Programme and Abstracts

PROGRAMME AND ABSTRACTS

Budapest 7-10 June 2017

4th International Symposium on Ranaviruses June 7 - 10, 2017 Budapest, Hungary

http://kabafalvi.wixsite.com/isvlv-2017 https://www.rana-2017.com/

Cover photo: Zsolt Sonnleitner

Thank You to our Sponsors!

TABLE OF CONTENTS

Welcome from the Organizers ...... 4 Contacts ...... 5 Welcome from the Director of the GRC ...... 6 The Global Ranavirus Consortium Board ...... 8 Scientific Committee ...... 10 Maps ...... 11 Budapest map ...... 11 University campus map ...... 12 Program and Overview ...... 12 Oral Presentations ...... 14 Poster Presentations ...... 20 Field Trips ...... 22 Oral Presentation Abstracts ...... 23 Poster Presentation Abstracts ...... 64 Author Index A-Z ...... 81 List of Participants ...... 84

WELCOME FROM THE ORGANIZERS

Dear Colleague,

We are delighted to welcome you to the 4th International Symposium on Ranaviruses (ISR) in Budapest. This is the first time that the ISR has been held outside the United States. Regular ISR participants may find certain conditions unusual, as Hungary is not one of Europe's most advanced or richest countries.

This is also the first time that the ISR is being held adjacent to the International Symposium on Viruses of Lower Vertebrates. The idea of hosting the two conferences here consecutively came to Rachel Marschang since there is a considerable overlap between the scientific interests of the usual audiences of the two symposia. Rachel was right indeed. More than half of the registered guests took advantage of the reduced fee for the combined participation. Over 100 delegates representing more than 20 countries from five continents are expected so that we had to choose a slightly larger lecture hall for the first, joint day.

We tried our best to keep up with the high standard of ISRs including the organization of hopefully intriguing field trips that provide insights into the problems and practices of nature conservation specific to Hungary.

Please enjoy the proximity of the campus to the real downtown of Pest, but do not forget to climb at least one hill (Gellért or Castle Hill) on the Buda side. The best panorama can be viewed around sunset.

We wish you a relaxing and fruitful meeting, and hope that you will enjoy your stay here as much as we have enjoyed the preparations. Special thanks are due to the members of the Scientific Committee and to the GRC Board for editing the abstract book and for technical and financial support, respectively.

Sincerely,

Mária Benkő, Balázs Harrach, Tibor Papp Local Organizers Rachel E. Marschang GRC Representative

CONTACTS

In case of questions or emergency, please do not hesitate to contact us Tibor Papp: +36 20 618 6114; [email protected] Mária Benkő: +36 30 569 8165; [email protected]

H-1143 Budapest, Hungária krt. 21. Hungary

WELCOME FROM THE DIRECTOR OF THE GRC

The mission of the Global Ranavirus Consortium (GRC) is to facilitate communication and collaboration among professionals and students interested in ranaviruses. The GRC is dedicated to: (1) advancing knowledge in all areas of ranavirus biology and disease, (2) facilitating multi-disciplinary research collaborations, (3) disseminating information on ranaviruses, and (4) providing technical guidance and training opportunities. The first GRC Board was elected and formed in April 2013, and consists of a Director (Dr. Matthew Gray), Associate Director (Dr. Jesse Brunner), Secretary/Treasurer (Dr. Amanda Duffus), and five continental representatives (Drs. Ellen Ariel, Rachel Marschang, Rolando Mazzoni, Yumi Une, and Thomas Waltzek). In addition, Dr. Greg Chinchar serves as an honorary advisor to the Board. During the 2017 International Symposium on Ranaviruses (ISR), the Board will transition to the following leadership: Past-Director (Dr. Matthew Gray), Director (Dr. Jesse Brunner), Associate Director (Dr. Tom Waltzek), Secretary/Treasurer (Dr. Amanda Duffus), and five continental representatives (Drs. Ellen Ariel, Marja Kik, Claudio Soto, Qiwei Qin, and Maria Forzan). Thank you to all of the officers that served for the past four years and congratulations to the incoming Board!

Since 2013, there have been several milestones for the GRC: (1) bylaws were drafted and approved, (2) the GRC became incorporated as a non-profit organization, (3) application for 501c tax-exempt status in the USA was submitted, (4) a new website with various resources was created and maintained (http://www.ranavirus.org/), (5) three international symposia were hosted, (6) a workshop on ranaviruses was organized in Harbin, China, (7) workshops on designing ranavirus studies, analyzing surveillance data, and diagnostic techniques were delivered at the 2015 ISR, (8) the Global Ranavirus Reporting System (GRRS, https://mantle.io/grrs) was created, (9) an open access eBook on ranaviruses was published (http://link.springer.com/book/10.1007%2F978-3-319-13755-1), and (10) an online course over ranaviruses was instructed; 27 GRC members contributed to instruction with 46 course participants from 7 countries.

Future activities of the GRC will be discussed during the 2017 ISR, but might include: (1) concerted efforts to populate the GRRS, (2) establishing continental discussion groups that meet online to discuss ongoing research, publications and opportunities for collaboration, and (3) organizing regional workshops and other outreach activities. As part of (3), we hope to offer the online course over ranaviruses again, but to allow participants the option to take portions of the full course that align most with their interests. We encourage all symposium attendees to become members of the GRC ($15 students, $30 professionals). Membership fees help maintain the GRC website, generate funds for travel grants and outreach activities, and defer costs of the biennial symposia. The GRC also maintains a LISTSERV ([email protected]) to enhance communication, which can be joined via the GRC website.

Factors responsible for the emergence of ranaviruses in ectothermic vertebrate communities are complex. In order to unravel the causes of ranavirus emergence and identify disease mitigation strategies, it requires teams of professionals with various areas of expertise. The GRC strives to foster development of new professional relationships that lead to the advancement of knowledge on ranaviruses. Please take a part in this mission by becoming a member of the GRC, serving on a GRC ad hoc committee, participating in a GRC continental discussion group, and attending future symposia. The GRC Board looks forward to interacting with you during this symposium and in the future!

All the Best— Matt Gray Director, Global Ranavirus Consortium, Inc. http://www.ranavirus.org/

THE GLOBAL RANAVIRUS CONSORTIUM BOARD 2013-2017:

DIRECTOR ASSOCIATE DIRECTOR SECRETARY-TREASURER

Matthew J. Gray, Ph.D. Jesse L. Brunner, Ph.D. Amanda L. J. Duffus Ph.D. University of Tennessee, USA Washington State University, Gordon State College, USA [email protected] USA [email protected] [email protected]

ASIA REPRESENTATIVE AUSTRALIA REPRESENTATIVE EUROPE AND AFRICA REPRESENTATIVE

Yumi Une, D.V.M., Ph.D. Ellen Ariel, Ph.D. PD Dr. vet. med. Rachel James Cook University, Marschang Azabu University, Japan Australia Laboklin GmbH & Co. KG, une@azabu‐u.ac.jp [email protected] Germany [email protected]

NORTH AMERICA REPRESENTATIVE SOUTH AMERICA HONORARY ADVISOR REPRESENNTATIVE

Thomas B. Waltzek, D.V.M., Rolando Mazzoni, D.V.M., V. Gregory Chinchar, Ph.D. Ph.D. Ph.D. University of Mississippi University of Florida, USA Universidade Federal de Goiás, Medical College, USA [email protected] Brazil [email protected] [email protected]

2017-2021

PAST DIRECTOR DIRECTOR ASSOCIATE DIRECTOR

Matthew J. Gray, Ph.D. Jesse L. Brunner, Ph.D. Thomas B. Waltzek, D.V.M., University of Tennessee, USA Washington State University, Ph.D. [email protected] USA University of Florida, USA [email protected] [email protected]

SECRETARY-TREASURER ASIA REPRESENTATIVE AUSTRALIA REPRESENTATIVE

Amanda L. J. Duffus Ph.D. Qin Qiwei, PhD Ellen Ariel, Ph.D. Gordon State College, USA South China Agricultural James Cook University, [email protected] University Australia [email protected] [email protected]

EUROPE AND AFRICA NORTH AMERICA REPRESENTATIVE SOUTH AMERICA REPRESENTATIVE REPRESENNTATIVE

Marja Kik, DVM, PhD María J. Forzán Claudio Soto Azat, DVM, PhD Utrecht University, Netherlands Cornell School of Veterinary [email protected] Medicine, USA Universidad Andrés Bello, Chile [email protected] [email protected]

SCIENTIFIC COMMITTEE

Chair: Rachel E. Marschang, PD Dr. vet. med., Dip. ECZM (herpetology) Laboklin GmbH & Co. KG Bad Kissingen, Germany and University of Hohenheim, Stuttgart, Germany

Ellen Ariel, PhD College of Public Health Medical and Veterinary Science James Cook University Townsville, QLD, Australia

Victor Gregory Chinchar, PhD University of Mississippi Medical Center Jackson, Mississippi, USA

David Lesbarréres, PhD Laurentian University Sudbury, Ontario, Canada

Amanda L. J. Duffus, PhD Gordon State College Barnesville, Georgia, USA

Tibor Papp, DVM, PhD Institute for Veterinary Medical Research Centre for Agricultural Research Hungarian Academy of Sciences Budapest, Hungary

MAPS Budapest map:

- Conference location (István street 2, Budapest H-1078)

- Kossuth Museum Boat, Vén Hajó Restaurant, at Széchenyi tér, Chain Bridge

- Starting point for the optional tour on Friday

wi-fi: univet, equus password: Budapest

University campus map:

WC WC

- Conference room (Tolnay Sándor)

- Conference room for the joint day, Wednesday (Hetzel Henrik)

- Lunch, Poster session(Equus club)

- Entrances of the campus

- Entrances of the auditoria

PROGRAM AND OVERVIEW

Oral Presentations WEDNESDAY, June 7th

JOINT SESSIONS ISVLV AND ISR

(All session in the Hetzl Henrik lecture hall)

8:00-9:00: Registration (Lobby)

9:00-9:15: Welcome and Opening of the 4th International Symposium on Ranaviruses

9:15-11:00: LARGE dsDNA VIRUSES I (Chair: Stephen J. Price)

9:15-10:00: Thomas B. Waltzek: Overview of the nucleo-cytoplasmic large DNA viruses (NCLDVs) (p. 23)

10:00-10:15: Sharon Clouthier*, Rachel Breyta, Gael Kurath, Eric Anderson: Sturgeon nucleo-cytoplasmic large DNA virus phylogeny (p. 24)

10:15-10:30: Samantha A. Koda*, Kuttichantran Subramaniam, Ruth Francis-Floyd, Roy P. Yanong, Salvatore Frasca, Jr., Joseph M. Groff, Shipra Mohan, William A. Fraser, Annie Yan, Thomas B. Waltzek: Phylogenomic characterization of a novel megalocytivirus lineage from archived ornamental fish samples (p. 25)

10:30-10:45: Tibor Papp*, Rachel E. Marschang: A member of the genus Iridovirus in reptiles and amphibians? (p. 26)

10:45-11:00: Kuttichantran Subramaniam*, Thomas B. Waltzek, Nicole I. Stac2, Claire Grosset, James F.X. Wellehan Jr.: Phylogenomic characterization of squamate erythrocytic iridoviruses (p. 27)

11:00-11:30: Coffee break

11:30-13:00: LARGE dsDNA VIRUSES II (Chair: Ellen Ariel)

11:30-11:45: Takafumi Ito*, Jun Kurita, Olga L. M. Haenen: Spread of various strains of cyprinid herpesvirus 2 (CyHV-2) through global trade of goldfish (p. 28)

11:45-12:00: Kyle A. Garver, Katja Leskisenoja, Laura M Hawley, Robert Macrae, Kuttichantran Subramaniam, Thomas B. Waltzek, Jon Richard, Caroline Josefsson, Rudolf Hoffmann, Theresia Fischer-Scherl, E. Tellervo Valtonen*: Discovery of a novel herpesvirus infection of European perch (Perca fluviatilis) in Finland (p. 29)

12:00-12:15: Francesco C. Origgi*, Benedikt R. Schmidt, Petra Lohmann, Patricia Otten, Ezgi Akdesir, Veronique Gaschen, Lisandra Bultet-Aguilar, 14

Thomas Wahli, Ursula Sattler, Michael H. Stoffel: Ranid herpesvirus 3 (RHV3), a novel virus associated with a proliferative skin disease in free-ranging wild common frogs (Rana temporaria) (p. 30)

12:15-12:30: Mikolaj Adamek*, Anna Oschilewski, Peter Wohlsein, Verena Jung- Schroers, Felix Teitge, David Gela, Veronika Piackova, Martin Kocour, Jerzy Adamek, Sven M. Bergmann, Dieter Steinhagen: Results from carp edema virus infections in differently susceptible common carp strains reveal differences in virulence between CEV genogroups (p. 31)

12:30-12:45: Mikolaj Adamek*, Felix Teitge, Martin Ganter, Ilka Baumann, Verena Jung-Schroers, David Gela, Veronika Piackova, Martin Kocour, Dieter Steinhagen: “Koi sleepy disease” as a pathophysiological consequence of branchial infection of common carp with carp edema virus (p. 32)

12:45-13:00: Joseph M. Groff, Kuttichantran Subramaniam, Robert W. Nordhausen, Thomas B. Waltzek*: Phylogenomic Characterization of a Novel Seahorse Poxvirus from Formalin-Fixed Paraffin-Embedded Tissues (p. 33)

13:00-14:30: Lunch (in the Equus Club)

14:30-15:45: RANAVIRUS EPIDEMIOLOGY (Chair: Jolianne M. Rijks)

14:30-14:45: Szilvia L. Farkas*,,Enikö Fehér, Andor Doszpoly, Balázs Horváth, Szilvia Marton, Barbara Forró, Krisztian Bányai, Tamás Juhász: Detection and complete genome analysis of ranaviruses causing mass mortality in brown bullheads (Ameiurus nebulosus) in Hungary (p. 34)

14:45-15:00: Stephen J. Price*, Alexandra Wadia, Owen Wright, Will Leung, Andrew A. Cunningham, Becki Lawson: How ranavirus brought Britain closer to the EU – molecular screening of a long-term tissue archive from herpetofauna (p. 35)

15:00-15:15: Paul M Hick*, Kuttichantran Subramaniam, Patrick M Thompson, Thomas B Waltzek, Joy Becker, Richard J Whittington: Molecular epidemiology of Epizootic haematopoietic necrosis virus (EHNV) (p. 36) 15:15-15:30: Bernardo Saucedo*, Joseph Hughes, Nicolas Suarez, Olga Haenen, Michal Voorbergen-Laarman Natasja Kruithof, Jolianne Rijks, Marc Schils, Annemarieke Spitzen, Maarten Gilbert, Andrea Gröne, Steven van Beurden: Comparative genomics and spatiotemporal characterization of two strains of Common midwife toad virus in the Netherlands (p. 37)

15:30-15:45: Hannah E. B. Stagg*, Sigríður Guðmundsdóttir, Niccolò Vendramin, Neil Ruane, Heiða Sigurðardóttir, Debes H. Christiansen, Argelia Cuenca Navarro, Petra E. Petersen, Eann Munro, Niels Jørgen Olesen: Isolation and characterisation of a new ranavirus isolated from lumpfish in the north Atlantic area (p. 38)

15:45-16:15: Coffee break

16:15-17:00 IRIDOVIRIDAE: TAXONOMY: Panel discussion led by Thomas B. Waltzek

19:00-22:00: Joint ISVLV/ISR Banquet (location: Kossuth Museum Boat, Vén Hajó Restaurant, 1052 Budapest, Vigadó Square dock, pier 2 – near the Chain Bridge, Pest side, Széchenyi tér)

THURSDAY, June 8th

(All sessions in the Tolnay Sándor lecture hall)

9:00-10:45: SURVEILLANCE AND ECOLOGY (Chair: Maria Forzán)

9:00-9:45: Trenton W. J. Garner, Stephen J. Price, Ellen Ariel: The European Ranaviruses (p. 39)

9:45-10:00: Bernardo Saucedo*, Joseph Hughes, Nicolás Suárez, Olga Haenen, Michal Voorbergen-Laarman, Andrea Gröne, Marja J L Kik, Steven van Beurden: Complete genome sequence of a Frog Virus 3-like ranavirus from an Oophaga pumilio frog imported from Nicaragua into the Netherlands (p. 40)

10:00-10:15 Matthew J. Gray*, Jennifer A. Spatz, E. Davis Carter, Debra L. Miller: Poor biosecurity could lead to ranavirus outbreaks in amphibian populations (p. 41)

10:15-10:30: Rebecca H. Hardman*, William B. Sutton, Dale McGinnity, K. J. Irwin, Sherri Reinsch, Ben Fitzpatrick, Philip Colclough, Marcy Souza, Michael Freake, Matthew. J. Gray, Debra L. Miller: Prevalence of ranavirus and Bd in hellbender polulations in Tennesee and Arkansas (p. 42)

10:30-10:45: Emily M. Hall, Caren S. Goldberg, Erica J. Crespi, Jesse L. Brunner*: Seasonal dynamics of ranavirus epidemics in wood frog populations (p. 43)

10:45-11:15: Coffee break

11:15-12:45: IMMUNOLOGY (Chair: Leon Grayfer)

11:15-11:45: Jacques Robert: The interplay between ranavirus and the amphibian immune system (p. 44) 16

11:45-12:00: Qiwei Qin*, Yepin Yu, Youhua Huang, Xiaohong Huang: Singapore grouper iridovirus (SGIV) TNFR homolog VP51 functions as a virulence factor by modulating cellular apoptosis and the host inflammatory response (p. 45)

12:00-12:15: Amulya Yaparla, Leon Grayfer*: Differentiation-dependent antiviral capacities of amphibian (Xenopus laevis) macrophages (p. 46)

12:15-12:30: Jacques Robert*, Eva-Stina Edholm, Maureen Banach, Timothy Kwan, Jazz Sanchez, Leta Yi, Francisco De Jesús Andino: Different antiviral T cell type immunity in tadpole and adult Xenopus (p. 47)

12:30-12:45: Lewis J. Campbell*, S. Austin Hammond, Stephen J. Price, Manmohan D. Sharma, Trenton W.J. Garner, Inanc Birol, Caren C. Helbing, Lena Wilfert, Angus Buckling, Amber G.F. Griffiths: A novel approach to transcriptomics in wild animals provides evidence for disease mediated differential expression and changes to the microbiome in amphibian populations (p. 48)

12:45-14:00: Lunch (in the Equus Club)

14:00-15:30: INFECTION AND DIAGNOSIS (Chair: Rachel E. Marschang)

14:00-14:15: María J. Forzán*, Erica Sloma: Increased detection of ranavirus in tissue using novel in situ hybridization technique (p. 49)

14:15-14:30: Alicia Maclaine*, Jennifer Scott, Wytamma Wirth, Narges Mashkour, Ellen Ariel: Pathogenesis of Bohle iridovirus in juvenile Eastern water dragons, Intellagama lesueurii lesueurii (p. 50)

14:30-14:45: Jesse L. Brunner*, Anjulie Olson, Jeremy G. Rice, Mitchel J. Le Sage, Jennifer A. Cundiff, Caren S. Goldberg, Allan P. Pessier: Performance of nonlethal methods of detecting ranavirus infections in captivity and trade (p. 51)

14:45-15:00: Debra L. Miller*, Ana Balseiro, Rosa Casais, Matthew J. Gray: Immunohistochemistry of hellbenders exposed to an FV3-like ranavirus, alone or with one stressor (p. 52)

15:00-15:15: Wytamma Wirth*, Jennifer Scott, Ellen Ariel: Dose-dependent morbidity of freshwater turtle hatchlings, Emydura macquarii krefftii, inoculated with Bohle iridovirus (Ranavirus sp, Iridoviridae) (p. 53)

15:15-15:30: Natalie K. Steckler, Salvatore Frasca Jr., Kuttichantran Subramaniam, Kamonchai Imnoi, Lacey Hopper, Jeffrey Powell, James Colee, Thomas

B. Waltzek*: The effect of water temperature on Frog virus 3 disease in young-of-the-year pallid sturgeon (Scaphirhynchus albus) (p. 54)

15:30-16:00: Coffee break

16:00-17:30: Regional meetings (location to be announced)

17:30-19:30: Poster session (in the Equus Club)

FRIDAY, June 9th

(All morning sessions in the Tolnay Sándor lecture hall)

9:00-11:00: ECOLOGY AND PATHOLOGY (Chair: Jesse Brunner)

9:00-9:15: Matthew J. Gray: The Global Ranavirus Consortium

9:15-9:30: Sieara C Claytor, Kuttichantran Subramaniam, V. Gregory Chinchar, Matthew Gray, Carla Mavian, Marco Salemi, Samantha Wisely, Thomas Waltzek*: Ranavirus phylogenomics: Signatures of recombination and inversions among ranaculture Isolates (p. 55)

9:30-9:45: E. Davis Carter*, Matthew J. Gray, Jenny A. Spatz, Debra L. Miller: Interaction of hydroperiod and ranavirus leading to possible amphibian population declines in the Great Smoky Mountains National Park (p. 56)

9:45-10:00: Angela Peace, Suzanne M. O'Regan, Jennifer A. Spatz, Patrick N. Reilly, Rachel D. Hill, E. Davis Carter, Rebecca P. Wilkes, Debra L. Miller, Matt Gray*: Importance of ranavirus transmission pathways mediated by density dependence (p. 57)

10:00-10:15: Roberto Brenes*, Jason T. Hoverman, Debra L. Miller, Matthew J. Gray: Presence of amplification hosts increases mortality of syntopic amphibians by ranaviral disease (p. 58)

10:15-10:30: Xiaohong Huang, Youhua Huang, Shina Wei, Yepin Yu, Qiwei Qin*: Comparative proteomic analysis show altered proteins regulated by ubiqutin proteasome system in fish iridovirus infection (p. 59)

10:30-10:45: Joe-Felix Bienentreu, Samantha A. Grant, Chris J. Kyle, Craig R. Brunetti, Danna M. Schock, David Lesbarrères*: Ranavirus in northern Canada: a low-diversity ecosystem for a better comprehension of disease dynamics? (p. 60)

10:45-11:00: Preeyanan Sriwanayos, Kuttichantran Subramaniam*, Natalie K. Steckler, Somkiat Kanchanakhan, Jaree Polchana, Thomas B. Waltzek: Phylogenomic characterization of ranaviruses detected in fish and amphibians in Thailand (p. 61)

11:00-11:30: Coffee break

11:30-13:00: Jesse L. Brunner: WORKSHOP: The Global Ranavirus Reporting System (GRRS) (p.62)

13:00-14:15: Lunch (in the Equus Club)

(Early afternoon session (14:15-15:30) in the Equus Club)

14:15-15:30: Breakout discussions (location: Equus Club):  Techniques for identification and classification of isolates  Diagnosis, Treatment, and Management (moderator: Paul M. Hick)  Epidemiology and Surveillance (moderator: David Lesbarrères)

15:30-16:00: Coffee break (Late afternoon session (16:00-18:00) in the Tolnay Sándor lecture hall)

16:00-17:00: Joint presentation and discussion of breakout group topics

17:00-18:00: The state of the GRC, GRC general meeting

Poster Presentations (Exhibited in the Equus Club)

Sthefany R. Alfaia, Cinthia R. Oliveira, Fernanda L. Ikari, Diego Sales, Ricardo L. Moro, Claudia Maris Ferreira*: Susceptibility of American bullfrog tadpoles (Lithobates castebeianus) to experimental infection by Frog Virus 3 (FV3) (p. 64)

Cinthia R. de Oliveira, Sthefany R. Alfaia, Fernanda L. Ikari, Patricia T. Coelho, Loiane S. Tavares, Ricardo L. Moro de Sousa, Claudia M. Ferreira*: Screening and molecular characterization of a ranavirus (Frog Virus 3) obtained from bullfrogs (Lithobates catesbeianus) from commercial farms in Southeast Brazil (p. 65)

Kateřina Matějíčková*, Stanislava Reschová, Pavel Kulich, Barbora Veselá, Tomáš Veselý: Is the angelfish Pterophyllum scalare susceptible to ranaviruses? (p. 66)

William T.M. Leung*, Laura Thomas-Walters, Trenton W.J. Garner1, Francois Balloux3, Chris Durrant1†, Stephen J. Price: Robust viral load estimation in ranavirus-infected amphibian, reptile, and fish tissues and cell cultures (p. 67)

Narges Mashkour*, Ellen Ariel: Development of green sea turtle (Chelonia mydas) primary cells to propagate aquatic animal viruses including Bohle Iridovirus (p. 68)

Jingguang Wei, Youhua Huang, Min Yang, Xiaohong Huang, Qiwei Qin*: Isolation and identification of Singapore grouper iridovirus Hainan (SGIV-HN) isolate (p. 69)

Augustino Alfred Chengula*, Christopher Kasanga, Stephen Mutoloki, Robinson Mdegela, Øystein Evensen: Molecular characterization of ranaviruses detected in Nile Tilapia (Oreochromis niloticus) in Tanzania (p. 70)

Sheng Zhou, Zhuxi Wang, Youhua Huang, Shaowen Wang, Xiaohong Huang, Qiwei Qin*: Envelope protein VP088 of Singapore grouper iridovirus is involved in viral attachment and entry into host cells (p. 71)

Lewis J. Campbell*, Trenton W.J. Garner, Giulia Tessa, Ben C. Scheele, Amber G.F. Griffiths, Lena Bayer-Wilfert, Xavier A. Harrison: Ranavirus infection potentially alters the age structure of infected populations (p. 72)

Marius von Essen, William T.M. Leung, Celia Serrano, Trenton W.J. Garner, Jaime Bosch, Cesar Ayres, Simon Pooley, Stephen J. Price*: Host range and local distribution of Bosca’s newt virus (p. 73)

Shina Wei, Youhua Huang, Jingguang Wei, Xiaohong Huang, Qiwei Qin*: Characterization of two novel cysteine protease inhibitors of cystatin B and cystatin C from grouper, Epinephelus coioides involved in SGIV infection (p. 74)

Youhua Huang, Xiaohong Huang, Shaowen Wang, Yepin Yu, Qiwei Qin*: Soft-shelled turtle iridovirus enters cells via cholesterol-dependent, clathrin-mediated endocytosis as well as macropinocytosis (p. 75)

Yvonne Black*, Anna Meredith, Stephen J. Price: Detection and reporting of ranavirus in amphibians: Evaluation of the roles of the World Organisation for Animal Health and the published literature (p. 76)

Lauren V. Ash*, C. Brandon Ogbunugafor, James Andrews, Aswini Cherukuri, Nicholas J. Gotelli: Detecting the emerging infectious disease ranavirus in amphibian communities of Vermont, USA (p. 77)

Maarten Gilbert*, Annemarieke Spitzen-van der Sluijs, Frank Pasmans, Richard Struijk, Marc Schils, Pieter Doornbos, Fleur van der Sterren, Jolianne Rijks, Marja Kik, Bernardo Saucedo, Wilbert Bosman, An Martel: Long-term monitoring of a recurrent Ranavirus outbreak in an isolated Pelobates fuscus population (p. 78)

Eloy Becares, Matthew J. Gray, E. Davis Carter, Irena Fernandez, Ana Balseiro, Debra L. Miller*: Negative qPCR for Ranavirus, Bd, and Bsal in surveillance samples from Castile and León Province, Spain (p. 79)

FIELD TRIPS TH SATURDAY, JUNE 10

1. Kiskunság National Park, Meadow Viper Conservation Centre, 30 EUR/person (including lunch), ca. 8:30 AM – 6 PM Starting in Budapest, we will take an hour long bus ride to the Hungarian Meadow Viper Conservation Centre. You can learn more about this flagship EU Life Project via the following link: http://www.rakosivipera.hu/en/. The morning program will take approx. 2hrs, and will be followed by a 20 min. ride to a local restaurant/resort for lunch. Before the 3 course menu, we will enjoy a local horse show. In the afternoon, we will travel on to the Upper-Kiskunság Plain (Felső-Kiskunsági Puszta), where we will do some bird watching, and also be able to see some traditional extensive animal husbandry with the typical ancient Hungarian domestic animals, including grey cattle and racka sheep. We will return to Budapest by around 6 PM in the evening.

2. Duna-Ipoly National Park, Sas-hegy (Eagles’s hill) Protected Area, Buda hills Nature Reserve Area, 15 EUR/person (no meal included), ca. 9:00 AM – 1 PM During this short field trip, you will stay within Budapest and learn about its natural habitats on its hilly Buda side. If you are lucky, you will be able to see/catch some representatives of the local herpetofauna. In the first 2 hours you will explore the Sas-hill nature trail. This enclosure within the city holds some unique flora and fauna elements on its dolomite slopes, and it offers a beautiful overview of the city. Later in the morning, in the forests of Buda outskirts, you will visit a hidden population of the fire salamander (S. salamandra). If the weather is rainy, then you can sample the adults for Bd with Judit Vörös. Otherwise, you will meet only the larvae.

ORAL PRESENTATION ABSTRACTS

Overview of the nucleo-cytoplasmic large DNA viruses (NCLDVs)

Thomas B. Waltzek 1* 1Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610

The Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs) are a diverse viral assemblage infecting a range of eukaryotes from unicellular algae and protists (e.g. phagocytic flagellates and amoebae) to multicellular invertebrates (e.g. sponges, corals, arthropods, cephlapods) and vertebrates (e.g. fish, amphibians, reptiles, birds, mammals). The NCLDVs include seven viral families: Ascoviridae, Asfarviridae, Iridoviridae, Marseilleviridae, Mimiviridae, Phycodnaviridae, and Poxviridae. They replicate entirely within the cytoplasm (e.g. Poxviridae) or involve both the nucleus and the cytoplasm (e.g. Iridoviridae). The large virion and genome sizes, a suite of conserved genes, and unique replication scheme has been argued to unite the NCLDVs families into a proposed order, the megavirales. To date, only iridoviruses have been established as significant pathogens of poikilothermic vertebrates (i.e. fish, amphibians, and reptiles). There are five genera within the family Iridoviridae: two contain members that infect invertebrates (Chloriridovirus, Iridovirus), whereas the remaining three contain viruses that infect only fish (Lymphocystivirus and Megalocytivirus) or fish, amphibians, and reptiles (Ranavirus). Recent phylogenomic analyses suggest erythrocytic iridoviruses of fish, amphibians, and reptiles form a monophyletic group (tentatively named the hemocytiviruses) branching off between the invertebrate and vertebrate iridoviruses. The recent partial genomic sequencing of a new branch of mimivirus, responsible for lethal diseases in endangered sturgeon, expands the known host range of the family Mimiviridae from protists to vertebrates. Phylogenetic analyses based on partial sequencing of poxviruses from Atlantic salmon (Salmo salar), common carp and koi (Cyprinus carpio), Ayu (Plecoglossus altivelis), and the cape seahorse (Hippocampus erectus) suggest fish poxviruses form a monophyletic group that represent a new genus (tentatively proposed piscipoxviruses) or subfamily. The success of NCLDVs, as evidenced by their abundance across varied hosts and environments, may be linked to their extraordinary genomic complexity and plasticity. Accumulating evidence suggests we are only now realizing the influence NCLDVs have on microbial communities and lower vertebrate biodiversity.

Contact: Thomas B. Waltzek, Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32608, USA, Phone: 530 574 2976, Email: [email protected]

Sturgeon nucleo-cytoplasmic large DNA virus phylogeny

Sharon Clouthier 1*, Rachel Breyta 2, Gael Kurath 3, Eric Anderson 4 1Freshwater Institute, Fisheries and Oceans Canada, Winnipeg, Manitoba, Canada; 2Oregon State University, Corvallis, Oregon, USA; 3United States Geological Survey, Western Fisheries Research Center, Seattle, WA, USA, 4Ste Anne, Manitoba, Canada

Namao virus (NV) is a sturgeon nucleo-cytoplasmic large DNA virus (NCLDV) that causes a lethal disease of the integumentary system in lake sturgeon Acipsenser fulvescens. Other sturgeon NCLDVs include white sturgeon iridovirus, Missouri River sturgeon iridovirus, shortnose sturgeon virus and isolates reported from sturgeon cultured in Italy and western Europe. Phylogeny performed using the major capsid protein (MCP) sequence from sturgeon NCLDVs in North America revealed that they form a distinct evolutionary lineage within the Megavirales. In this study, DNA was isolated from NV-infected sturgeon tissue and sequenced using the Illumina HiSeq platform. The sequence of three non-overlapping contigs comprising over 288,000 bp of the NV genome was assembled and annotated using the NCVOG and NCBI NR protein databases. Bayesian phylogenetic tree reconstructions were performed with the BEAST software package using nine conserved core orthologous proteins encoded by genes that have been mapped to the genome of the common ancestral virus of NCLDVs: MCP, DNA polymerase B elongation subunit, VVA18-type helicase, VVA32- type ATPase, VLTF3-like transcription factor, DNA-directed RNA polymerase II subunits 1 and 2, ribonucleotide reductase small subunit and the mRNA capping enzyme. A total of 47 taxa representing at least 6 virus families of the Megavirales were included in the analyses. Phylogeny with the MCP was repeated to include new sequences from European isolates of sturgeon NCLDVs. The tree topology confirmed the monophyly of this group of viruses and suggested that they share a common ancestor with phycodnaviruses and mimiviruses. Analyses with the other eight proteins revealed that NV was monophyletic or clustered with CroV and descended from a common ancestor of the Mimiviridae clade. The phylogenetic reconstruction performed with the ribonucleotide reductase small subunit (NCVOG0276) protein showed NV clustering with infectious spleen and kidney necrosis virus (Iridoviridae) and Trichoplusia ni ascovirus (Ascoviridae) in the Mimiviridae/Phycodnaviridae clade. This study provides more evidence that the epitheliotropic sturgeon NCLDVs are taxonomically most like viruses assigned to the extended Mimiviridae.

Contact email: [email protected]

Phylogenomic characterization of a novel megalocytivirus lineage from archived ornamental fish samples

Samantha A. Koda 1*, Kuttichantran Subramaniam 1, Ruth Francis-Floyd 2, Roy P. Yanong 3, Salvatore Frasca Jr. 4, Joseph M. Groff 5, Shipra Mohan 6, William A. Fraser 6, Annie Yan 6, Thomas B. Waltzek 1 1Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Florida, USA; 2Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Florida, USA; 3University of Florida School of Forest Resources and Conservation, Program in Fisheries and Aquatic Sciences Tropical Aquaculture Laboratory, Florida, USA; 4Connecticut Veterinary Medical Diagnostic Laboratory, University of Connecticut, Connecticut, USA; 5School of Veterinary Medicine, University of California Davis, California, USA; 6Florida Department of Agriculture and Consumer Services, Bronson Animal Disease Diagnostic Laboratory, Florida, USA

The genus Megalocytivirus is the newest member of the family Iridoviridae, and as such, little is known about the genetic diversity of these globally emerging fish pathogens. Using an Illumina MiSeq sequencer, we sequenced the genomes of two megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (keyhole cichlid, Cleithracara maronii and oscar, Astronotus ocellatus) and the three spot gourami Trichopodus trichopterus circulating in the ornamental fish trade in the early 1990s. Phylogenomic analyses revealed South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess nearly identical genomes and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2) previously reported from flatfish species reared for food in Asia (TRBIV Clade 1). The SACIV and TSGIV genomes were similar in size (111,347 and 111,591 bps, respectively), gene content (116 open reading frames for both), and %GC content (56.3 and 56.5, respectively) compared to other MCVs. However, both possess a unique truncated paralog of the major capsid protein (MCP) gene located immediately upstream of the full length parent gene. The MCP paralog likely arose through a gene duplication event and its function could be to increase antigenic diversity. Histopathological examination of archived oscar tissue sections revealed abundant cytomegalic cells characterized by basophilic granular cytoplasmic inclusions within various organs, particularly the anterior kidney, spleen and intestinal submucosa. A conventional PCR assay, designed to amplify and distinguish through Sanger sequencing all MCV genotypes, was partially validated and used to confirm the presence of SACIV DNA within archived formalin- fixed paraffin-embedded (FFPE) oscar tissues. TSGIV-infected grunt fin cells (GF) displayed cytopathic effect (e.g., cytomegaly, rounding, and refractility) as early as 96 hr post infection (pi). Ultrastructural examination revealed non-enveloped virus particles displaying hexagonal symmetry (120-144 nm) and an electron-dense core within the cytoplasm of infected GF cells, consistent with the ultrastructural morphology of a MCV. The sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

Contact: Samantha Koda, Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Florida, USA, Email: [email protected]

A member of the genus Iridovirus in reptiles and amphibians?

Tibor Papp 1, 2*, Rachel E. Marschang 2, 3 1Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary; 2Fachgebiet für Umwelt- und Tierhygiene, University of Hohenheim, Stuttgart, Germany; 3Laboklin GmbH, Bad Kissingen, Germany

Members of the genus Iridovirus are important pathogens of various invertebrates, mainly arthropods. However, variants of one of its members: the cricket iridovirus (syn. Gryllus bimaculatus iridovirus - GbIV), have been repeatedly detected by PCR and virus isolation from several snakes, lizards, and amphibians, as well as a scorpion and crickets from private owners, zoos, and the pet trade in Europe over the last two decades. In our studies we tested the hypothesis that an invertebrate iridovirus (IIV) isolate could infect reptiles. We established a sensitive and specific qPCR and an in situ hybridization (ISH) probe as new methods for the detection of these viruses. In per os and per coelom infection trials with bearded dragons (Pogona vitticeps), we were unable to induce disease in lizards. However, qPCR, nPCR, and virus isolation in cell culture were able to detect IIVs in non-digestive organs of per os infected lizards in several cases. No clinical signs or pathological or histopathological (including ISH, EM) changes were found in any of the lizards. In cricket bioassays, Koch’s postulates were fulfilled with 3 different isolates obtained from various reptiles. Although the bioassays showed some differences between the isolates, partial genome sequence analysis (15 genes, 14 kb) revealed very limited (up to 0.4%) variance between the different isolates. The sequence comparison of these partial genome sequences with the homologous parts of Chilo iridescent virus (CIV), the type species of the genus, showed long insertions/deletions and two areas of recombination which had not been previously reported. Complete genome sequencing, using NGS techniques followed by de novo assembly was then performed to elucidate further differences between CIV and one of our reptilian IIV isolates. A preliminary genome map based on this sequencing showed apparent colinearity between CIV and the reptilian IIV, with over 90% nt identity, while additional areas of insertion of unknown source as well as of recombination were detected. (Supports: OTKA K100163, Bolyai Research Fellowship of the Hungarian Acad. Sci. for TP)

Contact e-mail: [email protected]

Phylogenomic characterization of squamate erythrocytic iridoviruses

Kuttichantran Subramaniam 1, Thomas B. Waltzek 1, Nicole I. Stacy 2, Claire Grosset 3, James F.X. Wellehan Jr. 4 1Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA; 2Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA; 3Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA, USA; 4Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

Erythrocytic iridoviruses (EIV) have been documented in squamates within the families Gekkonidae, Phyllodactylidae, Scincidae, Cordylidae, Lacertidae, Pythonidae, Colubridae, Viperidae, Varanidae, Iguanidae, Phrynosomatidae, Agamidae, and Chamaeleonidae. Interestingly, similar viral agents have also been reported in more than 20 species of anadromous and marine fishes throughout the Atlantic and Pacific Oceans, as well as amphibians. However, the phylogenetic relationship of these viruses to other iridoviruses remains unclear to date. In this study, we compared the light microscopic abnormalities of infected cells, the ultrastructural morphology and phylogenetic relationship of EIVs to other iridoviruses. Recently, EIVs were partially characterized in a wild Peninsula ribbon snake (Thamnophis sauritus sackenii) and captive-bred inland bearded dragons (Pogona vitticeps). The Peninsula ribbon snake displayed two types of cytoplasmic inclusions in erythrocytes, polychromasia, anisocytosis, and hypochromasia, while the erythrocytes of the bearded dragon exhibited prominent blue-staining inclusions within normal appearing erythrocytes. Cytoplasmic inclusion bodies within erythrocytes of the Peninsula ribbon snake examined by transmission electron microscopy (TEM) revealed enveloped icosahedral particles morphologically consistent with iridoviruses. The complete genome of the EIV from Peninsula ribbon snake (Thamnophis sauritus sackenii; TsEIV) comprises 111,413 bp nucleotides which encodes 115 potential open reading frames (ORFs). Phylogenetic analysis based on 19 conserved genes shows that the squamate EIVs form a well-supported clade distinct from other established iridoviral genera, and likely represent a novel genus. We propose the genus Hemocytivirus for this new clade of iridoviruses to reflect their predilection for red blood cells.

Contact: Kuttichantran Subramaniam, Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32608, USA, Phone: 352 215 7278, Email: [email protected]

Spread of various strains of cyprinid herpesvirus 2 (CyHV-2) through global trade of goldfish

Takafumi Ito 1*, Jun Kurita 1 and Olga L. M. Haenen 2 1Tamaki Laboratory, Research Centre for Fish Diseases, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 224-1 Hiruda, Tamaki, Mie 519-0423, Japan; 2Wageningen Bioveterinary Research of Wageningen UR, P.O. Box 65, 8200 AB Lelystad, The Netherlands

The disease herpesviral haematopoietic necrosis (HVHN) has caused great economic losses to goldfish (Carassius auratus auratus) aquaculture in Japan since it was initially reported in 1992. The causative virus belongs to the genus cyprinid herpesvirus 2 (CyHV-2), has a double-stranded DNA genome approximately 290 kb in length, and belongs to the family Alloherpesviridae according to the rules for nomenclature of the International Committee on the Taxonomy of Viruses. The virus is now recognized as a major pathogen of goldfish, not only in Japan but also in the USA, Taiwan, Australia, New Zealand, the UK and France. However, recently, the virus has also been detected from Prussian carp (C. gibelio) and crucian carp (C. carassius) from European and Asian countries. To prevent spread of the causative virus to other areas, the investigation of risk factors of spread of this virus is important. In this study, eight batches of goldfish imported into the Netherlands by airfreight from third countries were investigated for presence of the virus, to evaluate the chance of spreading CyHV-2 by global trade of goldfish. CyHV-2 DNA was detected by PCR in the pooled kidneys of four of the eight imported goldfish batches, of which one from a CyHV-2 disease case at the Dutch importers quarantine. Sequence analysis of the CyHV-2 strains of this study and from previous reports showed, that there were at least six different lengths in the mA region. Additionally, virus isolation was positive for only one (AMS-1) of the eight samples after in vivo passage. The pathogenicity of the isolate (AMS-1) to naïve goldfish was similar to that of the Japanese (SaT-1) reference isolate. The viral titre of the AMS-1 isolate for GFF cells at several temperatures was also similar with that of the SaT-1 isolate. Our results prove that one of the routes of spread of various and virulent CyHV-2 strains is through global trade of virus-positive and sometimes clinically healthy goldfish, in this case from third countries into Europe.

Contact e-mail: [email protected]

Discovery of a novel herpesvirus infection of European perch (Perca fluviatilis) in Finland

Kyle A. Garver 1, Katja Leskisenoja 2, Laura M Hawley 1, Robert Macrae 3, Kuttichantran Subramaniam 4, Thomas B. Waltzek 4, Jon Richard 1, Caroline Josefsson 3, Rudolf Hoffmann 5, Theresia Fischer-Scherl 5 and E. Tellervo Valtonen * 1Pacific Biological Station, Fisheries and Oceans Canada, British Columbia, Canada; 2University of Jyväskylä, Department of Biological and Environmental Science, Jyväskylä, Finland; 3Vancouver Island University, Biology Department, British Columbia, Canada; 4University of Florida, Department of Environmental and Global Health, Gainesville, Florida, USA; 5Institut für Zoologie und Hydrobiologie, Munich, Germany;*Deceased

Herpesvirales is an order of viruses that are well represented throughout both terrestrial and aquatic ecosystems and share the commonality of possessing a DNA core surrounded by an icosahedral capsid. Members infecting fish and amphibians have been grouped into the family Alloherpesviridae which currently represents 12 species, divided among 4 genera that include aquatic herpesviruses of serious diseases such as channel catfish disease. Here we report on the discovery of a novel herpesvirus infection of European perch angled from lakes in Finland. Virus infected fish exhibited white nodules on the skin and fins that commonly occurred during the spring when prevalence reached nearly 40% in one of four lakes sampled. Transmission electron microscopy revealed virus particles with round or hexagonic morphology containing a core and two capsid membranes. Degenerate PCR targeting a conserved region of the polymerase gene of large DNA viruses amplified a 520 bp product in 5/5 perch skin tissues tested. Phylogenetic analysis of concatenated partial DNA polymerase and terminase gene sequences produced a well-supported tree grouping the European perch herpesvirus (PeHV2) with the alloherpesviruses infecting salmonid, ictalurid, acipenserid (Acipenserid herpesvirus 2, AciHV2), and esocid (esocid herpesvirus 1, EsHV1) fishes. The phenetic analysis of the PeHV2 partial DNA polymerase and terminase showed gene identity ranged from 34.6 to 63.9% and 39.6 to 59.4% to other Alloherpesviruses; respectively and confirms the identification of a novel species.

Contact e-mail: [email protected]

Ranid herpesvirus 3 (RHV3), a novel virus associated with a proliferative skin disease in free-ranging wild common frogs (Rana temporaria).

Francesco C. Origgi 1,6*, Benedikt R. Schmidt 2, Petra Lohmann 3, Patricia Otten 4, Ezgi Akdesir 1, Veronique Gaschen 5, Lisandra Bultet-Aguilar 6, Thomas Wahli 1, Ursula Sattler 1, Michael H. Stoffel 5 1Centre for Fish and Wildlife Health (FIWI), Department of Infectious Diseases and Pathobiology (DIP), University of Bern, Switzerland; [email protected]; 2KARCH, Passage Maximilien-de-Meuron 6, 2000 Neuchâtel & Department of Evolutionary Biology and Environmental Studies, University of Zurich, Switzerland; orcid.org/0000-0002- 4023-1001; 3P. Lohmann, Veterinarian, 8127 Forch, Switzerland; 4Fasteris SA, Geneva, Switzerland (PO); 5Division of Veterinary Anatomy, University of Bern, Switzerland; 6Institute of Veterinary Bacteriology, (DIP), University of Bern, Switzerland.

Amphibian pathogens have been recently considered important players contributing to the global amphibian decline. Fungal agents such as chytrids have been associated with significant amphibian losses worldwide. Similarly, among viruses, Ranavirus has been recognized as a serious primary pathogen of amphibians and is listed among those reportable by the OIE. However, other amphibian pathogens, including ranid herpesviruses, have received a relatively minor attention. Ranid herpesviruses are members of the family Alloherpesviridae and were discovered in the previous century. Ranid herpesvirus 1 is the etiologic agent of the Lucke renal adenocarcinoma in leopard frogs (Lithobates pipiens), whereas Ranid herpesvirus 2 has been associated with edema in leopard frogs infected as tadpoles. In the spring of 2015, two free-ranging, wild common frogs (Rana temporaria) were submitted to the Centre for Fish and Wildlife health of the University of Bern, Switzerland for full examination. The frogs showed prominent skin lesions consisting in multifocal to coalescent, raised grey patches scattered over the entire body but more abundant on the dorsum and the flanks. Similar lesions had been reported in free-ranging, wild frogs in Europe during the last 20 years but the conclusive etiology of the disease remained always elusive. Histological examination revealed that the grey skin areas corresponded to regions of moderate to severe epidermal hyperplasia with many cells showing the presence of eosinophilic to amphophilic intranuclear inclusions and variable glandular degeneration. Transmission electron microscopy showed the presence of intralesional electrondense particles consistent with herpesvirus. Next generation sequencing confirmed the presence of a novel herpesvirus sharing common features with the members of the genus Batrachovirus in the family Alloherpesviridae. The novel virus has been tentatively named Ranid herpesvirus 3 (RHV3).

Contact e-mail: [email protected]

Results from carp edema virus infections in differently susceptible common carp strains reveal differences in virulence between CEV genogroups

Mikolaj Adamek 1*, Anna Oschilewski 1, Peter Wohlsein 2, Verena Jung-Schroers 1, Felix Teitge 1, David Gela 3, Veronika Piackova 3, Martin Kocour 3, Jerzy Adamek 4, Sven M. Bergmann 5, Dieter Steinhagen 1 1Fish Disease Research Unit, Institute for Parasitology, University of Veterinary Medicine, Hannover, Germany; 2Department of Pathology, University of Veterinary Medicine, Hannover, Germany; 3Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia Ceske Budejovice, Vodnany, Czech Republic; 4Experimental Fish Farm in Zator, The Stanislaw Sakowicz Inland Fisheries Institute in Olsztyn, Poland; 5Institute of Infectology, Friedrich-Loeffler-Institut (FLI), Greifswald-Insel Riems, Germany

“Koi sleepy disease” (KSD) caused by infections with the carp edema virus (CEV) might pose a potential risk to carp aquaculture. The disease is associated with lethargic behaviour (hence the name), swollen gills, sunken eyes and skin alterations. Interestingly isolates of CEV from common carp and koi are not uniform. Sequence comparisons of virus infecting koi and common carp revealed the existence of two or three genogroups, one mostly associated with infections in koi (genogroup IIa) and a second predominantly isolated from cultured common carp (genogroup I). In a series of infection experiments, CEV from two different genogroups (I and IIa) was transmitted to several strains of naïve common carp via cohabitation with fish infected with CEV at 10 - 13 °C. Different genogroups of CEV were used to check for differences in infection biology of the virus while several carp strains were used in order to evaluate influences of the genetic background on the susceptibility to infection. For evaluations of virus load and virus replication qPCRs were performed for detection of CEV DNA and mRNA; furthermore, the development of the disease was monitored by observation of fish behaviour and histopathological changes in gills. In an infection experiment with CEV from genogroup I Amur wild carp (AS) and Ropsha carp (Rop) performed much better (were less susceptible to the infection) than Prerov scale carp (PS) or koi. When CEV from genogroup IIa was used all common carps (AS, PS, Rop) were far more resistant to the infection than koi. Analyses of behavioural, histopathological and molecular indicators of infection revealed differences in the virulence of the two CEV genogroups. Viruses showed higher virulence towards the same fish variety as the donor fish (koi or in common carp) inducing rapid onset of KSD. The results from the study show that resistance to CEV infection is dependent on the genetic background of carp. Furthermore significant differences in virulence and genetics of CEV genogroups rise questions about a better separation of these viruses by nomenclature.

Contact e-mail: [email protected]

“Koi sleepy disease” as a pathophysiological consequence of branchial infection of common carp with carp edema virus

Mikolaj Adamek 1*, Felix Teitge 1, Martin Ganter 2, Ilka Baumann 1, Verena Jung-Schroers 1, David Gela 3, Veronika Piackova 3, Martin Kocour 3, Dieter Steinhagen 1 1Fish Disease Research Unit, Institute for Parasitology, University of Veterinary Medicine, Hannover, Germany; 2Clinic for Swine and Small Ruminants, University of Veterinary Medicine, Hannover, Germany; 3Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia Ceske Budejovice, Vodnany, Czech Republic

Fish gills, with their involvement in water and ion transport, gas exchange and ammonia excretion are very important organs for fish metabolism; therefore, branchial diseases can cause severe complications for the fish’s homoeostasis. In common carp several viruses cause pathological changes in gills, among which the carp edema virus infection is mostly limited to gills, making it a potential model for a branchial disease in carp. Koi sleepy disease, caused by CEV infection, manifests itself with characteristic behavioural changes. Affected fish are lethargic, start lying at the bottom of the tank and, with progress of the disease, the activity of the fish decreases until nearly complete stillness, followed by death. We hypothesize that this behaviour is related to gill dysfunction from the infection and therefore, the pathophysiological impact of the infection was measured, including an analysis of the hydro-mineral balance, respiration and ammonia excretion. Furthermore the blood plasma metabolome of KSD affected fish was studied. The experimental setup included two strains of carp (AS - Amur wild carp and koi) with different susceptibilities to KSD. All carp were cohabitated with koi infected with CEV from genogroup IIa. During four infection experiments at 18 °C 100% of the koi developed severe KSD, which led to a complete immobilisation of the animals at the bottom of the tank between days 6 and 12 post infection (p.i.) with the peak at days 6 and 7 p.i.. In blood plasma collected at days 6 and 9 p.i. oxygen content was slightly reduced, sodium and calcium extremely decreased and ammonia levels severely increased. Analyses of over 2,500 metabolites, from which over 750 could be annotated, showed changes in the pyrimidine and urea cycle and the beta-alanine and the amino acid metabolism in blood plasma at day 6 p.i.. In the non-infected koi an increase of pyrimidine pathway and a decrease of amino acids synthesis were detected. At the same time a leukopenia had developed. These changes occurred only in koi while carp from the AS strain remained unaffected, which correlated with a much higher virus load and the onset of histopathological changes in gills of koi. The results from the study show that using differently susceptible strains of carp gives the opportunity for a deeper insight in to biology of the infection. Furthermore it confirms that sleepiness of the fish is related to severe osmotic imbalances and a perturbation of the waste removal from the amino acid metabolism which might lead to intoxication with ammonia.

Contact e-mail: [email protected]

Phylogenomic characterization of a novel seahorse poxvirus from formalin-fixed paraffin-embedded tissues

Joseph M. Groff 1,Kuttichantran Subramaniam 2, Robert W. Nordhausen 3, Thomas B. Waltzek 1* 1School of Veterinary Medicine, University of California Davis, Davis, CA; 2Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL; 3California Animal Health & Food Safety Laboratory System, Davis, CA

Double-stranded DNA viruses are important pathogens of homeothermic and poikilothermic vertebrates. However, only alloherpesviruses and iridoviruses are well studied among poikilothermic vertebrates (e.g. fish, amphibians, and reptiles). In 2002, moribund specimens of endangered Cape seahorse (Hippocampus capensis) from a managed population experiencing elevated mortality were submitted for histopathological examination. Histopathological examination revealed a cutaneous hyperplastic vacuolar dermatopathy. Ultrastructural examination revealed large and complex virions with spherical to reniform profiles (399 x 168 nm) within the cytoplasm of affected epidermal cells consistent with a poxvirus. The virions displayed a single lateral body as reported previously for poxviruses detected in koi (Cyprinus carpio), Ayu (Plecoglossus altivelis), and Atlantic salmon (Salmo salar). An Illumina Nextera XT DNA library kit was used to construct a DNA library from formalin-fixed, paraffin-embedded (FFPE) tissue DNA. The DNA library was sequenced using a v3 chemistry 600-cycle kit on an Illumina MiSeq sequencer. Assembly of the resulting reads followed by BLASTx analyses resulted in a number of contigs with significant homology to the salmon gill poxvirus. Phylogenomic analyses based on core poxvirus genes revealed the seahorse poxvirus (SHPV) grouped as the sister species to the salmon gill poxvirus within the fish poxvirus clade. The histological and ultrastructural pathology and phylogenetic analyses support SHPV as a novel poxvirus within the recently proposed genus of poxviruses from fish (i.e., Piscipoxvirus). This study confirms the utility of Next Generation Sequencing (NGS) technologies in obtaining phylogenetically useful viral genomic sequences from archived FFPE tissues.

Contact e-mail: [email protected]

Detection and complete genome analysis of ranaviruses causing mass mortality in brown bullheads (Ameiurus nebulosus) in Hungary

Szilvia L. Farkas 1, Enikő Fehér 1, Andor Doszpoly 1, Balázs Horváth 2, Mária Woynárovichné Láng 3, György Csaba 3, Ádám Dán 3, Szilvia Marton 1, Barbara Forró 1, Krisztián Bányai 1, Tamás Juhász 3 1Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary; 2Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary; 3Veterinary Diagnostic Directorate, National Food Chain Safety Office, Budapest, Hungary

During routine diagnostic work at the Veterinary Diagnostic Directorate ranaviruses of brown bullheads (Ameiurus nebulosus) had been detected in connection with mass mortality events in distant regions of Hungary between 2008 and 2016. These infections were characterized applying gross pathological, histopathological, traditional and modern virological methods. Post mortem evaluation of affected animals revealed hemorrhages in the skin all over the body, in the gills and the internal organs. Following homogenization of the liver, spleen and kidney samples virus isolation was performed on EPC (Epithelioma Papulosum Cyprini) and BF-2 (Bluegill fibroblast) cell lines. Cytopathogenic effect typical to ranaviruses (rounding of the infected cells and cytoplasmic inclusion bodies) could be observed on the second or third days post infection. Specific detection of ranaviruses in the isolates was performed applying polymerase chain reactions (PCR) targeting a portion of the iridoviral major capsid, DNA-polymerase and neurofilament triplet H1-like protein (NF-H1) coding genes, sequencing the PCR products and restriction endonuclease analysis of the NF- H1 gene’s PCR product. For complete genomic sequencing two strains, 13051/2012 and 14612/2012, isolated from samples collected in 2012 had been chosen to explore the genetic diversity of Hungarian ranaviruses and develop detection methods giving informative results for epidemiological studies and differentiation between virus strains. The complete genome sequences of the two strains were nearly identical with each other (99.9% nucleotide sequence identity) and were closely related to the European sheatfish (Silurus glanis) origin ranavirus (ESV, 99.7%-99.9% nucleotide sequence identity). The core genes proved to be highly conserved, point mutations were observed mainly in the non-coding regions. Complete genome sequencing of further isolates would provide more information about the genetic markers that could be used successfully for genetic characterization and molecular epidemiologic studies. These are the first reported cases of ranaviruses in Hungary.

Contact e-mail: [email protected]

How ranavirus brought Britain closer to the EU – molecular screening of a long-term tissue archive from herpetofauna

Stephen J. Price 1,2, Alexandra Wadia 2,3, Owen Wright 2,4, Will Leung 2, Andrew A. Cunningham 2, Becki Lawson 2 1UCL Genetics Institute, Gower Street, London WC1E 6BT, UK, 2Institute of Zoology, ZSL, Regents Park, London NW1 4RY, UK, 3 niversity of York, York, YO10 5DD, UK, 4School of Biosciences, Cardiff University, Cardiff, Wales, UK

Reports of disease outbreaks due to common midwife toad virus (CMTV)-like ranaviruses in mainland Europe are increasing and have much attention because of their severe impacts. In Britain, Frog virus 3 (FV3)-like ranaviruses have caused significant local declines of common frog populations and are thought to have been introduced recently. The British public has reported disease outbreaks to the Frog Mortality Project (FMP) since 1992, resulting in a 25- year tissue archive. We used molecular methods to screen this long-term archive for ranavirus and performed basic genetic characterisation of the ranaviruses detected. Ranavirus was detected in 90 of 458 individuals from 41 outbreaks in the north and south of England. Most detections involved common frogs but ranavirus was also detected in a second anuran, a caudate and a reptile. The majority of incidents were associated with FV3- like ranaviruses but two incidents, one in 1995 and another 300km away and 16 years later, involved CMTV-likes. The two British CMTV-like ranaviruses were more closely related to ranaviruses from mainland Europe than to one another; the estimated divergence time was at least 458 years ago. This evidence of CMTV-like ranavirus in Britain in 1995 raises important questions about the virulence and geographic distribution of CMTV-like ranaviruses and the history of Ranavirus in Britain. This study also demonstrates the role of citizen science projects in generating resources for research despite biases in the opportunistic sample used.

Contact: Stephen J. Price, UCL Genetics Institute, Gower Street, London WC1E 6BT, UK, Phone: +44 20 3108 4229, Email: [email protected]

Molecular epidemiology of Epizootic haematopoietic necrosis virus (EHNV)

Paul M Hick 1*, Kuttichantran Subramaniam 2, Patrick M Thompson 2, Thomas B Waltzek 2, Joy Becker 1, Richard J Whittington 1 1OIE Reference Laboratory for Epizootic Haematopoietic Necrosis Virus and Ranavirus Infection of Amphibians. Sydney School of Veterinary Science and School of Life and Environmental Sciences, The University Sydney, Australia. 2 Department of Infectious Disease and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

Epizootic haematopoietic necrosis virus (EHNV) has caused sporadic outbreaks of mass mortality of wild redfin perch (Perca fluviatilis) and less severe disease of farmed rainbow trout (Oncorhynchus mykiss) since it was first identified in 1984. Recurrence of disease each summer was observed initially as EHNV spread and became endemic in several water catchments. However, despite the experimental susceptibility of several species of fish and a broader distribution of the two natural hosts, EHNV has remained confined to south-eastern Australia. The aim of this study was to determine the genomic diversity of EHNV and evaluate historical inferences about spread of the pathogen based on molecular epidemiologic evidence. The complete genome was determined for 16 isolates selected to represent the full range of host, geographic and temporal origin of EHNV from the collection curated by the World Organization for Animal Health (OIE) reference laboratory. Isolates at less than 3 passages were grown on BF-2 cells and sequences were determined using libraries prepared with a Nextera XT DNA kit on a MiSeq platform (Illumina). Complete genomes were 125,591–127,487 nucleotides with 97.47% pairwise identity, 106–109 predicted genes and consistent structural organization. All isolates shared 101 core genes of a total of 121 different genes predicted within the pan-genome of this collection. There was very high conservation of the 90,181 nucleotide sequence of the core genes with isolates separated by average genetic distance of 3.43 x 10-4 substitutions per site. Evolutionary analysis of the core genome strongly supported historical epidemiological evidence of iatrogenic spread of EHNV to naïve hosts and establishment of endemic infection status in multiple epidemiologically discrete niches.

Contact: Paul M. Hick, School of Veterinary Science, J.L. Shute Building (C01) 425 Werombi Road, Camden, NSW, Australia. Phone: +61 2 9351 1608, Email: [email protected]

Comparative genomics and spatiotemporal characterization of two strains of Common midwife toad virus in the Netherlands

Bernardo Saucedo 1*, Joseph Hughes 2, Nicolas Suarez 2, Olga Haenen 3, Michal Voorbergen-Laarman 3 Natasja Kruithof 1, Jolianne Rijks 1, Marc Schils 4, Annemarieke Spitzen 4, Maarten Gilbert 4, Andrea Gröne 1 and Steven van Beurden 1 1Dutch Wildlife Health Centre (DWHC), Utrecht University, Utrecht the Netherlands; 2Center for Virus Research (CVR), MRC University of Glasgow, Glasgow, UK, National Reference Laboratory for Fish, Shellfish and Crustacean Diseases Wageningen Bioveterinary Research (former CVI) of Wageningen UR , Lelystad, The Netherlands,4Reptile Amphibian Fish Research in the Netherlands (RAVON), Nijmegen, The Netherlands

Common midwife toad viruse (CMTV) has been responsible for amphibian declines throughout Europe and China. In the Netherlands, an interdisciplinary retrospective analysis on amphibian mortality events (2011-2014) revealed the presence of two CMTV-NL virus strains based on phylogenetic analysis of seven genes. Strain I was shown to be the cause of an epidemic in the North, whereas strain II was associated with lower mortality in the Center-East and South of the country. In order to search for mutations that could give us further insight on short term evolution amongst closely related isolates or which could account for potential differences in pathogenicity amongst the two main virus strains, we performed complete genome sequencing of thirteen isolates belonging to either of the CMTV-NL strains from different sites and years. Additionally, to compare prevalence and mortality patterns of both virus strains we performed cross sectional monitoring and skin swabbing of water frogs (Pelophylax spp) and a few other amphibian species at two sites, one in the North where Strain I was present and another in the South, where Strain II was present. Eleven of the thirteen isolates were obtained from Northern provinces and showed a total nucleotide sequence similarity of over 99% amongst each other and compared to the original Dutch isolate from 2013. Mutations amongst these viruses were minor and mainly confined to genes with unknown functions composed of repetitive sequences. The two isolates from Central Eastern and Southern provinces clustered closely with the Andrias davidianus ranavirus (ADRV) clade from China and the Testudo hermanni ranavirus (THR) from Germany. These two isolates presented with deletions in genes involved in viral replication which could account for lower virulence. In regards to the monitoring study, ranavirus prevalence was shown to be higher in the Northern site than in the Southern site (14.2% versus 7 %). The higher ranavirus-associated mortality and involvement of adult life stages in the North (six Pelophylax spp) versus the involvement of only larvae in the South (two Pelophylax spp) could reflect higher pathogenicity or less effective immunity of animals exposed to Strain I. The data obtained from both complete genome sequencing and cross sectional monitoring supports the hypothesis that the two CMTV-NL strains differ in pathogenicity.

Contact: Bernardo Saucedo, Yalelaan 1 3584 CL, Faculty of Veterinary Medicine, Pathology Division, Utrecht University, Utrecht, The Netherlands, Phone:+31 302534370, Email: [email protected]

Isolation and characterisation of a new ranavirus isolated from lumpfish in the north Atlantic area

Hannah E. B. Stagg 1*, Sigríður Guðmundsdóttir 2, Niccolò Vendramin 3, Neil Ruane 4, Heiða Sigurðardóttir 2, Debes H. Christiansen 5, Argelia Cuenca Navarro 3, Petra E. Petersen 5, Eann S. Munro 1 & Niels Jørgen Olesen 3 1Marine Scotland Science, Aberdeen, Scotland, UK; 2Institute for Experimental Pathology, University of Iceland, Reykjavik, Iceland; 3Technical University of Denmark, National Veterinary Institute, Copenhagen, Denmark; 4Marine Institute, Galway, Ireland; 5 Food and Veterinary Authority, Torshavn, Faroe Islands

The commercial production of lumpfish, Cyclopterus lumpus, is expanding with the increased demand for their use as cleaner fish, to control sea lice numbers, at marine Atlantic salmon, Salmo salar L., aquaculture sites throughout northern Europe. A new ranavirus has been isolated from lumpfish at multiple locations in the north Atlantic area, initially isolated in 2014 in the Faroe Islands and subsequently the virus has been found in lumpfish from Iceland in 2015 and from Scotland and Ireland in 2016. The Icelandic lumpfish ranavirus was initially characterised by IFAT, optimal growth conditions and full major capsid protein (MCP) gene sequencing. Partial sequences of the MCP gene from all eight isolates to date showed high similarity between the lumpfish ranaviruses. Comparison to other described ranaviruses showed high homology with ranaviruses from cod, Gadus morhua, and turbot, Psetta maxima, isolated in Denmark in 1979 and 1999. Further sequencing targeting an additional three genomic regions: the DNA polymerase (DNApol) gene and the ribonucleoside diphosphate reductase alpha and beta subunit-like protein genes was carried out for the Scottish and Irish lumpfish ranavirus isolates. The subsequent phylogenetic analysis of concatenated sequences suggests this is a new ranavirus within the ATV/EHNV-like viruses lineage.

Contact: Hannah E. B. Stagg, Marine Scotland Science, 375 Victoria Road, Aberdeen, AB11 9DB, UK, phone: +44 1224 295509, email: [email protected]

The European ranaviruses

Trenton W.J. Garner 1, Stephen J. Price 1,2, Ellen Ariel 3 1Institute of Zoology, Zoological Society of London, Regent’s Park, NW1 4RY, London, U.K.; 2 UCL Genetics Institute, Gower Street, WC1E 6BT, London, U.K.; 3 James Cook University, College of Public Health, Medical and Veterinary Sciences, Townsville, Queensland, Australia

Europe hosts several lineages of ranaviruses, and with divergent pattern of host utilization. For example, isolates from marine and freshwater fishes appear to be fish specialists and are phylogenetically related to the ATV-like ranaviruses. CMTV-like ranaviruses infecting fish are only reported for asymptomatic pike perch (Sander lucioperca) in Finland. Conversely, epidemic ranavirosis affecting European amphibians was first detected in the UK in the late 1980s and attributed to FV3-like variants, while in the last decade cases affecting amphibians in six continental European countries were consistently associated with CMTV-like ranaviruses. FV3-like forms in the UK exhibit narrow amphibian host range when compared to continental CMTV-like ranaviruses, and in both cases introduction and spread is supported. Both lineages have been detected in wild European reptiles, but only CMTV-like forms have been isolated from diseased and dead animals. Phylogeographic patterns among European ranaviruses remain difficult to interpret: CMTV- like ranaviruses appear to represent a Eurasian lineage while FV3- and ATV-likes are more globally distributed. Insights about the spatial origins of lineages or patterns of broad-scale emergence remain speculative or absent. Despite considerable uncertainty about the evolutionary rates of these viruses, very conservative estimates of times to the most recent common ancestors of isolates within lineages (both FV3-like and CMTV-like) and the same country are in the order of hundreds of years. Such dates are clearly uncoupled from observations of disease outbreaks over the last four decades and this mismatch might reflect important changes in observer effort or the environment (including hosts) in combination with recent incidences of introduction and spread (above). Regardless, it is now clear that there is repeated overlap of the distributions of lineages which may yield co-infections and provide opportunities for exciting interactions between divergent viruses.

Contacts: [email protected]; [email protected]; [email protected];

Complete genome sequence of a Frog Virus 3-like ranavirus from an Oophaga pumilio frog imported from Nicaragua into the Netherlands

Bernardo Saucedo 1*, Joseph Hughes 2, Nicolás Suárez 2, Olga Haenen 3, Michal Voorbergen-Laarman 3, Andrea Gröne 1, Marja J L Kik 1 and Steven van Beurden 1 1Dutch Wildlife Health Centre (DWHC), Utrecht University, Utrecht the Netherlands; 2Center for Virus Research (CVR) , MRC University of Glasgow, Glasgow, UK, 3Wageningen Bioveterinary Research of WUR, Lelystad, The Netherlands

We obtained the whole genome sequence of a Frog Virus 3-like (FV3) ranavirus from two strawberry poison frogs (Oophaga pumilio) imported into the Netherlands. The specimens were imported from Nicaragua, via Germany, and died upon arrival to the Netherlands. Histopathology revealed intracytoplasmic basophilic inclusions in various organs, including bone marrow, and liver. Ranavirus infection was confirmed by conventional PCR using primers for the major capsid protein (MCP). Virus isolated from a 10% organ suspension was replicated on Epithelioma Papulosum Cyprini (EPC) cells, and purified by high speed ultracentrifugation through a 36% sucrose cushion. The virus was resuspended in ice cold PBS and the DNA was extracted with the QIAmp DNA Blood-minikit (Qiagen) according to manufacturer’s protocol. Afterwards, the DNA was sheared by sonification and a library was prepared using a KAPA library preparation kit. A MiSeq running v3 chemistry (Illumina) was used to generate 2x300nt paired-end sequencing reads. De novo assembly of the sequencing reads produced a contig of 107,183 bp with a total G+C content of 54.95%. The virus showed a nucleotide similarity of 98.22% to the original FV3 (GenBank accession no. AY548484) from the United States and a 97.96% nucleotide similarity to the Canadian isolate FV3 SMME (GenBank accession no. KJ175144). Phylogenetic characterization positioned the virus in the FV3 clade along with Soft-shelled turtle iridovirus and Rana grylio virus. In conclusion, we isolated FV3 from living specimen imported from South America to the Netherlands. This case highlights the risk of introduction of an exotic ranavirus species by means of international trade.

Contact: Bernardo Saucedo, Yalelaan 1 3584 CL, Faculty of Veterinary Medicine, Pathology Division, Utrecht University, Utrecht, The Netherlands, Phone: +31 302534370., Email: [email protected]

Poor biosecurity could lead to ranavirus outbreaks in amphibian populations

1 1 1 1,2 Matthew J. Gray *, Jennifer A. Spatz , E. Davis Carter , and Debra L. Miller 1Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, University of Tennessee Institute of Agriculture, Knoxville, TN 37996, USA; 2Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA

Outbreaks of ranavirus and chytrid fungus have contributed to amphibian population declines. It has been suspected that biologists could contribute to pathogen outbreaks through poor biosecurity practices during sampling. Biologists frequently co-house captured amphibians and do not change gloves between handling different individuals. We tested whether these poor biosecurity practices could facilitate transmission of ranavirus from infected to uninfected wood frog (Lithobates sylvaticus) tadpoles, and increase the likelihood of mortality. Co-housing tadpoles for only 15 minutes with 10% of individuals initially infected resulted in transmission and mortality of 50% of uninfected tadpoles. Not changing gloves between individuals when 10% were initially infected resulted in transmission of ranavirus and mortality of 70% of uninfected tadpoles. More extreme mortality was observed when tadpoles were co-housed for longer durations, or when the initial infection prevalence was >10%. Our results indicate that poor biosecurity practices can cause ranavirus transmission between individuals, which could lead to disease outbreaks and decrease survival in populations. Biologists should change gloves or decontaminate them between handling individuals, and not co-house animals.

Contact: Matthew Gray, Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, University of Tennessee Institute of Agriculture, 247 Ellington Plant Sciences Building, Knoxville, TN 37996, USA, Phone: +1-865-385-0772, Email: [email protected]

Prevalence of ranavirus and Bd in hellbender populations in Tennessee and Arkansas

Rebecca H. Hardman 1,*, William B. Sutton 2, Dale McGinnity 3, Kelly J. Irwin 4, Sherri Reinsch 5, Ben Fitzpatrick 6, Philip Colclough 7, Marcy Souza 8, Michael Freake 9, Matthew J. Gray 10 and Debra L. Miller 11 *1Center for Wildlife Health, University of Tennessee, Knoxville, Tennessee, USA; 2Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN, USA, 3Nashville Zoo at Grassmere, Nashville, Tennessee, USA; 4Nashville Zoo at Grassmere, Nashville, Tennessee, USA; 5Arkansas Game and Fish Commission, Arkansas, USA; 6Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, Tennessee, USA; 7Knoxville Zoo, Knoxville, Tennessee, USA; 8Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee, USA; 9Department of Biology, Lee University, Cleveland, TN, USA; 10Center for Wildlife Health, University of Tennessee, Knoxville, Tennessee, USA; 11Center for Wildlife Health, University of Tennessee, Knoxville, Tennessee, USA

The Hellbender (Cryptobranchus alleganiensis), is a large aquatic salamander containing two subspecies, Ozark Hellbender (C. a. bishopi), and Eastern Hellbender (C. a. alleganiensis), from the Ozark mountains and eastern U.S., respectively. Both subspecies have seen population declines over the past 25 years, especially in C. a. bishopi which is federally endangered. Habitat degradation alongside other factors may lead to secondary infections with amphibian pathogens such as Ranavirus and a chytrid fungus (Batrachochytrium dendrobatidis) or Bd. Other pathogens such as the emerging salamander chytrid (Batrachochytrium salamandrivorans or Bsal) are also of concern as potential primary or secondary causes of disease. Our objective was to determine prevalence of these pathogens in both subspecies to understand the role of emerging amphibian pathogens in C. alleganiensis declines. We collected tissue and swabs from C. a. bishopi and C. a. alleganiensis individuals from Arkansas and Tennessee respectively during the summers of 2011-2015. We used qPCR analysis to determine presence of Ranavirus and Bd from tail samples and skin swabs, respectively. In the latter two years we collected samples of microbiome and secretion analyses. Overall, for C. a. bishopi, we detected 32% prevalence of Bd and 8.6% ranaviral infections; for C. a. alleganiensis, we detected 15% prevalence of Bd and 3% prevalence of Ranavirus. We have not found any Bsal positive individuals but have discovered Bd consistently present in these populations. We are currently in our second phase of investigating morbidity and mortality in hellbenders by comparing host skin microbiomes with changes in clinical disease and host peptide production.

Contact: Center for Wildlife Health, University of Tennessee, Knoxville, Tennessee, USA, [email protected]

Seasonal dynamics of ranavirus epidemics in wood frog populations

Emily M. Hall 1, Caren S. Goldberg 2, Erica J. Crespi 1, Jesse L. Brunner 1 1Washington State University, School of Biological Sciences, P.O. Box 644236, Pullman, WA, 99164-4236; 2Washington State University, School of the Environment, P.O. Box 646410, Pullman, WA, 99164-2812

Epidemics in wildlife populations often display a striking seasonality. Ranaviruses, for instance, cause sudden mass mortality events in populations of wood frog (Lithobates sylvaticus) larvae in the summer, coincident with pond drying and stages nearing metamorphosis. While there are several types of explanations for this seasonality—from seasonal introductions of virus to environmental stressors to windows of susceptibility during development—most studies have focused on single factors in laboratory settings. Here we used environmental DNA (eDNA) and larval samples to characterize the time course of epidemics in a set of ephemeral wetlands in the Northeast USA and evaluated multiple hypotheses for seasonal die-offs. We found little support for the introduction of ranavirus to influence the timing of ranavirus epidemics and resulting die-offs; variation in when infections were detected varied between 4-8 weeks before mortality was observed. Moreover, prevalence reached high levels (≥75%) up to 6 weeks before mortality was observed, suggesting that ranavirus infections do not necessarily lead rapidly to death (in contrast to observations from laboratory experiments). Rather, die-offs coincided with rising water temperatures and larvae reaching developmental stages at the start of metamorphic climax, both of which have been shown to increase susceptibility to death in laboratory experiments. Rapid increases in prevalence suggest these factors may increase transmission as well as mortality. In summary, the occurrence and timing of ranavirus-related mortality events appears to be driven largely by seasonal and developmental changes in susceptibility, and temperature may play a dominant role.

Contact: Emily M. Hall, A5116 Medical Center North, Vanderbilt University Medical Center, Nashville, TN, USA. Phone: (615) 343 9449, email: [email protected]

The interplay between ranavirus and the amphibian immune system

Jacques Robert Department of Microbiology & Immunology, University of Rochester Medical Center Rochester, NY 14642, USA

Similar to mammalian immune responses against large double strand DNA virus such as poxviruses, adult amphibians have the ability to mount a rapid effective innate immune response against ranavirus pathogens including activation of granulocytes, macrophages and natural killer cells, releasing interferons and inflammatory cytokines (TNFα, IL-1β, etc.). This early response is followed by potent adaptive cytotoxic T cells and antibody responses that lead to viral clearance and ultimately control ranavirus infection. Importantly, primary ranavirus infection can lead to long lasting immunological memory. Furthermore, despite a weaker or more immature immune system, experimental and field observations suggest that urodeles and anuran tadpoles are not immunologically ignorant or deficient but rather have a distinct set of immune responses adapted for their life stages. Notably, tadpoles appear to rely more heavily on innate-like T cells. On the other side of the host-pathogen equation, ranavirus pathogens show a unique ability to evolve, evade immunity, persist in their host and cross species barriers. Thus, rather than an inherent weakness of amphibian immune systems, the increased prevalence of ranavirus infections may be more a consequence of factors affecting the immune function of host populations such as stress, pollution and/or genetic disequilibrium. Support: NSF IOS-1456213; NIH R24-AI-059830 grants

Contact: Jacques Robert, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA; Phone: +1 585 275-1722; e-mail: [email protected]

Singapore grouper iridovirus (SGIV) TNFR homolog VP51 functions as a virulence factor by modulating cellular apoptosis and the host inflammatory response

Qiwei Qin 1,2*,Yepin Yu 1, Youhua Huang 1, Xiaohong Huang 1 1Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China;2College of Marine Sciences, South China Agricultural University, Guangzhou, China

Virus encoded tumour necrosis factor receptor (TNFR) homologues are usually involved in immune evasion by regulating host immune response or affecting apoptotic cell death. Singapore grouper iridovirus (SGIV) is a novel ranavirus that causes great economic losses in the aquaculture industry. Here, a TNFR-like protein encoded by SGIV (VP51) was characterized using overexpression and knock out technology. Our results showed that overexpression of VP51 in vitro enhanced cell proliferation, and affected cell cycle progression via altering the G1/S transition. Furthermore, VP51 overexpression improved cell viability during SGIV infection by inhibiting virus-induced apoptosis, evidenced by the reduction of apoptotic bodies and the decrease of caspase-3 activation. In addition, overexpression of VP51 increased viral yields and the expression of the viral major capsid protein (MCP) and the cell proliferation promoting protein, ICP-18. In addition, we generated a VP51-knock out mutant (Δ51-SGIV) by replacing VP51 with GFP. We found that viral replication in Δ51-SGIV infected cells or grouper was significantly decreased compared to wild type SGIV (WT-SGIV). Moreover, deletion of VP51 significantly increased virus induced apoptosis, and reduced expression of pro-inflammatory cytokines and interferon related genes in vitro. In addition, the expression levels of several pro-inflammatory genes were also decreased in Δ51-SGIV infected grouper compared to WT-SGIV. Thus, we speculate that SGIV VP51 functions as a critical virulence factor by regulating host cell apoptosis and the inflammatory response during virus infection. (Support: Natural Science Foundation of China31330082 grant).

Contact: Qiwei Qin, Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China, Phone: +86-20-89023638, E-mail: [email protected]

Differentiation-dependent antiviral capacities of amphibian (Xenopus laevis) macrophages

Amulya Yaparla 1 and Leon Grayfer 1* 1Department of Biological Sciences, George Washington University, Washington, DC USA

The colony-stimulating factor-1 (CSF-1) cytokine was believed to be the principal macrophage (M) growth factor and was thought to be indispensable to the survival, proliferation and differentiation of M lineage cells. CSF-1 binds and signals through the colony stimulating factor-1 receptor (CSF-1R), which is expressed on committed M-lineage precursor cells and derivative populations. Intriguingly, despite sharing no sequence identity with CSF-1, the interleukin-34 (IL-34) cytokine is now known to serve as an alternate CSF-1R ligand. Moreover, in the anuran amphibian Xenopus laevis, this IL-34 cytokine gives rise to morphologically and functionally distinct Ms to those derived by CSF-1. In this respect, it is particularly notable that while the X. laevis bone marrow (BM)-derived, CSF-1-differentiated Ms are highly susceptible to the emerging Frog Virus 3 (FV3) ranavirus, the IL-34-derived Ms are particularly resistant to this viral pathogen. Considering that antiviral interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, we thus examined the expression of several select IFN genes in X. laevis BM-derived, CSF-1- and IL-34- differentiated Ms in order to account for the differences in the antiviral capacities of these M populations. As expected, IL-34 Ms exhibited robust gene expression of a number of these antiviral IFN cytokines as well as their respective receptors. By contrast, CSF-1 Ms exhibited modest IFN ligand and cognate receptor gene expression, presumably accounting for their less effective antiviral capacities. Since cellular resistance to viral replication is controlled by a plethora of intracellular mechanisms, collectively coined restriction factors, it was intuitive that the FV3-resistant X. laevis IL-34 Ms also possessed significantly greater expression of several of these pertinent restriction factor genes, as compared to the FV3- susceptible CSF-1 Ms. Finally, we demonstrated that the supernatants conditioned by IL-34 M contained factors capable of rendering the highly FV3-susceptible X. laevis A6 kidney cell line more resistant to this ranavirus. Together, our findings indicate that the antiviral capacities of X. laevis IL-34 M reflect their enhanced expression of important antiviral restriction factors and production of key IFN cytokines. Considering that the proportions of CSF-1 and IL-34 M may well dictate whether an animal clears a ranavirus infection or succumbs from it, a greater insight into the ontogeny of these respective M populations is important to understanding the facets of amphibian susceptibility and resistance to these emerging pathogens.

Contact: Grayfer L. 800 22nd St NW, Suite 600, Washington, DC 20052 USA. Phone: +1-202- 994-8076, E-mail: [email protected]

Different antiviral T cell type immunity in tadpole and adult Xenopus

Jacques Robert *, Eva-Stina Edholm, Maureen Banach, Timothy Kwan, Jazz Sanchez, Leta Yi, Francisco De Jesús Andino Department of Microbiology & Immunology, University of Rochester Medical Center Rochester, NY 14642, USA

We have established an instrumental amphibian Xenopus laevis/ranavirus FV3 model system to characterize host-ranavirus interactions. Increasing evidence using this model suggests distinct host-pathogen interactions between FV3 and the immune system of either tadpoles or adult frogs. Previous investigations have revealed a more prominent type III interferon (IFN)-based antiviral response in tadpoles versus a more type I IFN-based antiviral response in adults, as well as distinct macrophage polarizations between tadpole and adult Xenopus. Nevertheless, our recent experiments using chlodronate injection one-day prior to FV3 infection to deplete phagocytic macrophages, demonstrate that these cells are required for antiviral protection and survival of both life stages. Notably, besides a minor fraction of conventional T cells, we have shown that the majority of T cells in tadpoles is constituted by six subsets of innate-like (i)T cells. Conventional CD8 T cells dominating in adult frogs express a highly diversified T cell receptor (TCR) repertoire interacting with polymorphic classical MHC class I molecules presenting unique antigenic peptides, whereas iT cells preponderant in tadpoles express a more limited (often invariant) TCR interacting with non-polymorphic MHC class I-like molecules presenting conserved pathogenic patterns. Contrasting with the reliance on conventional CD8 T cells in adult frogs to clear FV3, tadpoles depend on a specific iT cell subset expressing the invariant TCR rearrangement Vα6-Jα1.43 and restricted by the MHC class I-like XNC10 molecule. XNC10 loss-of-function using RNA silencing or CRISPR/Cas9-mediated genome editing combined with transgenesis prevents the development of Vα6 iT cells. As a consequence, transgenic tadpoles lacking Vα6 iT cells are markedly more susceptible to FV3 infection. Despite efficient conventional anti-FV3 CD8 T cells, Vα6-iT cells remain critical in adult host response as evidenced by the delay in antiviral response, increased viral load and kidney damage in their absence. However, the mature adult immune system is sufficient to ultimately control the viral infection and clear FV3. Although not yet formally identified, our findings suggest that Vα6 iT cells recognize an FV3- derived pathogen pattern presented by XNC10. Indeed, infection with UV-irradiated FV3, as well as knock-out FV3 mutant deficient for the immediate-early gene 18K induce strong IFN and pro-inflammatory gene expression responses and significantly increased XNC10 gene expression, but do not recruit any Vα6 iT at the site of infection. Given the replication defect of irradiated or KO-recombinant FV3, it is likely that insufficient antigens are produced. The identification of an anti-viral host recognition/defense system based on an MHC class I-like molecule restricting an innate-like T cell subset implies an ancestral host adaptation toward ranavirus pathogens. (Support: NSF IOS-1456213; NIH R24-AI-059830 and R25-GM064133 (D. M.), F31CA192664 (M.B.) grants)

Contact: Jacques Robert, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA; Phone: +1 585 275-1722; e-mail: [email protected]

A novel approach to transcriptomics in wild animals provides evidence for disease mediated differential expression and changes to the microbiome in amphibian populations

Lewis J. Campbell 1,2*, S. Austin Hammond 4, Stephen J. Price 5,2, Manmohan D. Sharma 6, Trenton W.J. Garner 2, Inanc Birol 4, Caren C. Helbing 7, Lena Wilfert 6, Angus Buckling 1, Amber G.F. Griffiths 3 1.Environment and Sustainability Institute, University of Exeter, Penryn Campus, Penryn, Cornwall, TR11 9FE 2.Institute of Zoology, Zoological Society of London, Regents Park, London, NW1 4RY 3.FoAM Kernow, Studio E, Jubilee Warehouse, Commercial Road, Penryn, Cornwall TR10 8FG 4.Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 4S6, Canada. 5.UCL Genetics Institute, University College London, Darwin Building, Gower Street, London, WC1E 6BT 6.Centre for Ecology and Conservation, University of Exeter, Penryn Campus, Penryn, Cornwall, TR11 9FE 7.Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, STN CSC, Victoria, British Columbia, V8W 2Y2

It is becoming well established that ranavirus infections can have long-term detrimental impacts on amphibian populations. Despite this, relatively little is known about the mechanisms through which wild amphibian populations respond to ranaviral infection. The U.K is fortunate to have a well-studied field system of urban and suburban common frog (R. temporaria) populations that incorporates aspects of citizen science. Using this unique natural field system, we used RNA sequencing (RNA-Seq) to explore gene expression profiles among three R. temporaria populations with a positive history of ranaviral disease and three populations known never to have experienced ranaviral disease. In order to counter contamination problems inherent with performing RNA-Seq in wild populations, we employed a RNA read filtering protocol, previously utilised in clinical settings, in an ecological RNA-Seq study for the first time. This protocol uses Bloom filters to identify contaminant reads in a rapid and resource-efficient way. We have identified a suite of 407 transcripts that are differentially expressed between populations with a long-term history of ranavirus infection and populations that remain uninfected. This suite contains genes with functions related to immunity, development, protein transport and olfactory reception amongst others. However, we did not find significant over representation of any functional pathways. A large proportion of potential non-coding RNA transcripts present in our differentially expressed sets provides the first evidence of a possible role for long non-coding RNA (lncRNA) in the amphibian response to ranaviral infection. Our read-filtering approach also revealed a significant positive relationship between the disease history of a population and the proportion of putative bacterial reads per library. Further, the bacterial communities represented by these reads were distinct between ranavirus positive and negative populations. In this talk I will present an overview of our novel workflow as well as the key results from this work.

Contact: Lewis J. Campbell, Environment and Sustainability Institute, University of Exeter, Penryn, Cornwall, TR10 9FE, United Kingdom. Phone: +44 7787439639. Email: [email protected]

Increased detection of ranavirus in tissue using novel in situ hybridization technique

María J. Forzán 1*, Erica Sloma 2 1,2Cornell School of Veterinary Medicine, Animal Health Diagnostic Laboratory1 and Department of Biomedical Sciences2, New York, USA

Detection of ranaviruses in formalin-fixed paraffin-embedded tissues is a helpful tool for both research and diagnostic laboratories. The ability to visualize viral presence in specific tissues or cell types can greatly increase diagnostic capabilities and further our understanding of the pathogenesis of ranavirosis in various hosts. Ranavirus detection using immunohistochemical (IHC) staining has been successfully employed, particularly in cases of fatal infections and in animals sampled close to the infection’s end-point in time-course trials. IHC is not as effective in early stages of infection when viral loads are low, and it may also be ineffective in detecting carrier stages. In situ hybridization (ISH), based on detection of nucleic acids rather than proteins, is a more sensitive technique. ISH has long been plagued by poor tissue preservation: a harsh pre-staining treatment is necessary to break the bonds of double-stranded nucleic acid molecules and allow for probe attachment. Recently developed RNAscope technology, based on detection of single-stranded mRNA, has resulted in better tissue preservation without compromising sensitivity. RNAscope ISH staining using a probe based on a fragment of major capsid protein of Frog Virus 3 (FV3, GenBank FJ459783.1) was validated for use in amphibian tissues. The technique was then applied to tissues from adult wood frogs experimentally infected with Frog Virus 3 and killed at 0.25, 0.5, 1, 2, 4, 9 and 14 days post-infection and compared to IHC staining using anti- EHNV (a cross-reacting ranavirus) antibodies, presence of microscopic lesions, and FV3 detection via PCR. ISH was more sensitive than IHC and provided similar, high quality, tissue resolution. Implementation of this new technique will improve ranavirus research and diagnostic capabilities. (Support: Canadian Wildlife Health Cooperative, Canada, and Cornell Wildlife Health Laboratory, USA).

Contact: María J. Forzán, Cornell School of Veterinary Medicine, Animal Health Diagnostic Laboratory, Ithaca, NY 14853, USA, Phone: +1 607 253 3764, Email: [email protected]

Pathogenesis of Bohle iridovirus in juvenile Eastern water dragons, Intellagama lesueurii lesueurii

Alicia Maclaine *, Jennifer Scott, Wytamma Wirth, Narges Mashkour, Ellen Ariel College of Public Health, Medical and Veterinary Sciences, James Cook University, Australia

Ranaviruses infect and have been associated with mass mortality events in fish, amphibians and reptiles. The aim of this study was to investigate the development of ranaviral disease over two weeks in eleven orally inoculated juvenile Eastern water dragons, Intellagama lesueurii lesueurii, a species previously reported to be susceptible to infection with Bohle iridovirus (BIV). Cloacal swabs were collected at pre-determined time points throughout the study and a validated quantitative polymerase chain reaction (qPCR) was used to detect ranavirus in these samples. Tissues and lesions collected during necropsy were analyzed using histology, immunohistochemistry (IHC), and viral isolation. Necrosis, haemorrhage, inflammation, and intracytoplasmic inclusion bodies were observed histologically in the liver, spleen, kidney, gastrointestinal tract and lung. Additionally, infiltration of lymphocytes were observed in the epidermis and sub-dermis of the tongue in several animals in the early stages of infection. The severity of clinical signs and pathological changes increased over time, with animals exhibiting external lesions after day 8. IHC consistently confirmed ranavirus associated with lesions in tissues. BIV was isolated in culture from several tissues and ranaviral presence was confirmed by qPCR. This is the first ranaviral pathogenesis study in lizards.

Contact: Alicia Maclaine, College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, 4811 Queensland, Australia. Phone: +61 7 4781 6915 Email: [email protected]

Performance of nonlethal methods of detecting Ranavirus infections in captivity and trade

Jesse L. Brunner 1,*, Anjulie Olson 1, Jeremy G. Rice 1, Mitchel J. Le Sage 1, Jennifer A. Cundiff 1, Caren S. Goldberg 2, Allan P. Pessier 3 1School of Biological Sciences, Washington State University, Pullman, Washington, USA; 2School of the Environment, Washington State University, Pullman, Washington, USA; 3Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USA.

Ranaviruses are moved regionally and internationally with the trade of live animals and can then spillover into wild and captive populations (e.g., zoos, aquaculture). While they often cause little or no apparent mortality, even leading to chronic, sublethal infections, ranaviruses can also cause mass mortality events. For both reasons ranaviruses are now OIE- notifiable. But while surveillance should be routine, screening animals in captive settings is difficult and uncommon. Issues include the fact that nonlethal samples (i.e., tail or toe-clips, swabs) have relatively low sensitivity and are poorly validated in the context of detecting asymptomatic infections, and the prohibitively large sample sizes required to ensure a disease-free status. To address these issues, we empirically evaluated the diagnostic performance of tail or toe-clips, swabs, and environmental DNA (eDNA) samples, combined with a quantitative real-time PCR assay, to detect ranavirus infections relative to the gold standard of internal tissues in American bullfrogs (Lithobates catesbeianus). We then incorporated our empirical findings into a probabilistic framework with which to design and interpret sampling protocols, with an emphasis on detecting at least one infection in closed populations, such as would be found in captivity or trade. We found that sensitivity and specificity of all types of samples change throughout the course of infections, and even with holding temperature, and all were generally bad at detecting low-level infections. While eDNA was less sensitive than swabs and clips, it was also less prone to false positives. Moreover, because eDNA samples collect virus shed from the entire population, they are much more efficient at detecting rare, low-level infections in captive populations than more sensitive individual-level samples. We think that an eDNA-based approach to ranavirus surveillance (and perhaps other pathogens as well) can be efficiently and cost-effectively implemented in a variety of captive settings. (Support: CGF grant 14-1263 from the Association of Zoo & Aquariums with funds provided by the Disney Conservation Fund and a Zoological Medicine & Wildlife Health Research Grant from the American Association of Zoo Veterinarians).

Contact: Jesse L. Brunner, School of Biological Sciences, Washington State University, Pullman, Washington, USA, Phone: +1 509 335 3702, Email: [email protected]

Immunohistochemistry of Hellbenders exposed to an FV3-like Ranavirus, alone or with one stressor

Debra L. Miller 1*, Ana Balseiro 2, Rosa Casais 2, Matthew J. Gray 1 1Center for Wildlife Health, University of Tennessee, Knoxville, Tennessee USA; 2Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Gijon, Asturias Spain

Hellbenders are a charismatic, yet reclusive, member of the Cryptobranchidae that is experiencing devastating population declines. The factors contributing to population declines remain unclear, and one of our goals is to determine the contribution of environmental contaminants and pathogens to declines. For our initial studies, we chose to begin by challenging larval hellbenders with ranavirus (10 PFU/mL) to determine susceptibility, and then using that information to challenge juveniles with ranavirus alone or in combination with Batrachochytrium dendrobatidis (another amphibian pathogen) or a glyphosate herbicide (a commonly applied herbicide within hellbender habitat). Tissues of exposed hellbenders were examined for the presence of disease and immunohistochemical staining was used to detect the presence of ranavirus within the tissues. Only larvae exposed to ranavirus at 22 °C (vs 15) succumbed to disease and abundant positive staining for virus was seen within multiple tissues, including nervous system, skin, vascular endothelium, hematopoietic tissue, liver, kidney, spleen and to a lesser extent the digestive tract. Although more hellbenders died when exposed with concurrent stressors; no difference in distribution or staining intensity was noted with immunohistochemical staining for ranavirus. These findings provide us with insight into elucidating the effect and contribution of ranavirus and other stressors to disease progression (Support for the challenge studies was provided by Tennessee Wildlife Resources Agency).

Contact: Debra Lee Miller, Center for Wildlife Health, University of Tennessee, Knoxville, TN 37996 USA, Phone: +1 865 974 7948, Email: [email protected]

Dose-dependent morbidity of freshwater turtle hatchlings, Emydura macquarii krefftii, inoculated with Bohle iridovirus (Ranavirus sp, Iridoviridae)

Wytamma Wirth 1*, Jennifer Scott 1 and Ellen Ariel 1 1James Cook University College of Public Health, Medical and Veterinary Sciences.

Ranaviruses cause disease in wild and captive turtle species around the world, often resulting in high levels of mortality. Ranavirus infections in wild and captive turtles can result in high levels of mortality. Ranaviral pathology has been studied in turtles from the suborder Cryptodia, however, little research has gone into understanding this disease in species from the other living suborder of turtles, the Pleurodira. Turtle species from the Pleurodira inhabit South America, Africa and Australia. The Pleurodira turtles of Australia (family Chelidae) live in environments where ranaviruses have been detected and experimental infection has determined that these turtles are susceptible to a ranavirus, Bohle iridovirus (BIV), found in their environment. Despite the potential for disease in these Australian turtles little else is known about the pathogenesis of ranaviral disease and factors that influence its development. The development of an animal model is a crucial step in understanding how factors like dose influence pathogenesis. This will help researchers in modelling ranaviral disease in wild reptile populations and quantify the susceptibility of at- risk populations, such as the Australian freshwater turtle species. To determine the dose- dependent disease development in an Australian freshwater turtle species, the first step in establishing an Australian turtle species model, an experimental infection was performed. Captive-raised Emydura macquarii krefftii hatchlings (family Chelidae) were assigned to one 5.3 1.3 of five dose groups of tenfold dilutions from 10 to 10 TCID50. Members of each group received an intramuscular injection of BIV at their respective dose. Animals in the study were euthanised at the onset of clinical signs (15-28 days after injection). Infectivity status was determined using PCR. Statistical analysis was performed to determine the odds of morbidity and histological finds are reported. The research presented here represents the first step for developing an animal model for studying ranaviral disease in Australian freshwater turtle species.

Contact: Wytamma Wirth, James Cook University College of Public Health, Medical and Veterinary Sciences, Australia, 4814, Phone: +61423321989, Email: [email protected]

The effect of water temperature on Frog virus 3 disease in young-of- the-year pallid sturgeon (Scaphirhynchus albus)

Natalie K. Steckler 1, Salvatore Frasca Jr. 2, Kuttichantran Subramaniam 1, Kamonchai Imnoi 1, Lacey Hopper 3, Jeffrey Powell 4, James Colee 5, Thomas B. Waltzek 1* 1Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610; 2Connecticut Veterinary Medical Diagnostic Laboratory, University of Connecticut, Storrs, CT 06269; 3U.S. Fish and Wildlife Service, Bozeman Fish Health Center, Bozeman, MT 59718. 4U.S. Fish and Wildlife Service, Gavins Point National Fish Hatchery, Yankton, SD 57078. 5Department of Statistics, College of Liberal Arts and Sciences, University of Florida, Gainesville, FL 32611

Ranaviruses, a genus of large double-stranded DNA viruses within the family Iridoviridae, are being detected with increasing frequency among aquacultured and wild fishes. In the United States, multiple sturgeon hatcheries have experienced ranavirus epizootics resulting in significant morbidity and mortality in young-of-the-year (YOY). Repeated outbreaks of Frog virus 3 (FV3), the type species for the genus Ranavirus, in YOY pallid sturgeon (Scaphirhynchus albus) reared at a hatchery within the Missouri River Basin have resulted in economic losses exceeding $400,000. Water temperature and stocking density are known to influence the severity of ranavirus disease in ectothermic vertebrates. To determine the effect of water temperature on ranavirus disease in hatchery-raised YOY pallid sturgeon, we conducted FV3 challenges at two temperatures (17 and 23˚C) and compared cumulative mortality over a 28-day study period. Forty-three percent mortality (17/40 fish) was observed among sturgeon maintained at 23˚C, whereas no mortality was observed among sturgeon maintained at 17˚C. In a second challenge study, in progress at the time of abstract submission, we compared the effect of water temperature on disease progression by regularly sampling fish over the study period and evaluating lesions by histopathology and in situ hybridization, and by assessing viral load in external and internal tissues by virus isolation and qPCR. Preliminary results suggest temperature manipulation may be an effective mitigation strategy that sturgeon hatcheries can employ to prevent ranavirus outbreaks.

Ranavirus phylogenomics: Signatures of recombination and inversions among ranaculture isolates

Sieara C Claytor 1, Kuttichantran Subramaniam 2, V. Gregory Chinchar 3, Matthew Gray 4, Carla Mavian 5, Marco Salemi 5, Samantha Wisely 1, Thomas Waltzek 2* 1Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL, USA; 2Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, USA; 3Department of Microbiology, University of Mississippi, Jackson, MS, USA; 4Center for Wildlife Health, University of Tennessee, Knoxville, TN, USA; 5Department of Pathology, Immunology, and Laboratory Medicine, and Emerging Pathogens Institute, College of Medicine, University of Florida, Gainesville, FL, USA

Herein we describe the genomic sequencing of two ranaviruses isolated from North American bullfrogs at the same ranaculture facility during die-offs in 1998 and 2006. Our goal was to determine the identities of the etiological agents and to ascertain if the same or different viruses were responsible for these outbreaks. The genomes of the two ranaviruses were sequenced and phylogenomic analyses were used to compare them to 19 other ranaviruses. The 1998 outbreak was determined to be due to a common midwife toad virus (CMTV)-like ranavirus previously thought to be restricted to Europe and China. The 2006 outbreak was determined to be due to a chimeric ranavirus in which CMTV-like fragments were detected within a predominantly FV3 genomic background. Analyses revealed that bullfrogs in this ranaculture facility were infected with a highly virulent chimeric ranavirus that likely arose following recombination between CMTV- and FV3-like ranaviruses. Since the chimeric ranavirus is more virulent than wild-type FV3, the presence of CMTV-like sequence elements suggests that specific CMTV genes may be directly related to the increase in pathogenicity. Because bullfrogs are traded internationally, it is important to understand the role that ranaculture may play in generating novel highly virulent recombinant ranaviruses. Given the high density of amphibians in commercial ranaculture facilities and the broad host range of ranaviruses, these facilities may serve as venues for the generation of highly virulent recombinant ranaviruses. Thus, it is critical to consider the adverse effect that the movement of infected animals could have on aquatic animal industries and wildlife.

Interaction of hydroperiod and ranavirus leading to possible amphibian population declines in the Great Smoky Mountains National Park

E. Davis Carter 1*, Matthew J. Gray 1, Jenny A. Spatz 1 and Debra L. Miller 1,2 1Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, University of Tennessee Institute of Agriculture, Knoxville, TN 37996, USA; 2Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA

Within the United States, ranaviruses are the most common pathogen associated with disease-related amphibian mortality events. Great Smoky Mountains National Park is in the southern Appalachian Mountains of North America, and reoccurring ranaviral disease outbreaks have occurred at Gourley Pond (GP) in Cades Cove region since 1999. Ranavirus outbreaks and larval mortality have been observed in five amphibian species at GP: Ambystoma maculatum, A. opacum, Lithobates sylvaticus, Pseudacris crucifer, and P. ferriarum. Due to the ephemeral nature of GP and the limited environmental persistence of ranaviruses, it is unlikely that the pathogen is persisting outside of host species between outbreaks. To better understand ranavirus dynamics within Cades Cove, we designed a surveillance study to monitor ranavirus prevalence within the amphibian community at GP and Little Gourley Pond LGP; a small pond <100 meters from GP. Our goals were to determine ranavirus prevalence in the amphibian community at GP and LGP, estimate population sizes of larval and post-metamorphic amphibians, determine possible routes of ranavirus introduction, and determine potential environmental stressors that might contribute to ranavirus outbreaks and limited amphibian recruitment. We captured a total of 1972 adults of 16 amphibian species in pitfall traps between Feb – May 2016. We detected ranavirus infection at low prevalence in adult L. sylvaticus, Notophthalmus viridescens, and A. maculatum. Average daily capture of aquatic larvae was 9/m2 and comprised only two species (A. opacum, L. sylvaticus). GP went completely dry on 28 March and LGP on 21 April. Successful metamorphosis was not documented at GP, and recruitment was minimal for A. opacum at LGP. No disease outbreaks were documented prior to the ponds drying. From February-March 2017, we captured 102 adults of 11 amphibian species in pitfalls, but no larvae were captured because the ponds did not fill with water. Our results suggest that the intermittent hydroperiod is resulting in limited recruitment and possibly population declines at GP and LGP. In addition, ranavirus is being maintained in the adult cohort, therefore if hydroperiod is sufficient for egg laying and larval development, it is possible the virus could be shed by adults and amplified by highly susceptible larvae. We also documented significant human visitation to the sites, which is another possible route for ranavirus introduction. Our results suggest there is potential for amphibian population declines at GP and LGP due to the previously observed catastrophic losses of amphibian larvae during ranavirus outbreaks and years where limited recruitment occurs due to abbreviated hydroperiod.

Contact: E. Davis Carter, University of Tennessee, Knoxville. 2431 Joe Johnson Drive 37996, Knoxville, TN, United States, Phone:704 819 0708, Email: [email protected]

Importance of ranavirus transmission pathways mediated by density dependence

Angela Peace 1,2, Suzanne M. O'Regan 2, Jennifer A. Spatz 3, Patrick N. Reilly 4, Rachel D. Hill 3, E. Davis Carter 3, Rebecca P. Wilkes 5, Debra L. Miller 3,4, Matthew J. Gray 3* 1Department of Mathematics and Statistics, Texas Tech University, Lubbock, USA; 2National Institute for Mathematical and Biological Synthesis, University of Tennessee, Knoxville, USA; 3Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, Tennessee 37996 USA; 4College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 USA; 5Veterinary Diagnostic and Investigational Laboratory, University of Georgia, Tifton, GA, USA.

We developed Susceptible-Infection (SI) type compartmental disease models to examine transmission dynamics of ranavirus in a wood frog (Lithobates sylvaticus) population. Our models included three transmission pathways: direct transmission via contact, environmental transmission via shed virions in water, and transmission via necrophagy of morbid individuals. Empirical estimates of viral shedding rates, individual contact rates, and incubation period were used to parameterize the system of differential equations. Unlike previous models, we categorized individuals into multiple stages of infection (susceptible, latency, and infectious), where the probability of disease-induced mortality increased throughout the duration of infection following a gamma distribution with an integer shape parameter. Factoring in different stages of infection also allowed for changes in the probability of transmission to be accounted for as the state of disease progressed. Our numerical simulations showed that accounting for pathogen incubation improved model predictions when compared to survival data from controlled experiments. In addition, we found that the invasion probability for ranavirus (i.e., the basic reproductive number, R0) into a population of wood frog tadpoles under frequency-dependent transmission was driven by water-borne transmission from shed virions; however, under density-dependent transmission, R0 was driven by direct contact and necrophagy transmission. Our results highlight the importance of understanding transmission pathways for ranaviruses as well as the form of transmission in host-pathogen systems. Support: Support for this research was provided by the University of Tennessee Institute of Agriculture and the National Institute of Mathematical and Biological Synthesis of the U.S. National Science Foundation.

Contact: Angela Peace, Department of Mathematics and Statistics, Texas Tech University, Lubbock, TX, USA, Phone: +1 806 834 1014, Email: [email protected]

Presence of amplification hosts increases mortality of syntopic amphibians by ranaviral disease

Roberto Brenes 1, Jason T. Hoverman 2, Debra L. Miller 3,4, and Matthew J. Gray 3 1Carroll University, Department of Biology, Waukesha, WI 53186, Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907, 3Center for Wildlife Health and 4College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996

Declines in amphibian populations from disease outbreaks could be mediated by host susceptibility. Our objective was to determine if the outcome of a ranaviral disease outbreak in an amphibian community was dependent on which species was initially exposed in an experimental aquatic ecosystem. We created two amphibian communities: (1) an Appalachian community composed of wood frog (Lithobates sylvaticus), upland chorus (Pseudacris feriarum), and spotted salamander (Ambystoma maculatum) larvae, and (2) a coastal plain community composed of gopher frog (Lithobates capito), upland chorus frog (P. feriarum), and southern toad (Anaxyrus terrestris) larvae. The experiment was conducted outdoors in 320-L mesocosms, and treatments consisted one, all, or none of the species initially exposed to Frog virus 3. Initial exposure occurred under controlled laboratory conditions in 1-L water baths (103 PFU/mL) for 3 days prior to distribution of the larvae to the mesocosms. Mortality rates after 60 days depended on which species was initially exposed to the pathogen. In the Appalachian community, exposed wood frog tadpoles caused an outbreak of ranaviral disease in unexposed chorus frogs (40% mortality) and amplified mortality of spotted salamander larvae 2X greater than when this species was directly exposed to the virus. In the coastal plains community, all species were able to cause outbreaks of ranaviral disease (>40% mortality) in co-inhabitant unexposed species. Our results demonstrate that amphibian community composition can affect ranaviral disease outcomes. Support: The University of Tennessee institute of agriculture research grant

Contact: Roberto Brenes, Carroll University. 100 N. East Ave. Waukesha, WI. 53186, USA. Phone +1 262 951 3295, Email:[email protected]

Comparative proteomic analysis show altered proteins regulated by ubiqutin proteasome system in fish iridovirus infection

Xiaohong Huang 1,2, Youhua Huang 1,2, Shina Wei 1,2, Yepin Yu 1,2, Qiwei Qin 1,2,3* 1Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China;2University of Chinese Academy of Sciences, Beijing, China; 3College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.

The ubiquitin-proteasome system (UPS) has been demonstrated to be involved in mammalian viral infection at different stage of the viral replication. However, the roles of UPS in fish viral infection still remained uncertain. Here, the roles of UPS and proteins regulated by UPS during Singapore Grouper Iridovirus (SGIV) infection were investigated in grouper cell lines. Different proteasome inhibitors significantly reduced viral production, accompanied by the inhibition of virus assembly sites formation and the viral genes transportation, indicating that UPS was required for fish iridovirus infection in vitro. Furthermore, the altered proteins in SGIV-infected cells with or without MG132 were investigated by two dimensional electrophoresis and MALDI-TOF. A total of 62 differentially expressed spots were successfully identified, including 21 host proteins which were significantly down-regulated, and 7 host proteins that were dramatically up-regulated after SGIV infection. However, MG132 treatment had the opposite effect on most of the gene expression regulated by SGIV infection. MG132 treatment considerably regulated those cellular proteins involved in ubiquitin-mediated protein degradation, metabolism, cytoskeleton, macromolecular biosynthesis, and signal transduction. Moreover, 6 corresponding genes of the differentially expressed proteins were quantified using quantitative real-time PCR to examine their transcriptional profiles. Thus, this study provided useful protein-related information to further understand the underlying molecular mechanism of UPS during SGIV infection. (This work was supported by grants from the NationalNatural Science Foundation of China (31372566) and the National BasicResearch Program of China (973) (2012CB114402)).

Contact: Qiwei Qin, 164 West Xingang Road, Guangzhou, China.Phone:+86-20-89023638, Email:[email protected]

Ranavirus in northern Canada: a low-diversity ecosystem for a better comprehension of disease dynamics?

Joe-Felix Bienentreu 1, Samantha A. Grant 2, Chris J. Kyle 2, Craig R. Brunetti 2, Danna M. Schock 3, David Lesbarrères 1* 1Department of Biology, Laurentian University, Sudbury, Canada; 2Department of Biology, Trent University, Peterborough, Canada; 3Sciences and Environmental Technologies, Keyano College, Fort McMurray, Canada

Due to their complexity, host-pathogen systems are difficult to tease apart and infectious diseases are often studied as one host-one pathogen relationships. Yet, in many wildlife diseases, a pathogen can infect several hosts and a community ecology approach would improve our understanding of several epidemiologic factors. Among others, two pathogens threaten amphibians all around the globe: the chytrid fungus (Batrachochytrium dendrobatidis, Bd) and Ranavirus, a genus of viruses in the family Iridoviridae. Taking advantage of a low diversity ecosystem with only three amphibian hosts, the Wood Frog (Rana sylvatica), the Boreal Chorus Frog (Pseudacris maculata) and the Canadian Toad (Anaxyrus hemiophrys), this project aims to close important knowledge gaps on the epidemiology of Ranavirus and Bd above the 59th parallel in northern Canada and to better understand the effects and dynamics of these diseases. In 2015 and 2016, a total of 802 individuals were sampled across the three host species and while only 1% tested positive for Bd overall, 8.5% of adults and 35% of metamorphs tested positive for Ranavirus among Wood frogs and Boreal Chrorus frogs. However, no metamorph or adult Canadian toads tested positive. Additionally, we explored Ranavirus genetic variation in this species-poor community and compared it with a more southern and diverse area where the pathogen occurs. By comparing different Ranavirus variants and relating those to the region they occur, it will be possible to track the overall spread of virus in Canada and identify possible reservoirs species.

Contact: David Lesbarrères, Centre for Evolutionary Ecology and Ethical Conservation, Laurentian University, Sudbury, ON, P3E 2C6, Canada, Phone: 1(705)675-1151, Email: [email protected]

Phylogenomic characterization of ranaviruses detected in fish and amphibians in Thailand

Preeyanan Sriwanayos 1,2, Kuttichantran Subramaniam 1*, Natalie K. Steckler 1, Somkiat Kanchanakhan 2,3, Jaree Polchana 2, Thomas B. Waltzek 1 1Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32608; 2Aquatic Animal Health Research Institute, Department of Fisheries, Bangkok, Thailand 10900; 3Si Sa Ket Provincial Fishery Office, Department of Fisheries, Si Sa Ket, Thailand 33000

Ranaviruses are emerging pathogens associated with epizootics in farmed and wild poikilothermic vertebrates including fish, amphibians, and reptiles worldwide. In this study, we described the full genomes of seven ranaviruses isolated from cultured marbled sleeper goby (Oxyeleotris marmorata), goldfish (Carassius auratus), guppy (Poecilia reticulata), tiger frog (Hoplobatrachus tigerinus), Asian grass frog (Fejervarya limnocharis), and two East Asian bullfrogs (H. rugulosus) in Thailand. The full genomes of the above mentioned fish and amphibian isolates were sequenced using an Illumina MiSeq sequencer. The nucleotide alignments of the major capsid protein (MCP) sequences from the Thailand isolates compared to a Chinese isolate from tiger frog (H. tigerinus) were highly similar (99.9% nucleotide identity). Comparison of the Thailand isolate MCP sequences to other fully sequenced ranaviruses displayed a lower sequence identity ranging from 93.1-98.6%. Phylogenomic analyses based on the concatenation of 46 core ranavirus genes revealed that these eight Asian isolates formed a monophyletic group named the tiger frog (TFV) clade. Our findings confirm the transboundary movement of TFVs among Asian cultured fish and amphibians.

How to use the Global Ranavirus Reporting System (GRRS)

Jesse L. Brunner 1*, Deanna H. Olson 2, Amanda L.J. Duffus 3, Matthew J. Gray 4 1School of Biological Sciences, Washington State University, Pullman, Washington, USA; 2Forest Service, Pacific Northwest Research Station, Corvallis, Oregon, USA; 3Gordon State College, Barnesville, Georgia, USA; Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, Tennessee, USA..

The Global Ranavirus Reporting System (GRRS; https://mantle.io/grrs) is an online database and mapping system for reports of the occurrence (or absence) of ranaviruses across all taxa, in wild and captive populations, around the world. The system includes fields for the location, type of population, species involved, type of event (e.g., die-off or routine surveillance), method(s) of detection, and many other relevant pieces of information. It was designed to be a resource for individual researchers—users can create and manage records, import and export data, view tables and maps of reports, and control the visibility of their records—as well as a way for the broader community to better understand the geographic distribution and host range of these important viruses. The system has not been broadly used, however, in part because few know how to use it. This workshop will provide an overview of how the system works and might be used, and will then teach individual researchers to enter and manage their own records (including managing the visibility of records, correcting information, organizing studies). Participants should bring their own recently published or unpublished data (e.g., a record of a mortality event, a surveillance study) and a laptop if possible. (Support: Wildlife Disease Association Small Grant Competition).

Contact: Jesse L. Brunner, School of Biological Sciences, Washington State University, Pullman, Washington, USA, Phone: +1 509 335 3702, Email: [email protected]

POSTER PRESENTATION ABSTRACTS

Susceptibility of American bullfrog tadpoles (Lithobates castebeianus) to experimental infection by Frog Virus 3 (FV3)

Sthefany R. Alfaia ¹, Cinthia R. Oliveira ¹, Fernanda L. Ikari ¹, Diego Sales ¹, Ricardo L. Moro ², Claudia Maris Ferreira ¹* 1Instituto de Pesca, São Paulo, Brazil.²Faculdade de Medicina Veterinária e Zootecnia, Universidade São Paulo, Pirassununga,Brazil.

Reports of mass mortality outbreaks and morbidity of amphibian species have become increasingly frequent, reaching both wild populations and aquaculture. The causes include emerging diseases such as ranavirosis. The bullfrog (Lithobates catesbeianus) is the only species raised commercially for consumption in Brazil. Within frog farms, these animals are rearing in high densities increasing the possibility of diseases start. The objective of this work was to test the susceptibility of L. castebeianus tadpoles (stage 31 Gosner) at three different concentrations of FV3. The isolate was obtained from an outbreak in 2013 in adult frogs on a production farm. The tadpoles in this experiment, which were previously tested to ensure they were infection-free, were inoculated orally with 50 uL of the virus cultured in BF-2 cells and diluted to the following concentrations: 102.8 plaque-forming units (pfu)/mL; 104.8 pfu/mL and 106.8 pfu/mL, or with cell culture media as a negative control. Each treatment had four replicates with groups of 9 tadpoles housed in 9L of water. The experiment lasted for 21 days, with liver, spleen and kidney collected on the 14th day after exposure (p.e.) from a subset of 12 tadpoles, and again on the 21st day. The collected organs were pooled and screened for ranavirus infections with traditional PCR with primers targeting two regions in the conserved MCP gene (MCP-1, 321 bp) and (MCP2-2, 625 bp). Roughly 50% of the animals were positive for ranavirus DNA at the end of the experiment, regardless of the dose of inoculum. There were only 3 deaths during the experiment period (0, 6, and 16 days p.e.). The symptoms in ranavirus-exposed tadpoles included lethargy, lordosis in the caudal region and change in buoyancy, but there was no change in metamorphosis. Different symptoms were observed in the animals that died at days 6 and 16 p.e. including skin peeling, petechiae and edema. The low mortality suggests these viral doses were not sufficient to generate high mortalities, a cofactor such as an immunosuppressive agent may be necessary, or this strain circulating in the southeastern region of Brazil has low virulence. Thus, none of the treatments tested proved sufficient to generate a significant death. (Support: FAPESP 2015/24590-7)

Contact: Claudia Maris Ferreira, Divisão de Aquicultura, Instituto de Pesca, 05001-970, São Paulo, Brasil, Phone: +5511964916071 Email: [email protected]

Screening and molecular characterization of a ranavirus (Frog Virus 3) obtained from bullfrogs (Lithobates catesbeianus) from commercial farms in Southeast Brazil

Cinthia R. de Oliveira ¹, Sthefany R. Alfaia ¹, Fernanda L. Ikari ¹, Patricia T. Coelho ¹, Loiane S. Tavares ², Ricardo L. Moro de Sousa ², Claudia M. Ferreira ¹* ¹Instituto de Pesca, São Paulo, Brazil; ²Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, São Paulo, Brazil.

Amphibian populations are declining in the world and one of the causes is emerging diseases such as ranaviroses. The genus Ranavirus belongs to the family Iridoviridae that encompass viruses capable of infecting ectothermal animals and causing mass mortality among wild and captive amphibians. The aim of this study was to detect the presence of FV3 in commercial farms of the Southeast region of Brazil using molecular techniques. For that, 20 tadpoles of 7g +/- 1.2g were collected in five frog farms (C1 to C5) and 20 frogs of 120g +/- 8g in four frog farms (C1 to C4), totaling 180 specimens. The spleen, liver and one kidney of each animal were pooled and DNA extracted, followed by PCR with primers for the MCP (major capsid protein) gene and subsequent electrophoresis. Positive results were obtained from adults and tadpoles in the C2 ranch, affecting 50% of the local specimens in an apparently asymptomatic manner and without reported mortality outbreaks. The only abnormality observed in the specimens was vestibular disorder in some individuals, a clinical sign newly associated with ranavirus, but which may be associated with other diseases or nutritional deficiencies. One of the obtained PCR products was purified and subjected to nucleotide sequencing. The identity search of the sequence generated and edited with BioEdit v7.0.9 software was performed by the BLAST v2.6.1 program, obtaining 100% identity relative to sequences from other FV3 and/or ranavirus strains. In the phylogenetic reconstruction, by the maximum-likelihood method (IQ-TREE software), the sequence obtained was grouped with sequences of salamander ranavirus (Hynobius nebulosus and Pogona vitticeps) and wild-frog species (Pelophylax plancyi). Although the ranaviral infection is only subclinical in this farm, when associated with other pathogens and stressors, we believe that it may negatively impact the frog flocks and cause local extinctions of other species. In addition, the results of this study indicate the possible circulation in a commercial frog plant in the Brazilian Southeast of a Ranavirus variant similar to those found in species of reptiles from other countries, which requires further studies to better understand the epidemiology of Ranavirus infection in Brazil. Support: Fundação de Amparo a Pesquisa do Estado de São Paulo - FAPESP 2015/24590-7

Contact: Claudia Maris Ferreira, Divisão de Aquicultura, Instituto de Pesca, 05001-970, São Paulo, Brasil, Phone: +5511964916071 Email: [email protected]

Is the angelfish Pterophyllum scalare susceptible to ranaviruses?

Kateřina Matějíčková 1*, Stanislava Reschová 1, Pavel Kulich 1, Barbora Veselá 2, Tomáš Veselý 1 1Veterinary Research Institute, Brno, Czech Republic; 2Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Brno, Czech Republic

Ranaviruses belonging to the genus Ranavirus in the family Iridoviridae are responsible for serious diseases in wild and captive fish, amphibian and reptile species around the world. It has been suggested that the spread of Ranavirus may be facilitated by transcontinental movements of ornamental fish. Due to the lack of knowledge on the pathogenicity of ranaviruses to ornamental fish, experimental bath-infection trials were carried out in order to determine the susceptibility of the ornamental fish species angelfish Pterophyllum scalare to ranaviruses isolated from freshwater fish (Epizootic haematopoietic necrosis virus [EHNV], European sheatfish virus [ESV] and European catfish virus [ECV]). The angelfish were kept at two different temperatures, 20 °C and 28 °C, throughout the experimental period. Significant mortality was observed only in fish challenged with the ECV isolate at 20 °C. Viruses were successfully re-isolated from dead fish from all three challenged groups in cell culture with subsequent identification by electron microscopy, immunohistochemistry, ELISA, PCR and sequence analysis. No significant mortality was observed in the challenged groups at 28 °C and none of those viruses were re-isolated in cell culture or identified with the other methods. The results indicate that the water temperature may play an important role in the pathogenicity of ranaviruses. Furthermore, angelfish should be considered potential carrier species for ranavirus infection under certain conditions. (Support: FP6-6459 RANA, MZE-RO0517, LO1218 under the NPU1 program)

Contact: [email protected]

Robust viral load estimation in ranavirus-infected amphibian, reptile, and fish tissues and cell cultures

William T.M. Leung 1*, Laura Thomas-Walters 1,2, Trenton W.J. Garner 1, Francois Balloux 3, Chris Durrant 1†, Stephen J. Price 1,3 1Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY; 2 Durrell Institute of Conservation and Ecology, University of Kent, Canterbury CT2 7NZ; 3UCL Genetics Institute, Gower Street, London WC1E 6BT; †NatureMetrics Ltd, Ashford, Surrey, TW15 1UU

Ranaviruses are important pathogens of amphibians, reptiles and fish. To meet the need for rapid, cost-effective, robust and, importantly, validated tools for diagnostic testing, two standard-curve based quantitative-PCR assays (qPCR) were developed enabling viral load estimation across all these host groups. A virus qPCR was developed which exhibited specificity to amphibian-like ranaviruses (ALRVs) with high analytical sensitivity (5 copies per reaction), and high reproducibility (intra- and inter assay coefficient of variation below 4%) across a wide dynamic range (3x108 to 3). The qPCR showed 100% diagnostic specificity (n=78) compared to a gold standard. The qPCR also showed 100% diagnostic specificity (n=94) when applied to samples from a site with no history of ranavirus infection. Without appropriate normalisation, qPCR data is rendered only qualitative (i.e. presence/absence of infection) since the confounding effects brought on by variations in 1) amounts of starting material, 2) efficiencies of different DNA extraction methods, 3) and final elution volumes are not accounted for. Ranavirus quantities can be normalised by total DNA mass (e.g. pathogen genome copies per microgram of total DNA) but this approach 1) assumes that all of the genetic material is host derived, 2) is unsuitable for inter-species pathogen load comparisons when host genome sizes vary, and 3) its accuracy is affected by a lack of precision in photometric nucleic acid analysis. We provide here, a normalisation qPCR targeting an ultra-highly conserved host locus present as a single-copy across amphibian, reptile, and fish species. This locus is amplified across these host species with the single set of primers described in this study. Viral quantities can then be normalised by host cell number derived from the host qPCR to generate effective viral loads (virus per cell). We provide in silico and experimental evidence of single-copy status of this host locus across host species. The host qPCR also exhibits high analytical sensitivity (4 copies per reaction) and high reproducibility (intra- and inter assay coefficient of variation below 5%) across a wide dynamic range (3x109 to 3). Virus and host qPCRs were then applied to track ranavirus growth in cell culture. Used together, the two assays offer a robust approach to viral load estimation and the host qPCR can be paired with assays targeting other pathogens to study infection burdens.

Contact: William T.M. Leung, Nuffield Building, Institute of Zoology, Zoological Society of London, Outer Circle, Regent’s Park, London NW1 4RY; Phone +447933576667; Email: [email protected]

Development of green sea turtle (Chelonia mydas) primary cells to propagate aquatic animal viruses including Bohle Iridovirus

Narges Mashkour, Ellen Ariel * College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, Australia

Establishment of primary cultures from endangered animals is a new trend to preserve the genomic information and also cellular characteristics of the species for future biological studies. In terms of virological studies, primary cultures are valuable as alternatives to animal models. Primary cells provide a host cell spectrum to propagate viruses and may increase viral susceptibility and the chance to isolate viruses. In addition, primary cells of the same host as the sample origin are shown to be more susceptible in culturing viruses. The only commercially available chelonian cell line is from the terrestrial turtle (Terrapene Carolina). Therefore, establishment of primary cells for screening green sea turtle tissues for viruses would greatly enhance the opportunity to find such viruses. To establish primary cultures from Chelonia mydas, turtle eggs were collected form Heron Island (Queensland, Australia) nesting beaches and taken to James Cook University and incubated at 29°C in a moist atmosphere. From day 20 of incubation, the embryos were taken to establish primary cultures. In early stages of development, the whole embryos were taken for primary culture explants and closer to the hatching date the internal organs and muscles were specifically collected. The primary cells were established in Dulbecco's Modified Eagle Medium (DMEM, 90%) and foetal bovine serum (FBS, 10%). To check the cell growth and optimize viability critical growth factors were screened: (1) medium type, (2) FBS concentration, (3) Temperature, (4) pH. The optimal growth curve and the best cell maintenance were achieved in DMEM (90%), FBS (10%), 25°C and pH 7.2-7.4 respectively. According to American Type Culture Collection (ATCC) cell establishment, primary cultures should be documented with a fact sheet containing proof of cell origin. Karyotyping and molecular analysis of mtDNA D-loop gene were carried out for cell authentication. The primary cells were also tested to be free of mycoplasma contamination. Three primary cell lineages were chosen for viral isolation: CMEM1, CMEM2, CMEH. The susceptibility of CMEM2 to Bohle Iridovirus is the highest compared to CMEM1, CMEH, FHM and BF-2.

Contact: Narges Mashkour, College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, 4811 Queensland, Australia, Phone: +61 420 336 741 Email : [email protected]

Isolation and identification of Singapore grouper iridovirus Hainan (SGIV-HN) isolate

Jingguang Wei, Youhua Huang, Min Yang, Xiaohong Huang, Qiwei Qin * Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China

In our epidemic investigation of grouper from Hainan province, China, we found many groupers showing symptoms very similar to infections associated with the Singapore grouper iridovirus (SGIV), so we separated and identified the virus from the diseased groupers by co- cultivation of the affected tissue cells with grouper spleen cells (GS). Cytopathic effects (CPE) were visible 5 to 6 days post co-cultivation. The typical SGIV particles were observed under electron microscope. A 579 bp DNA fragment of the major capsid protein (MCP) gene was amplified by PCR and sequencing results indicated an homology of 99.31% with the reference strains of SGIV in GenBank. The evolutionary tree constructed based on the MCP amino acid sequences showed that the virus was clustered with SGIV, so the virus was named as Singapore grouper iridovirus Hainan (SGIV-HN) isolate. When groupers were 8.9 infected with 100 μl SGIV-HN at a concentration of 10 TCID50/ml by intraperitoneal injection, the same clinical signs as natural infection were observed, and the morbidity and mortality were 100% and 70%, respectively. These results demonstrate that the SGIV was isolated for the first time from a naturally infected fish in China. (Support: grant of Science and Technology and industrial development Marine fishery Project of Guangdong Province, China (A201501C01)).

Contact: Qiwei Qin, Ph D., Professor, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China. Tel:+86 20 89023638, Fax: +86 20 89023638. E-mail: [email protected]

Molecular characterization of ranaviruses detected in Nile Tilapia (Oreochromis niloticus) in Tanzania

Augustino Alfred Chengula 1,2*, Christopher Kasanga 2, Stephen Mutoloki 1, Robinson Mdegela 2 and Øystein Evensen 1 1Norwegian University of Life Sciences, Basic Sciences and Aquatic Medicine, Norway; 2Sokoine University of Agriculture, College of Veterinary and Medical Sciences, Tanzania

Ranaviruses are significant pathogens infecting all classes of ectothermic vertebrates (fish, reptiles, and amphibians) causing systemic necrotizing infections and mass mortalities in captive and wild populations across the globe. Ranaviruses are large DNA viruses belonging to the family Iridoviridae. There many published reports of ranaviruses infecting ectothermic vertebrates worldwide. However, based on our understanding there no reports of ranaviruses infecting Nile Tilapia in Tanzania. Therefore, the aim of this study was to identify and characterize ranaviruses infecting Nile Tilapia in selected natural water bodies and fish farms in Tanzania. Samples were collected from Lake Victoria (108 fish) in Mwanza and fish farms (120 fish) in Morogoro, Tanzania. Live fish were bought from the fishermen (at Lake Victoria) and from the owners (in Morogoro) who caught the fish from the ponds by netting. Lake Victoria was selected because is the main supplier of Nile Tilapia consumed and parent stock for the Nile Tilapia grown in the country. On the other hand, fish farms included in this study were selected purposely based on activity. Samples were collected in RNALater (livers, gills, spleen and heart) and in transport media (kidneys) for DNA extraction and virus re- isolation respectively. Degenerate primers targeting viral DNA polymerase were used during PCR amplification assays. The PCR products were then sequenced and formed the basis for designing virus specific primers for Real Time PCR. Real Time PCR was then used to screen all the remaining samples. Our findings show that wild Nile Tilapia in Tanzania contained ranaviruses. Phylogenetic analysis of the sequences suggests that the isolates in these fish were closer to those of Rana grylio virus (96%), Soft-shelled turtle iridovirus (96%) and Frog virus 3 (95%) both from the genus Ranavirus. These findings complement the previous hypotheses of the possible host-switch of ranaviruses from one host to another and vice versa. Based on our knowledge, this is the first report of molecular characterization of ranaviruses in Nile Tilapia from Tanzania and Africa. (This work was funded by NORAD through the Project on "Capacity building for training and research in aquatic and environmental health in Eastern and Southern Africa" (TAN-13/0027).)

Contact: Augustino Alfred Chengula, P.O.Box 3037, Morogoro, Tanzania. Phone: +255767605098, Email: [email protected]

Envelope protein VP088 of Singapore grouper iridovirus is involved in viral attachment and entry into host cells

Sheng Zhou 1,3, Zhuxi Wang 2, Youhua Huang 1,3, Shaowen Wang 1,3, Xiaohong Huang 1,3, Qiwei Qin 1,3* 1Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China, 2College of Marine Science, Hainan University, Experimental Teaching Demonstration Center of Marine Biology, Key Laboratory of Tropical Aquatic Biotechnology of Hainan Province, Haikou 570228, China, 3College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China

Iridovirus infections are a great threat to agriculture. Singapore grouper iridovirus (SGIV) is highly infectious to groupers and increasingly threatens mariculture. SGIV intervention is not available because little is known about key players and their precise roles in SGVI infection. Envelope proteins are the first molecules of viruses to interact with host cells. VP088 was identified as an envelope protein and is known to play a key roleinfection. The rVP088 protein can bind to the membrane of the host cell and has been found to block the entry of virus into cells. In this study, we report the construction of a recombinant SGIV (SGIV-lacO- VP088) containing the inducible lac repressor/operator system. The recombinant was a conditional lethal mutant in which the expression of VP088 was regulated by IPTG. The expression level of VP088, the ability of the virus to cause plaque formation, and the virus titers were strongly reduced in the absence of IPTG. The amount of recombinant virus entering into the host cells was strongly reduced comparing to the wild-type virus. The recombinant virus resulted in a remarkable reduction in the death of grouper compared to the wild-type virus in animal experiments. Electron microscopy showed that there were no obvious changes in the morphology of the virus. Together, our results indicated that VP088 of SGIV is involved in viral attach and entry into host cells. Reducing the expression of VP088 strongly reduced the infectivity of SGIV. Support: Grant from National Natural Science Foundation of China Youth Found (31502207)

Contact: Qiwei Qin, College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China, Phone: +8620 890 23638, Email: [email protected], [email protected]

Ranavirus infection potentially alters the age structure of infected populations.

Lewis J. Campbell 1,2*, Trenton W.J. Garner 2, Giulia Tessa 3, Ben C. Scheele 4, Amber G.F. Griffiths 5, Lena Bayer-Wilfert 6, Xavier A. Harrison 2 1Environment and Sustainability Institute, University of Exeter, Penryn Campus, Penryn, Cornwall, TR11 9FE, U.K 2Institute of Zoology, Zoological Society of London, Regents Park, London, NW1 4RY, U.K 3Life science and Systems Biology Department, Università degli Studi di Torino, via Accademia Albertina 13, 10123, Torino, Italy 4Fenner School of Environment and Society, Australian National University, Canberra, ACT 2601, Australia 5FoAM Kernow, Studio E, Jubilee Warehouse, Commercial Road, Penryn, Cornwall TR10 8FG 6Centre for Ecology and Conservation, University of Exeter, Penryn Campus, Penryn, Cornwall, TR11 9FE

Amphibians are the most threatened group of vertebrates on the planet. Along with ubiquitous challenges such as climate change, habitat destruction and over-harvesting, an important threat that they face is infectious disease. Besides increased mortality and morbidity, a secondary impact of infectious diseases is their potential to alter the demographic structure of populations in which they are present. Demographic shifts can allow populations to reduce the impacts of infection but changes in the stable demographic structure can also contribute to the threat that a population faces. Despite this potential additional threat, very little work has yet been done on how disease can impact population demography in amphibians. It has been shown that infection with chytrid fungus results in a truncated population age structure in amphibians, heightening their susceptibility to recruitment failure. However, this work provides, to our knowledge, the first investigation of the influence of Ranavirus infection on amphibian population demography. Using the unique, comparative nature of the U.K field system for the study of the impacts of Ranavirus on wild common frog (R. temporaria) populations, we investigated the demographic structure of 5 Ranavirus positive and 5 Ranavirus negative populations. We attended each population during peak breading season and sampled as many individuals as possible from in and around the breeding pond. We took morphological measurements as well as toe clips for aging by skeletochronology. We found that Ranavirus infection does not seem to impact the growth rates or age at sexual maturity of individuals. However, there was a shift in the age structure of populations. Breeding ponds at Ranavirus positive populations contain a higher proportion of younger individuals than do their Ranavirus negative counterparts. Though the exact mechanisms of this change remain to be elucidated, this is the first evidence of Ranavirus infection having an impact on the age structure of infected populations, and is likely to prove an important springboard for further research in this area. In this poster we will present an overview of our methodologies and the key results from this work.

Contact: Lewis J. Campbell, Environment and Sustainability Institute, University of Exeter, Penryn, Cornwall, TR10 9FE, United Kingdom. Phone: +44 7787439639. Email: [email protected]

Host range and local distribution of Bosca’s newt virus

Marius von Essen 1,2, William T.M. Leung 2, Celia Serrano 2,3, Trenton W.J. Garner 2, Jaime Bosch 3, Cesar Ayres 4, Simon Pooley 1 and Stephen J. Price 2,5 1Imperial College London, Buckhurst Road, Ascot, Berks, SL5 7PY, UK; 2Institute of Zoology, ZSL, Regents Park, London NW1 4RY, UK; 3Museo Nacional de Ciencias Naturales, CSIC, José Gutiérrez Abascal 2, 28006, Madrid, Spain; 4Asociacion Herpetologica Espanola, Jose Gutierrez Abascal 2, 28006 Madrid, Spain; 5UCL Genetics Institute, Gower Street, London WC1E 6BT, UK

Since 2010, recurring mass mortality events in Bosca’s newts (Lissotriton boscai) and marbled newts (Triturus marmoratus) have been reported at a reservoir in Galicia, Spain. A novel common midwife toad virus (CMTV)-like virus – Bosca’s newt virus – has been associated with these disease outbreaks but little is known about the effects on other species in the amphibian and reptile community or the spatial distribution of this virus. We conducted an extensive survey of the potential host community and local area in 2016. We detected BNV in a broad range of amphibian and reptile hosts and at several other sites in the local area. We also observed disease in other species in addition to the two caudates and one squamate previously reported. The reservoir is part of a large network of waterways and the site of a watersports training centre and could function as a source of ranavirus dispersal, both regionally and nationally.

Contact: Stephen J. Price, UCL Genetics Institute, Gower St, London WC1E 6BT, UK. Phone: +44 20 3108 4229 , Email: [email protected]

Characterization of two novel cysteine protease inhibitors of cystatin B and cystatin C from grouper, Epinephelus coioides, involved in SGIV infection

Shina Wei 1, Youhua Huang 1, Jingguang Wei 1, Xiaohong Huang 1, Qiwei Qin 1,2* 1Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; 2College of Marine Sciences, South China Agricultural University, Guangzhou, China

Cystatins are a superfamily of proteins that act as reversible inhibitors of cysteine proteinases, a group of proteins which play essential roles in diverse pathological processes such as viral infection. Singapore grouper iridovirus (SGIV) is a major viral pathogen of grouper aquaculture, and has caused heavy economic losses in China and South-east Asia. In this study, the cystatin B (Ec-cysB) and cystatin C (Ec-cysC) genes from grouper were cloned and characterized and their effects against SGIV infection were investigated. Quantitative PCR showed that Ec-cysB was abundant in head kidney, gill and intestine, whereas Ec-cysC was strongly expressed in spleen, brain, head kidney and kidney. Both Ec-cysB and Ec-cysC were differentially upregulated after SGIV infection. Subcellular localization analysis showed that Ec-cysB was distributed in the cytoplasm and nucleus, while Ec-cysC was distributed predominantly in the cytoplasm. Overexpression of Ec-cysC in grouper cells significantly delayed the occurrence of the cytopathic effect (CPE) induced by SGIV, and inhibited viral gene transcription. However, overexpression of Ec-cysB in grouper cells could promote SGIV replication. Further experiments will elucidate the role of Ec-cysB and Ec-cysC during SGIV infection and perhaps lead to the development of new control and therapeutic strategies. (Support: Natural Science Foundation of China 41506176 grant).

Soft-shelled turtle iridovirus enters cells via cholesterol-dependent, clathrin-mediated endocytosis as well as macropinocytosis

Youhua Huang 1,2, Xiaohong Huang 1,2, Shaowen Wang 1,2, Yepin Yu 1,2, Qiwei Qin 1,2,3 * 1Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China;2University of Chinese Academy of Sciences, Beijing, China; 3College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.

Iridoviruses are nucleo-cytoplasmic large DNA viruses (NCLDV) that contribute to marked economic losses in aquaculture and are a global threat to ecological diversity. However, the molecular mechanisms of iridovirus entry are largely unknown. In this study, using specific inhibitors of different endocytic pathways and fluorescence tagged recombinant virus, we investigated the cellular entry routes used by soft-shelled turtle iridovirus (STIV). Treatment with chlorpromazine and sucrose, inhibitors which block clathrin-mediated endocytosis, significantly decreased STIV infection, suggesting STIV was able to use clathrin-mediated endocytosis to enter cells. The depletion of cellular cholesterol with methyl-β-cyclodextrin significantly inhibited STIV entry, but neither filipin III nor nystatin did, suggesting that STIV entry was cholesterol dependent, but caveola-independent. Interestingly, inhibition of a key regulator of macropinocytosis, the Na+/H+ exchanger, by EIPA revealed that macropinocytosis might be an alternative pathway used by STIV to enter cells. Moreover, treatment with IPA-3, ML-7 or cytochalasin D all significantly inhibited STIV infection, indicating that Rac GTPase and myosin II activity were required for macropinocytosis-like entry as well as actin polymerization. In addition, the marked inhibitory effect of chloroquine and bafilomycin A1 on STIV infection suggested that STIV entry was pH-dependent. Together, this study identified multiple routes of STIV entry and identified several cellular factors involved in this process. (This work was supported by grants from the National Natural Science Foundation of China (31330082, 31172445), the National Basic Research Program of China (973) (2012CB114402), and the Knowledge Innovation Program of the Chinese Academy of Sciences (SQ201014))

Contact: Qiwei Qin, 164 West Xingang Road, Guangzhou, China.Phone:+86-20-89023638, Email:[email protected]

Detection and reporting of ranavirus in amphibians: Evaluation of the roles of the World Organisation for Animal Health and the published literature

Yvonne Black 1,2,5, Anna Meredith 2 and Stephen J. Price 3,4 1Centre for Systems Studies, Hull University Business School, University of Hull, Hull HU6 7RX, UK; 2Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK; 3Institute of Zoology, ZSL, Regents Park, London NW1 4RY, UK; 4UCL Genetics Institute, Gower Street, London WC1E 6BT, UK

Pathogens of wildlife can have direct impacts on human and livestock health. They also impact on biodiversity, as causative factors in population declines and extinctions. The World Organization for Animal Health (OIE) seeks to facilitate rapid sharing of information about animal diseases to enable up-to-date risk assessments of translocations of animals and animal products. The OIE also produces manuals of recommended methods to standardise diagnostic testing. Ranaviruses are important amphibian pathogens that may have spread through international trade, and infections became notifiable to the World Organization for Animal Health (OIE) in 2009. We carried out surveys and reviewed published literature to gather data on sampling, diagnostic testing and reporting of Ranavirus during the period 2009-2014. We also investigated attitudes and awareness of the OIE and its recommendations for best practice. We found that sampling effort is uneven, and concentrated in the northern hemisphere. We also identified activities carried out by citizen science projects which have the potential to improve the quantity and quality of data available concerning the incidence of Ranavirus infection and the circumstances surrounding disease outbreaks. We found reporting of infection to be inconsistent: reporting was split between the published literature (where it was subject to a two-year lag) and the OIE with little overlap, results of negative diagnostic tests were under-reported in the literature, and scientific researchers lacked awareness of the role of the OIE. Approaches to diagnostic screening were poorly harmonized, and often heavily reliant on molecular methods. These flaws in the mechanisms of Ranavirus detection and reporting hamper the construction of a comprehensive disease information database.

Contact: Yvonne Black, Centre for Systems Studies, Hull University Business School, University of Hull, Hull HU6 7RX, UK, Phone: 01482 347500, Email: [email protected]

Detecting the emerging infectious disease ranavirus in amphibian communities of Vermont, USA

Lauren V. Ash *, C. Brandon Ogbunugafor , James Andrews, Aswini Cherukuri, Nicholas J. Gotelli Department of Biology, University of Vermont, Burlington, Vermont, USA

Ranaviruses are a group of emerging pathogens negatively impacting amphibian communities around the globe. This disease results in the hemorrhaging, ulceration, and organ necrosis of susceptible individuals and has the capability of causing sudden and mass amphibian mortality events; yet, its distribution and natural variation are not entirely understood. Virtually no Ranavirus studies have been conducted in the natural amphibian communities of Vermont, USA, and so we do not know whether it is present in the state. The goal of this study was to estimate ranavirus prevalence and host abundance in northwestern Vermont and determine which viral species are present. We collected tissue from a total of 1,822 amphibians across 18 sites every other week from May to August 2016. Toe tissue was collected from adult frogs and tail tissue was collected from tadpoles and salamanders. Samples were obtained from a total of 10 amphibian species, with Green Frogs (Lithobates clamitans) and Eastern Newts (Notophthalmus viridescens) being the most abundant. A subset of samples (n=400) was tested for virus using quantitative PCR to amplify a conserved region in the major capsid protein of the virus. We estimated species richness and abundance at each site and time point to better understand the effects of the pathogen on host community structure. In addition, we used the quantitative PCR results to obtain prevalence estimates in the state. No mass mortality events were witnessed throughout the summer, however our results indicate ranavirus was present in 4 of the 18 sites, with higher prevalence in July and August. Ranavirus was found in four species, including Green Frogs, Eastern Newts, Wood Frogs, and Northern Leopard Frogs, and in both larval and adult life stages. We hope our findings inform amphibian conservation efforts by identifying disease hotspots in the state and potentially reducing the amount of human-driven ranavirus transmission. To better understand the complexities of this disease, it is imperative for its distribution and prevalence to be identified in locations it has not been previously found and for its impacts on host populations to be revealed.

Contact: Lauren V. Ash, Department of Biology, University of Vermont, 109 Carrigan Drive, Burlington, VT 05405, USA, Phone +1 813 310 0130, Email: [email protected]

Long-term monitoring of a recurrent Ranavirus outbreak in an isolated Pelobates fuscus population

Maarten Gilbert 1,2*, Annemarieke Spitzen-van der Sluijs 1, Frank Pasmans 3, Richard Struijk 1, Marc Schils 4, Pieter Doornbos 5, Fleur van der Sterren 6, Jolianne Rijks 7, Marja Kik 7, Bernardo Saucedo 7, Wilbert Bosman 1, An Martel 3 1Reptile, Amphibian & Fish Conservation Netherlands (RAVON), Nijmegen, The Netherlands; 2Department of Infectious Diseases & Immunology, Utrecht University, Utrecht, The Netherlands; 3Department of Pathology, Bacteriology and Avian Diseases, Ghent University, Merelbeke, Belgium; 4Gentiaan 50, Nieuwleusen, The Netherlands; 5Waterschap Groot Salland, Zwolle, The Netherlands; 6Dokkumertrekweg 40B, Leeuwarden, The Netherlands; 7Dutch Wildlife Health Centre (DWHC), Utrecht, The Netherlands

The development and progression of a ranavirus-induced outbreak, varies greatly depending on the host species and geographic location. Not much is known about the effects of prolonged ranavirus-induced mortality on the long-term survival of an affected population. From 2012 to 2015, the recurrent course of an outbreak of common midwife toad virus (CMTV) was monitored in an isolated population of the threatened common spadefoot toad (Pelobates fuscus) in the Netherlands. Dead animals were examined for ranavirus infection by necropsy, histological examination, and a PCR screening targeting the gene encoding the major capsid protein (MCP). After an initial mass mortality of late-stage larvae in 2012, no mass mortalities were recorded in 2013 and 2014. In 2015, however, a recurrent outbreak of the virus caused high mortality rates among late-stage larvae and one adult. No obvious decline in the maximum number of calling adult common spadefoot toads was observed in the period 2009-2015, which could be explained by the mortality rates being high amongst larvae and low among adults and subadults. However, ongoing monitoring is needed to determine the impact of ranavirus-induced mortality on the long-term survival of this population. In the Netherlands, the common spadefoot toad has a highly fragmented distribution which makes the species prone to local extinction. In conclusion, this longitudinal study shows that long-term monitoring is invaluable to determine the effect of ranavirus-induced mortality on amphibian population size and trend.

Contact: Maarten Gilbert, Reptile, Amphibian & Fish Conservation Netherlands (RAVON), Toernooiveld 1, PO Box 1413, 6501 BK Nijmegen, The Netherlands, Phone: +31247410600, Email: [email protected]

Negative qPCR for Ranavirus, Bd, and Bsal in surveillance samples from Castile and León Province, Spain

Eloy Becares 1, Matthew J. Gray 2, E. Davis Carter 2, Irena Fernandez 1, Ana Balseiro 3, Debra L. Miller 2 1Department of Biodiversity and Environmental Management, Universidad de León, León, Spain, 2Center for Wildlife Health, University of Tennessee, Knoxville, Tennessee USA; 3Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Gijon, Asturias Spain

Recent outbreaks of ranavirus in Spain and concern about Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) emergence have sparked concern regarding the distribution of these pathogens in Spain. Surveillance in areas with high international human traffic (e.g., along the Camino de Santiago pilgrimage) is of particular concern. In June of 2016, 140 anurans (tadpoles, metamorphs, and juveniles) were collected from wetlands in the Castile and León Province in northern Spain to test for infection by ranavirus, Bd and Bsal. Wetlands included Laguna de Chozus de Arriba, Small Santiz, Large Santiz, Lago Grande de Babia and Casares. Species collected included Pelobates cultripes, Pelophylax perezi, Hyla arborea and Bufo bufo. Animals were collected by dipnet, identified to species, weighed and measured. Swabs were collected from each animal to test for Bd and Bsal. Tail (tadpoles and metamorphs) or toe (juveniles) clips were collected for ranavirus testing. Genomic DNA was extracted from the samples and all samples were tested for the pathogens using quantitative real-time PCR (qPCR). All animals were qPCR-negative. Geographic areas exposed to high international human traffic are at increased risk for pathogen emergence. Therefore, continued vigilance is warranted to ensure our ability to detect and effectively manage emergence and outbreaks of these pathogens when they occur.

Contact: Debra Lee Miller, Center for Wildlife Health, University of Tennessee, Knoxville, TN 37996 USA, Phone: +1 865 974 7948, Email: [email protected]

AUTHOR INDEX A-Z

A Csaba...... 35 Cundiff ...... 52 Adamek J ...... 32 Cunningham...... 36 Adamek M ...... 32, 33 Akdesir ...... 31 D Alfaia ...... 65, 66 Anderson ...... 25 Dán ...... 35 Andino ...... 48 Doornbos ...... 79 Andrews ...... 78 Doszpoly ...... 35 Ariel ...... 40, 51, 54, 69 Duffus ...... 63 Ash ...... 78 Durrant ...... 68 Ayres ...... 74 E

B Edholm ...... 48 Balloux ...... 68 Essen ...... 74 Balseiro ...... 53, 80 Evensen ...... 71 Banach ...... 48 Bányai ...... 35 F Baumann ...... 33 Becares ...... 80 Farkas ...... 35 Becker ...... 37 Fehér ...... 35 Bergmann ...... 32 Fernandez ...... 80 Beurden...... 38, 41 Ferreira ...... 65, 66 Bienentreu ...... 61 Fischer-Scherl ...... 30 Birol ...... 49 Fitzpatrick ...... 43 Black ...... 77 Floyd ...... 26 Bosch ...... 74 Forró ...... 35 Bosman ...... 79 Forzán ...... 50 Brenes ...... 59 Frasca Jr...... 26, 55 Breyta ...... 25 Fraser ...... 26 Brunetti ...... 61 Freake ...... 43 Brunner ...... 44, 52, 63 Buckling ...... 49 G Bultet-Aguilar ...... 31 Ganter ...... 33 Garner ...... 40, 49, 68, 73, 74 C Garver ...... 30 Campbell ...... 49, 73 Gaschen ...... 31 Carter ...... 42, 57, 58, 80 Gela ...... 32, 33 Casais ...... 53 Gilbert ...... 38, 79 Chengula ...... 71 Goldberg ...... 44, 52 Cherukuri ...... 78 Gotelli ...... 78 Chinchar ...... 56 Grant ...... 61 Christiansen ...... 39 Gray ...... 42, 43, 53, 56, 57, 58, 59, 63, 80 Claytor ...... 56 Grayfer ...... 47 Clouthier ...... 25 Griffiths ...... 49, 73 Coelho ...... 66 Groff ...... 26, 34 Colclough ...... 43 Gröne ...... 38, 41 Colee ...... 55 Grosset ...... 28 Crespi ...... 44 Guðmundsdóttir ...... 39 81

H M

Haenen ...... 29, 38, 41 Maclaine ...... 51 Hall ...... 44 Macrae ...... 30 Hammond ...... 49 Marschang ...... 27 Hardman ...... 43 Martel ...... 79 Harrison ...... 73 Marton ...... 35 Hawley ...... 30 Mashkour ...... 51, 69 Helbing ...... 49 Matějíčková ...... 67 Hick ...... 37 Mavian ...... 56 Hill ...... 58 McGinnity ...... 43 Hoffmann ...... 30 Mdegela ...... 71 Hopper ...... 55 Meredith ...... 77 Horváth ...... 35 Miller ...... 42, 43, 53, 57, 58, 59, 80 Hoverman ...... 59 Mohan ...... 26 Huang X ...... 46, 60, 70, 72, 75, 76 Moro ...... 65 Huang Y ...... 46, 60, 70, 72, 75, 76 Munro ...... 39 Hughes ...... 38, 41 Mutoloki ...... 71

I N

Ikari ...... 65, 66 Navarro ...... 39 Imnoi ...... 55 Nordhausen ...... 34 Irwin ...... 43 Ito 29 O J Ogbunugafor ...... 78 Olesen ...... 39 Josefsson ...... 30 Oliveira ...... 65, 66 Juhász ...... 35 Olson ...... 52, 63 Jung-Schroers ...... 32, 33 O'Regan ...... 58 Origgi ...... 31 K Oschilewski ...... 32 Otten ...... 31 Kanchanakhan ...... 62 Kasanga ...... 71 P Kik ...... 41, 79 Kocour ...... 32, 33 Papp ...... 27 Koda ...... 26 Pasmans ...... 79 Kruithof ...... 38 Peace...... 58 Kulich ...... 67 Pessier ...... 52 Kurath ...... 25 Petersen ...... 39 Kurita...... 29 Piackova ...... 32, 33 Kwan ...... 48 Polchana ...... 62 Kyle ...... 30, 61 Pooley ...... 74 Powell ...... 55 L Price ...... 36, 40, 49, 68, 74, 77 Lawson ...... 36 Q Le Sage ...... 52 Lesbarrères ...... 61 Qin ...... 46, 60, 70, 72, 75, 76 Leskisenoja ...... 30 Leung ...... 36, 68, 74 R

Reilly ...... 58 Reinsch ...... 43

Reschová ...... 67 Thomas-Walters ...... 68 Rice ...... 52 Thompson ...... 37 Richard ...... 30 Rijks ...... 38, 79 V Robert ...... 45, 48 Ruane ...... 39 Valtonen ...... 30 Vendramin ...... 39 S Veselá ...... 67 Veselý ...... 67 Salemi...... 56 Voorbergen-Laarman ...... 38, 41 Sales ...... 65 Sattler ...... 31 W Saucedo ...... 38, 41, 79 Scheele ...... 73 Wadia ...... 36 Schils ...... 38, 79 Wahli ...... 31 Schmidt ...... 31 Waltzek ...... 24, 26, 28, 30, 34, 37, 55, 56, 62 Schock ...... 61 Wang S ...... 72, 76 Scott ...... 51, 54 Wang Z ...... 72 Serrano ...... 74 Wei J ...... 70, 75 Sharma ...... 49 Wei S ...... 60, 75 Sigurðardóttir ...... 39 Wellehan Jr...... 28 Sloma ...... 50 Whittington ...... 37 Sousa ...... 66 Wilfert ...... 49, 73 Souza ...... 43 Wilkes ...... 58 Spatz ...... 42, 57, 58 Wirth ...... 51, 54 Spitzen ...... 38, 79 Wisely ...... 56 Sriwanayos ...... 62 Wohlsein ...... 32 Stacy ...... 28 Woynárovichné Láng ...... 35 Stagg...... 39 Wright ...... 36 Steckler ...... 55, 62 Steinhagen ...... 32, 33 Y Sterren ...... 79 Stoffel ...... 31 Yan ...... 26 Struijk ...... 79 Yang ...... 70 Suarez ...... 38 Yanong ...... 26 Suárez ...... 41 Yaparla ...... 47 Subramaniam ...... 26, 28, 30, 34, 37, 55, 56, 62 Yi 48 Sutton...... 43 Yu 46, 60, 76

T Z

Tavares ...... 66 Zhou ...... 72 Teitge ...... 32, 33 Tessa ...... 73

LIST OF PARTICIPANTS

# LAST NAME FIRST NAME E-MAIL COUNTRY

1 Ariel Ellen [email protected] Australia

2 Ash Lauren V [email protected] USA

3 Benkő Mária [email protected] Hungary

4 Böszörményi Kinga [email protected] Hungary

5 Brenes Roberto [email protected] USA 6 Brunner Jesse [email protected] USA

7 Campbell Lewis J [email protected] UK 8 Carter E. Davis [email protected] USA

9 Dale Ole B [email protected] Norway 10 Forzan Maria J [email protected] USA

11 Garner Trenton WJ [email protected] UK

12 Gilbert Maarten [email protected] Netherlands

13 Gilson Bryce F [email protected] USA 14 Gray Matthew [email protected] USA 15 Grayfer Leon [email protected] USA 16 Hall Emily M [email protected] USA

17 Hardman Rebecca [email protected] USA

18 Harrach Balázs [email protected] Hungary

19 Herczeg Dávid [email protected] Hungary

20 Hick Paul M [email protected] Australia

21 Huang Xiaohong [email protected] China

22 Huang Youhua [email protected] China

23 Irizzary Ivette Nikol [email protected] USA

24 Juhász Thomas [email protected] Hungary

25 Kaján Győző [email protected] Hungary

26 Koda Samantha [email protected] USA

27 Kolesnik Ekaterina [email protected] Germany

28 Lesbarrères David [email protected] Canada

29 Leung William TM [email protected] UK

30 Maclaine Alicia L [email protected] Australia

31 Marschang Rachel E [email protected] Germany

32 Mashkour Narges (Mrs) [email protected] Australia

33 Matějíčková Kateřina [email protected] Czech Rep. 34 Miller Debra Lee [email protected] USA

35 Mosterio Claudia MF [email protected] Brazil

36 Mugimba Kizito K [email protected] Norway

37 Origgi Francesco [email protected] Switzerland

38 Papp Tibor [email protected] Hungary

39 Pénzes Zoltán [email protected] Hungary

40 Price Stephen J [email protected] UK

41 Qin Qiwei [email protected] China

42 Rijks Jolianne J [email protected] Netherlands

43 Robert Jacques [email protected] USA

44 Saucedo Bernardo [email protected] Netherlands

45 Scherbatskoy Elizabeth [email protected] USA 46 Stagg Hannah [email protected] UK

47 Subramaniam Kuttichantran [email protected] USA

48 Tarján Zoltán L [email protected] Hungary

49 Vesely Tomas [email protected] Czech Rep.

50 Vidovszky Márton [email protected] Hungary

51 Vörös Judit [email protected] Hungary

52 Waltzek Thomas [email protected] USA

53 Wei Jingguang [email protected] China

54 Wei Shina [email protected] China

55 Wirth Wytamma T [email protected] Australia

56 Zhou Sheng [email protected] China