of virus to the haemolymph of developing pupae favours the replication of a virulent strain of the virus that we designated DWVV. This virus is best described as a recombinant between two previously described, closely related strains, Varroa Destructor Virus type 1 (VDV-1) and Deformed Wing Virus. There are no honey bee cell lines and the virus does not infect a wide range of other insect cell lines tested. During our analysis of the biology of DWV we generated nominally full-length cDNAs but were unable to recover them after direct injection of honey bee pupae maintained in vitro. However, when using next-generation sequencing to characterise variation in the virus population we identified a discrete 5 stem-loop structure, absent from our cDNA or any published sequences of DWV or related viruses. We engineered the missing sequence onto the cDNA and made synonymous changes to the region encoding the virus polyprotein to generate tagged virus genomes that we demonstrated could be recovered after direct injection of RNA to honey bee pupae in vitro. The additional sequences include nucleotides characteristic of viruses in which replication is primed by a VPg-like protein-nucleotide primer. In subsequent studies we have synthesised full-length VDV-1 and DWV variants, luciferase and GFP-encoding replicons and are using these to characterise the replication and pathogenesis of DWV. Current studies include the analysis of the ability of these RNAs to replicate in a range of insect cell lines after direct transfection, and the generation of pure virus stocks for transmission, pathogenesis, challenge and protection studies. Europic 2016 119

120 WEDNESDAY 07/09/2016 E04 COXSACKIEVIRUS B1 INFECTIONS ARE ASSOCIATED WITH INSULIN-DRIVEN AUTOIMMUNE PROCESS IN CHILDREN WITH INCREASED RISK OF TYPE 1 DIABETES IN FINLAND (DIPP STUDY) Amirbabak Sioofy Khojine, Jussi Lehtonen, Noora Nurminen, Olli H. Laitinen, Sami Oikarinen, Heikki Hyoty University of Tampere BACKGROUND: The specificity of the first appearing autoantibody in prediabetic children categorizes them into two immunophenotypes (IAA and GADA) depending on the genetic susceptibility to type 1 diabetes and age at autoantibody seroconversion. It is possible that these phenotypes also have different etiology. Coxsackie B virus infections have been linked to the initiation of islet autoimmunity but it is not known whether they correlate with these immune phenotypes. AIM: The aim was to study if Coxsackie B viruses are associated with a specific first appearing islet autoantibody in children who were prospectively followed up from birth. METHODS: The participating children were initially recruited to the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study. The current study cohort included 332 case children who became persistently positive for multiple islet autoantibodies during prospective observation, and 651 control children who were matched with the cases by age, gender, HLA-II conferred risk of T1D, and registered study center. A total number of 91 children developed IAA and 78 children became GADA positive as the single first-appearing autoantibody. Infections by each Coxsackievirus B serotype were identified by detecting the seroconversion to virus-specific neutralizing antibodies between serial follow up serum samples collected before the autoantibodies appeared. RESULTS: Coxsackievirus B1 infections were associated with the appearance of IAA as the first autoantibody (OR 2.4, 95%CI 1.4-4.2, P=0.003) while no such association was seen with the appearance of GADA as the first autoantibody (OR 1.05, 95%CI 0.6-1.8, P=0.85). This risk association was modulated by prior infections with other Coxsackievirus B serotypes in a manner which fits with immunological cross protection against Coxsackievirus B1 as well as by the presence of maternal antibodies in cord blood against this virus. CONCLUSION: The results suggest that infection by Coxsackievirus B1 in early childhood may induce an autoimmune response against insulin leading to the manifestation of clinical type 1 diabetes. 120 Europic 2016

121 WEDNESDAY 07/09/2016 E05 ARE ENTEROVIRUSES ASSOCIATED WITH TYPE 1 DIABETES? REPORTS FROM THE NPOD-V CONSORTIUM AND THE TEDDY STUDY Richard Lloyd1, Heikki Hyoty2, Joseph Petrosino1, Nadim Ajami1, Matthew Wong1, Xiangjun Tian1, Alberto Pugliese3, Kendra Vehik4 1 Baylor College of Medicine, 2University of Tampere, 3University of Miami, 4Univeristy of South Florida Enteroviruses (EVs) have been linked to the pathogenesis of Type 1 Diabetes (T1D) through serological and epidemiological data, but there remains a need to more directly determine if enteroviruses can trigger T1D, and if so, what range of viral serotypes trigger and what the pathogenic mechanisms are in humans. Two investigations working towards this end have made progress and initial findings will be reported. The nPOD virus working group has determined that human pancreata from T1D or autoantibody positive persons contain significantly higher incidences of islets with beta cells staining for enterovirus VP1 antigen, and also high levels of MHC Class 1 expression. Viral RNA has been isolated from several cases and sequenced, suggesting that multiple viruses may infect pancreata simultaneously. The TEDDY Study is a large ongoing prospective birth-cohort study where monthly stools were collected and sequenced for complete virome analysis in children who developed T1D and in their matched controls. We will discuss the clustering of various enteroviruses from children among the six geographically distinct clinical centers in US and in Europe and associations with the development of autoantibody positivity and TID. Europic 2016 121

122 WEDNESDAY 07/09/2016 E06 OUTBREAK OF TYPE 1 VACCINE-DERIVED POLIOVIRUS IN LAO PEOPLES DEMOCRATIC REPUBLIC IN 2015-2016 Hiroyuki Shimizu1, Phengta Vongphrachanh2, Bouaphanh Khamphaphongphane2, Bounthanom Sengkeopraseuth2, Tomofumi Nakamura3, Yorihiro Nishimura1, Minetaro Arita1, Junko Wada1, Hiromu Yoshida1, Walter William Schluter4, Siddhartha Sankar Datta5 1 National Institute of Infectious Diseases, 2National Centre for Laboratory and Epidemiology, Lao PDR, 3The Research Foundation for Microbial Diseases of Osaka University (BIKEN), 4Expanded Programme on Immunization, World Health Organization, Regional Office for the Western Pacific, 5Office of the WHO Representative in the Lao Peoples Democratic Republic BACKGROUND: In Lao Peoples Democratic Republic (Lao PDR), a polio-free status has been maintained for nearly 15 years and the last polio case due to a type 1 wild poliovirus was identified in 1996. According to the Lao PDR national polio end-game plan, IPV was introduced in October 2015, and the switch from trivalent to bivalent OPV was performed in April 2016 in Lao PDR to minimize the risk for an outbreak of type 2 vaccine-derived polioviruses (VDPVs). METHODS: Polioviruses were isolated from stool samples (approximately 630 samples as of May 25, 2016) of acute flaccid paralysis (AFP) cases and individuals in contact with these cases in Lao PDR in L20B and RD cell lines according to standard protocols. The poliovirus isolates were screened for VDPVs using real-time RT-PCR and were finally confirmed by sequencing the capsid VP1 region. RESULTS AND DISCUSSION: In October 2015, a type 1 poliovirus was isolated from a child who had the AFP onset on Sep 7, 2015 in the Bolikhamxay Province in Lao PDR at National Institute of Infectious Diseases, Japan. The isolate was identified as a type 1 vaccine-derived poliovirus (VDPV) with 30 nucleotide differences from the parental Sabin 1 strain (3.3% nucleotide diversity) by VP1 sequencing. Under intensified surveillance activities following the detection of the first case, a growing number of type 1 circulating VDPV (cVDPV1) isolates were identified from AFP cases and contacts in three geographically distinct areas in Lao PDR. Eleven confirmed cVDPV1 cases (two patients died) were aged between 8 months and 44 years (average, 15 years) and had generally inadequate OPV immunization history, suggesting immunity gaps even in adults in the affected areas/ communities in Lao PDR. Phylogenetic analysis based on the VP1 sequences of 26 type 1 VDPV isolates from the AFP cases and contacts revealed a distinct genetic cluster with considerable genetic diversity among the cVDPV1 isolates (2235 VP1 nucleotide differences from the Sabin strain). Recombination with non-polio enteroviruses (species C) or Sabin 2/Sabin 3 strains has not been identified for type 1 VDPVs in Lao PDR. The date of onset of the last case of laboratory confirmed type 1 cVDPV is January 11, 2016; thereafter, type 1 cVDPV has not been detected from any AFP case. The government of Lao PDR has conducted multiple supplemental immunization activities covering wide age group across all provinces. These results highlight the remaining risk for polio outbreaks due to VDPVs in areas/communities with low immunization coverage with OPV or IPV including the current setting following the switch from trivalent to bivalent OPV. 122 Europic 2016

123 WEDNESDAY 07/09/2016 E07 DETECTION OF ENTEROVIRUS D68 BASED ON THE NATIONAL EPIDEMIOLOGICAL SURVEILLANCE OF INFECTIOUS DISEASES SYSTEM IN JAPAN FROM 2005 TO 2015 Hitomi Kinoshita, Yuzo Arima, Tomimasa Sunagawa, Keiko Tanaka-Taya, Nozomu Hanaoka, Tsuguto Fujimoto, Kazunori Oishi, Hiroyuki Shimizu National Institute of Infectious Diseases OBJECTIVES: In 2014, a nationwide outbreak of enterovirus D68 (EV-D68) infections was reported in the USA. These infections mainly occurred among children and were associated with severe respiratory illness and possible neurological manifestations, including acute flaccid myelitis. Although national surveillance and laboratory diagnosis systems have not been established specifically for EV-D68 infections in Japan, we aimed to describe the epidemiological and clinical characteristics of EV-D68 infections in Japan from 2005 to 2015 based on data from the National Epidemiological Surveillance of Infectious Diseases (NESID) system. METHODS: Under the Infectious Diseases Control Law in Japan, EV-D68 infections are not notifiable infectious diseases. However, information on patient samples that were positive for EV-D68 is available from the NESID database. Local public health institutes have been conducting ad hoc laboratory testing for EV-D68 for clinical specimens of patients with a diverse clinical spectrum, including respiratory illness. Accordingly, we reviewed and described EV-D68-positive cases (n=538) detected from 2005 to 2015, in terms of clinical diagnosis/symptoms, epidemiological information, detection methods, and the type of sample. RESULTS:The number of EV-D68-positive cases was more than 100 in 2010 (n=129), 2013 (n=122), and 2015 (n=258) higher than those in other years from 2005 to 2015 (n=09), suggesting periodic EV-D68 epidemics in Japan. The number of EV-D68-positive cases peaked in September during the EV-D68 endemic years in 2010, 2013, and 2015. Approximately 80% of EV-D68-positive cases were 6 years of age or younger. Among EV-D68- positive cases, the majority was patients with respiratory illness (40% from lower respiratory infections, 19% from upper respiratory infections, 16% from bronchial asthma); however, EV-D68 was also detected from patients with neurologic syndromes including encephalitis (n=4) and paralysis (n=8). The majority of EV-D68-positive clinical samples were throat swabs (97%), but EV-D68 was also detected from stool (n=10), blood (n=3), or conjunctival (n=1) specimens. Diagnosis was made mainly by molecular detection methods. DISCUSSION: In September 2015, the number of EV-D68-positive cases rapidly increased, resulting in the largest number in 2015 (n=258) from 2005 to 2015 in Japan. Almost simultaneously, a growing number of cases with acute flaccid paralysis were reported nationwide in Japan and some of them were identified as EV-D68-positive by the NESID system. Although reporting and laboratory diagnosis biases should be carefully considered, our results provide insights into the epidemiological and clinical aspects of EV-D68 infections. Europic 2016 123

124 WEDNESDAY 07/09/2016 E08 AMBULATORY PAEDIATRIC SURVEILLANCE OF HAND, FOOT AND MOUTH DISEASE: OUTBREAK OF CV-A6 INFECTIONS IN FRANCE, 2014-2015 Audrey Mirand1, Franois Vi le Sage2, Bruno Pereira3, Robert Cohen4, Corinne Levy4, Christine Archimbaud5, Hlne Peigue-Lafeuille5, Jean-Luc Bailly6, Ccile Henquell5 1 CHU Clermont-Ferrand, 2Association Franaise de Pdiatrie Ambulatoire, AFPA, http://www.afpa.org/, 3CHU Clermont-Ferrand, Biostatistics Unit (DRCI), 4Association Clinique et Thrapeutique Infantile du Val de Marne, ACTIV, F-94100 St Maur des Fosss, France, 5 CHU Clermont-Ferrand, Virology laboratory, National Reference Centre for Enteroviruses and parechoviruses-associated laboratory, 6 Universit dAuvergne, Laboratoire Epidmiologie, Pathogense des Infections Entrovirus Hand, foot and mouth disease (HFMD) and herpangina (HA) are common childhood diseases mostly associated with human enteroviruses (EV). Although usually benign illnesses, neurological complications may be observed during large epidemics when enterovirus A71 (EV-A71) is involved. While EV-A71 mainly circulates in Asia, coxsackievirus A6 (CV-A6) has emerged as the most predominant type associated with outbreaks of HFMD in Europe and the United States and has been associated with more severe and atypical HFMD. To date, the clinical impact of EV associated with HFMD remains widely unknown outside Asia and the prevalence of EV-A71 may be underestimated. OBJECTIVES: We set up a prospective community-based surveillance of HFMD to monitor EV infections associated with HFMD/HA in France and to describe the epidemiological and clinical spectrum of HFMD/HA. METHODS: The sentinel surveillance was performed by 47 paediatricians in 20/22 French administrative regions. Throat or buccal swabs were collected in children clinically diagnosed with HFMD and/or HA and prospectively tested for the detection of EV genome and genotyping. Physical examinations at presentation were recorded on a standardised form. The data collected by the sentinel paediatricians were compared with those collected through a national hospital-based surveillance. RESULTS: From 1 April 2014 to 31 March 2015, of the 659 children recruited, 523 (79.3%) were EV-infected. Two epidemic waves, dominated by CV-A6 (54%), occurred from week 25 to week 27 and in Mid-October. Only 6 EV-A71 (1.1 %) infections were detected. No neurological complication was reported. CV-A6 infections were more frequently associated with atypical HFMD characterised by extended eruptions to limbs and face. Through the hospital-based surveillance, 118 HFMD/HA cases were reported and CV-A6 accounted for 64.6 % (64/99 cases with identified EV type). Among the 27 CV-A6 infected and hospitalised patients, 5 presented with neurological signs. CONCLUSIONS: This study contributes to a more comprehensive view of the epidemiology HFMD/HA in France and of the clinical spectrum of HFMD/HA associated with EV, in particular with CV-A6. Syndromic surveillance of HFMD/HA by ambulatory paediatricians with prospective and standardised collection of clinical data in combination with EV testing and genotyping are useful to monitor the epidemiology of EV infection, to timely detect peaks of highest activity and to determine the EV types involved, leading to a better detection of the emergence of EV-A71 or any other EV that could be associated with severe usual clinical features. 124 Europic 2016

125 WEDNESDAY 07/09/2016 E09 COXSACKIEVIRUS B1 IS ASSOCIATED WITH TYPE 1 DIABETES RESULTS FROM VIRUS ANTIBODY SURVEY IN DIFFERENT EUROPEAN POPULATIONS Amirbabak Sioofy Khojine1, Sami Oikarinen1, Didier Hober2, Bernadette Lucas2, Hanna Honkanen1, Jorma Ilonen3, Mikael Knip4, Heikki Hyoty1 1 University of Tampere, 2University Lille and CHR, Lille, France, 3University of Turku, Turku, Finland, 4Childrens Hospital, University of Helsinki, Helsinki, Finland BACKGROUND: Enteroviruses have been linked to type 1 diabetes (T1D) in various studies. Our recent observations in different European populations suggest that one specific enterovirus type namely Coxsackievirus B1 (CBV1) is associated with the increased risk of T1D. In the present study, we analyzed further this association by adding new countries to the study and by analyzing neutralizing antibodies against several CBV1 strains in newly diagnosed T1D patients and matched controls. STUDY DESIGN AND POPULATION: This case control study included 345 patients with newly diagnosed type 1 diabetes and 345 control children matched for the time of sampling, gender, age, and the country including Finland (102 pairs), Sweden (24 pairs), England (13 pairs), France (42 pairs), Greece (31 pairs), Estonia (33 pairs), and Lithuania (100 pairs). Serum samples were collected within two weeks after the diagnosis of diabetes. Plaque neutralization assay was used to detect neutralizing antibodies against 12 different CBV1 strains. Conditional logistic regression analysis was used to calculate odd ratios (OR) and 95% confidence intervals (CI). RESULTS: Neutralizing antibodies against CVB1 were more frequent in T1D cased than matched controls. Taking together all study countries, being positive to any CVB1 strain was associated with higher risk of T1D in cases versus controls (OR 1.62, CI 1.08-2.41, P=0.019). This association was higher when Lithuania was excluded from the analysis due to high positivity rates compared to other countries (OR 1.89, CI 1.2-2.99, P=0.006). Interestingly, this association was the greatest in Finland and Sweden (OR 2.44, CI 1.13-5.31, 0.024), where the incidence of T1D is high. CONCLUSION: The results suggest that CVB1 infection is associated with type 1 diabetes in European populations. However, this association was not seen in Lithuania suggesting that it may vary according to time and place and possibly depend on the epidemiology of enterovirus infections in a given population. Europic 2016 125

126 WEDNESDAY 07/09/2016 E10 THE CROSS-NEUTRALIZING CAPACITY OF SERA FROM DUTCH DONORS PREDICTS PROTECTION AGAINST ASIAN ENTEROVIRUS 71 OUTBREAK STRAINS Sabine van der Sanden1, Gerrit Koen2, Hetty W. van Eijk3, Sylvie M. Koekkoek3, Menno D. De Jong3, Katja Wolthers3 1 Medical Microbiology, Academic Medical Center, Amsterdam, 2Academic Medical Center, Amsterdam., 3Academic Medical Center, Amsterdam Outbreaks of human enterovirus 71 (EV71) in Asia coincide with high child morbidity and mortality. To gain insight in the potential threat for the European population, we determined the neutralizing activity in immunoglobulin (IVIg)-batches and individual sera from Dutch donors against EV71 strains isolated in Europe and from outbreaks in Asia. All IVIg-batches and 41%, 79% and 65% of sera from children (

127 WEDNESDAY 07/09/2016 E11 ENTEROVIRUS D68 INFECTIONS IN SPAIN, 2014-2016 Maria Cabrerizo1, Jordi Reina2, Maria Pilar Romero3, Antonio Moreno-Docn4, Maitane Aranzamendi5, Ana Martnez-Sapia6, Juan Pablo Garca-Iiguez6, Paula Madurga6, Andrs Antn7, Carlos Rodrigo7, Alfons Macaya7, Sonia Prez-Castro8, Nuria Rabella9, Almudena Otero10 1 Institute of Health Carlos III, 2Hospital Son Espases, Palma de Mallorca, 3Hospital La Paz, Madrid, 4Hospital Virgen de la Arrixaca, Murcia, 5Hospital Cruces, Bilbao, 6Hospital Miguel Servet, Zaragoza, 7Hospital Vall DHebrn, Barcelona, 8Hospital Meixoeiro, Vigo, 9 Hospital de Santa Creu i Sant Pau, Barcelona, 10Institute of Health Carlos III, Madrid INTRODUCTION: Enterovirus 68 (EV-D68) is member of Enterovirus genus (species D). Since it first detection in 1972, is a common cause of respiratory illness. In last years, however, the circulation of this EV type has increased in different parts of the world. Specifically, during 2014, several EV-D68 outbreaks were described in North America, causing substantial hospitalization of children with severe respiratory diseases. Acute flaccid paralysis (AFP) following infection has been also reported. In Spain studies of EV-D68 infections are scarce. OBJECTIVES: To characterize the type of EV detected in hospitalized patients with respiratory and/or severe neurological illnesses and to investigate the epidemiology of EV-D68 in Spain during a 2-year study period, including clinical presentations, seasonality, distribution patterns and phylogenetic relationships. STUDY DESIGN: EV-positive respiratory samples (N=202) collected between March 2014 and May 2016 from hospitalized patients with respiratory symptoms were sent to the Enterovirus Laboratory (CNM) for genotyping. On the other hand, AFP cases were studied through national AFP surveillance system by the Enterovirus Laboratory following WHO protocols. EV genotypes were identified by specific RT-PCRs of species EV-A, B, C and D which amplify 3-VP1 region, sequencing and phylogenetic analysis. RESULTS: EV were confirmed in 186 (92%) of the total of 202 respiratory specimens analyzed while 16 were found to be RV. EV-D68 was the most frequently detected type, accounting for 14% (28/202). Other 25 EV types from EV species A and B were also identified, being the 3 most prevalent coxsackievirus (CV)A6 (19/202, 9%), echovirus (E)-6 (11/202, 5%) and CV-B4 (10/202, 5%). All EV-D68-infected patients but 2 were children between 1 month and 13 years of age (age mean 3.83.7 years). The 2 infected adults were >65 years. Only 2 EV- D68-positive children had coinfections (with AdV and RV, respectively). Clinically, 8 patients (29%) had upper respiratory tract infections and 20 were diagnosed with lower airway disease, where bronchospasm (13 cases, 46%), bronchiolitis, (3 cases, 11%), bronchitis or pneumonia (2 cases, 7% each) was reported. Eight (29%) EV- D68-positive patients required admission to ICU, but none had further complications. In addition, EV-D68 was detected in respiratory and stool samples from 3 AFP cases occurred between December 2015 and March 2016. No EV-D68 was detected in CSF samples. Clinical presentations were romboencephalitis with tetraparalysis (4 years-old boy and 2 years-old girl) and acute flaccid myelitis (22 months boy). The 3 children had residual paralysis 60 days after the onset of symptoms. EV-D68 infections showed a seasonal distribution identifying during the cool months (from October to April) coinciding with the influenza season. Phylogenetic analysis showed all EV-D68 sequences studied belonged to the clade B formed by most of the American and European strains circulating currently. CONCLUSIONS: Surveillance of EV in Spain has revealed ongoing circulation of EV-D68 causing primarily mild/severe respiratory illnesses. In addition, EV-D68 was detected in AFP cases, which supports the putative association between this EV and severe neurological disease. Further surveillance studies are needed to improve our knowledge about the epidemiology and pathologies associated with EV-D68 infections. Europic 2016 127

128 WEDNESDAY 07/09/2016 E12 THE SUCKLING MOUSE AS A MODEL FOR STUDYING ENTEROVIRUS D68 PATHOGENESIS Jennifer L. Anstadt1, Shane E. Smithee2, Kandice L. Lightner2, W. Allan Nix2, William C. Weldon2, Mark A. Pallansch2, M. Steven Oberste2 1 CDC/NCIRD/DVD, 2Division of Viral Diseases, Centers for Disease Control and Prevention Human enterovirus infections cause a wide range of illness, from mild to severe, and target several diverse organ systems, with paralytic poliomyelitis being perhaps the most devastating of enterovirus-associated diseases. Enterovirus D68 (EV-D68) is an emerging human pathogen causing an increasing number of acute respiratory infections in children. During the summer and fall of 2014, more than 1100 cases of severe pediatric respiratory illness were confirmed positive for EV-D68 infection. Concurrent with this outbreak, unusual clusters of neurologic illness (acute flaccid myelitis; AFM) were also reported, suggesting a possible link with EV-D68 infection. Patients experienced symptoms with clinical indications similar to enterovirus-induced viral myelitis. Although more than 100 AFM cases were reported, no causative agent has been identified. Currently, the study of EV-D68 pathogenesis is limited due to the absence of a suitable animal model. Since infection of suckling mice has proven useful as an acutely sensitive model for a number of pathogens, including many enteroviruses, we tested the ability of EV- D68 to replicate in suckling mice. Two- to three-day-old BALB/c mice were inoculated intracranially with either PBS or EV-D68 strain 14-18949, a major strain isolated during the 2014 respiratory outbreak. An additional group was inoculated intracranially with filtered cerebrospinal fluid (CSF) collected from AFM patients during the 2014 outbreak. Mice were monitored daily for signs of disease, and tissues were collected at two times post- inoculation. While no signs of illness were observed in CSF-inoculated animals, paralysis was observed in mice inoculated with EV-D68 strain 14-18949. Furthermore, viral RNA was detected in brain tissue resected from the paralyzed mice. Though further work is needed, our results suggest that suckling mice may be a useful model to study EV-D68 pathogenesis. A comparative study using several EV-D68 strains may identify key differences in tropism, pathology, and disease progression. 128 Europic 2016

129 WEDNESDAY 07/09/2016 E13 FURTHER STUDIES ON THE EVOLUTION OF SENECA VALLEY VIRUS Nick Knowles, Jemma Wadsworth, Katarzyna Bachanek-Bankowska The Pirbright Institute Seneca Valley virus (SVV) is a picornavirus belonging to the species Senecavirus A (formerly named Seneca Valley virus) in the genus Senecavirus. SVV was first isolated from pigs in North Carolina and Minnesota in March and July 1988, respectively. From about 1997, SVV was occasionally associated with idiopathic vesicular disease in pigs. However, early experimental infection of pigs failed to reproduce any disease (John Landgraf and James House, personal communication, 2003). Recently, there has been an increased incidence of the isolation of SVV from cases of porcine IVD and an apparent spread of both the virus and the disease to Canada, Brazil and China. Experimental infection of pigs with SVV has now been shown to result in vesicular lesions (Montiel et al. 2016; Emerg. Infect. Dis. 22: 1246-1248). We have previously determined the genome sequences, using Illumina MiSeq technology, of seven porcine SVVs isolated between 1988 and 2001. Recently the complete (or nearly complete) genome sequences of over 20 SVVs (isolated from pigs in the United States, Brazil and China) have been determined by other researchers. We have performed phylogenetic analyses (Maximum Likelihood and Bayesian reconstruction) showing that all currently identified SVVs belong to a single lineage which has a common ancestor dating to around 1985. These results suggest that SVV probably entered the US pig population in a single event around that date from an unknown source and has spread throughout the country and then later to other countries (Canada, Brazil and China). The source could have been from pigs from another, as yet unidentified, country or from another host species, e.g. rodents. Europic 2016 129

130 WEDNESDAY 07/09/2016 E14 SAFFOLD VIRUS IN VIVO PASSAGES DRIVE THE STRUCTURAL EVOLUTION OF THE CAPSID PROTEIN FOR ENHANCING REPLICATION FITNESS IN NEURAL CELLS. Osamu Kotani1, Masaru Yokoyama2, Naoko Iwata-Yoshikawa2, Tadaki Suzuki2, Hideki Hasegawa2, Hiroyuki Shimizu2, Hironori Sato2, Noriyo Nagata2 1 National Institute of Infectious Diseases,Pathogen Genomics Center, 2National Institute of Infectious Diseases Saffold virus (SAFV), a human cardiovirus, is occasionally detected in infants with neurological diseases, including meningitis, acute flaccid paralysis and cerebellitis. We previously reported that SAFV type 3 (SAFV-3) isolates shared potential to infect glial cells and neural progenitor cells, but not large neurons in the brain of mice (Kotani et al., 2016). More recently, we obtained an in vivo passaged SAFV-3 strain using cerebella of neonatal mice. The passaged strain gained higher levels of replication capability in the brain with increased neurovirulence than the original isolate, especially in the cerebellum. In this study, we biologically, genetically, and structurally characterized the passaged strain to gain molecular insights into neurotropism of SAFV-3. The in vivo passaged SAFV-3 gained enhanced replication capability not only in mouse brain but also human neural cell lines, such as U-251 MG and ONS-76 cells. Genome-wide tracing of mutations revealed that the passaged strain acquired three amino acid substitutions only in the capsid protein as compared with the original isolate: Q239R and H160R in the VP2 and K62M in the VP3. Although these substitutions were located distantly in the primary sequences, they were closely positioned after protein folding; they were arranged on the exposed loop regions near the putative receptor-binding surface reported for the Theilers murine encephalomyelitis virus. Molecular modeling of the capsid proteins predicted that the VP2-H160R and VP3-K62M mutations altered the structural dynamics of the viral receptor binding surface via formation of a novel hydrophobic interaction between the VP2 puff B and VP3 knob regions. These results suggest that the changes in the structural flexibility in the receptor-binding surface is a critical mechanism in the regulation of neurotropism of SAFV-3. Reference: Kotani O, Naeem A, Suzuki T, Iwata-Yoshikawa N, Sato Y, Nakajima N, Hosomi T, Tsukagoshi H, Kozawa K, Hasegawa H, Taguchi F, Shimizu H, Nagata N. 2016. Neuropathogenicity of Two Saffold Virus Type 3 isolates in Mouse Models. PLoS One. 11: e0148184 130 Europic 2016

131 WEDNESDAY 07/09/2016 E15 EMERGING RECOMBINANT NEW GENOGROUP OF COXSACKIEVIRUS A16 HAS RAPIDLY DISPERSED IN FRANCE, 20112014 Jean-Luc Bailly1, Chervin Hassel2, Audrey Mirand2, Elena Terletskaia-Ladwig3, Agnes Farkas4, Sabine Diedrich5, Hartwig Huemer6, Hlne Peigue-Lafeuille2, Christine Archimbaud2, Ccile Henquell2 1 Universit dAuvergne, 2Laboratoire Epidmiologie, Pathogense des Infections Entrovirus, Universit dAuvergne, CHU Clermont- Ferrand, France, 3Prof. Gisela Enders & Kollegen MVZ GbR and Institute of Virology, Stuttgart, Germany, 4Division of Virology, National Center for Epidemiology, Budapest, Hungary, 5National Reference Center for Poliomyelitis and Enterovirus, Robert Koch Institute, Berlin, Germany, 6Austrian Agency for Health and Food Safety, Vienna, Austria Coxsackievirus A16 (CV-A16) is one of the most predominant enterovirus (EV) types reported in children with hand-foot-and-mouth disease (HFMD) and mainly associated with mild forms of this muco-cutaneous illness. Co- circulation and sequential circulation with other EV types were described during large HFMD outbreaks in countries of South East Asia. CV-A16 and EV-A71 co-infections were reported within individuals and recombination was described between these two EV types. CV-A16 strains were grouped into three genogroups by comparative analysis of VP4 nucleotide sequences, but the evolutionary and epidemiological patterns need further investigation on the basis of comprehensive sequence samples. Our recent study of the phylogeography of EV-A71 (Hassel et al, 2015) highlighted the importance of spatial migration of viral strains in the epidemiology of EV-A71 infections in Europe. OBJECTIVES: This collaborative study provides an evolutionary analysis of CV-A16 to investigate the circulation pattern of this virus over time and across countries. METHODS: A virus sample was obtained during sentinel surveillance of HFMD in France (20102014, n=129) and clinical surveillance of EV infections in Austria (20002004, n=3), France (20022007, n=23), Germany (20032010, n=8), and Hungary (20042010, n=8). The VP1 sequences were determined for the overall sample and analysed with 416 CV-A16 publicly available sequences of various geographic origins. A sample of complete genomes was obtained (CV-A16, n=25; CV-A10, n=3) to analyse the recombination origin of CV-A16 lineages. RESULTS: On the basis of the VP1 sequence diversity, the CV-A16 strains can be assigned to 3 major clades: genogroups A and B, and a previously unreported clade (genogroup C). The shape of the CV-A16 phylogenetic tree showed co-circulation of genetically distinct virus populations over time and across geographic regions worldwide. The time origin of contemporaneous CV-A16 strains was estimated in 1866 (1784-1928) and that of genogroup C in 2004 (20012007). The precise geographic origin of this genogroup was not determined although a VP1 sequence assigned to this clade was first reported in Peru (2009). The virus was then reported in France from 2011 to 2014. The global CV-A16 phylogeny is consistent with frequent virus migration over long distance (Europe/China) and displayed a balanced shape (as opposed to ladder-like shape). Although two amino acid changes were determined within the VP3 capsid protein (-sheet G, GH loop) between genogroups B and C, the overall phylogenetic data suggested that the virus is not subject to strong immune selection or other directional selection. Most of the other amino acid changes clustered within non-structural proteins 2A and 3D. Comparisons of complete genomes showed evidence of intratypic and intertypic recombination (between genogroups B and C, and CV-A16 and CV-A10 respectively). CONCLUSIONS: The global phylogeny of CV-A16 provides evidence for recent emergence of a previously unreported recombinant genogroup. Phylogenetic data combined with epidemiological surveillance in France showed sustained dissemination of this genetic variant but the phylogeny is consistent with multiple virus introductions. The combination of sentinel surveillance and comprehensive viral phylogenies provide important epidemiological and evolutionary information on EV infections, such as epidemic spread and spatio-temporal dynamics. Europic 2016 131

132 WEDNESDAY 07/09/2016 E16 SCREENING AND ANALYSIS OF SAFFOLD VIRUS IN SWEDEN Helena Vandesande1, Kjell Edman1, Anna Svneby2, Elin Rondahl3, Lena Serrander3, A. Michael Lindberg1 1 Linnaeus University, 2Dynamic Code, 3Linkping University Virus-associated diarrhoea is a major source of morbidity and mortality, predominantly caused by so-called NOROAD viruses (Norovirus, Rotavirus, and Adenovirus). Approximately a third of these cases, however, are, as of yet, unexplained in their aetiology. The in 2007 discovered human Cardiovirus Saffold virus (SAFV) may prove to be a plausible candidate to elucidate these unknown cases of viral gastroenteritis. This virus, a member of the Picornaviridae family and closely related to the murine viruses Encephalomyelitis virus and Theilers Murine Encephalomyelitis virus, is a widespread pathogen and causes infection early in life. In this study, we managed to fully adapt an infectious SAFV 3 type strain to a HeLa-SOH cell line, thus supporting high-titre growth of the virus and gaining more insight in its replicative properties. Screening of 238 faecal or vomitus samples obtained from NOROAD-negative, elderly patients with acute gastroenteritis at the University Hospital of Linkping showed that SAFV appears to be present in low abundance in at least some patient samples; however, these data do not provide indisputable proof, and require further confirmation by, for example, semi-nested PCR. The apparent existence of SAFV in the patient samples raises the hope of isolating a Swedish SAFV strain and, in a later stadium, elucidating its relation to gastroenteritis. Keywords: Picornaviridae, Cardiovirdae, Saffold virus. 132 Europic 2016

133 WEDNESDAY 07/09/2016 E17 CHRONIC ENTEROVIRUS INFECTION IN A PATIENT WITH MYALGIC ENCEPHALOMYELITIS/ CHRONIC FATIGUE SYNDROME (ME/CFS) CLINICAL, VIROLOGIC AND PATHOLOGICAL ANALYSIS John Chia1, David Wang2, Andrew Chia2, Rabiha El-Habbal2 , EV Med Research 1 2 OBJECTIVES: A 23 y/o Caucasian male developed prolonged, recurrent gastrointestinal symptoms, followed by onset of severe ME/CFS (CDC criteria, ICC). At initial evaluation, Echovirus 11 antibody titer was 1:640 (normal

134 WEDNESDAY 07/09/2016 E18 PHENOTYPIC ANALYSIS OF THE 2014 EPIDEMIC ENTEROVIRUS D68 STRAIN FROM NORTH AMERICA IN RESPIRATORY EPITHELIAL CELLS Debra Lugo1, Paul Krogstad2 1 UCLA Department of Pediatric, Infectious Diseases, 2Department of Pediatrics, David Geffen School of Medicine at UCLA Enteroviruses exhibit rapid genetic mutability due to nucleotide substitution and recombination, sometimes resulting in dramatic shifts in phenotypic presentation. Enterovirus D68 (EV-D68) was initially identified in 1962 with the Fermon strain causing mild respiratory illness. In 2014, a widespread outbreak of EV-D68 infections occurred, characterized by intractable bronchospasm and severe respiratory tract manifestations; a temporal association with acute flaccid myelitis was also noted in some cases. Although recombination events and specific nucleotide changes have been identified in the emergent EV-D68 strains, the phenotypic properties of these new isolates have not been described. We examined the replication of the prototype Fermon strain and 2014 U.S. isolates of EV-D68 in BEAS-2B cells, a bronchial epithelial cell line. Infection was confirmed by immunofluorescence, and quantified using limiting dilution infectivity assays. BEAS-2B cells showed susceptibility to EV-D68 infection, but with a slower progression of infection and lower titer than in A549 and RD cells. Cytopathic effect during infection was demonstrated with 2014 strain showing increased destruction of cells. Faster progression of infection and higher titer occurred at 34C for all EV-D68 strains, compared to 37C. We performed acid neutralization experiments, and both strains of EV-D68 showed a significant decrease in titer (~100 fold) after 1 hr incubation with low pH. Thus we have confirmed that the recent epidemic strain demonstrates phenotypic properties common to rhinoviruses, which has previously been demonstrated with the prototype Fermon strain. We are currently studying the activation of type I interferon responses during enterovirus infection as a possible explanation for the recent increase in pathogenicity of EV strains. Significant changes in the gene activation and chemokine response when comparing older strains with currently circulating strains would suggest this may be the underlying mechanism of increased pathogenicity. Using microarray analysis, variability in gene expression will be analyzed to identify target pathways activated which correlate with innate immunity. We will also interrupt the infection at different time points and analyze with cytokine multiplex assays to map the timeline of expression of proinflammatory cytokines known to be involved in bronchospasm. 134 Europic 2016

135 WEDNESDAY 07/09/2016 E19 VIRULENCE OF RECENT COXSACKIEVIRUS B2 ISOLATES IN A NEONATAL MOUSE MODEL Noriyo Nagata1, Waka Ushioda1, Tomofumi Nakamura1, Masanobu Agoh2, Setsuko Iizuka3, Osamu Kotani1, Naoko Iwata-Yoshikawa1, Hiroyuki Shimizu1, Hideki Hasegawa1 1 National Institute of Infectious Diseases, 2Nagasaki Prefectural Institute of Environmental Science and Public Health, 3Shimane Prefectural Institute of Public Health and Environmental Science Coxsackievirus B (CVB) infection causes aseptic meningitis, encephalitis, acute diabetes, and myocarditis in humans. Neonatal mice show similar susceptibility to CVB, and experimental infection of these mice causes damage to brain, muscles, and pancreas. Thus, this animal model is both convenient and useful for the study of CVB pathogenesis. In 2013, some cases of maternal-fetal transmission of CVB2 were reported in Japan. Here, we evaluate the virulence of recent CVB2 isolates in a neonatal mouse model. ddY neonatal mice were experimentally infected with Ohio-1, a prototype strain, or with 17 CVB2 isolates obtained in Japan from 2008 to 2013. Mice received 103 or 106 CCID50 doses of CVB2 strains via intracerebral or intraperitoneal inoculation, and were placed under observation for clinical signs for 3 weeks. Animals were then sacrificed and subjected to histopathologic examination. An in-house polyclonal antibody against CVB3 was used to detect virus antigens on formalin-fixed paraffin-embedded tissues by immunohistochemistry (1). The nucleotide sequence of the partial VP1 region of CVB2 isolates was determined, based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). Intracerebral inoculation with CVB2 isolates, but not with Ohio-1, resulted in spastic paralysis and death. Two isolates caused paresis, and one isolate from a gastroenteritis case induced flaccid paralysis. After intraperitoneal inoculation with the isolates, but not with Ohio-1, most neonatal mice became moribund as a result of peritonitis with acute pancreatic necrosis. These isolates infected neurons, skeletal muscle, and/or the pancreas. Comparative sequence analysis of the partial VP1 region revealed a low degree of genetic identity between the isolates and Ohio-1. Phylogenetic analysis indicated that the isolate that induced flaccid paralysis in neonatal mice clustered with Philippine isolates from 2009, whereas the others clustered with Japanese isolates from 2010. The virulence (early death, high mortality, and high infectivity) of isolates in neonatal mice seems to correlate with the severity of symptoms observed in humans. In addition, recent CVB2 isolates may be highly neurovirulent and more infectious than Ohio-1 in neonatal mice. A study combining animal studies and genetic analyses could provide new insights into coxsackievirus infections. 1. Kotani O, Iwata-Yoshikawa N, Suzuki T, Sato Y, Nakajima N, Koike S, Iwasaki T, Sata T, Yamashita T, Minagawa H, Taguchi F, Hasegawa H, Shimizu H, Nagata N. 2015. Establishment of a panel of in-house polyclonal antibodies for the diagnosis of enterovirus infections. Neuropathology. 35:107-121. Europic 2016 135

136 WEDNESDAY 07/09/2016 13:30 Session 6 ANTIVIRAL STRATEGIES Chairs: Kimberley Benschop & Katja Wolthers 15:45 K08: TOWARDS THE DEVELOPMENT OF HIGHLY POTENT RHINO- AND Johan Neyts (University of 13:30 ENTEROVIRUS INHIBITORS Leuven, Belgium) F01: AN ENTEROVIRUS INFECTION MOUSE MODELS TO STUDY ANTIVIRAL Els Scheers (KU Leuven 14:05 THERAPY AND THE POTENTIAL DEVELOPMENT OF RESISTANCE University of Leuven, Belgium) F02: THE TALE OF HOW AN IN SILICO DESIGNED COXSACKIEVIRUS B3 PROTEASE Rana Abdelnabi (KU Leuven 14:20 INHIBITOR TURNED OUT TO BE A CAPSID BINDER WITH A NOVEL MECHANISM University of Leuven, Belgium) INSTEAD Anabel Guedan (Imperial College 14:35 F03: PROTEIN KINASE D INHIBITORS BLOCK PICORNAVIRUS REPLICATION London, United Kingdom) Scott Dessain (Lankenau F04: CROSS-NEUTRALIZATION OF MULTIPLE POLIOVIRUS TYPES BY HUMAN 14:50 MONOCLONAL ANTIBODIES Institute for Medical Research, United States of America) F05: EXPLORING THE PERMISSIVITY OF GENETIC EXCHANGES IN THE 2C REGION Carmen Mirabelli (KU Leuven 15:05 OF RHINOVIRUS TO ESTABLISH A MODEL OF INFECTION FOR RV-C University of Leuven, Belgium) F06: TARGETING THE HOST: ROLE OF N-MYRISTOYLTRANSFERASES IN THE Irena Corbic Ramljak (Medical 15:15 INFECTIVITY OF COXSACKIEVIRUS B3 (CVB3) University of Vienna, Austria) F07: THERMOSTABLE, IMMUNOGENIC POLIOVIRUS VLPS OF ALL THREE Helen Fox (NIBSC, Unite 15:25 SEROTYPES Kingdom) Konstantin Chumakov (US Food F08: HUMAN MONOCLONAL ANTBODIES AS POTENTIAL THERAPEUTIC AGENTS 15:35 AGAINST POLIOMYELITIS and Drug Administration, United States of America) COFFEE BREAK 136 Keynote Lecture Communication (12 + 3 min) Short Communication (8 + 2 min) Europic 2016

137 WEDNESDAY 07/09/2016 POSTER SESSION 6 - ANTIVIRAL STRATEGIES F09: EFFICACY OF ANTIVIRAL DRUGS IN A HUMAN IN VITRO NASAL EPITHELIUM MODEL (MUCILAIRTM) Bernadett Boda (Epithelix, Switzerland) F10: COMPARATIVE ANALYSIS OF THE MOLECULAR MECHANISM OF RESISTANCE TO VAPENDAVIR ACROSS A PANEL OF PICORNAVIRUS SPECIES Kristina Lanko (KU Leuven University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium) F11: SELECTIVE INHIBITION OF ENTEROVIRUS 71 BY A NOVEL FAMILY OF TRYPTOPHAN DENDRIMERS Liang Sun (Rega institute, KU Leuven, Belgium) F12: A STRUCTURE-ACTIVITY RELATIONSHIP STUDY OF ITRACONAZOLE, A NOVEL ANTIVIRAL COMPOUND THAT TARGETS THE OXYSTEROL BINDING PROTEIN Lisa Bauer (University Utrecht, Netherlands) F14: STRUCTURAL DATA OF CVB3-DRUG COMPLEX James Geraets (University of Helsinki, Finland) F15: ANTIVIR - AUTOMATED PLAQUE-BASED SCREENING FOR HOST DIRECTED INHIBITORS Luca Murer F16: TARGETING THE HOST: ROLE OF N-MYRISTOYLTRANSFERASES IN THE INFECTIVITY OF COXSACKIEVIRUS B3 (CVB3) Irena Corbic Ramljak F17: THERMOSTABLE, IMMUNOGENIC POLIOVIRUS VLPS OF ALL THREE SEROTYPES Helen Fox F18: RESULTS FROM AIROPICO, THE EU NETWORK DEDICATED TO HUMAN PICORNAVIRUSES Katja Wolthers Europic 2016 137

138 WEDNESDAY 07/09/2016 K08 TOWARDS THE DEVELOPMENT OF POTENT RHINO- AND ENTEROVIRUS INHIBITORS Johan Neyts, Laboratory of Virology (www.antivirals.be) Rega Institute, University of Leuven, Belgium Antiviral drugs are available for the treatment of infections caused by herpesviruses, the influenza virus, HIV, HBV and HCV. However, despite their enormous medical and socio-economical impact, there is still no approved antiviral therapy for the treatment of infections caused by entero-/rhinoviruses. Such drugs are needed for the treatment and prophylaxis of rhinovirus induced exacerbations of asthma and COPD, for the treatment of infections such as those caused by EV71 and EV68 and in the polio endgame. Typical capsid binding compounds such as pleconaril and vapendavir (that bind into a hydrophobic pocket within VP1) as well as 3C protease inhibitors have been well studied but other targets remain largely underexplored. We identified recently a class of molecules that target enterovirus entry by a mechanism that is different from that of the classical capsid binding compounds and that are not cross-resistant with the latter inhibitors. We also demonstrate that viral proteins such as the 2C helicase and the 3D RNA-dependent RNA polymerase are excellent targets for inhibition of viral replication. Interestingly the anti-influenza compound Favipiravir (T-705), a compound endowed with activity against multiple other viruses, also inhibits in vitro enterovirus replication. We demonstrate that K159 in the F1 motif of the viral RdRp, an amino acid highly conserved in +ssRNA viruses, is a key residue in the mechanism of action of T-705. Precise understanding of the interaction of the compound with the enzyme may possibly allow to design broad- spectrum +ssRNA virus inhibitors. An important factor that should be considered during drug development is the barrier to resistance; indeed compounds to be developed for clinical use should preferably have a high barrier to resistance to reduce the possibility that drug-resistant variants may easily develop. Also drug-resistant variants should ideally have a markedly reduced fitness. When developing drugs, sufficient attention should as well be given to test their efficacy against panels of representative clinical isolates (for example against EV71 and EV68). Finally relevant (small) animal models are needed to assess efficacy of novel antiviral compounds for enterovirus infections of for example the respiratory tract and the (central) nervous system. Animal models should also allow to easily assess the potential of compounds to select from drug-resistant variants in the in vivo situation. 138 Europic 2016

139 WEDNESDAY 07/09/2016 F01 AN ENTEROVIRUS INFECTION MOUSE MODELS TO STUDY ANTIVIRAL THERAPY AND THE POTENTIAL DEVELOPMENT OF RESISTANCE Els Scheers, Leen Delang, Johan Neyts KU Leuven University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy OBJECTIVES: We report the development of a relevant enterovirus mouse infection model that is ideally suited to (i) assess the antiviral efficacy of enterovirus inhibitors and (ii) monitor the possible in vivo development of antiviral drug-resistance. METHODS: SCID mice were infected intraperitoneally (ip) with 105 TCID50 Coxsackievirus B4 (CVB4) and weight loss was monitored as a symptom of disease. CVB4 RNA and infectious particles in serum and organs were quantified by means of qRT-PCR and end-point titration respectively. Viral RNA was genotyped by Sanger sequencing. RESULTS: As of day 3 post infection (dpi), weight loss was observed in infected mice which all had to be euthanized at 5 dpi. High viral RNA titers (~109 genome equivalents (GE)/100mg tissue) were detected in the pancreas. Titers in heart, lung, liver, spleen and brain were ~106-107 GE/100mg tissue. High titers of both viral RNA (109 GE/mL) and infectious particles (106 TCID50/ml) were detected in serum. To explore whether this model can be used to assess the efficacy of antiviral compounds, the effect of compound A (a proprietary 2C-targeting enterovirus inhibitor) was studied. CVB4-infected mice were treated for either 5 or 12 consecutive days with twice daily (BID) dosing of 20 mg/kg. After stop of treatment, the mice remained healthy until the end of the experiment (60 dpi) and no virus was detectable in serum or pancreas. Next, infected mice were treated with suboptimal regimens: either 20 mg/kg once daily during 15 consecutive days or 20 mg/kg once daily during 20 consecutive days (treatment was initiated at day of infection). The mortality rate of both groups was 40% and the mean day of death 45 12 dpi and 48 10 dpi respectively. The 60% of mice that survived were euthanized at 77 dpi; no virus was detectable in serum or organs indicating that they were cured from infection. To assess whether the (delayed) mortality in the 40% of mice was due to the emergence of drug-resistant variants, viral RNA was isolated from serum, heart and pancreas and the 2C gene was sequenced. Interestingly, in nearly all samples one substitution i.e. 2C_A239T/V was identified. This mutation did not result in a reduced sensitivity to the inhibitor as assessed in vitro and in mice that had been inoculated with these variants. Full genome sequencing revealed the presence of additional mutations in the viral genome for which it is being explored whether these, and the 2C_A239T/V, represent mouse adaptive mutations. CONCLUSIONS: We developed a robust CVB4-infection mouse model to assess the in vivo efficacy of novel antiviral agents. We demonstrate that a potent antiviral agent is able to cure immunodeficient mice from this aggressive enterovirus infection. It was next explored whether suboptimal dosing (that delays but does not prevent mortality of all infected mice) may lead to the selection of drug-resistant variants. For this particular inhibitor, no drug-resistant variants developed. This CVB4/SCID model provides a powerful tool to study population dynamics over time in the presence or absence of antiviral pressure in the infected host. Europic 2016 139

140 WEDNESDAY 07/09/2016 F02 THE TALE OF HOW AN IN SILICO DESIGNED COXSACKIEVIRUS B3 PROTEASE INHIBITOR TURNED OUT TO BE A CAPSID BINDER WITH A NOVEL MECHANISM INSTEAD Rana Abdelnabi1, Ajay Kumar Timiri2, Venkatesan Jayaprakash2, Leen Delang3, Johan Neyts3, Pieter Leyssen3 1 KU Leuven University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, 2Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi-835215, Jharkhand, India., 3KU Leuven University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium OBJECTIVES: An in silico molecular docking study on the Coxsackievirus B3 (CVB3) 3C-protease guided the synthesis of a novel benzene sulfonamide derivative (i.e. compound 17) that was shown to inhibit the in vitro replication of CVB3. Our aim was to use virus-cell-based assays to confirm the 3C-protease as a target for the compound and to study the particular characteristics of its antiviral activity. METHODS: Cell-based antiviral assays (CPE-reduction, virus yield and plaque assays) and molecular biology tools (reverse-engineering, RT-PCR and sequencing). RESULTS: Compound 17 proved to inhibit the in vitro replication of CVB3 (strain Nancy) as well as of CVB1, CVB4, CVB5 and CVB6 with EC50 values ranging between (0.7-37) M. Surprisingly, the compound did not show any antiviral activity against CVB2 and other viruses from different enteroviruses groups. In contrast to what is expected for a protease inhibitor, a time-of-drug-addition study pointed out that compound 17 interfered with an early step in the CVB3 replication cycle. A thermo-stability assay provided an additional indication of an interaction between compound 17 and the CVB3 virus particle. This latter mechanism of action was confirmed by the genotyping of independently selected compound 17-resistant CVB3 variants, which all carried mutations in the VP1 gene (F76C, E78G, A98V and D133G). Compared to the wild-type (WT), the reverse-engineered VP1 F76C, E78G, A98V and D133G mutants proved to be 18, 21, 3 and 38-fold less sensitive to the antiviral effect of compound 17, respectively. Interestingly, the mutated VP1 residues are located outside the common drug-binding pocket for capsid binders such as pleconaril. Moreover, the D133 residues of all five VP1 units are arranged in the form of ion channel at the 5-fold axis. Plaque phenotyping revealed that the VP1 F76C, E78G and D133G mutations resulted in a smaller plaque size than WT. CONCLUSION: Compound 17, originally designed in silico to inhibit the viral 3C-protease, is a potent inhibitor of CVB3 replication. Surprisingly, study of the mechanism of action revealed that the compound is a capsid binder with an entirely new target on the virus particle. Further experiments are ongoing to explore the precise mechanism by which compound 17 interacts with CVB3 capsid and to develop more potent and broader-spectrum analogs. 140 Europic 2016

141 WEDNESDAY 07/09/2016 F03 PROTEIN KINASE D INHIBITORS BLOCK PICORNAVIRUS REPLICATION Anabel Guedan1, David Swieboda1, Aurelie Mousnier1, Marie Toussaint1, Sebastian Johnston1, Mark Charles2, Tony Raynham3, Amin Asfor4, Anusha Panjwani4, Toby Tuthill4, Roberto Solari1 1 Imperial College London, 2Cancer Research Technology, 3University of East London, 4The Pirbright Institute Human rhinovirus (HRV), the virus responsible for the common cold, is the most frequent cause of asthma and chronic obstructive pulmonary disease (COPD) exacerbations. Respiratory infections are a major healthcare problem and the lack of vaccines and antiviral therapies, mainly due to the high ability of mutation of the virus and the large range of serotypes, points out a real unmet medical need. HRV belongs to the family Picornaviridae which includes important human and animal pathogens such as poliovirus (PV), enterovirus and foot and mouth disease virus (FMDV). These single-stranded positive-sense RNA viruses exploit host intracellular membranes such as the endoplasmic reticulum and the Golgi apparatus for their replication. Several interactions between viral non-structural proteins and cellular proteins involved in the host secretory pathway have been described. Phosphatidylinositol 4-kinase III (PI4KIII) and oxysterol-binding protein (OSBP) have been published as potential targets for anti-picornaviral drugs as their knock-down or inhibition by small molecules block viral replication. Protein kinase D (PKD), a serine-threonine kinase involved in the transport and fission of vesicles from the trans-Golgi network to the plasma membrane, is an upstream regulator of both PI4KIII and OSBP. Moreover, PKD is recruited to the Golgi by ADP-ribosylation factor 1 (Arf1). Brefeldin A, a fungal metabolite that inhibits Arfs GEF, has also been described as anti-viral, thus we hypothesised that PKD may be involved in the viral replication complex. A link between PKD and picornaviral replication has not been previously explored although PKD has been implicated in respiratory syncytial virus and hepatitis C virus infection. Using HeLa cells as an in vitro model, we showed that PKD is phosphorylated both in its activation loop and its autocatalytic site during HRV infection. We tested four structurally different PKD inhibitors and all were able to inhibit viral replication. We also tested one of these inhibitors against different picornaviruses and showed that it blocks PV and FMDV replication. In order to explore the mechanism of action of PKD inhibitors we revealed that this compound is acting in an early stage of the viral replication cycle by showing it is affecting viral mRNA transcription and viral protein translation. The PKD inhibitor induces changes to the architecture of the Golgi but does not appear to inhibit endocytosis and time-of-addition studies suggest it is not affecting viral entry. Previous studies have proposed a role for PKD in down-regulating the type I interferon (IFN) signalling pathway thus suggesting PKD inhibitors might be antiviral by enhancing the IFN response. However, our studies are not consistent with the antiviral properties of PKD inhibitors acting through enhancing IFN signalling. These observations therefore reveal a new potential anti-picornaviral host target, although the mechanism in which PKD is involved during viral infection still needs further elucidation. Europic 2016 141

142 WEDNESDAY 07/09/2016 F04 CROSS-NEUTRALIZATION OF MULTIPLE POLIOVIRUS TYPES BY HUMAN MONOCLONAL ANTIBODIES Scott Dessain1, Rama Devudu Puligedda1, Diana Kouiavskaia2, Chandana Devi1, Konstantin Chumakov2 1 Lankenau Institute for Medical Research, 2FDA/CBER BACKGROUND: An essential requirement for eradication of poliomyelitis is the elimination of reservoirs of immunodeficiency-related vaccine-derived poliovirus (iVDPV), which are chronically excreted by individuals with immune dysfunction and may be reintroduced into the general population. In addition, circulating VDPV (cVDPV) strains may cause outbreaks of flaccid paralysis following the transition to universal IPV vaccination. Monoclonal antibodies (mAbs) specific for poliovirus (PV) may contribute to clearance of iVDPV and control of cVDPV outbreaks. Here, we describe a panel of human mAbs isolated from PV-immune volunteers, that have potent neutralizing activity against Sabin and wild type PV. Some of mAbs neutralize more than one PV type. OBJECTIVES: Characterize the human neutralizing antibody response to PV in PV immune subjects by cloning mono-specific and cross-specific PV mAbs Select candidate therapeutic mAbs on the basis of neutralizing activity against a series of wild type and vaccine-derived PV strains Begin to assess the binding and structural correlates of cross-specific neutralization METHODS: We isolated mAbs from OPV-vaccinated subjects using a novel cell fusion method. We screened the mAbs by ELISA using whole Sabin PV virions, and assessed neutralization against Sabin and wild type PV strains. We assessed PV type-specific binding by competitive binding studies and through the study of escape mutant PV strains, using as a basis for comparision a previously described chimpanzee mAb, A12, which neutralizes PV types 1 and 2 and reduces viral excretion in animal studies. We assessed the ability of the cross-neutralizing mAbs to neutralize a panel of patient-derived, type 1 PVs that included WT strains, iVDPVs and an ambiguous VDPV strains (aVDPV). RESULTS: We isolated a panel of mAbs with potent neutralization activities against PV wild-type (WT) and Sabin strains, including some with activity against two or three PV types. Escape mutant studies demonstrated that single-serotype specific 7E2 and 8F9 mAbs interact with residues in antigenic site 1, whereas the type 1/type 2 cross-neutralizing 1E4 and 12F8 mAbs bind to residues in the canyon domain, the site of interaction with the PV cellular receptor, CD155. We further assessed the therapeutic potential of the cross specific mAbs, 12F8 and 1E4. Both mAbs neutralized A12 escape mutant PV strains, identifying at least 2 distinct cross-neutralizing epitopes between type 1 and 2 PV. 12F8 and 1E4 also neutralized a panel of patient-derived, type 1 PVs that included WT strains, iVDPVs and an aVDPV. Ongoing studies of a triple-specific mAb (types 1, 2, and 3), additional escape mutant data, and epitope binning studies will be presented. CONCLUSIONS: We isolated a panel of potent cross-neutralizing human mAbs that may be candidates for a PV therapeutic to clear PV from immunodeficient subjects. Our in vitro data support animal testing of human, cross- neutralizing mAb combinations to reduce PV secretion. Study of cross-neutralizing mAbs may elucidate principles of cross-neutralization that are generalizable among PV and other picornaviruses. 142 Europic 2016

143 WEDNESDAY 07/09/2016 F05 EXPLORING THE PERMISSIVITY OF GENETIC EXCHANGES IN THE 2C REGION OF RHINOVIRUS TO ESTABLISH A MODEL OF INFECTION FOR RV-C Carmen Mirabelli1, Leen Delang2, Dirk Jochmans2, Johan Neyts2 1 KU Leuven university. Department of Microbiology of Immunology, Rega Institute for Medical Research, 2KU Leuven University of Leuven, Department of Microbiology of Immunology, Rega Institute for Medical Research OBJECTIVE: Rhinovirus C (RV-C) is one of the leading causes of upper and lower respiratory tract infections and it has been correlated by genetic analysis with asthma exacerbation in children. No antiviral or vaccine is available for the treatment or prevention of RV-C infection and the lack of a simple method to culture the virus hinders the development of antiviral strategies. Here, we report attempts to develop a model of RV-C infection in cell culture that should allow assessing the efficacy of small molecules of RV replication which target the 2C protein. METHODS: Chimeric RVs in the region encompassing the viral protein 2C were constructed by fusion PCR. In particular, the 2C region derived from RV-C15 was inserted in a RV-B14 or RV-A16 background. Replication kinetics of the chimeric viruses were analyzed by specific qRT-PCR. RV-B14, RV-A16 nano-luc-replicons and their chimeric counterpart carrying the 2C from RV-C15 were engineered and the replication capacity of such chimeric constructs was assessed. RESULTS: RV-B14_2C(RV-C15) and RV-A16_2C(RV-C15) viruses were obtained by transfection of PCR fusion product in MRC-5 or Hela cells. The in vitro fitness of these chimeric viruses appeared sub-optimal. Both constructs resulted in a slight CPE in MRC-5 at 5 days p.i. or at latter times p.i. Also growth curves revealed a sub-optimal virus production. A temperature shift binding assay showed that the entry of these chimeric viruses was not impaired. Indeed the proportion intracellular/bound viral particle was comparable to that of the parental viruses. Interestingly, nano-Luc chimeric sub-genomic replicons replicated 100-fold less efficiently than the parental replicons. In the presence of a RV 3C protease targeting compound (Rupintivir analogue, compound I) and a 2C targeting compound, complete inhibition of RV-B14 and RV-A16 replicons was observed. The sensitivity of the chimeric replicons is currently under investigation together with canonical antiviral assays. CONCLUSION: The permissivity of intertypic exchanges in the 2C region among RVs has been evaluated by engineering the chimeric viruses RV-B14_2C(RV-C15) and RV-A16_2C(RV-C15). The replication of these viruses was impaired in vitro. For this reasons, such constructs appear not suitable as surrogates to study the potential effect of 2C-targeting antivirals. Europic 2016 143

144 WEDNESDAY 07/09/2016 F06 & POSTER F16 TARGETING THE HOST: ROLE OF N-MYRISTOYLTRANSFERASES IN THE INFECTIVITY OF COXSACKIEVIRUS B3 (CVB3) Irena Corbic Ramljak1, Julia Stanger2, Dominik Dreier3, Marko D. Mihovilovic4, Wolfgang Fischl5, Dieter Blaas2, Karin Klingel6, Heinrich Kowalski2 1 Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria, 2Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Vienna, Austria, 3Institute of Applied Synthetic Chemistry, Technical University of Vienna, Vienna, Austria, 4Institute of Applied Synthetic Chemistry, Technical University of Vienna, VIenna, Austria, 5The Haploid Genetics Company, Campus Vienna Biocenter 3, Vienna, Austria, 6Department for Molecular Pathology, University Hospital Tbingen, Tbingen, Germany N-myristoylation is an important mostly co-translational modification of many cellular and an appreciable number of viral proteins. The 14-carbon saturated fatty acid myristate (C14:0) is covalently attached to an absolutely conserved N-terminal glycine in the context of a GlyXXXSer/Thr myristoylation signal. Transfer of the acyl group is catalyzed by the myristoyl-CoA:protein N-myristoyltransferase (NMT), which in higher eukaryotes is achieved by two isozymes (NMT1 and NMT2). Notably, most picornaviruses carry a myristate moiety within their capsid attached to each of the 60 copies of the small protein VP4. It is acquired during translation of the polyprotein, a precursor of all structural and non-structural viral proteins. Based on high-resolution X-ray structures of several enteroviruses paired with evidence mostly from poliovirus with altered myristoylation signal, the C14:0 group has been variably implicated in capsid stabilization/assembly, aberrant polyprotein processing, diminished replication, hindered maturation of viral particles and inefficient RNA uncoating. In this study we revisited the role of myristoylation in the virus life cycle particularly of coxsackievirus 3B (CVB3), a member of the genus enterovirus, by specifically targeting the host factors responsible for this modification. In concert with their partially overlapping activity, CVB3 production is only slightly diminished in human hNMT1 or hNMT2 ablated HAP1 haploid cells, matching results obtained by knock down of hNMT1 or hNMT2 expression in HeLa cells with specific siRNAs. Acute inactivation of both NMTs was achieved with a novel NMT pan-inhibitor (compound 1), which rapidly led to drastic reduction of myristoylation in cells as shown by metabolic tagging/ bioorthogonal labeling. Compound 1 potently inhibited production of infectious CVB3 particles across different cell-lines with similar effect on other enteroviruses, while Aichivirus-1 (genus Kobuvirus) lacking a myristoylation signal was insensitive. Employing eGFP-CVB3 and a RLuc-CVB3 replicon we demonstrate that viral attachment, polyprotein synthesis/processing and replication are not significantly perturbed by the drug. Surprisingly, viral particles with slightly increased thermal stability were still formed at 10% of mock-treated cells, but are marked by an up to 3 log drop in specific infectivity. Replication could be rescued by co-infection with normal CBV3 and to a lesser extent, with RV-A2. We speculate that the endosomal membrane-destabilizing activity of VP4 expelled from the helper virus during conversion to the 135S subviral particle is exploited for cytosolic transfer of RNA genome from co-internalized CVB3 lacking functional (sufficiently myristoylated) VP4. This indicates for the first time, that a properly modified VP4 may act in trans. Altogether, our data suggest that limiting myristoylation in CVB3-infected cells translates to inefficient virion assembly compounded by impaired RNA release into the cytosol. Mutants resistant to compound 1 have recently been obtained implying the intriguing possibility that myristoylation might have become dispensible. 144 Europic 2016

145 WEDNESDAY 07/09/2016 F07 & POSTER F17 THERMOSTABLE, IMMUNOGENIC POLIOVIRUS VLPS OF ALL THREE SEROTYPES Helen Fox1, Philip Minor1, Sarah Knowlson1, Johanna Marsian2, George Lomonossoff2, Nicola Stonehouse3, David Rowlands3, David Stuart4, Elizabeth Fry4, Ian Jones5, Toby Tuthill6, Andrew Macadam1 1 NIBSC, 2John Innes Centre, 3University of Leeds, 4University of Oxford, 5University of Reading, 6Institute for Animal Health, Pirbright Thermostable poliovirus VLPs (empty capsids), have been designed using information obtained by revertant analysis of poliovirus assembly mutants and produced by introducing multiple stabilising changes into the same construct. A ts assembly lesion was inserted into wild-type infectious clones, RNA transcripts were introduced into cell cultures and assembly competent mutants were selected after culturing at elevated temperatures. Analysis of viable viral progeny by deep sequencing revealed a range of capsid protein amino acid changes suitable for inclusion in stabilised particles and also provided an indication of the most favourable modifications from the mutation frequency data in multiple independent selections. The particles are extremely stable, for long periods, and generate high levels of protective antibodies in animal models. In vaccine lot-release assays the VLPs were superior to the current inactivated polio vaccine (IPV). A recombinant polio VLP vaccine, which would not require any live virus in its production, would be very useful in the post-eradication, high containment world. Europic 2016 145

146 WEDNESDAY 07/09/2016 F08 HUMAN MONOCLONAL ANTBODIES AS POTENTIAL THERAPEUTIC AGENTS AGAINST POLIOMYELITIS Konstantin Chumakov1, Diana Kouiavskaia2, Olga Mirochitchenko2, Eugenia Dragunsky2, Crystal Y. Chen3, Dan Huang3, Richard Wang3, Alvin Z Zhang3, Enzhuo Yang3, Ling Shen3, Zheng Chen3 1 US Food and Drug Administration, 2Food and Drug Administration, 3University of Illinois College of Medicine In April of 2016 global polio eradication campaign has entered a new phase when the use of trivalent Oral Polio Vaccine (OPV) has stopped and was replaced by a combination of bivalent (without type 2 virus) vaccine and at least one dose of Inactivated Polio Vaccine (IPV). There is a considerable uncertainty about the impact of this change on population immunity, especially on the ability of wild and vaccine-derived polioviruses to spread. Immunodeficient individuals chronically infected with poliovirus provide a potential source for restarting poliovirus circulation, and the magnitude of this problem is still not fully understood. Therefore development of therapeutic agents effective against poliovirus remains a high priority, and is pursued in two directions. The first one is development of small-molecule antiviral compounds, and the second one is passive immunotherapy based on human monoclonal antibodies. Both could be used to treat chronic virus excretors, as well as (in combination with vaccines) for emergency response to potential resurgence of poliomyelitis. We are pursuing the second direction and study A12 monoclonal antibodies that are effective against type 1 and type 2 poliovirus. Experiments in transgenic mice susceptible to poliovirus have demonstrated that the antibodies can protect against paralysis even if administered one day after lethal challenge with the virus. Co-administration of A12 antibodies with IPV did not prevent mice from developing immune response, suggesting their potential use for emergency post-exposure prophylaxis. Using the newly developed Rhesus monkey model of oral poliovirus infection we have demonstrated that A12 antibodies protect 100% of the animals against paralysis, completely block virus secretion in tonsils, and significantly decrease the duration of virus excretion in stool and the amount of virus shed. In contrast with our IPV immunization experiments in mice, co-administration of antibodies with live virus prevented development of immunity in Rhesus monkeys, despite productive infection resulting in virus shedding in stool. This suggests that immunization with live virus may either require active replication in tonsils, or involves virus migration from the sites of replication in the gut to immune organs where it can trigger immune response. Potential use of human monoclonal antibodies in the end-game of polio eradication and beyond will be discussed. 146 Europic 2016

147 WEDNESDAY 07/09/2016 F09 EFFICACY OF ANTIVIRAL DRUGS IN A HUMAN IN VITRO NASAL EPITHELIUM MODEL (MUCILAIRTM) Bernadett Boda1, Sacha Benaoudia1, Rosy Bonfante1, Song Huang1, Laurent Kaiser2, Caroline Tapparel2, Samuel Constant1 1 Epithelix, 2Laboratory of Virology, Division of Infectious Diseases, Geneva University Hospitals OBJECTIVE: Respiratory viral infections cause mild to severe diseases worldwide, such as common cold, bronchiolitis and pneumonia and are associated with huge costs for society. To test new molecules for shortening, alleviating the diseases or to develop new therapies, relevant human models are mandatory. Interestingly, MucilAir, a human reconstituted standardized nasal epithelia holds in vitro specific mechanisms to counter invaders comparable to the in vivo situation, such as mucus production, mucociliary clearance, and secretion of defensive molecules. Here we took advantage of these unique properties to perform a proof of concept study designed to screen antiviral compounds. METHODS: Clinically relevant Rhinovirus (A16, C15), Enterovirus (EV68) and Influenza A virus (H1N1, H3N2) strains were added to the apical side of fully differentiated MucilAirPool cultures (mix of 14 donors). Apically released viral genome copy number, overall mucin secretion, cilia beating frequency, velocity of mucociliary clearance and tissue integrity were assessed daily during 4 days. Basal media of the MucilAir cultures was used for cytokine as well as toxicity measurements. Different concentrations of antiviral treatments were tested in parallel and all endpoints were compared to control conditions. RESULTS: MucilAir cultures showed excellent host characteristics and high rate of replication for all tested viruses, including difficult-to-culture Rhinovirus C15. The use of MucilAir cultures allowed us to describe the early viral pathological mechanisms in the nasal epithelia for the different viruses studied. Rupintrivir efficiently inhibited the replication of HRV-A16 and HRV-C15, in a dose and time dependent manner and restored the mucociliary clearance impaired by EV68. Oseltamivir reduced the replication of H1N1 and H3N2 and restored the impaired barrier function monitored by transepithelial electrical resistance. CONCLUSION: Altogether, our results demonstrate that MucilAir is a robust, reliable and relevant tool for antiviral drug development. Europic 2016 147

148 WEDNESDAY 07/09/2016 F10 COMPARATIVE ANALYSIS OF THE MOLECULAR MECHANISM OF RESISTANCE TO VAPENDAVIR ACROSS A PANEL OF PICORNAVIRUS SPECIES Kristina Lanko1, Liang Sun1, John Vernachio2, Pieter Leyssen1, Leen Delang1, Johan Neyts1 1 KU Leuven University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium, 2Aviragen Therapeutics, Inc., Alpharetta, GA 30009 United States OBJECTIVES: Vapendavir (BTA798), a capsid binder in development by Aviragen Therapeutics, is a selective inhibitor of the replication of many picornavirus species (i.e. poliovirus, enterovirus D68, enterovirus A71, rhinoviruses, etc.). The drug is currently in Phase II clinical trial in adults with moderate to severe asthma that suffer from symptomatic human rhinovirus infection. The purpose of this study was to perform a detailed comparative analysis of the mechanism of resistance between different picornavirus species. METHODS: Virus isolates with reduced sensitivity to the antiviral effect of vapendavir were obtained by means of a five-step clonal resistance selection protocol. The genotype of these compound-resistant variants was determined and, for hRV14 and PV1, reverse engineering was performed to confirm that the mutations were responsible for the resistant phenotype. A comparative modelling study was performed by AutoDock Vina software to explore the role of the amino acid substitutions in resistance to the drug and the pH sensitivity of wild-type (WT) and compound-resistant virus variants was assessed. RESULTS: While for the WT viruses EC50 values of 0.6 0.1 M, 1.4 0.5 M, 0.07 0.01 M and 0.044 0.003 M were obtained respectively for PV1 (Sabin), EV68 (CU70), hRV14 and hRV02, all compound-resistant virus isolates were proven to be insensitive to the antiviral effect of vapendavir up to 20 M, the highest concentration tested. Following genotyping of the virus isolates, the amino acid substitutions I194F and V196I were observed in PV1 (Sabin) and C199I\R in hRV14. These mutations are located in the pocket of VP1 in which vapendavir is known to bind. For both hRV02 and EV68, however, the amino acid substitutions in VP1 (G149C for hRV02 and K177E for EV68) are located outside the binding pocket. Of interest was to observe that some of the resistant virus variants (hRV14-C199R, EV68-K177E) were also cross-resistant to other capsid binders (pirodavir and pleconaril) and that the infectivity of the hRV02-G149C mutant increased in the presence of vapendavir, suggesting that the compound still physically interacts with the mutant virus. Molecular modelling studies are ongoing to investigate changes in the binding mode of vapendavir to the mutant capsids. Because some of the additional amino acid substitutions, which were also identified in vapendavir- resistant hRV02 (S164P and R260H in VP2, D58G in VP3) are located at the pentameric interface known to be linked with pH sensitivity of picornaviruses, pH-dependency assays were performed with the compound-resistant virus variants. No changes in pH sensitivity of the resistant virus variants were observed compared to WT. CONCLUSIONS: Taken together, these results confirm that amino acid substitutions in VP1 are the major contributors to resistance to vapendavir. Surprisingly, these amino acid substitutions need not necessarily to be located inside the drug binding pocket, but may also be situated at some distance from this pocket, suggesting the possibility that the molecular mechanism behind compound-resistance is different between virus species and dependent on the structural properties of VP1. 148 Europic 2016

149 WEDNESDAY 07/09/2016 F11 SELECTIVE INHIBITION OF ENTEROVIRUS 71 BY A NOVEL FAMILY OF TRYPTOPHAN DENDRIMERS Liang Sun1, Eva Rivero-Buceta2, Leen Delang1, Ana Ana San-Flix3, Johan Neyts1, Pieter Leyssen1 1 Rega institute, KU Leuven, 2Instituto de Tecnologa Qumica (UPV-CSIC), 3nstituto de Qumica Mdica (CSIC) BACKGROUND AND OBJECTIVE: In particular in Asia, enterovirus 71 (EV71) infections are on the rise and are considered to be a major public health problem. Still, to date, no antiviral drugs are available for treatment or prevention of the infection. Hence, potent and safe anti-EV71 drugs are urgently needed. METHODS: Tryptophan dendrimers were evaluated for their antiviral activity in a virus-cell-based multi-cycle CPE reduction assay. Viral RNA levels were quantified by means of RT-qPCR. RESULTS: This study reports that a series of large molecular weight tryptophan dendrimers, which have recently been shown to inhibit in vitro HIV replication (by binding to gp120 and gp41), also inhibits the in vitro replication of enterovirus 71. Several of these molecules exerted potent (at low micromolar concentrations) antiviral activity against the BrCr lab strain of EV71. Exploration of the structure-activity relationship demonstrated that 9 or 12 Trp residues were crucial for antiviral activity. Based on this information, compound 12 was synthesized. This compound inhibited the BrCr lab strain at sub-micromolar concentrations. Surprisingly, the compound proved to be 250- to 3800-fold more potent against a panel of clinical isolates (representative for each subgenogroup) of EV71 with EC50 values ranging between 0.097 and 1.121 nM. In a time-of-drug-addition study, the compound behaved identical to pirodavir, i.e., it lost its activity when first added after infection. CONCLUSION: A novel class of highly potent, selective and early-stage inhibitors of enterovirus 71 replication has been discovered. The precise mechanism of action of these class compounds is currently being explored. Europic 2016 149

150 WEDNESDAY 07/09/2016 F12 A STRUCTURE-ACTIVITY RELATIONSHIP STUDY OF ITRACONAZOLE, A NOVEL ANTIVIRAL COMPOUND THAT TARGETS THE OXYSTEROL BINDING PROTEIN Lisa Bauer, Lucian Albulescu, Jeroen R.P.M Strating, Frank J.M. van Kuppeveld University Utrecht OBJECTIVES: Recently, we discovered that itraconazole (ITZ) is a broad-spectrum inhibitor of picornaviruses from the genera Enterovirus and Cardiovirus, as well as of hepatitis C virus (HCV). ITZ is a commonly used FDA-approved antifungal drug that acts through the fungal enzyme CYP51, a key enzyme in the synthesis of the lipid ergosterol. In addition, ITZ is under investigation for its anticancer activity, which it exerts via multiple cellular signaling pathways (i.e. Hedgehog , mTOR and VEGFR2 pathways). We discovered that the known targets of ITZ could not explain the antiviral activity of ITZ and instead identified the oxysterol binding protein (OSBP) as a novel target of ITZ through which the antiviral activity is mediated. In uninfected cells, OSBP localizes to membrane contact sites between the endoplasmic reticulum and the Golgi apparatus by binding to the ER protein VAP-A and phosphatidylinositol 4-phosphate (PI4P) lipids at the Golgi. At ER-Golgi membrane contact sites, OSBP transports cholesterol to the Golgi in exchange for PI4P. OSBP plays a key role in the replication of enteroviruses, cardioviruses and HCV by shuttling cholesterol to the replication organelles of those viruses. ITZ directly binds to OSBP and inhibits its lipid shuttling activities, thus interfering with the replication of viruses that require OSBP. A structure-activity relationship study was conducted to determine which structural features of ITZ are important for its antiviral activity. METHODS: We investigated the antiviral effects of ITZ using the cardiovirus encephalomyocarditis virus (EMCV) as model virus. In parallel, we tested the activity of the ITZ analogs towards OSBP by studying the accumulation of OSBP at the Golgi as a read out for inhibition of lipid shuttling. Inhibitors of OSBP activity prevent the cholesterol/ PI4P exchange and cause an increase in PI4P at the Golgi, thus inducing an accumulation of OSBP at the Golgi. RESULTS: We observed that all stereoisomers of ITZ were able to inhibit virus replication. Despite the importance of the triazole moiety for the antifungal effect, this structure is not important for antiviral activity. Furthermore, the core region of ITZ was required for efficient inhibition of virus replication. Importantly, we observed a good correlation between antiviral activity OSBP redistribution, confirming previous conclusion that ITZ exerts its antiviral activity through OSBP. Homology based modeling of ITZ into the lipid binding domain of the yeast homolog Osh4 indicates that the molecule occupies the lipid binding pocket and thereby, inhibits lipid shuttling. CONCLUSIONS: This structure activity relationship study determined the important chemical features of ITZ towards virus inhibition This can be used to generate ITZ-derived compounds that are specific towards OSBP, and do not affect fungal proteins or cellular signalling pathways. 150 Europic 2016

151 WEDNESDAY 07/09/2016 F14 STRUCTURAL DATA OF CVB3-DRUG COMPLEX James Geraets1, Rana Abdelnabi2, Shabih Shakeel1, Ausra Domanska1, Dirk Jochmans2, Sarah Butcher1 1 University of Helsinki, 2KU Leuven Coxsackievirus B3 (CVB3) is a small, single-stranded RNA virus belonging to the enterovirus B species of the picornavirus family, and is a leading etiological agent for human viral myocarditis that can result in both acute and chronic heart failure. The structure of mature CVB3 has been determined by X-ray crystallography to be fairly similar to other picornaviruses, containing the structural proteins VP1, VP2, VP3, and VP4. Importantly, it has been shown to have a hydrophobic pocket in VP1, which has been used as a drug target for enteroviruses. This is notably the case for the antiviral pleconaril, but as the drug failed clinical trials, there is a need to develop other capsid-recognising drugs for enteroviruses. Here we present structural data of CVB3 in complex with a novel benzene sulfonamide derivative that has been identified to inhibit CVB3. This study will help to identify the binding site, the mode of drug action, and make predictions of its effect on strain variations. Europic 2016 151

152 WEDNESDAY 07/09/2016 F15 ANTIVIR AUTOMATED PLAQUE-BASED SCREENING FOR HOST DIRECTED INHIBITORS Murer L1*, Andriasyan V1*, Yakimovich A2*, Georgi F1*, Witte R1*, Rudnicka A1, Yamauchi Y1, Greber UF1+ 1 Institute of Molecular Life Sciences (IMLS), University of Zurich, Switzerland 2 MRC Laboratory for Molecular Cell Biology, University College London, United Kingdom * These authors contributed equally to this work + Corresponding author: [email protected] Viruses pose a serious threat to humans and livestock. Their medical, societal, humanitarian, and economical impact is devastating in many parts of the world. Rhinovirus (RV) is the common cold virus, and the cause of the majority of upper respiratory tract infections in humans. RV infections are linked to asthma and severe chronic obstructive pulmonary disease (COPD). The World Health Organisation WHO estimates that COPD is the third leading cause of death in 2030, behind ischemic heart disease and strokes (http://www.who.int/respiratory/ copd/en/). So far, there are no adequate anti-rhinovirus viral therapies available. Conventional screens for anti- viral drugs have been limited by the fact that there have been no high throughput assays available scoring the full viral infection cycle, comprising virus entry, replication, assembly and release from infected cells. Here we are using our recently developed automated high-throughput image analysis framework Plaque2.0 to probe the entire rhinovirus life cycle by scoring viral gene products by immunohistochemistry. The Plaque2.0 framework can be readily fed with screening-scale high throughput images. It extracts features of single cells, such as infection status, and object features, such as plaque area, shape, infection density and intensity. Using Plaque2.0, we are screening an established library of 1280 small molecules (Prestwick Library) for inhibitors of respiratory virus spread to expand the number of antiviral drug targets. The Prestwick Library consists of FDA approved compounds, most of them with a known mode-of-action. Targeting the viral host is crucial for sustained anti-viral therapy, since viruses inherently provide few targets for attack, and viral proteins are difficult targets for broad and long lasting inhibitors due to emerging viral resistance. By simultaneously screening the library against three human respiratory pathogens, adenovirus, Influenza A and rhinovirus, we aim for broad anti-viral compounds and new insights into generic host modules supporting respiratory viral infection. 152 Europic 2016

153 WEDNESDAY 07/09/2016 F18 RESULTS FROM AIROPICO, THE EU NETWORK DEDICATED TO HUMAN PICORNAVIRUSES Katja Wolthers1 and Stella Koppel2, on behalf of AIROPico partners3 1 Consortium Coordinator and 2Project Manager, Department of Medical Microbiology, Laboratory of Clinical Virology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3 Prof S. Butcher, University of Helsinki, Helsinki, Finland; Dr J. Koskinen, ArcDia International Oy Ltd, Turku, Finland; Dr P. Susi, University of Turku, Turku, Finland; Prof J. Neyts, Catholic University of Leuven, Leuven, Belgium; Dr O. Landt, TIB Molbiol, Berlin, Germany; Dr M. Jara, AbBcn S.L., Barcelona, Spain; Dr K. Palm, Protobios, Talinn, Estonia. WWW.AIROPICO.EU Picornaviruses such as enteroviruses (EV), rhinoviruses (RV) and parechoviruses (HPeV) represent the most commonly encountered infectious agents in mankind. The abundance of virus subtypes and variety of clinical symptoms hamper studies on prevalence, transmission, and pathogenesis of which many aspects are still largely unknown. In addition, therapy is not available against human picornavirus infections. AIROPico is a FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP) network of four academic partners and four industrial partners funded for 4 years (2014-18) to explore and unravel mechanisms of human picornavirus pathogenesis and to develop fast diagnostics, novel treatment options and additional therapy strategies. Within the present AIROPico project, the partners combine by intersectoral exchanges the disciplines of pathogenesis, diagnostics and therapy development in order to understand disease and to develop effective antiviral treatments. MAIN AIMS: 1) To investigate biological features of clinically relevant EV/RV/HPeV types to understand basic mechanism of host pathogen interactions; 2) To develop faster diagnostic detection methods both for routine detection of EV/RV/HPeV and for easy-to-use typing tools; 3) To generate knowledge and tools necessary to fight these infections by antibodies and small antiviral compounds. RESULTS Research within AIROPico focuses on 3D virus structure and binding to (neutralizing) antibodies, virus- host interaction by the use of 3D human organ cultures, development of rapid point of care (POC) testing and faster genotyping methods for faster diagnostics of EV/RV/HPeV, and identification of small compounds and/or monoclonal antibodies with potential to develop as antiviral therapy. A summary of the results obtained from the collaborative network research will be presented. This project has received funding from the European Unions Seventh Framework People Programme under REA grant agreement no 612308 Europic 2016 153

154 THURSDAY 08/09/2016 VACCINES, DISEASE PREVENTION AND CONTROL, 8:45 Session 7 ERADICATION 12:30 Chairs: David Rowland & Andrew Macadam K09: CAN NEW VACCINES HELP MAINTAIN A POLIO-FREE WORLD AFTER Andrew Macadam (NIBSC, South 08:45 ERADICATION? Mimms, Herts, United kingdom) Ken Fujii (Tokyo Metropolitan G01: THE VP1 AMINO ACID RESIDUE 145 OF EV71 IS A VIRULENCE DETERMINANT 09:10 IN SCARB2-DEPENDENT INFECTION Institute of Medical Science, Japan) G02: MOLECULAR BASIS OF POLIOVIRUS VACCINE ATTENUATION IN NON-HUMAN 09:20 PRIMATES AND POLIOVIRUS-SUSCEPTIBLE TRANSGENIC MICE ASSESSED BY Begona Valdazo-Gonzalez DEEP SEQUENCING ANALYSIS Ekaterina Viktorova (University of Maryland, and Virginia-Maryland 09:30 G03: VECOTRED VACCINE AGAINST POLIOMYELITIS Regional College of Veterinary Medicine, United States of America) G04: PLANT-MADE POLIOVIRUS-LIKE PARTICLES: DEVELOPING A SYNTHETIC Johanna Marsian (John Innes 09:40 POLIO VACCINE Centre, United Kingdom) G05: PHENOTYPE OF CONGO-2010 POLIOVIRUS THAT IS POORLY NEUTRALIZABLE Alexander Lukashev (Chumakov 09:50 BY SERA FROM VACCINE RECIPIENTS SUGGESTS THE REASONS FOR ITS Institute of Poliomyelitis and Viral EMERGENCE AND EXTINCTION Encephalitides, Russia) G06: NOVEL HIS-TAG MARKER FOOT-AND-MOUTH DISEASE VIRUS VACCINE Elizabeth Rieder (USDA, ARS, 10:00 BOUND TO NANOLIPOPROTEIN ADJUVANT VIA METAL IONS: UTILITY ON VACCINE United States of America) AND DIAGNOSTIC DEVELOPMENTS Heli Harvala (European Centre G07: EUROPEAN NON-POLIO ENTEROVIRUS SURVEILLANCE AND LABORATORY 10:10 DETECTION ARE WE PREPARED TO DETECT AN ENTEROVIRUS OUTBREAK? for Disease Prevention and Control (ECDC), United Kingdom) Kimberley Benschop (National G08: ENVIRONMENTAL ENTEROVIRUS SURVEILLANCE IN THE NETHERLANDS: 10:20 POLIO AND BEYOND Institute for Public Health and the Environment, Netherlands) COFFEE BREAK Ann Palmenberg (University 11:00 MEETING REPRISE of Wisconsin-Madison, United States of America) James Gern (University of ALBERT SABIN LECTURE 11:45 DETERMINANTS OF RHINOVIRUS ILLNESS SEVERITY Wisconsin-Madison, United States of America) SELECTED VISITS OR DEPARTURE WITH LUNCH BOXES 154 Keynote Lecture Sabin Lecture Short Communication (8 + 2 min) Europic 2016

155 THURSDAY 08/09/2016 POSTER SESSION 7 - VACCINES, DISEASE PREVENTION AND CONTROL, ERADICATION G10: A CONTAMINATED ENVIRONMENT IS AN EFFICIENT ROUTE OF TRANSMISSION FOR FOOT AND MOUTH DISEASE VIRUS Claire Colenutt (The Pirbright Institute, United Kingdom) G11: FIRST WHO INTERNATIONAL STANDARD FOR ANTI-EV71 HUMAN SERUM Javier Martin (NIBSC, United Kingdom) G12: COLD-ADAPTED VIRAL ATTENUATION (CAVA): HIGHLY TEMPERATURE SENSITIVE POLIOVIRUSES AS NOVEL VACCINE STRAINS FOR A NEXT GENERATION INACTIVATED POLIOVIRUS VACCINE Barbara Sanders (Janssen, Netherlands) G13: M5BT PROTEIN INDUCED M CELL ACTIVATION AGAINST FMDV Dong-Jun An (Animal and Plant Qurantine Agency, South Korea) G14: EPITOPE ANALYSIS OF MONOCLONAL ANTIBODIES FOR MEASUREMENT OF D ANTIGEN CONTENTS IN THE INACTIVATED POLIOVIRUS VACCINE Tomofumi Nakamura (The Research Foundation for Microbial Diseases of Osaka University (BIKEN), Japan) G15: ENVIRONMENTAL SURVEILLANCE FOR POLIOVIRUS IN THE UK. THE USE OF A DEEP SEQUENCING APPROACH. Manasi Majumdar (NIBSC, United Kingdom) G16: IMMUNOLOGICAL DIVERSITY AMONG TYPE 1 STRAINS OF WILD AND VACCINE-DERIVED POLIOVIRUS Konstantin Chumakov (US Food and Drug Administration, United States of America) G17: GLUTATHIONE: A POTENTIAL STABILIZER FOR THE ORAL POLIOMYELITIS VACCINE Rana Abdelnabi (KU Leuven University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, ) G18: INACTIVATED POLIO VACCINE AND THE ENDGAME OF POLIO ERADICATION Javier Martin (NIBSC, United Kingdom) G19: DEVELOPMENT OF NON-POLIO ENTEROVIRUS VACCINES BASED ON THE POLIOVIRUS VACCINE PRODUCTION PLATFORM Dinja Oosterhoff (Intravacc, Netherlands) G20: STRATEGIES TO STABILISE POLIOVACCINE GENOMES AGAINST RECOMBINATION Matt Smith (NIBSC, United Kingdom) G21: INVESTIGATION OF A CHIMERIC SAT2 FOOT-AND-MOUTH DISEASE VIRUS EXHIBITING AN INCREASE IN CAPSID THERMOSTABILITY Julian Seago (The Pirbright Institute, United Kingdom) G22: MODIFIED SABIN 2 POLIOVIRUSES FOR USE AS AN ORAL-LIVE ATTENUATED VACCINE POST-ERADICATION Matthijn de Boer (Intravacc, Netherlands) G23: ATTENUATION OF PICORNAVIRUS GROWTH BY ENGINEERED POLYMERASE VARIANTS Olve Peersen (Colorado State University, United States of America) G25: TOLL LIKE RECEPTOR [TLR] 3 AGONIST POLY I:C AS AN ADJUVANT IN FMD VACCINE ENHANCED PROTECTION OF CATTLE AGAINST VIRULENT VIRUS CHALLENGE Satya Parida (The Pirbright Institute, United Kingdom) G28: IMMUNOGENICITY OF ORAL POLIO VACCINE AGAINST XINJIANG IMPORTED TYPE 1 WILD POLIOVIRUS Dongmei Yan (Institute for viral disease control and prevention, Chinese center for disease control and prevention, China) G29: IS CAPSID PROTEIN VP4 AN ANTIGENIC SWITCH IN FMDV? ENGINEERING A MATURATION CLEAVAGE TO GENERATE RECOMBINANT PENTAMERS WITH DIFFERENT PROPERTIES Joseph Newman (The Pirbright Institute, United Kingdom) G31: VIRAL SHEDDING PATTERNS IN ENTEROVIRUS MENINGITIS IN CHILDREN AND ADULTS OVER A THREE YEARS EPIDEMIOLOGICAL SURVEILLANCE PERIOD IN SWITZERLAND Manuel Schibler (Geneva University Hospitals, Switzerland) Europic 2016 155

156 THURSDAY 08/09/2016 G32: PREVENTION OF FOOT-AND-MOUTH DISEASE IN CATTLE USING A PRIME-BOOST-VACCINATION STRATEGY Graham Belsham (DTU National Veterinary Institute, Denmark) G33: ENVIRONMENTAL SURVEILLANCE IN SEWAGE SAMPLES WITH ULTRA-DEEP SEQUENCING: RESULTS OF A ONE-YEAR PILOT STUDY (CLERMONT-FERRAND, FRANCE, 2014-2015). Maxime Bisseux (EA4843-EPIE, Universit dAuvergne - LMGE-CNRS 6023, Universit Blaise Pascal, France) G34: VISUALIZING PATTERNS OF SEQUENCE EVOLUTION: EXAMPLES AND APPLICATION TO LARGE POLIO AND OTHER ENTEROVIRUS DATABASES Jaume Jorba (CDC, United States of America) G35: THE BACULOVIRUS AVENUE FOR VLP-BASED FOOT-AND-MOUTH DISEASE VIRUS VACCINES Claudine Porta (The Pirbright Institute, United Kingdom) G36: DETECTION OF ANTIBODIES AGAINST CONSERVED CAPSID EPITOPES PROVIDES A UNIVERSAL SEROLOGY ASSAY FOR DIAGNOSIS OF FMDV Amin Asfor (Pirbright Institute, United Kingdom) G37: THE LAST MILE: DIAGNOSTIC ASSAY DEVELOPMENT FOR THE FINAL PHASE OF POLIO ERADICATION Everardo M. Vega (Centers for Disease Control and Prevention, United States of America) G39: EVOLUTIONARY DYNAMICS OF POLIOVIRUS NEUTRALIZING ANTIGENIC SITES AND OTHER CAPSID FUNCTIONAL DOMAINS DURING A LARGE AND PROLONGED OUTBREAK Cara Burns (Centers for Disease Control and Prevention, United States of America) 156 Europic 2016

157 THURSDAY 08/09/2016 K09 CAN NEW VACCINES HELP MAINTAIN A POLIO-FREE WORLD AFTER ERADICATION? Andrew Macadam, National Institute for Biological Standards and Control, UK Wild type polio has been nearly eradicated; nevertheless there will be a need to continue immunisation programmes for some time because of the possibility of re-emergence and the existence of long term excreters of poliovirus. The vaccines we have are not ideal for a post-eradication world. All vaccines in current use depend on growth of large quantities of virus and most of the inactivated vaccines involve wild type viruses known to cause poliomyelitis so production itself carries biosecurity risks. Sabins live-attenuated strains are known to evolve into essentially wild-strains so their long-term use is incompatible with eradication. However, inactivated vaccines are less effective in breaking transmission of poliovirus so live-attenuated strains are valuable for future outbreak control. Potential solutions to these problems are being sought. New strains have been developed for IPV production with significantly lower risk of escape into the human population. Furthermore, the capsid proteins of poliovirus have been engineered to produce VLPs which are stable enough to allow vaccine production using recombinant expression. Genetically stable live-attenuated strains have been designed which have reduced potential for regaining wild-type properties. If these advances can be translated into clinical reality they could make a significant contribution to safeguarding the achievements of the Eradication Programme. The development of Sabins live-attenuated viral vaccines involved selection of virus variants by the judicious, but empirical, use of a range of cell substrates, hosts and growth conditions. Unsurprisingly these variants evolve back towards wild-type properties during replication in, and transmission between, their natural hosts. An understanding of the molecular basis of these pathways, and other aspects of virus replication, has led to new vaccine design approaches including genome alterations to restrict evolutionary potential and control infectivity, genetic stability and particle thermostability. Europic 2016 157

158 THURSDAY 08/09/2016 G01 THE VP1 AMINO ACID RESIDUE 145 OF EV71 IS A VIRULENCE DETERMINANT IN SCARB2-DEPENDENT INFECTION Ken Fujii1, Yui Sudaka1, Ayumi Imura1, Ayako Takashino1, Chikako Kataoka2, Tadaki Suzuki3, Naoko Iwata-Yoshikawa3, Osamu Kotani3, Yasushi Ami3, Hiroyuki Shimizu3, Noriyo Nagata3, Satoshi Koike1 1 Tokyo Metropolitan Institute of Medical Science, 2Hokkaido University, 3National Institute of Infectious Diseases Enterovirus 71 (EV71) belongs to the species Enterovirus A of the genus Enterovirus. EV71 is a causative agent of hand-foot-mouth disease and it sometimes causes severe or fatal neurological complications. Studies using neonatal mice suggested that EV71 strains with glutamic acid at VP1-145 (E virus) are more virulent than those with glycine (G virus). However, it is unclear whether VP1-145 is involved in EV71 virulence in humans because neonatal mouse does not express authentic human EV71 receptor. We found that human Scavenger receptor class B, member 2 (hSCARB2) is the functional receptor for EV71 and generated a hSCARB2 transgenic (hSCARB2-tg) mouse model. To determine the involvement of VP1-145 for EV71 virulence in hSCARB2-dependent infection, we analyzed virological features of VP1-145 variants in vitro and compared their virulence in hSCARB2-tg mice and cynomolgus monkeys. We prepared E or G viruses from the infectious clones of three virus strains; Isehara (subgenogroup C2), C7/Osaka (B4) and SK-EV006 (B3). In each strain, E virus showed a large plaque phenotype while G virus exhibited a small plaque phenotype. The E viruses grew similarly or less efficiently in RD cells and L-929 expressing hSCARB2 cells compared to G viruses. The binding efficiency of E and G viruses to hSCARB2 was similar as judged by pull-down assay. We infected E or G viruses of three strains intravenously and intracerebrally to hSCARB2-tg mice. E virus-infected mice were paralyzed at high rates but G viruses-infected mice were rarely paralyzed. On the contrary to in vitro cell culture, E viruses replicated more efficiently in the mice than G viruses. E virus was recovered from the CNS of the G virus-infected mice, suggesting that a mutation from G to E at VP1-145 occurred and VP1-145E variants were selected in the course of infection. Four cynomolgus monkeys were intravenously inoculated with E or G viruses of the Isehara strain. In the E virus-inoculated group, two monkeys exhibited neurological symptoms and pathological changes. None of the G virus-inoculated monkeys had apparent neurological manifestations and pathological changes. The viruses were detected in some of the clinical samples and tissue homogenates from the E virus-inoculated monkeys. On the other hand, the viruses were detected in G virus-infected monkeys at a low frequency, but the recovered viruses were found to be E viruses. The results suggested that E at VP1-145 is a neurovirulence determinant in both hSCARB2-tg mice and monkeys. We investigated why E viruses have advantage in replication in vivo. We found that E viruses exhibited more refractory to neutralization than G viruses in in vitro neutralization assay using anti-EV71 rabbit polyclonal antiserum. Our analysis suggests that VP1-145 may be important for virus replication efficiency in vivo and evasion of the host immune responses. 158 Europic 2016

159 THURSDAY 08/09/2016 G02 MOLECULAR BASIS OF POLIOVIRUS VACCINE ATTENUATION IN NON-HUMAN PRIMATES AND POLIOVIRUS-SUSCEPTIBLE TRANSGENIC MICE ASSESSED BY DEEP SEQUENCING ANALYSIS Javier Martin, Begoa Valdazo-Gonzalez, Philip Minor, Glynis Dunn, The National Institute for Biological Standards and Control, The Medicines , UK Poliomyelitis is a crippling disease caused by a poliovirus. It has been threatening children with acute flaccid paralysis since the antiquity. Nowadays the disease has been eradicated in all countries except Pakistan and Afghanistan thanks to the use of oral poliovirus vaccines (OPV) containing attenuated poliovirus strains (Sabin 1, 2 and 3) developed in the 50s. The passage in cell culture of these strains required for the production of vaccines might cause mutations in specific and well described sites of the virus genome reverting the attenuated strains to pathogenic viruses that can cause disease in humans and animal models. Therefore a key factor for the use of these vaccines is safety and quality control. Currently, there are three standard procedures to check the pathogenicity of these vaccines before releasing them to the market. One is the laboratorial technique Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC), whilst the other two are in vivo tests: the monkey neurovirulence test and the transgenic mice neurovirulence test (using TgPVR21 mice, susceptible to poliovirus). MAPREC is a technically challenging and time consuming test which calculates the percentage of mutants at specific well described sites when compared to standards of known percentage of mutants which are included for the validation of the test. Animal tests are not only expensive and time-consuming but they also represent ethical issues. Moreover, some discrepancies between the monkey and the mouse neurovirulence tests have been described for some vaccines. The objective of this study is the comparison of these three reference tests with deep sequencing (DS) using a set of 20 historical OPV type 3 vaccines in order to ascertain the use of DS for safety and quality control purposes, as well as to understand the genomic cause of the disagreement between the two animal tests. A shot-gun approach after the viral RNA extraction was used to reverse-transcribe and amplify the virus genome. Another approach based on the amplification of the 5 UTR fragment of the genome where the pathogenic sites detected by MAPREC are present was used in parallel to check the robustness of the method. After DNA purification and library preparation, samples were run on an Illumina platform (MiSeq). Sequencing data was analysed using Geneious. There was a good correlation between the results of DS and MAPREC. DS was also able to detect changes in nucleotide 2493 (coding for VP1-6 amino acid) which cause increased pathogenicity in the transgenic mice model but not in the monkey neurovirulence test. Preliminary results suggest that DS seems to be as reliable as MAPREC for safety and quality control of OPV independently of the approach for DNA generation. Moreover, full genome DS will help to detect other genomic sites causing changes in the pathogenicity and immunogenicity of the virus. Further international collaborative studies to analyse sets of different vaccine samples are required to establish this technique as a standard procedure. Europic 2016 159

160 THURSDAY 08/09/2016 G03 VECOTRED VACCINE AGAINST POLIOMYELITIS Ekaterina Viktorova1, Diana Kouiavskaia2, Eugenia Dragunsky2, Siba Samal1, Konstantin Chumakov2, George Belov1 1 Department of Veterinary Medicine, University of Maryland, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, MD 20742, 2Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, FDA, Silver Spring, MD 20993 Global Polio Eradication Initiative resulted in more than 99% reduction of polio morbidity worldwide, but repeatedly missed the eradication deadlines. The suboptimal properties of existing anti-poliovirus vaccines, inactivated vaccine (IPV) and oral live attenuated vaccine (OPV), are one of the major hurdles in the campaign. Both of these vaccines, and their derivatives currently under development, have intrinsic flaws that jeopardize achieving the ultimate eradication goal. Production and administration of IPV is prohibitively expensive in low-income countries, and it does not induce adequate mucosal immunity necessary to stop the virus transmission. OPV can induce vaccine associated poliomyelitis upon administration, and the genetically unstable vaccine strains can establish circulation in human populations and regain pathogenic phenotype resulting in outbreaks of vaccine derived poliomyelitis. Moreover, immunocompromised individuals can sustain replication and excrete vaccine-derived viruses for years. Thus, a novel approach to anti-poliovirus vaccine development is needed. We developed a prototype live vectored vaccine against poliovirus based on Newcastle disease virus (NDV). NDV is a negative strand RNA virus restricted to avian hosts. Its genome codes for six genes which are transcribed individually by the viral RNA dependent RNA polymerase starting from the 3 end of the genomic RNA. We inserted a cassette for expression of the poliovirus capsid protein precursor P1 between the second and the third genes of NDV, and the one for expression of protease 3CD necessary for P1 processing before the last gene. This arrangement of poliovirus-specific inserts allows for a high level of expression of P1 and a relatively low expression of the protease. Co-expression of P1 and 3CD has been shown to produce virus-like particles (VLP) which can induce protective immunity against poliovirus infection. Importantly, in contrast to other VLP- based vaccines which rely on mass production and purification of VLPs, administration of our NDV-based vaccine generates poliovirus VLPs in situ. These VLPs are presented to the immune system in the context of active NDV replication which serves as a natural adjuvant and has been shown to promote induction of strong mucosal immune response. The recombinant NDV propagates similarly to the wt virus in cell culture and in embryonated eggs. Moreover, it retains expression of poliovirus inserts over multiple passages. Accumulation of poliovirus P1 proteins in recombinant NDV-infected cells is similar to that observed in poliovirus-infected cells by the end of infectious cycle. Importantly, we demonstrated that co-expression of P1 and 3CD from the NDV vector results in proper processing of the P1 polyportein and massive production of VLPs which were detected by conformation- specific A12 monoclonal antibody. Thus, NDV-vectored anti-polio vaccine is absolutely safe since it is not based on live poliovirus at any stage. NDV propagates to high titers in Vero cells and embryonated eggs, established systems for industrial vaccine production. Importantly, since NDV is a respiratory virus, its administration is performed via nasal route, eliminating the requirement for trained medical personnel. Thus, NDV-vectored vaccine against poliomyelitis provides an attractive alternative to traditional vaccine development. 160 Europic 2016

161 THURSDAY 08/09/2016 G04 PLANT-MADE POLIOVIRUS-LIKE PARTICLES: DEVELOPING A SYNTHETIC POLIO VACCINE Johanna Marsian1, George Lomonossoff1, Andrew Macadam2, Helen Fox2, Phillip Minor2, Mohammad Bahar3, David Stuart3, Ian Jones4, Oluwapelumi Adeyemi5, Nicola Stonehouse5, Toby Tuthill6, Elizabeth Fry3, Luigi De Colibus3, David Rowlands5 1 John Innes Centre, 2NIBSC, 3University of Oxford, 4University of Reading, 5University of Leeds, 6The Pirbright Institute The initiative by the World Health Organisation (WHO) to eradicate poliomyelitis has been very successful, leading to almost complete eradication of the disease. However, the existing Sabin oral polio vaccine (OPV) and the parentally administered Salk inactivated polio vaccine (IPV) have major disadvantages and their continued deployment is incompatible with eradication. The WHO is therefore seeking novel polio vaccines, and we propose the generation of a virus-free polio vaccine based on the production of recombinantly expressed virus like particles (VLPs) of polio, which do not contain any viral genome. The overall aim of this WHO-funded project is to produce a synthetic vaccine against poliovirus using a variety of expression system including plants. In particular, it is aimed at the generation of non-infectious poliovirus-like particles (PV VLPs) that have the capability of eliciting a protective immune response against polio. To enable plant-based transient expression of PV-VLPs, stabilised mutant versions of the poliovirus gene P1, encoding the precursor of the structural genes and the proteinase 3CD, were cloned into separate pEAQ plant expression vectors and transformed into Agrobacterium tumefaciens. Recombinant strains were co-infiltrated into Nicotiana benthamiana and the leaves harvested 5 days after infiltration. VLPs were purified by Nycodenz gradient centrifugation giving a purified PV VLP yield of 70 mg/kg fresh weight tissue. Cryo-EM analysis revealed that the atomic structure of stabilised PV VLPs closely resemble the native wild-type poliovirus. Furthermore, mice carrying the gene for the human PV receptor were protected against wild-type PV when immunised with the plant-produced PV VLPs. This represents the first report of the production of immunologically effective non- infectious poliovirus-like particles in plants. In order to make the product more attractive to the vaccine industry, tobacco BY-2 cells have been successfully tested for the transient expression of the above-mentioned PV mutant VLPs using the cell-pack method. Work is currently ongoing on examining the properties of these PV VLPs. BY-2 cells grow in suspension culture and have no limit for mass production which is a potential advantage over whole plants. Europic 2016 161

162 THURSDAY 08/09/2016 G05 PHENOTYPE OF CONGO-2010 POLIOVIRUS THAT IS POORLY NEUTRALIZABLE BY SERA FROM VACCINE RECIPIENTS SUGGESTS THE REASONS FOR ITS EMERGENCE AND EXTINCTION Alexander Lukashev1, Anatoly Gmyl1, Elena Shustova1, Marina Kolesnikova2, Diana Kouiavskaia3, Konstantin Chumakov3 1 Chumakov Institute of Poliomyelitis and Viral Encephalitides, [email protected], 3U.S. Food and Drug Administration Poliovirus outbreak in Congo in 2010 was caused by wild-type poliovirus 1 and involved 445 cases with 47% case-fatality rate, and has affected mostly young adults. Many of the victims were duly immunized with OPV and had robust antibody titers against all three poliovirus types. Sera of vaccinated individuals from Germany, Russia, and the United States poorly neutralized the virus. 20% to 50% of vaccinated adults who had antibodies against vaccine strains had no detectable neutralizing antibodies against Congo-2010 virus. Phenotypic properties of PV1 strain Congo-2010 were studied in attempts to explain high mortality associated with the virus. It was neurovirulent in PVR transgenic mice. The 50% paralytic doze (PD50) of Mahoney and Congo-2010 strains were close (3.95 and 4.4 log TCID50, respectively) and significantly differed from that for the reference attenuated Sabin 1 strain (PD50 = 6.87 log TCID50). Therefore high case-fatality rate cannot be explained by a higher intrinsic virulence of Congo-2010 strain because it was not more neurovirulent than the reference Mahoney strain. Another unexpected observation was that Congo-2010 strain replicated at a reduced rate at permissive conditions. Virus yield in RD cell culture after 8 hours at 37C was 8.5 log TCID50/100l for most wild strains, but only 8.0 log for Congo-2010 and 7.75 log for Sabin 1 strain. At 40C, Congo-2010 replicated about as well as WT strain Mahoney. In a competition experiment in RD cell culture at 37C Congo-2010 was consistently overgrown by wild strains Mahoney and Bar65. After eight passages, virus ratio changed from 1:1 to 1:100 in favor of conventional wild strains. On the other hand, Congo-2010 readily overgrew Sabin 1 strain at these conditions, suggesting that its fitness was intermediate between wild and attenuated strains. When virus mixtures were passaged in the presence of pooled serum from vaccine recipients, Congo-2010 quickly overgrew any other PV1 strain. After 3-4 passages of virus mixtures in the presence of immune serum, Mahoney, Sabin1 and Bar65 strains were completely eliminated from the mixture. This suggests that increased resistance of Congo-2010 strain to neutralization may have provided it a selective advantage in vaccinated populations. High population immunity may have facilitated the emergence of this strain with partial immune escape phenotype necessary to survive under high immune pressure. Reduced replicative capacity of the virus may reflect the fitness cost of the immune escape phenotype and be one of the reasons why this virus lineage ultimately did not establish circulation and became extinct. The ability of this virus to be neutralized by vaccine-induced antibodies is about 10 times lower than of conventional wild and vaccine strains. Therefore waning immunity in young adults created a significant cohort susceptible to this immunologically abnormal strain, triggering the outbreak. Its high case mortality rate may be explained by the unusual high age of the victims, since poliomyelitis is more severe in adults compared to children. These findings reiterate the need to maintain high population immunity in all age cohorts. 162 Europic 2016

163 THURSDAY 08/09/2016 G06 NOVEL HIS-TAG MARKER FOOT-AND-MOUTH DISEASE VIRUS VACCINE BOUND TO NANOLIPOPROTEIN ADJUVANT VIA METAL IONS: UTILITY ON VACCINE AND DIAGNOSTIC DEVELOPMENTS Elizabeth Rieder1, Devendra Rai2, Fayna Diaz-San Segundo2, Elizabeth Schafer2, Luis Rodriguez2, Teresa de los Santos2, Paul Hoeprich3 1 USDA, ARS, 2Plum Island Animal Disease Center, ARS, USDA, Greenport, NY, USA, 3Lawrence Livermore National Laboratory, Livermore, California, USA Foot-and-Mouth disease (FMD) is considered an economical important disease of livestock, is an OIE notifiable disease and it remains a global threat to national and international trade in livestock and livestock products. Control of FMD by means of vaccination, relies strongly on the chemical inactivation of complete viral particles. Improvements on FMDV vaccine and diagnostic assay are warranted on several fronts of development. In this study, we engineered novel FMDV vaccines by an approach involving (1) mutant FMDV containing six histidine residues (6X-His) inserted into FMDV genome between the C-terminus of capsid protein VP1 and non-structural protein 2A (2) 6xHis FMDVs readily assembled into antigen:adjuvant complexes in solution with Ni2+-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). This marker 6xHis FMDV exhibited in vitro growth profiles, virus titers and neutralizing (r1) values similar to the parental virus. Concentration of the viruses by Co2+ affinity columns and ELISA assays confirmed the functional expression of His tags on the marker virus capsid surface. Electron microscopic imaging and biochemical assays showed that the mutant virus binds to a Ni-chelating nanolipoprotein monophosphoryl acid adjuvant (NiNLP-MPLA) in a concentration dependent manner. Animals Immunized with BEI-inactivated 6xHis-FMDV:MPLA:NiNLP formulated vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone, using an experimental mouse model. This technology has broad applications to facilitate antigen purification and to induce effective immune responses to other relevant picornaviruses. Europic 2016 163

164 THURSDAY 08/09/2016 G07 EUROPEAN NON-POLIO ENTEROVIRUS SURVEILLANCE AND LABORATORY DETECTION ARE WE PREPARED TO DETECT AN ENTEROVIRUS OUTBREAK? Heli Harvala1, Aftab Jasir2, Pasi Penttinen2, Lucia Pastore Celentano2, Donato Greco2, Eeva Broberg2 , European Centre for Disease Prevention and Control (ECDC) 1 2 BACKGROUND: Enteroviruses (EVs) are known to cause large and severe outbreaks, as recently demonstrated by EV-D68 in USA and Europe. Another type, EV-A71, is also known for its ability to cause geographically widespread and clinically significant hand, foot, and mouth disease (HFMD) outbreaks. Although EV-A71 outbreaks have been mostly described in Asia so far, the virus is already known to circulate in Europe and has been occasionally linked to the fatal outcomes. We have evaluated the European preparedness for detection and characterisation of non- polio EVs in order to improve our response for (re)-emergencing EVs linked to severe disease. METHODS: An on-line survey on non-polio enterovirus surveillance and enterovirus typing/characaterisation was submitted to all EU/EEA Member States (MS) national coordinating competent bodies. RESULTS: A total of 29 MS from 30 responded to the survey. Twenty-seven countries conduct non-polio enterovirus surveillance based on reporting of enteroviruses detected from clinical specimens, two of them without further characterisation of EV-positive samples. Almost all countries include typing of EV-positive samples obtained from individuals with neurological infections (n=24) in their EV surveillance, whereas HFMD and respiratory infections are included in the EV surveillance less frequently (n= 18 and 16, respectively). Three countries have also initiated specific surveillance for HFMD, and eleven for EV-D68. EV-D68 surveillance has been established via sentinel influenza surveillance (n=7), by typing EV-positive respiratory samples (n=10) and/or via acute flaccid paralysis (AFP) surveillance (n=5). Virus isolation is performed in all except one country, whereas molecular methods are used by all. Non-polio enterovirus typing is performed in 26 MS; ten MS type/characterise only culture-positive EV isolates, whereas the remaining MS subject also PCR-positive samples to typing/characterisation. Nineteen MS have introduced sequencing based EV typing, whereas neutralisation assay is used by 16 MS and seven of them rely entirely on it. Over 5000 EV-positive specimens were successfully characterised/typed in the EU/EEA region in 2015. The estimated number of typed EV specimens was

165 THURSDAY 08/09/2016 G08 ENVIRONMENTAL ENTEROVIRUS SURVEILLANCE IN THE NETHERLANDS: POLIO AND BEYOND Kimberley Benschop, Harrie van der avoort, Harry Vennema, Erwin Duizer National Institute for Public Health and the Environment OBJECTIVES: The aim of the study was to gain insight in the relevance of environmental enterovirus (EV) surveillance (EEVS) for poliovirus exclusion and enterovirus surveillance in the Netherlands as used in combination with clinical EV surveillance (CEVS). METHODS: Sewage samples (1 Liter) were collected from sewages (n= 856, 2005-February 2015) originating from a secondary school or from a residential district (with or without primary school). In addition sewage samples were collected from a primary refugee center (Ter Apel, n= 186, November 2013-February 2015), where refugees from OPV using/PV endemic countries enter the Netherlands. Sewage samples were centrifuged and concentrated to a volume of 1-3 mL by ultrafiltration using Amicon ultrafiltration membranes PM10 in Amicon stirred ultrafiltration. Clinical Enterovirus Surveillance (CEVS, January 2007- February 2015) included Enterovirus positive samples (n=2816) from Dutch virology laboratories sent to the RIVM for exclusion of poliovirus and/ or further characterization of non-polio enteroviruses. Poliovirus is excluded by inoculation of the EV-positive sewage and clinical samples on L20B cells. Non-polio enteroviruses are characterized by sequencing the VP1 gene as described by Nix et al 2006 RESULTS: The EEVS-Bible Belt yielded 483 isolates of which 446 (92.3%) could be typed: EV-A, 422 (94.6%) EV- B, 14 (2.9%) EV-C and 1 (0.2%) EV-D. Poliovirus was found in 2006 (OPV2) and in 2008 (OPV1). Ter Apel yielded 177 EVs of which 158 (88.7%) could be typed: 16 (10.2%) were EV-A, 119 (75.8%) EV-B, and 23 (14.0%) EV-C. OPV-1 was found once in January 2015. For the CEVS, 2076 (94.2%) could be typed; 451 (21.7%) were EV-A, 1565 (75.3%) EV-B, 38 (1.8%) EV-C and 24 (1.2%) EV-D. Poliovirus was found in 25 samples: 11 OPV1, 5 OPV2, and 9 OPV3, all from children who had a documented history of OPV vaccination abroad or who had recently traveled to a country in which OPV is used for routine vaccination. EV-B viruses were frequently detected in the Bible belt and CEVS, while in Ter Apel EV-C viruses were frequently found. Analysis of the monthly distribution of Echovirus 11 found in sewage and clinical showed that clinical summer peaks are preceded by peaks in sewage. CONCLUSIONS: EEVS is supplementary to the CEVS and can be used to predict most likely candidates for the yearly clinical summer peaks. However, more data are needed to investigate how and for what EV types sewage surveillance can be a valuable tool for early warning of new or re-emerging EV types. Europic 2016 165

166 THURSDAY 08/09/2016 ALBERT SABIN LECTURE DETERMINANTS OF RHINOVIRUS ILLNESS SEVERITY James Gern University of Wisconsin-Madison, United States of America Rhinoviruses are best known for causing the common cold, but can cause lower respiratory illnesses and acute episodes of wheezing in some individuals. Risk factors for more severe illnesses include factors related to the virus, host, and environment. The rhinovirus C species is most closely associated with more severe illnesses. This virus does not grow in traditional tissue culture, and this delayed its discovery until 2006, 50 years after detection of the first rhinoviruses. Recent advances have led to development of culture techniques and molecular analysis of RV-C viruses, and insights into their pathogenesis. Host factors affecting illness severity include age, genetics, and the presence of chronic respiratory disease. Environmental factors, including seasonal factors and airway bacteria, have a major influence on illness severity. New developments in understanding rhinovirus molecular virology, host immune responses and environmental determinants of illness severity have led to renewed efforts to prevent or inhibit illnesses caused by this clinically important pathogen. 166 Europic 2016

167 THURSDAY 08/09/2016 G10 A CONTAMINATED ENVIRONMENT IS AN EFFICIENT ROUTE OF TRANSMISSION FOR FOOT AND MOUTH DISEASE VIRUS Claire Colenutt1, Emma Brown1, Noel Nelson2, Jose Gonzales3, Simon Gubbins1 1 The Pirbright Institute, 2Met Office, 3Central Veterinary Institute OBJECTIVES: Foot and Mouth disease virus (FMDV) is a contagious pathogen of cloven hooved animals, which can be transmitted via numerous routes. Transmission through direct contact with infected animals is the most common route of spread of FMDV, but can be prevented by implementation of control measures, such as culling and movement restrictions. The role of indirect contact and fomite transmission is less well understood and more complex to apply blanket control measures to. This study used a series of challenge experiments to determine and quantify the risk of FMDV transmission posed by environmental contamination. METHODS: A series of FMDV transmission experiments were performed in high containment facilities. Seven environmental challenges were undertaken with pairs of calves exposed for 24 hours to environments previously housing FMDV-infected calves. This included one challenge room in which infected calves were housed only during their incubation period. Samples were taken from the contaminated rooms during and for a week after challenge to assess the level of potential exposure and the length of FMDV survival in the environment. RESULTS: Five out of seven environmental challenges resulted in successful transmission of FMDV, including the challenge using an environment contaminated by preclinical, infected calves. In one case where environmental transmission did not occur, no infectious virus was isolated from environmental samples. The second unsuccessful environmental challenge used a contaminated room that was unoccupied for 24 hours between holding clinically diseased calves and the challenge of nave calves. FMDV RNA was detected at consistently high levels over a one week period. Although levels dropped substantially in the same time period, infectious virus could still be detected for six days. CONCLUSIONS: The outcome of these challenge experiments suggests that the environment is an efficient route of transmission for a short period of time. Consequently, an appreciable proportion of transmission within a farm could occur via a freshly contaminated environment, as well as through direct contact. In addition, virus can survive in the environment for an appreciable length of time, although survival time scales will vary depending on environmental conditions. Therefore, risk of environmental transmission alone could be reduced in a short period of time, reduced further if disinfectants are applied. Taken together, our results highlight the need for effective biosecurity on the farm to reduce transmission through environmental routes and thorough decontamination and disinfection of premises prior to restocking with FMDV-susceptible livestock. Europic 2016 167

168 THURSDAY 08/09/2016 G11 FIRST WHO INTERNATIONAL STANDARD FOR ANTI-EV71 HUMAN SERUM Javier Martin1, Gillian Cooper2, Qunying Mao3, Laura Crawt2, Yiping Wang3, Zhenglun Liang3, Miao Xu3, Philip Minor2, Junzhi Wang3 1 NIBSC, 2NIBSC, UK, 3NIFDC, China Hand, foot and mouth disease (HFMD) is a highly contagious disease caused by intestinal virus mostly seen in young children. HFMD is typically characterized by a mild fever followed by a rash of spots and bumps that may blister, involving the skin of the hands, feet, mouth, and occasionally the buttocks and genitalia. The virus is transmitted by oropharyngeal secretions and the faecaloral route. HFMD severe neurologic symptoms are often associated with enterovirus 71 (EV71). EV71 epidemics with significant numbers of fatalities have been reported in Southeast and East Asia. Several EV71 vaccines are under development so the need to establish anti-EV71 serum standards was recognized, to support methods used to measure anti-EV71 antibody levels. They would be useful for vaccine efficacy and seroprevalence studies. A number of serum samples from human donors were used to prepare candidate serum materials (lyophilised and filled in ampoules). Serum samples were used in a collaborative study. Laboratories were asked to characterise them in cell culture neutralization assays using a C4 EV71 reference strain. The samples were also analysed against a panel of EV71 isolates at NIBSC. All three candidate serum samples analysed exhibited good levels of neutralizing antibodies against a wide range of EV71 strains of various genotypes. Between laboratory variations in neutralization titres were significantly reduced when values were expressed relative to those of either of the two candidate sera. Candidate 14/140 was established as the 1st WHO IS for anti-EV71 serum (Human) with an assigned unitage of 1,000 International Units (IU) of anti-EV71 neutralizing antibodies per vial, 14/138 was recommended as a potential replacement for 14/140 in the future, with an assigned unitage of 1,090 IU per vial. Sample 13/238 was established as a WHO Reference Reagent, serving as an assay control for EV71 neutralization assays with an assigned value of 300 IU per ampoule. 168 Europic 2016

169 THURSDAY 08/09/2016 G12 COLD-ADAPTED VIRAL ATTENUATION (CAVA): HIGHLY TEMPERATURE SENSITIVE POLIOVIRUSES AS NOVEL VACCINE STRAINS FOR A NEXT GENERATION INACTIVATED POLIOVIRUS VACCINE Barbara Sanders1, Isabel de los Rios1, Vladimir van Hoek1, Viki Bockstal1, Yutong Song2, Jeronimo Cello2, Eckard Wimmer2, Gillian Cooper3, Laura Crawt3, Javier Martin3, Jerome Custers1, Hanneke Schuitemaker1, Diana Edo Matas1 1 Janssen, 2Stony Brook University, 3NIBSC OBJECTIVE: The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. Our aim was to generate novel attenuated poliovirus strains as a basis for a next generation IPV. METHODS: We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains, by serial passage at low temperature and subsequent genetic engineering, where 31 mutations, identified during cold-adaptation, were combined into one genome as well as incorporation of the capsid sequences of cIPV strains. The three resulting CAVA vaccine candidates were characterized by infectious titer determination, RT-qPCR, western blot, electron microscopy, in vivo neurovirulence and in vivo immunogenicity. RESULTS: The CAVA viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30C, they could be propagated to high titers (9.4 9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non- structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. CONCLUSIONS: The unprecedented temperature sensitivity observed for the CAVA strains is induced by synergistic effect of (a subset of) 14 mutations located in the IRES and Non-structural proteins. Further research is required to pinpoint the exact mechanism of temperature sensitivity. Nonetheless, the inability of CAVA strains to replicate at 37C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV. Europic 2016 169

170 THURSDAY 08/09/2016 G13 M5BT PROTEIN INDUCED M CELL ACTIVATION AGAINST FMDV Dong-Jun An, Se-Eun Choe Animal and Plant Qurantine Agency Foot-and-mouth disease virus (FMDV) has an essential RGD in GH loop which infect vesicles (blisters) in the mouth and feet of bovids, suids, ovids, caprids and other cloven-hoofed animals. The 80 amino acids continuously with RGD motif has make a specific a-helix peptide and it could be cloned representative 5 peptides (amino acid position: 136-162) on a basis of GenBank nucleotides database in this study. Fifteen amino acids on non-structure protein (3A) of FMDV also performed cloning for T-cell activation. M5BT subunit vaccine candidate have made continuously connection representative 5 peptides and fifteen amino acids of 3A protein using pET21a system. This protein was purified to His-Tag peptide and removed lipopolyshaccaride using LPS removal kit. M5BT protein injected each three pigs with twice interval 2 weeks using IM and oral routes. The commercial FMD vaccine also inoculated three pigs as positive control group. Pigs serum with oral route was check all negative to the PrioCHECK FMDV NS ELISA (PI, 50 positive) but the serum of pigs injected IM route showed positive at 2 weeks after first vaccination (PI results: 63, 54, and 20) and at 2 weeks after second vaccination (PI results: 83, 72, and 83). The commercial FMDV vaccine has similarly high PI values at 2 weeks after first vaccination (PI results: 64, 57, and 57) and at 2 weeks after second vaccination (PI results: 74, 55, and 86). Our findings demonstrate that M5BT protein could be induce seropositive for FMDV structure protein to IM route but could not be elicit for M cell activation to oral route. 170 Europic 2016

171 THURSDAY 08/09/2016 G14 EPITOPE ANALYSIS OF MONOCLONAL ANTIBODIES FOR MEASUREMENT OF D ANTIGEN CONTENTS IN THE INACTIVATED POLIOVIRUS VACCINE Tomofumi Nakamura, Daisuke Yokouchi, Keiko Suzuki, Akiko Watanabe, Waka Ushioda, Kazuyoshi Ikuta, Susumu Ochiai The Research Foundation for Microbial Diseases of Osaka University (BIKEN) OBJECTIVES: According to the progress of global poliovirus (PV) eradication program, planned and stepwise switch from oral PV vaccine (OPV) to inactivated PV vaccine (IPV) have been continuing. At the quality control of IPV vaccine manufacturing process, accurate measurement of D antigen level, which induces neutralizing antibody (NAb) response against vaccinee, is most important element. In our foundation, D antigen content of Sabin IPV (sIPV) has been determined by original sandwich ELISA method with in-house prepared specific monoclonal antibodies (MAbs). In this study, we attempted to analyze the epitope sites of MAbs on the surface of PV capsid by utilizing mutant viruses escaped from neutralizing reactions of the serotype specific MAb. METHODS: Specific MAbs against each PV serotypes were obtained from hybridoma cells established by versatile PEG method. The escape mutant viruses were isolated from plaques that formed after two times neutralizing reaction between original viruses and MAbs. Subsequently, we sequenced capsid-coding nucleotides (VP1-VP3) to identify mutated amino acid residues. We generated original and mutant viruses from reverse genetics (RG) technique according to the sequencing results and compared their NAb titer against each specific MAbs. RESULTS: We isolated 28 mutant viruses having 14, 6 and 8 amino acid mutations for PV1, PV2 and PV3 capsid proteins respectively. Then, we have successfully generated total 5 original and 26 mutant viruses from RG technique. The comparison of NAb titer of these viruses showed that at least 14 mutants dramatically lost the interaction between specific MAbs. These results strongly suggested that mutated positions of these strains were the neutralizing epitopes of MAbs. CONCLUSIONS: We could identify several precise recognition sites of specific MAbs. These findings will provide scientific evidence to our ELISA method and quality control of IPV manufacturing. Moreover, we hope that these will accelerate international harmonization of sIPV D antigen assay. Europic 2016 171

172 THURSDAY 08/09/2016 G15 ENVIRONMENTAL SURVEILLANCE FOR POLIOVIRUS IN THE UK. THE USE OF A DEEP SEQUENCING APPROACH. Manasi Majumdar, Dimitra Klapsa, Jackie OBrien, Glynis Dunn, Javier Martin NIBSC The UK has been polio-free for more than 35 years thanks to excellent vaccine coverage and disease surveillance. However, the 2013 annual report of the European Regional Certification Commission identified the risk of extensive transmission of polio in the UK following importation as intermediate. Moreover extensive circulation of wild poliovirus has been recently demonstrated in Israel in the absence of any recognized clinical cases and in a context of high vaccination coverage with IPV, potentiating the value of environmental monitoring for polio. Its use is recommended by WHO and forms an important part of endgame strategies for the global polio eradication program. With this in mind, a pilot study was set up for the surveillance of sewage samples from two different sites in the UK considering various demographic and epidemiological factors to provide greater assurance of the polio-free status. Sewage samples were concentrated using standard protocols and added on to L20B and RD cell lines for virus culture. The resulting isolates were then subjected to deep sequencing analysis using random primers on the Illumina platform (MiSeq). Deep sequencing data analysis strategies were then applied for identifying the viruses producing cytopathic effect. During 14 months surveillance a total of 27 sewage samples were processed. No poliovirus was detected. However, 80% of the sewage samples were positive for non-polio enteroviruses. Higher numbers of enteroviruses were found in sewage samples collected in the winter months. Coxsackievirus B4 and B5 were identified from both sites throughout the surveillance period, while the presence of Echoviruses peaked during the winter months. Coxsackievirus B4, B5, Echovirus 6, 7 and 11 were the most common viruses identified, with the percentage positivity of 21%, 19%, 21%, 12% and 17%, respectively. The presence of Coxsackievirus B2 and Echovirus 3 was appreciated in two samples each. The highest number of viruses (n=5) identified from a single sample showed the presence of Coxsackievirus B2 and B4, and Echovirus 3, 7 and 11. The deep sequencing strategy was found to be very useful in identifying nearly whole genome virus sequences even in samples containing virus mixtures and helped us better understanding the molecular epidemiology of non-poliovirus transmission in humans. In the absence of poliovirus circulation, non-polio enteroviruses, which are potential human pathogens, need to be monitored and therefore it is important to have ongoing surveillance system to correlate epidemiological and clinical data and ensure that this surveillance system will also act as an early detection of poliovirus transmission, should this occur. 172 Europic 2016

173 THURSDAY 08/09/2016 G16 IMMUNOLOGICAL DIVERSITY AMONG TYPE 1 STRAINS OF WILD AND VACCINE-DERIVED POLIOVIRUS Konstantin Chumakov1, Diana Kouiavskaia2, Olga Mirochnitchenko2, Eugenia Dragunsky2, Stephanie Troy2, Liubov Kozlovskaya3, Alexander Lukashev3 1 US Food and Drug Administration, 2Food and Drug Administration, 3Chumakov Institute of Poliomyelitis and Viral Encephalitides The worldwide campaign against poliomyelitis has entered its critical phase taking the world into an uncharted territory. The circulation of wild polioviruses of serotype 2 has been stopped, and the worldwide immunization policy shifted from the use of trivalent Oral Polio Vaccine (OPV) to bivalent OPV containing only two serotypes of poliovirus, 1 and 3. To properly predict the consequences of such a significant change we need to better understand the immunologic relatedness of different vaccine strains. Recent studies of human immune response unexpectedly revealed a significant immunological overlap between three serotypes, with multiple human monoclonal antibodies being able to neutralize more than one serotype of poliovirus, some neutralizing all three. Understanding the role of these cross-neutralizing antibodies in vaccine-induced immunity is very important. On the other hand some reports suggest that highly-divergent poliovirus strains may emerge to resist neutralization by vaccine-induced immunity. How wide is the antigenic diversity within one serotype, and does it have a measurable impact on protection of immunized populations against the disease? In this study we have attempted to address some of these questions. A representative panel of wild and vaccine-derived polioviruses of serotype 1 belonging to several lineages (genotypes) were neutralized with human and animal sera, as well as monoclonal antibodies. When measured against different strains, neutralization titers of the sera differed as much as 30 fold. The heterogeneity among the study population was statistically significant, and the average neutralizability of individual strains differed up to 8-fold. Type 1 wild poliovirus isolated in Congo during 2010 outbreak characterized by unusually high mortality was among the poorly neutralized strains. All 26 subjects in a group of polio-immunized individuals had detectable neutralizing antibodies against Sabin 1 (Geometric Mean Titer = 1:156) and Mahoney (GMT = 1:117) strains, but 12 of them (41%) had no detectable neutralizing antibodies (1:8 or higher) against Congo/2010 strain. The GMT among those who were positive was 1:39. To further investigate the antigenic divergence of the strain we have repeatedly immunized three groups of mice with Mahoney, Sabin 1, and Congo/2010, and used the sera for cross-neutralization. Neutralization titers in homologous neutralization tests were 1:2,500, 1:1,006, and 1:423 for Mahoney, Sabin 1 and Congo/2010, respectively. Despite that, no cross-neutralization was observed between Sabin 1-induced serum and Congo/2010, and Congo/2010-induced serum and Sabin 1 virus. There was a limited cross neutralization of Congo/2010 strain by Mahoney serum (1:14), and Mahoney strain by Congo/2010 serum (1:8). Additional data will be presented to document the virtual absence of cross-neutralization between mouse sera to Sabin 1 and Congo/2010, including virus challenge experiments in Tg mice with antibodies to the strains. While the degree of antigenic divergence of Congo/2010 strain in humans appears to be significant but less dramatic than in mice, the results suggest that caution must be exercised when interpreting the results of seroprevalence studies and drawing conclusions about the protection of human populations. Europic 2016 173

174 THURSDAY 08/09/2016 G17 GLUTATHIONE: A POTENTIAL STABILIZER FOR THE ORAL POLIOMYELITIS VACCINE Rana Abdelnabi, Leen Delang, Johan Neyts KU Leuven University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy OBJECTIVES: Glutathione (GSH) is the most abundant thiol peptide in animal cells which plays a critical role in antioxidation, detoxification of xenobiotics and maintenance of immune system functions. GSH was also reported to have potential therapeutic effects in disease conditions such as cancer, Parkinsons disease and some viral infections such as with HIV. We showed earlier that GSH is an essential host factor for stabilization of the enterovirus particle [including Coxsackievirus B3 (CVB3) and poliovirus (PV)] during viral morphogenesis (Thibaut et al., PLOS Pathogen 2014). We here studied whether GSH has the potential to act as a thermal-stabilizer for oral poliomyelitis vaccine (OPV) formulations. METHODS: Viral infections were performed in BGM cells (end-point titration) and in SCID mice. RESULTS: In an initial experiment, the dose-response effect of GSH on thermal-stability of the three types of poliovirus Sabin strains (PVs) was determined by incubating a high titered stock of the virus for 30 min at 48C. The three types of PV were significantly protected from heat-inactivation in a concentration-dependent manner at GSH concentration ranging from 2-50 mM. Next, the effect of GSH on the thermal-stability profile of the PVs was further explored by incubating PV1, PV2 and PV3 Sabin strains at temperatures ranging between 42C and 56C for 2 min in the presence or absence of 20mM GSH. GSH markedly protected the three strains from heat- inactivation at all tested temperatures, whereas at temperatures 53, 56 and 51C the the titers of the untreated PV1, PV2 and PV3 (respectively) were reduced to under the detection limit. To study whether the stabilizing effect of GSH was also still observed with longer incubation periods, the PV1 strain was incubated at 44, 46, 48 and 50C for 6 hours in the presence or absence of 20mM GSH. GSH markedly protected PV1 from heat-inactivation at 44C and 46C and an effect was still observed although less pronounced at 48C and 50C. Next, CVB3 was used as a surrogate for PV to evaluate whether the GSH-stabilized virus remained infectious in vivo. SCID mice that had been infected with virus heated in the presence of GSH developed disease as rapidly as mice that had been inoculated with the control unheated virus [with the virus being detectable in the target organ]. On the other hand, mice infected with virus heat-inactivated in the absence of GSH did not develop disease. Surprisingly, the combination of GSH with high concentrations of the commercial OPV stabilizer i.e. MgCl2 resulted in antagonistic effect and partial loss of GSH stabilizing activity. CONCLUSION: GSH is a potent thermal-stabilizer for different poliovirus Sabin strains. The facts that GSH is naturally present in human cells and that it has many beneficial therapeutic applications make it a safe and efficient candidate stabilizer for OPV formulations. Formulations of OPV with GSH may allow to use OPV out of the cold chain for longer periods of time. 174 Europic 2016

175 THURSDAY 08/09/2016 G18 INACTIVATED POLIO VACCINE AND THE ENDGAME OF POLIO ERADICATION Javier Martin, Thomas Wilton, Alison Tedcastle, Laura Crawt, Gillian Cooper, Philip Minor NIBSC With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine (OPV) assume greater importance. For this reason, the use of inactivated polio vaccine (IPV) will become more relevant and universally used. We have characterised both conventional IPV (cIPV) based on wild poliovirus strains and Sabin IPV (sIPV) based on OPV strains in terms of their antigenicity and immunogenecity with a view to standardise potency assays and estimate vaccine efficacy against recently isolated wild and vaccine-derived poliovirus (VDPV) strains and viruses with highly modified antigenicity. Various batches of cIPV and sIPV from different manufacturers were analysed. OPV, wild and VDPV strains of the three poliovirus serotypes were used. Standard cell culture neutralization and ELISA tests were used to study the reactivity of viruses and vaccines with anti-polio monoclonal antibodies (MAbs). In vivo potency assays in rats were used to measure vaccine immunogenecity. The efficacy of IPV at preventing paralysis induced by selected viruses was evaluated using an immunisation-challenge model in transgenic mice expressing the human poliovirus receptor (Tg21-bx). The degree of antigenic modification induced by formaldehyde varied between IPV products which means that the use of different MAbs for D-Ag measurement can result in remarkable differences in relative potencies. There is a need to further standardize in vitro potency assays for IPV so different products can be compared. Both cIPV and sIPV products induced low levels of neutralizing antibodies in rats and Tg21-bx mice against PAK5388, iVDPV-102050 and FIN23127 poliovirus strains containing various antigenic changes. Both vaccines protected immunised mice against challenge with paralytic doses of virus but protection was incomplete against virus strains iVDPV-102050 and FIN23127 and less efficient with sIPV vaccination. It is likely that current IPV products protect the population against paralytic disease caused by any poliovirus strain but it is also possible that antigenically atypical viruses could circulate in populations only using IPV as recently described in Israel for wild type 1 poliovirus. IPV based on the Sabin strains, encouraged by WHO for reasons of environmental safety, appears to be less effective. It is important to test the efficacy of IPV products against poliovirus strains with different antigenic profiles in pre-clinical and clinical studies as immune responses against some of these strains seem to vary significantly. Europic 2016 175

176 THURSDAY 08/09/2016 G19 DEVELOPMENT OF NON-POLIO ENTEROVIRUS VACCINES BASED ON THE POLIOVIRUS VACCINE PRODUCTION PLATFORM Dinja Oosterhoff, Aart van t Oever, Bella Monica, Maarten de Vries, Wouter van den Braak, Matthijn de Boer, Yvonne Thomassen, Wilfried Bakker Intravacc Intravacc has a long standing experience in the production of poliovirus vaccines. The poliovirus belongs to the group of enteroviruses which is a member of the Picornavirus family. So far, more than 100 types of enteroviruses have been isolated from man, and several of these enteroviruses, like EV-A71, CV-A16 and EV-D68, can cause serious diseases. Since all enteroviruses have a comparable structure and genetic composition, we investigated whether non-polio enteroviral vaccines could be easily developed using the poliovirus vaccine production process as a platform process. A panel of human enteroviruses belonging to different species, was selected and it was determined if these viruses could replicate on Vero cells, an African Green Monkey cell line that has been approved for the production of viral vaccines. After the production of viral working seed lots, Vero cells were grown on micro-carriers in bioreactors, infected at an MOI of 0.01 and virus containing medium was harvested when full cytopathic effects were observed. The enteroviruses were concentrated, purified by size-exclusion chromatography and inactivated with formaldehyde. We could conclude that all enteroviruses capable of growing on Vero cells could be produced into vaccines using the poliovirus vaccine production process as a starting point, although production conditions of vaccines for each individual virus need optimization. In a follow up study an infectious clone of EV-A71 was constructed and vaccines are being produced from a 10L bioreactor that will be tested in vivo in mice. Altogether, these data suggest that it is possible to use the poliovirus vaccine production process as a platform process for fast-track production of other non-polio enterovirus vaccines. 176 Europic 2016

177 THURSDAY 08/09/2016 G20 STRATEGIES TO STABILISE POLIOVACCINE GENOMES AGAINST RECOMBINATION Matt Smith1, Erika Bujaki1, Ming Te Yeh2, Raul Andino2, Andrew Macadam1 1 NIBSC, 2University of California We have previously shown it is possible to genetically stabilise the key attenuating mechanism in Sabins polio vaccine strains (a) against mutation by altering the sequence of RNA domain V in the 5UTR and (b) against loss through recombination by relocating the essential cis-acting replication (cre) element upstream of domain V. Using a cell culture recombination assay this strategy has been shown to significantly increase the genetic stability of an attenuated virus replicating in the presence of a wild type strain. Conditions used were those under which the related Sabin vaccine strain reverted to wild-type either by mutation or by replacing its 5end with that of the wild-type. Other recombination events occurred to similar degrees in all strains but none resulted in replacement of the native or disabled cre in the 2C gene. Polymerase modifications, selected for their impact on recombination and fidelity, are also under assessment in this model. Live attenuated polio vaccines remain part of the strategy for control of outbreaks if they occur after eradication; genetically-stable strains, less capable of evolution into wild-type viruses, will provide a safer option than current vaccines. Europic 2016 177

178 THURSDAY 08/09/2016 G21 INVESTIGATION OF A CHIMERIC SAT2 FOOT-AND-MOUTH DISEASE VIRUS EXHIBITING AN INCREASE IN CAPSID THERMOSTABILITY Julian Seago1, Abhay Kotecha2, Eva Perez3, Fuquan Zhang3, Serban Ilca2, Dave Stuart2, Elizabeth Fry2, Francois Maree4, Katherine Scott4, Michiel Harmsen5, Valrie Mioulet3, Nick Juleff3, Bryan Charleston3 1 The Pirbright Institute, 2Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive Oxford OX3 7BN, United Kingdom, 3The Pirbright Institute, Woking, Surrey, GU24 0NF, United Kingdom, 4Transboundary Animal Disease Programme, ARC-Oderstepoort Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa, 5Central Veterinary Institute Wageningen UR, Division Virology, P.O. Box 65, 8200 AB Lelystad, The Netherlands Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock causing foot-and-mouth disease (FMD) and severe economic impact. FMDV exists as seven distinct serotypes with numerous subtypes within each serotype dictating the requirement to match vaccine strains to those circulating in the field. In addition FMDV, particularly the South African Territories (SAT) serotypes, are thermally unstable and the viral capsid readily dissociates into non-immunogenic pentameric subunits which can compromise the effectiveness of FMD vaccines. Recombinant DNA technology facilitates the development of infectious FMDV clones that can be genetically manipulated to overcome such obstacles. Here we report the construction of a chimeric clone between the SAT2 and O serotypes. Characterisation of the chimeric virus showed growth kinetics equal to that of the parental SAT2 virus but a comparative increase in thermostability. Residues that mediate capsid stability of the chimeric virus were identified within the VP4 structural protein. 178 Europic 2016

179 THURSDAY 08/09/2016 G22 MODIFIED SABIN 2 POLIOVIRUSES FOR USE AS AN ORAL-LIVE ATTENUATED VACCINE POST-ERADICATION Matthijn de Boer1, Andrew Macadam2, Raul Andino3, Cara Burns4, Wilfried Bakker1 1 Intravacc, 2NIBSC, 3UCSF, 4CDC Poliovirus is a crippling and potentially fatal infectious human disease for which adequate vaccines are available. Considering these facts the Global Polio Eradication Initiative was launched in 1988 to eradicate and contain wild, vaccine-related and Sabin polioviruses worldwide. Trivalent Oral Poliovirus Vaccine (tOPV) has been instrumental in this polio eradication program since it is safe, effective, and induces long-lasting immunity. Strains in this live OPV replicate in the human gut and are excreted for several weeks after immunization. However, during this period the attenuating mutations in the vaccine strains may revert and vaccine-derived poliovirus (VDPV) may emerge, spread and, in rare cases, cause vaccine-associated paralytic poliomyelitis (VAPP). Therefore, in the post-eradication era OPV will be replaced by Inactivated Poliovirus Vaccines (IPV). For poliovirus type 2 the switch has been made in April 2016, coordinated by the WHO. In case of a poliovirus outbreak in the post- eradication era stockpiles of OPV need to be available to contain and eliminate the outbreak efficiently. Current OPV is not preferred due to possible VDVP release post-eradication, and IPV is not preferred for outbreak control due to the higher costs per dose and the more complex administration route. Therefore, new OPV type 2 strains are developed that are extremely genetically stable and hyper-attenuated and are considered as replacement candidates of the current Sabin OPV type 2. In this contribution, these new oral poliovirus concepts, provided to Intravacc by partner institutes, are produced and evaluated. The concepts are evaluated, for example, using deep sequencing to confirm genetic stability after passaging in Vero cell culture. The production of the pre-seed, from a single plaque, and preparation of master- and working virus seeds are discussed. The vaccine candidate virus seed lots were produced using an innovative animal component free Vero platform production process under cGMP for evaluation in clinical trials. This work was kindly supported by the Bill and Melinda Gates Foundation (Grant numbers: OPP1118489 and OPP1119267). Europic 2016 179

180 THURSDAY 08/09/2016 G23 ATTENUATION OF PICORNAVIRUS GROWTH BY ENGINEERED POLYMERASE VARIANTS Olve Peersen1, Seth McDonald1, Elizabeth Caine2, Stphanie Beaucourt3, Jorge Orosio2, Marco Vignuzzi3 1 Colorado State University, 2University of Wisconsin, 3Institut Pasteur Positive strand RNA viruses replicate via virally encoded RNA-dependent RNA polymerases (RdRPs) that employ a unique palm domain movement to close their active sites and establish the canonical geometry needed for a two-metal catalytic mechanism (Gong & Peersen, PNAS, 2010). This palm-based movement enables the viruses to readily fine-tune their replication kinetics in order to create the appropriate quasispecies distribution of genetic variants. Prior work by our group has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background (Campagnola et al., J. Virology, 2015). Interestingly, either effect attenuates virus growth in vivo, explicitly pointing out the importance of a maintaining a proper quasispecies distribution for virus pathogenesis. In the work presented here we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel. Inspection of polymerase structures in the open and closed states of the active site led us to targeted mutagenesis of Phe364, an absolutely conserved residue in enteroviral polymerases that forms an important structural interaction within Motif D and undergoes a sliding motion during active site closure and. We generated a panel of Phe364 mutations in coxsackievirus B3 3Dpol and analyzed their effects by crystallography, stopped- flow kinetics, quench-flow reactions, and infectious virus studies with sequencing of the progeny population (McDonald et al., J. Biol. Chem, 2016). Mutating Phe364 to tryptophan resulted in a genetically stable high-fidelity coxsackievirus B3 variant with significantly reduced pathogenesis in mice. We have now extended the biochemical analyses of this mutant to several other enteroviral polymerases, including enterovirus 71, coxsackievirus A6 and A16, type C human rhinovirus, and poliovirus, and in all cases the biochemical data predict F364W to be a fully functional but high-fidelity polymerase. Infectious virus studies in a mouse-adapted EV71 background show that the mutant also significantly reduces pathogenesis in that virus. The data demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis and further illustrate the importance of the palm domain movement for RdRP active site closure. Data from coxsackievirus B3 and EV71 suggest that mutating the conserved Phe364 to tryptophan may significantly attenuate multiple enteroviruses. For structural and biochemical reasons this engineered mutation presents a very high barrier to reversion that likely explains its genetic stability, making it a strong candidate for live-attenuated vaccine development. 180 Europic 2016

181 THURSDAY 08/09/2016 G25 TOLL LIKE RECEPTOR [TLR] 3 AGONIST POLY I:C AS AN ADJUVANT IN FMD VACCINE ENHANCED PROTECTION OF CATTLE AGAINST VIRULENT VIRUS CHALLENGE Satya Parida1, Katie Lloyd-Jones2, Mana Mahapatra2, Rebeca Herbert2, Krupali Parekh2, Aravindh Babu3, David Paton2, Geraldine Taylor2, Satya Parida2 1 The Pirbright Institute, 2TPI, 3NIAB Virus neutralizing (VN) antibody is important for protection of animals against foot-and-mouth disease (FMD) but antibody titres are not fully predictive of protection. Furthermore, we have shown that FMDV vaccines that induce similar levels of neutralizing antibodies can have different levels of T-cell responses and confer different degrees of protection against FMDV infection. We have shown a positive correlation between IFN- response and vaccine induced protection as well as reduction of long term persistence of FMD virus was observed. When combining this IFN- response in re-stimulated blood with VN antibody titers in serum on the day of challenge, a better correlation of vaccine-induced protection with IFN- and VN antibody was predicted. Our investigations showed that CD4+ T-cells are the major proliferating phenotype and IFN- producing cells.Recently in our CIDLID (BBSRC and DFID) funded grant incorporating a molecular adjuvant (Toll like receptor [TLR] 3 agonist polyinosinic:polycytidylic acid [Poly I:C]) in to the existing formulation of FMD vaccine (A serotype) we demonstrated enhanced CD8+ and CD4+ T cell responses. In addition, all the vaccinated animals were protected in this group, whereas the conventional vaccine formulated with an oil-in-water adjuvant (ISA206) did not protect following challenge. Similarly, a significant increase in VN antibody titer (VNT) was observed for the poly I:C group in comparison to other adjuvant groups including the conventional vaccine group (ISA-206). To measure the protective effect of adjuvant in these experiments we had reduced the antigen pay load from 10 g to 2.5 g in each experimental group. Therefore, from our above recent work it is evident that incorporation of poly I:C as an adjuvant into the existing FMD vaccine formulation, the potency and longevity can be significantly increased. In conclusion, we have identified a molecular adjuvant for FMD vaccine that enhances immunity. Therefore we are currently investigating further the longevity of immunity provided by new vaccine formulation. Europic 2016 181

182 THURSDAY 08/09/2016 G28 IMMUNOGENICITY OF ORAL POLIO VACCINE AGAINST XINJIANG IMPORTED TYPE 1 WILD POLIOVIRUS Dongmei Yan, Dongyan Wang, Yong Zhang, Shuangli Zhu, Wenbo Xu Institute for viral disease control and prevention, Chinese center for disease control and prevention OBJECTIVES: An outbreak of type 1 wild-type poliovirus (WPV) imported from Pakistan occurred during July- October, 2011, in Southern Xinjiang, China. Although both the rapid oral polio vaccine (OPV) coverage survey and the seroprevailence of poliovirus neutralization antibody against Sabin 1 strain (vaccine strain of OPV) showed that local immunity status of OPV was not extremely concerning, type 1 WPV finally broke the OPV immunity barrier and caused 21 WPV cases. Therefore, we aimed to investigate whether the immunity induced by OPV was adequate to protect against Xinjiang type 1 wild poliovirus in populations. METHODS: Neutralization antibody titres against Xinjiang type 1 wild poliovirus and Sabin 1 were measured in 121 sera from 2 groups of children (Group 1 for Xinjiang children and Group 1 for Heilongjiang children) and 1 group of adults (Group 3). Additionally, 50 sera from one of these two child groups after OPV booster and 26 sera from the adult group after IPV booster were analyzed. The neutalization assay against poliovirus was performed according to WHO guidelines. RESULTS: The neutralization antibody titres against Xinjiang type 1 wild poliovirus in all 197 measured sera, regardless of grouping and pre- or post- vaccine booster, were significantly lower than those against Sabin 1. Notably, 28.9%, 9.4% and 41.7% persons respectively from Group 1, 2 and 3 had antibody titres against Xinjiang type 1 wild poliovirus less than 1:4, thus presuming they were not protected, although the majority of the measured titres against Sabin 1 ranged between 1:128 and 1:1024. Compared with the 2 children groups, the adult group had the lowest seroprevalence and geometic mean titers (GMT) in both WPV1 (41.7% and 4.8) and Sabin 1 (83.3% and 33.9) analysis. CONCLUSION: The low or undetectable of neutralizing antibodies against Xinjiang type 1 wild poliovirus in children and adults with a history of OPV might be involved with type 1 wild poliovirus infection. Although the infected individuals do not necessarily develop poliomyelitis due to immunity memory, non-inhibited virus excretion could pose a risk of the wild poliovirus transmission in population. 182 Europic 2016

183 THURSDAY 08/09/2016 G29 IS CAPSID PROTEIN VP4 AN ANTIGENIC SWITCH IN FMDV? ENGINEERING A MATURATION CLEAVAGE TO GENERATE RECOMBINANT PENTAMERS WITH DIFFERENT PROPERTIES Joseph Newman, Tobias Tuthill The Pirbright Institute OBJECTIVES: The Aphthovirus, foot-and-mouth disease virus (FMDV) assembles capsids from 60 copies of a structural protein precursor known as P1-2A. This precursor is processed by the viral 3C protease into the capsid proteins VP0, VP3 and VP1 which remain associated as a protomer, and multimerise into a pentameric intermediate on the pathway to assembling into an icosahedral capsid. The maturation of the virus occurs after a cleavage on the inside of the capsid which generates the structural proteins VP4 and VP2 from the precursor VP0. Upon virus entry into cells, capsids disassemble into pentamers that no longer contain VP4. Pentamers from the disassembly pathway are therefore thought to have different properties than pentamers found on the assembly pathway. Inactivated virus vaccines that have dissociated into pentamers without VP4 have an altered antigenic profile, which correlates with a corresponding lack of protection conferred by the vaccine. The objective of this work was to generate pentamers with and without VP4 from recombinant material and investigate whether the loss of VP4 changed their sedimentation and antigenic characteristics. METHODS: We have developed a cell-free assay for analysing the assembly of pentameric capsid intermediates by expressing FMDV structural precursors in mammalian cell lysates followed by addition of purified 3Cpro derived from expression in E. coli. A maturation-like cleavage event was engineered into recombinant FMDV pentamers by the introduction of a new cleavage recognition site (from the picornavirus, human rhinovirus) into the VP4/2 junction, to allow processing at this site to be controlled. The sedimentation and antigenic properties of assembly products have been analysed by sucrose density gradient sedimentation, and immuno-asssay, using a panel of monoclonal antibodies to probe the antigenic epitopes on the pentamer surface. RESULTS: The HRV 3C protease cleavage recognition site engineered at the FMDV VP4/2 junction allowed cleavage to be controlled independently of processing at the FMDV 3C protease cleavage sites. Cleavage at the engineered site resulted in the loss of VP4 from pentamers and an altered pentamer sedimentation from that expected of assembly pentamers (14S) to that expected of disassembly pentamers (12S). Probing with a panel of monoclonal antibodies also showed altered antigenic characteristics of these 12S pentamers. CONCLUSIONS: These experiments reveal that the VP4/2 junction is accessible to exogenous protease when present in an assembled pentamer and that cleavage at this site released VP4 from the pentamer creating a pentamer with sedimentation expected of pentamers from the capsid disassembly pathway. These pentamers also had altered antigenic properties suggesting that the loss of VP4 is the reason for reduced protection conferred by vaccine preps with a high proportion of 12S material. Europic 2016 183

184 THURSDAY 08/09/2016 G31 VIRAL SHEDDING PATTERNS IN ENTEROVIRUS MENINGITIS IN CHILDREN AND ADULTS OVER A THREE YEARS EPIDEMIOLOGICAL SURVEILLANCE PERIOD IN SWITZERLAND Samuel Cordey1, Manuel Schibler1, Arnaud G. LHuillier1, Nomie Wagner1, Ana Rita Gonalves1, Juan Ambrosioni2, Sandra Asner3, Lara Turin1, Klara Posfay-Barbe1, Laurent Kaiser1 1 Geneva University Hospitals, 2Hospital Clinic Barcelona, 3Centre Hospitalier Universitaire Vaudois Several enterovirus (EV) genotypes can result in aseptic meningitis, but their routes of access to the central nervous system remain to be elucidated and may differ between the pediatric and adult populations. In this study, we assess the pattern of viral shedding in pediatric and adult subjects with acute EV meningitis and provide EV surveillance data for Switzerland. All pediatric and adult subjects admitted to the University Hospitals of Geneva with a diagnosis of EV meningitis between 2013 to 2015 were enrolled. A quantitative EV real-time reverse transcriptase (rRT)-PCR was performed on the cerebrospinal fluid (CSF), blood, stool, urine and respiratory specimens to assess viral shedding and provide a comparative analysis of pediatric and adult populations. EV genotyping was also systematically performed. EV positivity rates differed significantly between pediatric and adult subjects; 62.5% of pediatric cases (no adult case) were EV-positive in stool and blood for subjects for whom these samples were all collected. Similarly, the viral load in blood was significantly higher in pediatric subjects. C-reactive protein levels and the number of leucocytes/mm3 in the CSF were lower and higher in non-viremic than in viremic pediatric subjects, respectively. A greater diversity of EV genotypes was observed in pediatrics cases, with echovirus 30 and coxsackievirus B5 representing the two most frequent genotypes detected in both populations. In contrast to adults, EV-disseminated infections are predominant in pediatric subjects and show different patterns of EV viral shedding. This observation may be useful for clinicians and contribute to modify current practices of patient care. 184 Europic 2016

185 THURSDAY 08/09/2016 G32 PREVENTION OF FOOT-AND-MOUTH DISEASE IN CATTLE USING A PRIME-BOOST- VACCINATION STRATEGY Graham Belsham1, Maria Gullberg1, Louise Lohse1, Anette Btner1, Gerald McInerney2, Alison Burman3, Terry Jackson3, Charlotta Polacek1 1 DTU National Veterinary Institute, 2Karolinska Institutet, 3The Pirbright Institute Foot-and-mouth disease (FMD) is one of the most economically important infectious diseases of production animals globally. Vaccination can help to control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in mammalian cell culture under high containment. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a single cycle packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. In cattle vaccinated once with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV- FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge. Following challenge with FMDV, these cattle were protected against disease and no FMDV RNA was detected in their sera. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed and clinical disease occurred. This prime-boost system, using reagents that can be generated outside of high-containment facilities, offers significant advantages to achieve control of FMD by vaccination. Europic 2016 185

186 THURSDAY 08/09/2016 G33 ENVIRONMENTAL SURVEILLANCE IN SEWAGE SAMPLES WITH ULTRA-DEEP SEQUENCING: RESULTS OF A ONE-YEAR PILOT STUDY (CLERMONT-FERRAND, FRANCE, 2014-2015). Maxime Bisseux1, Jonathan Colombet2, Audrey Mirand3, Jean-Luc Bailly4, Ccile Henquell3, Didier Debroas2 1 EA4843-EPIE, Universit dAuvergne - LMGE-CNRS 6023, Universit Blaise Pascal, 2LMGE-CNRS 6023, Universit Blaise Pascal, 3 EA4843-EPIE, Universit dAuvergne - CHU de Clermont-Ferrand, Centre National Rfrence des entrovirus, 4EA4843-EPIE, Universit dAuvergne In certified polio-free regions, environmental surveillance is a supplemental strategy to complete the certification process of poliomyelitis eradication and can substantially improve our knowledge of the epidemiological dynamics of the non-polio enterovirus (EV) types. OBJECTIVE: We set up a global methodology of EV surveillance in waste water to explore the dynamics of EV in a French urban community. METHODS: Raw and treated waste water from a sewage treatment plant (capacity of 425,000 people) in Clermont- Ferrand (France) was sampled two weeks apart during 12 consecutive months (October 2014-September 2015). The method of viral concentration included tangential flow ultrafiltration and polyethylene glycol precipitation. All the raw water samples were positive for RNA EV with an in-house qRT-PCR (Volle et al, 2012) and the EV VP1 sequence was analysed through gene amplification using oligonucleotide primers derived from those described earlier (Nix et al, 2006). Amplicon sequencing was performed with Illumina MiSeq technology, 2 x 250 nucleotides paired-end sequencing. The data were processed by a pipeline derived from PANAM (Phylogenetic Analysis of Next-generation Amplicon) developed in the laboratory (Taib et al, 2013). Sequences having successfully pass the length and quality filters were clustered in representative OTUs with a threshold of 2% of difference. Taxonomic affiliation was performed with a tree based method using a homemade sequence database containing the largest known EV diversity. The analyses of treated water samples are in process. RESULTS: The Illumina MiSeq generated > 1.3 million sequences and 4393 OTUs. OTU sequences were assigned to three EV species (A, B and C) over the 12 consecutive months, within all water samples. The overall 48 distinct EV types were distributed as follows: EV-A species (n=13), EV-B (n=23), and EV-C (n=12); mean number of EV types per sample (n=27). CV-A16, CV-B5 and CV-A22 were the most frequent types in species A, B and C, respectively. The distribution of EV types in sewage samples was compared with that determined in clinical samples during the same period for the same population. Coxsackievirus B5 was detected over the whole sampling period and was the most frequent EV type involved in acute meningitis during the 2015 summer season. By contrast, during the same study period, CV-A16 was not the most predominant type identified through the surveillance of hand, foot and mouth disease. Similarly, EV-C associated infections (EV-C99 and C109) were only detected twice among hospitalised patients. CONCLUSION: NGS analysis combined with tangential flow filtration ensures consistency in the detection of enteric viruses in sewage water without cell culture. Monitoring of EV excretion using analytical NGS can provide important spatial and temporal trend data in three important contexts: providing essential evidence for the absence of poliovirus transmission in the general population, exploring the global epidemiological patterns of non-polio EV types and their relevance to epidemic diseases, and addressing challenges in relating water contamination to human health. 186 Europic 2016

187 THURSDAY 08/09/2016 G34 VISUALIZING PATTERNS OF SEQUENCE EVOLUTION: EXAMPLES AND APPLICATION TO LARGE POLIO AND OTHER ENTEROVIRUS DATABASES Jaume Jorba, Kun Zhao, Kelley Bullard, Jane Iber, Beth Henderson, Steve Oberste, Cara Burns CDC Worldwide circulation of wild poliovirus (WPV) has reached an all-time low. Only a few transmission chains from a single WPV1 genotype remain to be stopped. However, virus surveillance remains a critical tool as a progress indicator, including environmental surveillance for both WPV and vaccine-derived polioviruses (VDPVs). For about 20 years, genetic analysis of the VP1 capsid region has been used for deducing transmission pathways. Learning from this experience, we have developed bioinformatics pipelines and applications that improve integration of large genetic and epidemiologic databases and extend the analysis to wider sequence intervals. We obtained two critical outcomes: first, we automated the integration and analysis of genetic, temporal, and geographic information from large databases, decreasing preparation time and second, we were able to unify a wide range of different database formats. We also developed a reliable Matlab script wrapped around an easy-to-use graphical user interface (GUI) that improves the visualization of patterns of sequence evolution in a phylogenetic context. The script reveals the substitution dynamics at the nucleotide and amino acid level and accurately tracks the evolutionary history along phylogenetic trees. In summary, we have applied new bioinformatics approaches to study the phylodynamics and phylogeography of large polio and other enterovirus databases. For example, we implemented automated phylogeographic analysis for identifying local reservoirs and cross border transmission of WPV1 endemic to Pakistan and Afghanistan. Results from these analyses are frequently updated and provide critical support for targeting immunization campaigns in the region. Europic 2016 187

188 THURSDAY 08/09/2016 G35 THE BACULOVIRUS AVENUE FOR VLP-BASED FOOT-AND-MOUTH DISEASE VIRUS VACCINES Claudine Porta1, Holger Hoenemann2, Eva Perez1, Silvia Loureiro3, Alexandra Jimnez Melsi2, Liz Fry4, David Stuart4, Ian Jones3, Erwin van den Born2, Bryan Charleston1 1 The Pirbright Institute, 2MSD Animal Health, 3University of Reading, 4University of Oxford OBJECTIVES: Amongst picornaviruses foot-and-mouth disease virus (FMDV) remains a challenge for vaccine manufacture. Due to its highly infectious nature and airborne transmissibility, high containment facilities are required for virus growth, before chemical inactivation is undertaken. Virus-like particles (VLPs) offer the prospect of safe alternative vaccines that could be made in standard production facilities. Construction of FMDV VLPs is however dependent on the ability to synthesise capsids from inherently unstable particles, in the absence of a stabilising viral RNA content. In addition their expression system needs to be scalable in a cost-effective manner to allow substitution for the currently utilised killed virus preparations. Expression of empty particles in insect cells, mediated by baculovirus amplification, could satisfy production cost requirements, as well as offer the freedom to engineer capsids for enhanced stability since they no longer need to allow for genome release. METHODS: The capsids derived from 3 serotypes of FMDV were produced in insect cells infected with appropriate recombinant baculoviruses. They comprise a single capsid stabilising mutation devised through analysis of the three-dimensional structure of the corresponding virions. Crude preparations were used for antigen detection and quantitation by immunoassays. After formulation with adjuvant, VLP-based vaccines were used to immunise groups of 5 guinea pigs. Purified VLPs were analysed by electron microscopy (EM). RESULTS: Production of VLPs was undertaken in several insect cell lines. VLP assembly was confirmed by EM following particle purification. Selective/differential ELISA before and after heat treatment allowed demonstration of the stability of the engineered VLPs. The sera of guinea pigs immunised with VLPs yielded neutralising antibodies at titres comparable to those of control groups vaccinated with inactivated virus. CONCLUSION: Results obtained in experimental animals pave the way for immunisation trials in a target species (cattle). Yields of VLPs indicate that the synthesis process would be compatible with industrial production. Extension to further strains/challenging serotypes can be undertaken in order to create targeted multivalent vaccines. 188 Europic 2016

189 THURSDAY 08/09/2016 G36 DETECTION OF ANTIBODIES AGAINST CONSERVED CAPSID EPITOPES PROVIDES A UNIVERSAL SEROLOGY ASSAY FOR DIAGNOSIS OF FMDV Amin Asfor1, Santina Grazioli2, Emiliana Brocchi Brocchi2, donald King3, Satya Parida Parida4, Toby Tuthill5 1 Pirbright Institute, 2Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia Romagna, Brescia, Italy, 3The Pirbright Institute, 4 The pirbright institute, 5The pirbright Institute Foot-and-mouth disease virus (FMDV) causes one of the most economically important livestock diseases worldwide. FMDV exists as 7 serotypes and evolves rapidly so that circulating viruses display high levels of antigenic variation. Diagnosis of FMD employs a range of approaches including serological tests to detect FMDV specific antibodies. Conventional serology based diagnostics are reliable and rapid but require tests specific to each virus serotype. A number of monoclonal antibodies (mAbs) have previously been reported with cross- reactivity against multiple FMDV serotypes. Some of these mAbs have been mapped to the highly conserved N terminus of FMDV capsid protein VP2, suggesting that such conserved sequences might be useful diagnostic reagents. The aim of this study was to assess the potential of conserved N-terminal sequences of capsid proteins VP2 and VP4 as universal epitopes for the detection of FMDV specific antibodies against multiple FMDV serotypes. Synthetic peptides of various lengths (15 to 45) were used to represent the conserved target epitopes at the N terminus of VP2 (VP2N) and N terminus of VP4 (VP4N). A panel of mAbs with existing evidence for cross-serotype activity and sera from cattle infected with each of the 7 serotypes of FMDV were tested for reactivity against the peptides by indirect peptide ELISA. Three mAbs showed strong reactivity to VP2N peptides, including to the shortest peptide tested (the first N-terminal 15 amino acids) suggesting this contained the epitope for these antibodies. Cattle sera against all 7 serotypes of FMDV reacted strongly with VP2N peptides and also with VP4N peptides demonstrating the peptides are indeed able to function as universal detection reagents for FMDV specific antibodies. This study demonstrates that conserved peptide epitopes in the FMDV capsid can be used as serotype-independent antigens for FMD serology. This may have utility in the development of new universal and rapid laboratory or field- based diagnostic tests. Europic 2016 189

190 THURSDAY 08/09/2016 G37 THE LAST MILE: DIAGNOSTIC ASSAY DEVELOPMENT FOR THE FINAL PHASE OF POLIO ERADICATION Nancy Gerloff1, Hong Sun2, Chelsea Maher2, Mark Mandelbaum2, William A. Nix2, Sohail Zaidi3, Shahzad Shaukat3, Lerato Seakamela4, Mark S. Oberste2, Everardo M. Vega2 1 Centers for Disease Control and Prevention, 2Centers for Disease Control and Prevention, Polio and Picornavirus Laboratory Branch, 3 National Institute of Health-Pakistan, 4National Institute for Communicable Diseases-South Africa Poliovirus is on the verge of global eradication. In 2015, there were 74 cases of wild poliovirus cases in the world, all occurring in the endemic countries of Afghanistan and Pakistan. As of May 2016 there were a total of 16 confirmed wild poliovirus cases, restricted to the same two countries. The primary tool for polio eradication has been the live, attenuated, oral polio vaccine (OPV). However, attenuating sites in the vaccine strains may revert, resulting in neurovirulent vaccine-derived polioviruses (VDPV) that have caused outbreaks in a number of countries. Of the three wild poliovirus (WPV) types, only type 1 is circulating. WPV type 3 was last detected in November 2012 in Africa. WPV2 was last detected in Asia in 1999 and was declared eradicated in 2015, allowing removal of type 2 from the oral poliovirus vaccine in 2016 to prevent emergence of type 2 VDPVs. Diagnostic testing for poliovirus has reached a critical stage where the eradication program cannot afford to miss any WPV or VDPV. As circulating polioviruses continue to evolve, there is a need to redesign the molecular diagnostic assays that are used in ~100 laboratories in the Global Polio Laboratory Network (GPLN). In addition, the removal of live type 2 poliovirus from OPV means it is urgent to detect any type 2 poliovirus, to facilitate a rapid response. In order to improve diagnostic sensitivity and specificity, the standard polio diagnostic assays were redesigned to identify the wild type 1-2015 variant, and all type 2 polioviruses. The wild poliovirus type 1 assay was designed in silico to detect all wild PV1s circulating in Afghanistan and Pakistan, and the type 2 assay was designed to detect vaccine, VDPV, and the WPV2 strain used in production of inactivated polio vaccine. In addition, the run conditions were re-optimized. Assays, in a full kit format and using a commercial real-time RT-PCR mastermix, were empirically tested against a standard virus panel (n = 184) representing all types, including WPV, VDPV, non- reverted vaccine-related strains as well as non-polio enteroviruses and negative culture supernatants. The assays were also evaluated at NICD-South Africa (n = 67) and NIH-Pakistan (n = 170). Results for the new assays were excellent. All WPV1 were detected and there was no cross-reactivity with vaccine viruses or VDPV. In addition, the run conditions identified more consistently than the previous run conditions. The PV2 assay was designed to identify all vaccine-derived poliovirus type 2 strains as well as MEF-1 used in vaccine production facilities. The new type 2 assay was tested against the CDC standard panel and a panel of 19 type 2 VDPVs isolated from immunodeficient patients from Asia, Europe, and North America (iVDPVs can result from long-term replication of vaccine viruses in persons with antibody deficiencies). The type 2 assay detected all immunodeficient VDPVs, although the most divergent iVDPV, >100 nt changes in VP1, had a low fluorescence signal. The new assays are being distributed to GPLN laboratories starting in May 2016. 190 Europic 2016

191 THURSDAY 08/09/2016 G39 EVOLUTIONARY DYNAMICS OF POLIOVIRUS NEUTRALIZING ANTIGENIC SITES AND OTHER CAPSID FUNCTIONAL DOMAINS DURING A LARGE AND PROLONGED OUTBREAK Cara Burns1, Jing Shaw1, Jaume Jorba2, Kun Zhao2, Jane Iber2, Qi Chen2, Festus Adu3, Adekunle Adeniji3, David Bukbuk4, Marycelin Baba4, Elizabeth Henderson2, Nicksy Gumede-Moeletsi5, M. Steven Oberste2, Olen Kew2 1 Centers for Disease Control and Prevention, 2CDC, 3University of Ibadan, 4University of Maiduguri, 5World Health Organization AFRO office We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. These outbreak isolates were derived from 23 independent cVDPV2 emergences, at least 7 of which established circulating lineage groups. Analysis of the evolutionary dynamics of cVDPV2s provides an opportunity to document differences and similarities with evolution of circulating wild polioviruses. Four different functional domains were analyzed: neutralizing antigenic (NAg) sites and VP1 residues 132. Amino acid replacements mapped to 37 of 43 positions across all 4 NAg sites; the most variable and polymorphic residues were in NAg sites 2 and 3b. The most divergent of the 120 NAg variants had no more than 5 NAg site differences from Sabin 2. Residues 132 of VP1 were highly variable (133 different variants; 06 replacements/isolate; 5/32 invariant positions), with residues 118 predicted to form a well-conserved amphipathic helix. Two additional functional domains were selected for analysis: residues binding the poliovirus receptor (PVR) and the capsid structural core. As expected, the PVR-binding residues were less variable (25 different variants; 02 replacements/isolate; 30/44 invariant positions), with the most variable residues also forming parts of NAg sites 2 and 3a. Replacement events were dated by mapping them onto the branch structure of time-scaled phylogenetic trees. Rates of amino acid replacement varied widely across positions and followed no simple substitution model. Replacements into the structural core were the most conservative and were fixed at an overall rate ~20-fold lower than rates for the NAg sites and VP1 132, and ~5- fold lower than the rate for the PVR-binding sites. Only VP1-143-Ile, a non-NAg site surface residue associated with attenuation of neurovirulence, appeared to be under strong negative selection. Europic 2016 191

192 INDEX OF AUTHORS A uthor A bstract N umber P age A. Joyce, Michael B12 56 Abdelnabi, Rana F02, F14, G17 140, 151, 174 Abrescia, Nicola A23 35 Acevedo, Ashley D17 102 Adeyemi, Oluwapelumi A04, G04 18, 161 Adu, Festus G39 191 Agoh, Masanobu E19 135 Agol, Vadim I. D20 105 Ajami, Nadim E05 121 Albulescu, Lucian F12 150 Altan-Bonnet, Nihal K02, B07 26, 51 Ambrosioni, Juan G31 184 Ami, Yasushi G01 158 An, Dong-Jun G13 170 Ana San-Flix, Ana F11 149 Anastasina, Maria C11 83 Andino, Raul D15, D17, G20, G22 100, 102, 177, 179 Androletti, Laurent D18 103 Andriasyan, Vardan A08 21 Anstadt, Jennifer L. B19, C08, E12 63, 80, 128 Antn, Andrs E11 127 Aranzamendi, Maitane E11 127 Archimbaud, Christine E08, E15 124, 131 Arima, Yuzo E07 123 Arita, Minetaro E06 122 Arnberg, Niklas B11 55 Arnold, Jamie B07, D14, D17, D19, D25 51, 99, 102, 104, 110 Arzt, Jonathan B20 64 Asare, Emmanuel A15 29 Asfor, Amin A20, A19, F03, G36 33, 32, 141, 189 Ashley, Robert A09, A13 22, 27 Asner, Sandra G31 182 Aw Yong, Kam Leng C10 82 Baba, Marycelin G39 191 Babu, Aravindh G25 181 Bachanek-Bankowska, Katarzyna D27, E13 112, 129 Baggen, Jim B10, B11 54, 55 Bahar, Mohammad G04 161 Bailly, Jean-Luc D24, E08, E15, G33 109, 124, 131, 186 Bakker, Arjen A22 34 Bakker, Wilfried G19, G22 176, 179 Balbirnie, Katharin E03 119 Barbezange, Cyril D22 107 192 Europic 2016

193 INDEX OF AUTHORS A uthor A bstract N umber P age Brcena, Montserrat B02, B16 45, 60 Barton, David D07 92 Basnet, Sarmila D03 89 Bauer, Lisa F12 150 Beaucourt, Stphanie D22, G23 107, 180 Beaumont, Tim A22 34 Belov, George B06, G03 50, 160 Belsham, Graham John A14, G32 28, 185 Benaoudia, Sacha B09, F09 53, 147 Benschop, Kimberley B11, G08 55, 165 Bentley, Kirsten D04 90 Berryman, Stephen A05 19 Bessaud, Mal D12, D24 97, 109 Bisseux, Maxime G33 186 Blaas, Dieter B14, F06 58, 144 Blanc, Herve D22 107 Blomen, Vincent A. B10 54 Blondel, Bruno D12, D24 97, 109 Bochkov, Yury A01, D03 15, 89 Bockstal, Viki G12 169 Boda, Bernadett F09 147 Boehr, David B07 51 Bonfante, Rosy F09 147 Borderia, Antonio D01 87 Bordera, Antonio D22 107 Borghese, Fabian C06 78 Bosch, Albert D02 88 Btner, Anette G32 185 Bouin, Alexis D18 103 Broberg, Eeva G07 164 Brocchi, Emiliana Brocchi G36 189 Brown, Emma G10 167 Brummelkamp, Thijn R. B10 54 Bujaki, Erika G20 177 Bukbuk, David G39 191 Bullard, Kelley G34 187 Burman, Alison G32 185 Burns, Cara C08, G22, G34, G39 80, 179, 187, 191 Buskens, Christianne E02 118 Butcher, Sarah A02, A22, C11, F14 16, 34, 83, 151 Cabrerizo, Maria E11 127 Cacciabue, Marco B22 66 Caglar, Mehmet B01 44 Europic 2016 193

194 INDEX OF AUTHORS A uthor A bstract N umber P age Caine, Elizabeth G23 180 Cameron, Craig B01, B07, D14, D17, D19, D25 44, 51, 99, 102, 104, 110 Capaldi, Elizabeth A09 22 Carrau, Lucia D22 107 Carrillo, Elisa B22 66 Castro, Christina E01 117 Cello, Jeronimo G12 169 Chan, Yan B07 51 Chan, Yoke Fun C10 82 Chandler, Courtney B06 50 Chandler-Bostock, Rebecca A04 18 Chapman, Nora D09, D13, D26 94, 98, 111 Charles, Mark F03 141 Charleston, Bryan A23, G21, G35 35, 178, 188 Charlton, Bethany A18 31 Chen, Crystal Y. F08 146 Chen, Qi G39 191 Chen, Xian A07 20 Chen, Zheng F08 146 Chen, Zhenguo A01 15 Cherkasova, Elena D20 105 Chern, Shur-wern E01 117 Chia, Andrew E17 133 Chia, John E17 133 Chio, Chi-Chong C05 77 Choe, Se-Eun G13 170 Chou, Chia-Jung C05 77 Chua, Tiing Tiing B12 56 Chumakov, Konstantin D20, F04, F08, G03, G05, G16 105, 142, 146, 160, 162, 173 Ciomperlik, Jessica B19 63 Clare-Salzler, Michael D13 98 Clevers, Hans E02 118 Cockburn, Joseph A02 16 Cohen, Robert E08 124 Colenutt, Claire G10 167 Colombet, Jonathan G33 186 Constant, Samuel B09, F09 53, 147 Conway, James A09, A13 22, 27 Conzemius, Rick B14 58 Cooper, Gillian G11, G12, G18 168, 169, 175 Corbic Ramljak, Irena F06 144 Cordey, Samuel G31 184 Cottam, Eleanor M. A03 17 194 Europic 2016

195 INDEX OF AUTHORS A uthor A bstract N umber P age Crawt, Laura G11, G12, G18 168, 169, 175 Cremer, Paul B07 51 Croft, Sarah C04 76 Cullen, John M. C01 73 Custers, Jerome G12 168 D. Tyrrell, Lorne B12 56 DAndrea, Luca D02 88 Dang, Minghao C07 79 de Boer, Matthijn G19, G22 176, 179 de Bruin, Jost W. B10 54 De Clercq, Kris A11 24 De Colibus, Luigi G04 161 De Groot, Raoul C09 81 De Jong, Menno D. E10 126 de los Rios, Isabel G12 169 De los Santos, Teresa B15, G06 59, 163 De Miranda, Joachim Rodrigues A26 38 De Vleeschauwer, Annebel A11 24 de Vries, Maarten G19 176 Debroas, Didier G33 186 Dekker, Nynke D14, D25 99, 110 Delang, Leen F01, F02, F05, F10, F11, G17 139, 140, 143, 148, 149, 174 Delpeyroux, Francis D12, D24 97, 109 Delwart, Eric D08 93 Depken, Martin D14 99 Derisi, Joe B15 59 Dessain, Scott F04 142 Devi, Chandana F04 142 Diaz-San Segundo, Fayna B15, G06 59, 163 Diedrich, Sabine E15 131 Domanska, Ausra A22, F14 34, 151 Domsgen, Erna A25 37 Dong, Xiangchi A23 35 Dorobantu, Cristina B17 61 Dragunsky, Eugenia F08, G03, G16 146, 160, 172 Drappier, Melissa C03 75 Dreier, Dominik F06 144 Drost, Jarno E02 118 Dryden, Kelly A09 22 Dufour, Antoine B08 52 Duizer, Erwin B11, G08 55, 165 Dulin, David D14 99 Dunn, Glynis D05, G02, G15 91, 159, 172 Europic 2016 195

196 INDEX OF AUTHORS A uthor A bstract N umber P age Durrani, Kulsoom A09 22 Dykeman, Eric A02 16 Edman, Kjell E16 132 Edo Matas, Diana G12 169 El-Habbal, Rabiha E17 133 Ellis, Shane B06 50 Ernst, Robert B06 50 Essaidi-Laziosi, Manel B09 53 Evans, David D04, D17, E03 90, 102, 119 Fannon, Jess E03 119 Farkas, Agnes E15 131 Feng, Zongdi A07 20 Fernandes-Rocha, Melanie B09 53 Fessler, Evelyn E02 118 Fischl, Wolfgang F06 144 Flanegan, James D13 98 Flather, Dylan B05 48 Flodstrm-Tullberg, Malin A25, B21 37, 65 Fontes, Magnus D01, D22 87, 107 Ford-Siltz, Lauren B06 50 Fornes, Paul D18 103 Fox, Helen F07, G04 145, 161 Fry, Elizabeth A19, A23, A27, C07, F07, G04, G21, G35 32, 35, 39, 79, 145, 161, 178, 188 Fuchs, Renate B14 58 Fujii, Ken G01 158 Fujimoto, Tsuguto E07 123 Gao, Qiang C07 79 Ganjian, Haleh B14 58 Garca Nez, Mara Soledad B22 66 Garca-Iiguez, Juan Pablo E11 127 Geiser, Johan A32 40 Georgi, Fanny A08 21 Georgiev, Oleg A10 23 Geraets, James F14 151 Gerloff, Nancy G37 190 Gern, James K11, D03 165, 89 Gershon, Paul D. B05 48 Ghildyal, Reena C04 76 Gismondi, Mara Ins B22, B24 66, 68 Gmyl, Anatoly G05 162 Gndinger, Simon A10 23 Gold, Sarah A11, A12, A19, B23, D11, D21 24, 25, 32, 67, 96, 106 Gonalves, Ana Rita G31 184 196 Europic 2016

197 INDEX OF AUTHORS A uthor A bstract N umber P age Gonzales, Jose G10 167 Gonzalez-Lopez, Olga A07 20 Gorbalenya, Alexander D08, D16 93, 101 Gouandjika, Ionela D24 109 Grazioli, Santina G36 189 Greber, Urs A08, A10, B13 21, 23, 57 Greco, Donato G07 164 Greninger, Alexander B15 59 Groppelli, Elisabetta B23 67 Grubman, Marvin B15 59 Gubbins, Simon G10 167 Guedan, Anabel F03 141 Gullberg, Maria G32 185 Gulyaeva, Anastasia D16 101 Gumede-Moeletsi, Nicksy G39 191 Guo, Feng B01 44 Gusachenko, Olesya E03 119 H.G. Steenbergern, Rineke B12 56 Haapakoski, Marjo A24 36 Hafenstein, Susan A09, A13 22, 27 Hanaoka, Nozomu E07 123 Harak, Christian A05 19 Harmsen, Michiel G21 178 Harris, Mark A17 30 Harvala, Heli G07 164 Hasegawa, Hideki E14, E19 130, 135 Hassel, Chervin E15 131 Hayashi, Yohei C06 78 Haydon, Dan A03, D23 17, 108 Henderson, Elizabeth G34, G39 187, 191 Henningsson, Rasmus D01, D22 87, 107 Henquell, Ccile E08, E15, G33 124, 131, 186 Hensley, Lucinda C01 73 Heraud, Jean-Michel D24 109 Herbert, Rebeca G25 181 Herod, Morgan A12, A17, D21 25, 30, 106 Hill, Marchel A01, D03 15, 89 Hirai-Yuki, Asuka C01 73 Hober, Didier E09 125 Hoenemann, Holger G35 188 Hoeprich, Paul G06 163 Hogle, James B23 67 Honkanen, Hanna E09 125 Europic 2016 197

198 INDEX OF AUTHORS A uthor A bstract N umber P age Hovi, Tapani D08 93 Howes, Emma A11 24 Hsu, Nai-Yun B07 51 Hu, Fengyu B03 46 Hu, Zhongyu C07 79 Huang, Dan F08 146 Huang, Hsing-I B25, C05 69, 77 Huang, Song F09 147 Huang, Tony B01 44 Huemer, Hartwig E15 131 Huiskonen, Juha A19, A23 32, 35 Hyoty, Heikki A25, B04, E04, E05, E09 37, 47, 120, 121, 125 Hytnen, Vesa A25, B21 37, 65 Iber, Jane G34, G39 187, 191 Iizuka, Setsuko E19 135 Ikuta, Kazuyoshi G14 170 Ilca, Serban A23, G21 35, 178 Ilonen, Jorma E09 125 Imura, Ayumi G01 158 Ivanova, Olga D20 105 Iwata-Yoshikawa, Naoko E14, E19, G01 130, 135, 158 Jskelinen, Anne D28 113 Jackson, Terry A05, A11, D10, D11, G32 19, 24, 95, 96, 185 Jae, Lucas T. B10 54 Jagdeo, Julienne B08 52 Jan, Eric B08 52 Janissen, Richard D25 110 Jaramillo, Lee D26 111 Jasir, Aftab G07 164 Jayaprakash, Venkatesan F02 140 Jha, Babal K. C03 75 Jiang, Ping A15 29 Jimnez Melsi, Alexandra G35 188 Jochmans, Dirk F05, F14 143, 151 Joffret, Marie-Line D12, D24 97, 109 Johnston, Sebastian F03 141 Jones, Ian F07, G04, G35 145, 161, 188 Jones, Sin D04 90 Jorba, Jaume G34, G39 187, 191 Juleff, Nick G21 178 Jurgeit, Andreas A10 23 Kaiser, Laurent B09, F09, G31 53, 147, 184 Kallio-Kokko, Hannimari D28 113 198 Europic 2016

199 INDEX OF AUTHORS A uthor A bstract N umber P age Kalynych, Sergei A26 38 Kapell, Sebastian A25, B21 37, 65 Kataoka, Chikako G01 158 Kauppinen, Anni D28 113 Kempf, Brian D07 92 Kew, Olen G39 191 Khamphaphongphane, Bouaphanh E06 122 Khojine, Amirbabak B04 47 Kibe, Adite E03 119 King, Andrew M.Q. D08 93 King, Don P. A03, D23 17, 108 King, Donald A11, D10, D11, G36 24, 95, 96, 189 Kinoshita, Hitomi E07 123 Kirkegaard, Karla K03 43 Kivil, Anri C11 83 Kjr, Jonas A14 28 Klapsa, Dimitra D05, G15 91, 172 Klingel, Karin F06 144 Kloc, Anna B15 59 Klose, Thomas A01 15 Knip, Mikael E09 125 Knowles, Nick D08, D10, D11, D27, E13 93, 95, 96, 112, 129 Knowlson, Sarah F07 145 Knudsen, Giselle B15 59 Koekkoek, Sylvie M. E02, E10 118, 126 Koen, Gerrit E02, E10 118, 126 Koh, Mia Tuang C10 82 Koike, Satoshi G01 158 Kolehmainen, Pekka D28 113 Kolesnikova, Marina G05 162 Kondratiev, Nikolai D16 101 Knig, Guido B24 68 Koning, Roman A22 34 Koppel, Stella F18 153 Korotkova, Ekaterina D20 105 Koster, Abraham J. A22, B02, B16 34, 45, 60 Kotani, Osamu E14, E19, G01 130, 135, 158 Kotecha, Abhay A19, A23, A27, G21 32, 35, 39, 178 Kouiavskaia, Diana F04, F08, G03, G05, G16 142, 146, 160, 162, 173 Kowalski, Heinrich F06 144 Kozlovskaya, Liubov G16 173 Krause, Karl-Heinz A32 40 Krogstad, Paul E18 134 Europic 2016 199

200 INDEX OF AUTHORS A uthor A bstract N umber P age LHuillier, Arnaud G. G31 184 Laassri, Majid D20 105 Laitinen, Olli A25, B21, E04 37, 65, 120 Langereis, Martijn C09 81 Lanko, Kristina F10 148 Larsson, Pr A25 37 Lasecka, Lidia A03, A12, D10, D11, D21 17, 25, 95, 96, 106 Lauber, Chris D16 101 Lawrence, Paul B20 64 Lee, Cheri D14 99 Lee, Hyunwook A13 27 Lehtonen, Jussi E04 120 Lemon, Stanley M. A07, B03, C01 20, 46, 73 Lo, James K05 72 Leonov, German A04 18 Leontovich, Andrey D16 101 Lvque, Nicolas D18 103 Levy, Corinne E08 124 Levy, Hazel B23 67 Leyssen, Pieter F02, F10, F11 140, 148, 149 Li, Sixing B01 44 Liang, Zhenglun G11 168 Lightner, Kandice L. E12 128 Limpens, Ronald W. A. L. B16 60 Lin, Jhao-Yin B25 69 Lin, Jing-Yi B18 62 Lin, Kai-Zhe B18 62 Lindberg, A. Michael D08, E16 93, 132 Linnekamp, Janneke F. E02 118 Liu, Yue A01, B10 15, 54 Lloyd, Richard E05 121 Lloyd- Jones, Katie G25 181 Logan, Grace A03, D10, D11 17, 95, 96 Lohmann, Volker A05 19 Lohse, Louise G32 185 Lomonossoff, George F07, G04 145, 161 Looney, Benjamin D13 98 Ltzerich, Mark A10 23 Loundras, Eleni-Anna A17 30 Loureiro, Silvia G35 188 Lu, Jia-Ying B18 62 Lucas, Bernadette E09 125 Lugo, Debra E18 134 200 Europic 2016

201 INDEX OF AUTHORS A uthor A bstract N umber P age Lukashev, Alexander G05, G16 162, 173 Luke, Garry D11 96 Lyoo, Heyrhyoung B17 61 Macadam, Andrew K09, F07, G04, G20, G22 157, 145, 161, 177, 177 Macaya, Alfons E11 127 Madurga, Paula E11 127 Mahapatra, Mana G25 181 Maher, Chelsea G37 190 Maher, Kaija E01 117 Mahkov, Alexander A09 22 Majumdar, Manasi G15 172 Makhov, Alexander A13 27 Malik, Nayab A19 32 Mandelbaum, Mark G37 190 Mao, Qunying G11 168 Mao, Zhangming B01 44 Maree, Francois G21 178 Marjomki, Varpu A24, B04 36, 47 Marrero, Rubn B22, B24 66, 68 Marsian, Johanna F07, G04 145, 161 Martikainen, Mari B04 47 Martin, Javier A18, D05, G02, G11, G12, G15, G18 31, 91, 159, 168, 169, 172, 175 Martnez-Sapia, Ana E11 127 Matadeen, Rishi A22 34 McDonald, Seth G23 180 McInerney, Gerald G32 185 McKnight, Kevin L. A07, B03 20, 46 Medema, Jan Paul E02 118 Medina, Gisselle N B15 59 Melia, Charlotte B02, B16 45, 60 Meng, Bo A04 18 Michiels, Thomas C02, C03, C06 74, 75, 78 Mihovilovic, Marko D. F06 144 Minor, Philip D05, F07, G02, G04, G11, G18 91, 145, 159, 161, 168, 175 Mioulet, Valrie G21 178 Mirabelli, Carmen F05 143 Mirand, Audrey E08, E15, G33 124, 131, 186 Mirochitchenko, Olga F08 146 Mirochnitchenko, Olga G16 173 Mistry, Nitesh B11 55 Mitchell, Jonathan K. B03 46 Mittl, Peer A10 23 Moffat, Christopher E03 119 Europic 2016 201

202 INDEX OF AUTHORS A uthor A bstract N umber P age Moffat, Katy A05 19 Monica, Bella G19 176 Montmayeur, Anna E01 117 Moran, Joseph B07 51 Morasco, Joan D13 98 Moratorio, Gonzalo D01, D22 87, 107 Morelli, Marco D23 108 Moreno-Docn, Antonio E11 127 Mounce, Bryan D22 107 Mousnier, Aurelie F03 141 Moustafa, Ibrahim B07 51 Mugavero, Joann A15 29 Mullapudi, Edukondalu A26 38 Mller-Planitz, Felix C02 74 Murer, Luca F15 152 Myllynen, Mira A24 36 Nagata, Noriyo E14, E19, G01 130, 135, 158 Nakamura, Tomofumi E06, E19, G14 122, 135, 170 Nchoutmboube, Jules B06 50 Nelson, Noel G10 167 Newman, Joseph A03, G29 17, 183 Neyts, Johan K08, F01, F05, F10, F11, G17 138, 139, 143, 148, 149,174 Ng, Terry E01 117 Nguyen, Yohan D18 103 Nicol, Clare B23 67 Nishimura, Yorihiro E06 122 Nix, William A. E01, E12, G37 117, 128, 190 Njouom, Richard D24 109 Norte, Aysen D25 110 Nurminen, Anssi B21 65 Nurminen, Noora E04 120 OBrien, Jackie G15 172 Oberste, Mark S. B19, C08, E01, E12, G34, G37, G39 63, 80, 117, 128, 187, 190, 191 Ochiai, Susumu G14 171 Ogram, Sushma D13 98 Oh, Hyung-Suk D14 99 Oikarinen, Sami E04, E09 120, 125 Oishi, Kazunori E07 123 Oosterhoff, Dinja G19 176 Organtini, Lindsey A09, A13 22, 27 Orosio, Jorge G23 180 Orton, Richard D23 108 Otero, Almudena E11 127 202 Europic 2016

203 INDEX OF AUTHORS A uthor A bstract N umber P age Overall, Christopher B08 52 Pacheco, Juan B20 64 Plkov, Lenka A26 38 Pallansch, Mark A. D08, E01, E12 93, 117, 128 Palm, Kaia C11 83 Palmenberg, Ann K10, A01, D03, D08 15, 89, 93 Pang, Daniel B12 56 Panjwani, Anusha A20, F03 33, 141 Parekh, Krupali G25 181 Parida, Satya Parida G25, G36 181, 189 Pastore Celentano, Lucia G07 164 Paton, David D23, G25 108, 181 Paul, Aniko A15 29 Paxton, Robert A26 38 Pearson, Ashley D04 90 Peersen, Olve D07, G23 92, 180 Peeters, Michael C02 74 Peigue-Lafeuille, Hlne E08, E15 124, 131 Penttinen, Pasi G07 164 Pereira, Bruno E08 124 Perez, Eva G21, G35 178, 188 Prez-Castro, Sonia E11 127 Prez-Rodrguez, Francisco-Javier D02 88 Petrosino, Joseph E05 121 Petrovskaya, Svetlana D20 105 Pihlak, Arno C11 83 Pint, Rosa D02 88 Piuz, Isabelle A32, B09 40, 53 Pizzorno, Marie A09 22 Plevka, Pavel A26 38 Poirier, Enzo D22 107 Polacek, Charlotta G32 185 Porta, Claudine G35 188 Posfay-Barbe, Klara G31 184 Poulsen, Line Dahl A14 28 Pidal, Antonn A26 38 Pugliese, Alberto E05 121 Puligedda, Rama Devudu F04 142 Rabella, Nuria E11 127 Rabouw, Huib C09 81 Rai, Devendra B20, G06 64, 163 Rao, Zihe A27, C07 39, 79 Rasmussen, Thomas Bruun A14 28 Europic 2016 203

204 INDEX OF AUTHORS A uthor A bstract N umber P age Raynham, Tony F03 141 Razafindratsimandresy, Richter D24 109 Reina, Jordi E11 127 Ren, Jingshan A19, A27, C07 32, 39, 79 Renois, Fanny D18 103 Reuter, Gabor D08 93 Rieder, Elizabeth B15, B20, B22, G06 59, 64, 66, 163 Rivero-Buceta, Eva F11 149 Roberts, Richard B10, B11 54, 55 Rodionova, Elvira D20 105 Rodrigo, Carlos E11 127 Rodriguez, Luis G06 163 Rogers, Shannon E01 117 Romero, Maria Pilar E11 127 Rondahl, Elin E16 132 Rossmann, Michael A01, B10 15, 54 Roulin, Pascal A10, B13 23, 57 Rowlands, David J A02, A04, A12, A20, B23, D21, F07, G04 16, 18, 25, 33, 67, 106, 145, 161 Royston, Lna A32 40 Ruokolainen, Visa A24 36 Ryabov, Eugene E03 119 Ryan, Martin A11, D11 24, 96 Sachs, Norman E02 118 Sadeuh-Mba, Serge D24 109 Sam, Jamal I-Ching C10 82 Samal, Siba G03 160 Samborskiy, Dmitry D16 101 Sanders, Barbara G12 169 Sankar Datta, Siddhartha E06 122 Sato, Hironori E14 130 Svneby, Anna E16 132 Schafer, Elizabeth G06 163 Scheers, Els F01 139 Schibler, Manuel G31 184 Schluter, Walter William E06 122 Schuitemaker, Hanneke G12 169 Scott, Alison B06, G21 50, 178 Seago, Julian A23, G21 35, 178 Seakamela, Lerato G37 190 Seiler, Daria A10 23 Sejvar, James E01 117 Semler, Bert L. B05 48 Sengkeopraseuth, Bounthanom E06 122 204 Europic 2016

205 INDEX OF AUTHORS A uthor A bstract N umber P age Serrander, Lena E16 132 Shakeel, Shabih A02, A22, F14 16, 34, 151 Shaukat, Shahzad G37 190 Shaw, Jing G39 191 Shen, Ling F08 146 Shengjuler, Djoshkun B07 51 Shields, Justin B12 56 Shih, Shin-Ru K07, B18 116, 62 Shimizu, Hiroyuki E06, E07, E14, E19, G01 122, 123, 130, 135, 158 Shingler, Kristin A09, A13 22, 27 Shustova, Elena G05 162 Sidorov, Igor D16 101 Sijapati, Shakher D03 89 Silverman, Robert H. C03 75 Simmonds, Peter D08, D11, G07 93, 96, 164 Sioofy Khojine, Amirbabak E04, E09 120, 125 Siponen, Anu D28 113 Skern, Tim D08 93 kubnk, Karel A26 38 Slager, Jasper B11 55 Smith, Matt G20 177 Smithee, Shane C08, D09, D26, E12 80, 94, 111, 128 Smura, Teemu D28 113 Solari, Roberto F03 141 Song, Yutong G12 169 Sorgeloos, Frederic C02 74 Springer, Timothy A23 35 Spurn, Radovan A26 38 Stanger, Julia F06 144 Stanway, Glyn D08 93 Staring, Jacqueline B10 54 Stehle, Thilo B11 55 Stenfeldt, Carolina B20 64 Stockley, Peter A02, A04 16, 18 Stone, Virginia A25 37 Stonehouse, Nicola J. A12, A17, A20, D21, F07, G04 25, 30, 33, 106, 145, 161 Strating, Jeroen R.P.M B02, F12 45, 150 Strauss, Mike B23 108 Stuart, Dave G21 178 Stuart, David A19, A23, A27, C07, F07, G04, G35 32, 35, 39, 79, 145, 161, 188 Sudaka, Yui G01 158 Sun, Eileen B23 67 Sun, Hong G37 190 Europic 2016 205

206 INDEX OF AUTHORS A uthor A bstract N umber P age Sun, Liang F10, F11 148, 149 Sun, Simou B07 51 Sun, Yao C07 79 Sunagawa, Tomimasa E07 123 Suzuki, Keiko G14 171 Suzuki, Tadaki E14, G01 130, 158 Svedin, Emma A25, B21 37, 65 Swanson, Jessica A20 33 Swieboda, David F03 141 Takashino, Ayako G01 158 Tanaka-Taya, Keiko E07 123 Tanis, Pieter J. E02 118 Tapparel, Caroline A32, F09 40, 147 Tapparel Vu, Caroline B09 53 Tauriainen, Sisko D28 113 Taylor, Geraldine G25 181 Te Yeh, Ming G20 177 Tedcastle, Alison G18 175 Terletskaia-Ladwig, Elena E15 131 Thibaut, Hendrik Jan B10, B11 54, 55 Thomassen, Yvonne G19 176 Tian, Xiangjun E05 121 Timiri, Ajay Kumar F02 140 Todd, Daniel D27 112 Toussaint, Marie F03 141 Tracy, Steven D09, D26 94, 111 Troy, Stephanie G16 173 Tulloch, Fiona D11 96 Turin, Lara G31 184 A03, A11, A12, A19, A20, A27, B23, D10, 17, 24, 25, 32, 33, 39, 67, 95, 96, 106, 141, Tuthill, Tobias D11, D21, F03, F07, G04, G36, G29 145, 161, 189, 183 Twarock, Reidun A02, A04 16, 18 Uchida, Akira B07 51 Ushioda, Waka E19, G14 135, 171 Valdazo-Gonzlez, Begoa A11, G02 24, 159 van t Oever, Aart G19 176 van Bruggen, Armando B02 45 van den Born, Erwin G35 188 van den Braak, Wouter G19 176 van der avoort, Harrie G08 165 van der Sanden, Sabine E02, E10 118, 126 van der Schaar, Hilde B02, B16, B17 45, 60, 61 van Eijk, Hetty W. E10 126 206 Europic 2016

207 INDEX OF AUTHORS A uthor A bstract N umber P age van Geenen, Maartje B02 118 van Hoek, Vladimir G12 168 van Kuppeveld, Frank K04, B02, B10, B11, B16, C09, F12 49, 45, 54, 55, 60, 81, 150 van Laar, Theo D14 99 van Vliet, Arno B10, B11 54, 55 Vandesande, Helena E16 132 Vapalahti, Olli D28 113 Vega, Everardo M. G37 190 Vehik, Kendra E05 121 Vennema, Harry G08 165 Vernachio, John F10 148 Vertommen, Didier C02 74 Veselkov, Michaela A26 38 Vi le Sage, Franois E08 124 Vignuzzi, Marco K06, D01, D22, G23 86, 87, 107, 180 Viktorova, Ekaterina B06, G03 50, 160 Vinther, Jeppe A14 28 Visser, Linda C09 81 Volle, Romain D24 109 Vongphrachanh, Phengta E06 122 Wada, Junko E06 122 Wadsworth, Jemma E13 129 Wagner, Nomie G31 184 Walker, Erin C04 76 Walter, Thomas A27, C07 39, 79 Wang, David E17 133 Wang, Dongyan G28 182 Wang, Junzhi G11, C07 167, 79 Wang, Quan A23 35 Wang, Richard F08 146 Wang, Xiangxi A27, C07 39, 79 Wang, Yiping G11 168 Ward, Joseph A17, D21 30, 106 Warner, Emma D21 106 Watanabe, Akiko G14 171 Watters, Kelly A01, D03 15, 89 Wehbe, Michel D18 103 Weiss, Susan R. C03 75 Weldon, William C. B19, E12 63, 128 Westerhuis, Brenda A22 34 White, Simon A02, A04 16, 18 Whitmire, Jason K. C01 73 Wilke, Claus B01 44 Europic 2016 207

208 INDEX OF AUTHORS A uthor A bstract N umber P age Wilton, Thomas G18 175 Wimmer, Eckard K01, A15, G12 14, 29, 169 Witte, Robert A08 21 Wolthers, Katja A22, C11, E02, E10, F18 34, 83, 118, 126, 153 Wong, Matthew E05 121 Woodman, Andrew D04, D17, D19, D25 90, 102, 104, 110 Wright, Caroline A03, A11, A12, D10, D11, D21, D23 17, 24, 25, 95, 96, 106, 108 Wynn, Nhien T. C08 80 Xiao, Yinghong D15 100 Xie, Ling A07 20 Xu, Miao G11 168 Xu, Wenbo G28 182 Yakimovich, Artur A08 21 Yamashita, Teruo A27, D08 39, 93 Yan, Dongmei G28 182 Yang, Enzhuo F08 146 Yokouchi, Daisuke G14 171 Yokoyama, Masaru E14 130 Yoshida, Hiromu E06 122 Yuan, Shuai A27, C07 39, 79 Zagorodnyaya, Tatiana D20 105 Zaidi, Sohail G37 190 Zakharov, Matvey D16 101 Zell, Roland D08 93 Zhang, Alvin Z F08 146 Zhang, Bo C07 79 Zhang, Fuquan G21 178 Zhang, Yong G28 182 Zhao, Kun G34, G39 187, 191 Zhu, Ling A27, C07 39, 79 Zhu, Shuangli G28 182 Zhuang, Xiaowei B23 67 Zocher, Georg B11 55 208 Europic 2016

209 FEMS 2017 7TH CONGRESS OF EUROPEAN MICROBIOLOGISTS JULY 9-13, 2017 VALENCIA, SPAIN In Association with 26th Congress of the Spanish Society for Microbiology www.fems-microbiology2017.kenes.com

210 PATHOGENS AND DISEASE Editor-in-Chief Patrik M. Bavoil Next generation host-pathogen research 2.403 Impact Factor 2014 Available in >160 34 days average time from countries worldwide submission to first decision International Editorial Board Free to Gold Open publish Access (non Open options Access papers) available Highly cited Continuous Publication papers published online immediately thematic issues Income from our journals goes back into science through grants, awards, and the FEMS Congress www.femspd.oxfordjournals.org

211 LIST OF PARTICIPANTS L astname F irstname C ountry Abdelnabi Rana Belgium Altan Bonnet Nihal United States of America An Dong-Jun South Korea Anastasina Maria Finland Andino Raul United States of America Anstadt Jennifer L. United States of America Archimbaud Christine France Arnold Jamie United States of America Asare Emmanuel United States of America Asfor Amin United Kingdom Baggen Jim Netherlands Bailly Jean-Luc France Bakker Wilfried Netherlands Barcena Montserrat Netherlands Barton David United States of America Bauer Lisa Netherlands Belov George United States of America Belsham Graham Denmark Benschop Kimberley Netherlands Bentley Kirsten United Kingdom Berryman Stephen United Kingdom Bessaud Mal France Bisseux Maxime France Blaas Dieter Austria Blomqvist Soile Finland Boda Bernadett Switzerland Bosch Albert Spain Bouin Alexis United States of America Burns Cara United States of America Butcher Sarah Finland Cabrerizo Maria Spain Cagno Valeria Switzerland Cameron Craig United States of America Chan Yoke Fun Malaysia Chandler-Bostock Rebecca United Kingdom Chapman Nora United States of America Charlton Bethany United Kingdom Chia John United States of America Chua Tiing Tiing Canada Chumakov Konstantin United States of America Ciomperlik Jessica United States of America Colenutt Claire United Kingdom Corbic Ramljak Irena Austria Europic 2016 211

212 LIST OF PARTICIPANTS L astname F irstname C ountry Croft Sarah Australia de Boer Matthijn Netherlands Dekker Nynke Netherlands Dessain Scott United States of America Edinger Thomas Switzerland Essaidi-Laziosi Manel Switzerland Evans David United Kingdom Feenstra Femke Netherlands Flanegan James United States of America Flather Dylan United States of America Fletcher Sarah United Kingdom Flodstrm-Tullberg Malin Sweden Fox Helen United States of America Francis DELPEYROUX France Fujii Ken Japan Geraets James Finland Gern James United States of America Gismondi Mara Ins Argentina Gold Sarah United Kingdom Gorbalenya Alexander Netherlands Greber Urs Switzerland Groppelli Elisabetta United Kingdom Guedan Anabel United Kingdom Hafenstein Susan United States of America Harvala Heli Switzerland Hayashi Yohei Belgium Henningsson Rasmus Sweden Herod Morgan United Kingdom Hogle James United States of America Hovi Tapani Finland Howes Emma United Kingdom Huang Hsing-I Taiwan Hyde Byron Canada Hyoty Heikki Finland Jagdeo Julienne Canada James Leo United Kingdom Janissen Richard Netherlands Jorba Jaume United States of America Kaiser Laurent Switzerland Kapell Sebastian Sweden Kelly James United Kingdom Kim Su-Young South Korea Kinoshita-Yamaguchi Hitomi Japan 212 Europic 2016

213 LIST OF PARTICIPANTS L astname F irstname C ountry Kirkegaard Karla United States of America Kjr Jonas Denmark Klapsa Dimitra United Kingdom Knowles Nick United Kingdom Koike Satoshi Japan Kotani Osamu Japan Kotecha Abhay United Kingdom Kowalski Heinrich Austria Lanko Kristina Belgium Lemon Stanley United States of America Lin Jing-Yi Taiwan Lin Jhao-Yin Taiwan Lindberg Michael Sweden Lipton Howard L United States of America Liu Yue United States of America Lloyd Richard United States of America Loundras Eleni-Anna United Kingdom Lugo Debra United States of America Lukashev Alexander Russia Luke Garry United Kingdom Lyoo Heyrhyoung Netherlands Macadam Andrew United Kingdom Majumdar Manasi United Kingdom Marjomki Varpu Finland Marsian Johanna United Kingdom Martin Javier United Kingdom McKnight Kevin United States of America Medina Gisselle N United States of America Melia Charlotte Netherlands Michiels Thomas Belgium Mirabelli Carmen Belgium Mirand Audrey France Moratorio Gonzalo France Murer Luca Switzerland Nagata Noriyo Japan Nakamura Tomofumi Japan Newman Joseph United Kingdom Neyts Johan Belgium Ochiai Susumu Japan Oosterhoff Dinja Netherlands Organtini Lindsey United States of America Pallansch Mark United States of America Palmemberg Ann United States of America Europic 2016 213

214 LIST OF PARTICIPANTS L astname F irstname C ountry Parida Satya United Kingdom Peersen Olve United States of America Peeters Michael Belgium Pint Rosa Spain Plevka Pavel Czech Republic Porta Claudine United Kingdom Rieder Elizabeth United States of America Roos Raymond United States of America Rowlands David United Kingdom Royston Lna Switzerland Ruokolainen Visa Finland Ryan Martin United Kingdom Sanders Barbara Netherlands Sato Hironori Japan Scheers Els Belgium Schibler Manuel Switzerland Seago Julian United Kingdom Selvarangan Rangaraj United States of America Semler Bert United States of America Shakeel Shabih Finland Shengjuler Djoshkun United States of America Shih Shin-Ru Taiwan Shimizu Hiroyuki Japan Simmonds Peter United Kingdom Sioofy Khojine Amirbabak Finland Smith Matt United Kingdom Smithee Shane United States of America Solano hermosillH Belen Netherlands Solari Roberto United Kingdom Sonobe Yoshifumi United States of America Stockley Peter United Kingdom Stonehouse Nicola United Kingdom Strauss Mike Germany Stuart David United Kingdom Sun Liang Belgium Svedin Emma Sweden Swanson Jessica United Kingdom Tapparel Caroline Switzerland Tauriainen Sisko Finland Thibaut Hendrik Jan Netherlands Tseligka Eirini Switzerland Tulloch Fiona United Kingdom Tuthill Toby United Kingdom 214 Europic 2016

215 LIST OF PARTICIPANTS L astname F irstname C ountry van der Sanden Sabine Netherlands van der Schaar Hilde Netherlands van Kuppeveld Frank Netherlands Vandesande Helena Sweden Vega Everardo United States of America Vignuzzi Marco France Viktorova Ekaterina United States of America Visser Linda Netherlands Volle Romain France Ward Joseph United Kingdom Watters Kelly United States of America Wimmer Eckard United States of America Witte Robert Switzerland Wolthers Katja Netherlands Woodman Andrew United States of America Wright Caroline United Kingdom Xie Lili United States of America Yan Dongmei China Europic 2016 215

216 SPONSORS The Organizing Committee of the 19th International Picornavirus Meeting would like to thank the following companies and institutions for their support ABBOTT www.abbott.com Educational sponsoring BIOMRIEUX www.biomerieux.com Travel grant sponsoring ELSEVIER www.journals.elsevier.com/antiviral-research/ EPITHELIX www.epithelix.com 216 Europic 2016

217 SPONSORS Federation of European Microbiological Societies www.fems-microbiology.org Travel grant sponsoring GILEAD www.gilead.com JANSSEN www.janssen.com Educational sponsoring Pharma Consulting Marion Senn GmbH www.pharmaconsulting.ch Europic 2016 217

218 SPONSORS QIAGEN www.qiagen.com ROCHE www.roche-diagnostics.ch RUWAG www.ruwag.ch UNIVERSITY OF GENEVA www.unige.ch 218 Europic 2016

219 GENERAL INFORMATION Congress Venue La Maison des Congrs 5 minutes walking distance from the Eurotel Victoria Hotel Lunches and Dinners Location (according to the programme) Restaurant of the Eurotel Victoria Hotel Registration desk opening hours Sunday September 4: 16:00-18:30 (in the Eurotel Victoria Lobby) Monday September 5: 08:00-12:00 13:30-18:30 Tuesday September 6: 08:15-12:00 13:30-19:00 Wednesday September 7: 08:15-12:00 10:00-16:00 Thursday September 8: 08:15-12:00 WIFI Free WIFI is available in the congress center. The code is available at the registra- tion desk. For the participants who are staying at the Eurotel Victoria Hotel, please use the same WIFI than in the hotel. Europic 2016 219

220 GENERAL INFORMATION Gala Dinner on Wednesday September 7 at 19:00 Discover a Swiss typical evening. Bring warm clothes! 10 minutes walking distance from the Eurotel Victoria. Excursions on September 8 at 13:00 Departure in front of the congress venue (Maison des Congrs) For the visits, the participants must be equipped with walking shoes, warm clothes and sunglasses Visit 1 Parc des Diables Les Diablerets treetop adventure park! Venture along obstacle trails set-up high in the trees. Experience the 3 different courses and negociate the obstacles, ladders and bridges that lead you high up into the trees. Rate: CHF 90 per person (VAT not included) the rate includes the guides, the parc entrance and refreshments Duration of the tour: 2-3 hours back to Les Diablerets at 15h30 220 Europic 2016

221 GENERAL INFORMATION Visit 2 Hiking Les Ormonts Hiking from les Diablerets to Vers lEglise and visit of the typical mountain village and the charming museum. The current exhibition illus- trates the story of the A.S.D Aigle Spey Diablerets trainline, 100 years of conquering the mountain! Rate: CHF 60 per person (VAT not included) the rate includes the guides, the museum entrance and refreshments Duration of the tour: walk around 1 hour back to Les Diablerets at 15h30 Visit 3 Glacier Experience 24 peaks over 4000m can be seen from the top of the glacier! A variety of activities are organ- ised surrounded by this breath-taking panora- ma. 13h: transfer by minibus to Col du Pillon 13h20: Ascension by cable-car to the Glacier 3000 Treck on the Glacier (1h): accompanied by 2 mountain guides, who shall explain the glacier geography Ride the Alpine Coaster: highest roller-coaster in the world, 1km descent, 100m drop in altitude, 11 waves, 3 jumps, 2 bridges and a maximum of speed of 40km per hour! Peak Walk: The Peak Walk by Tissot is the first suspension bridge (107m long and 80cm wide) between two points, connecting the View Point with the main peak Scex Rouge . Schnaps offered at the end of the bridge! 16h00: Descent with the cable-cars to the Col du Pillon 16h30: Back to the village Les Diablerets Rate: CHF 180 per person (VAT not included) the rate includes, the bus trans- fert, the guides, the cable car, the alpine coaster, the Peak-Walk and refreshments Europic 2016 221

222 GENERAL INFORMATION About Les Diablerets Les Diablerets, an important centre for adven- ture sports, lies between Lake Geneva and Gstaad at an altitude of 1200 metres. Even in summer, the Glacier 3000 ski region in the heart of the Vaud Alps offers skiing and glacier enjoy- ment over an expansive area. In the Middle Ages, the rock wall of the mountain massif which surrounds the small village was regarded as a dangerous and cursed place where the devil did his worst. Accordingly, the place name of Les Diablerets also derives from the French le Diable, the devil. But no one need harbour any fears in Les Diablerets today, at the most some courage for the multitude of adventurous sports on offer. Highlights Glacier 3000 snow fun, cross-country skiing and savouring the outstanding vista in the Gastronamik panorama restaurant. Aigle viticultural museum the viticultural museum in the canton of Vaud presents nearly 2000 years of the history of viticulture. Bex salt works salt mine with a gallery length of over 50 km. Part of the underground labyrinth can be visited on a tour. Gstaad-Saanenland the neighbouring region in the Bernese Oberland is a meeting point for high society as well as unadul- terated nature. Narrow-guage railway network from Aigle and Bex the lines to Leysin, Les Diablerets and Villars/Bretaye provide access to the region without a car of your own. 222 Europic 2016

223 GENERAL INFORMATION Free Access Card The Free Access Card gives you a large selection of free deals during your stay. Each ho- tel guest receives a Free Access Card on arrival at their accom- modation. This card allows un- limited use of up to 30 different mountain transports (trains, gondolas, buses), as well as different sporting and fun ac- tivities. More information at the hotel reception Train Schedule Les Diablerets Aigle Europic 2016 223

224 GENERAL INFORMATION Tourist board office Address: Chemin du Collge 2, 1865 Les Diablerets Website : www.diablerets.ch Congress secretariat Rue Rousseau 30 1201 Geneva Switzerland Email : [email protected] 224 Europic 2016

225 NOTES Europic 2016 225

226 NOTES 226 Europic 2016

227 Rapidly mutating HIV-1 continues to challenge single target viral load tests, especially whentargeting a region subject to selective drug pressure. Stay one step ahead with the innovative HIV-1 Dual Target assay from Roche Molecular Diagnostics. 2015 Roche Roche Diagnostics (Schweiz) AG 6343 Rotkreuz Visit us at www.dual-target.com for more information.

228 Accurate HCV viral load monitoring is essential for current and future HCV treatments: to evaluate virological response, guide treatment duration, and decide on futility. This demands an HCV RNA assay that can correctly distinguish true signals from background noise. See what truly matters with the innovative HCV Dual Probe assay from Roche Molecular Diagnostics. 2015 Roche Roche Diagnostics (Schweiz) AG 6343 Rotkreuz Visit us at www.hcvdualprobe.com for more information.

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