Diversity of Ostreid Herpesvirus and its Impact on Oyster Farming. Kimberly S. Reece

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1 Diversity of Ostreid Herpesvirus and its Impact on Oyster Farming Kimberly S. Reece

3 Molluscan Herpes-like Virus in USA The first herpes-like virus detected in bivalves was described in the 190 s by Farley et al. 192 in Maine (USA East Coast, Crassostrea virginica) Ostreid herpesvirus (OsHV-1) found associated with mortality in C. gigas hatchery larvae in France (1992)-typically see mortality in larvae and juveniles. Same as the virus described by Farley? No other molluscan herpes-like viruses were identified in North America until the early 2000 s (OsHV), although C. gigas mortality events (larvae and juveniles) occurring in California (west coast) since The virus is found frequently in Tomales and Drakes Bays in California

4 Ostreid Herpesvirus in Tomales Bay, California Initially for these mortality events in juveniles no disease agent was identified-it was called summer mortality and attributed to stress. Summer mortality has often been reported in adults many locations associated with various stressors including reproduction. A virus (OsHV) was identified in 2002 and has since been associated with mortalities on several occasions Analysis was done on archived samples and the virus was present as early as 1995 (Friedman laboratory)

OsHV USA Survey (2002 2006) PCR survey conducted by Friedman and Reece

5 OsHV USA Survey ( ) PCR survey conducted by Friedman and Reece laboratories (Friedman et al. 2005) *Species tested: C. gigas, C. sikamea and C. virginica

0 1999 Dafen River, Beihai, China 50 1 2.0 2002 Dafen River, Beihai, China 1 2 11.8 2002 Chengcun, China 24 3 12.

6 OsHV in Asia (Moss et al. 200) In some area it was found in more than 20% of the oysters # individuals # OsHV Prevalence Year Location Screened pos. % 1999 Itoki River, Ariake Bay, Kyushu, Japan Yellow River, China Yamen River, Zhuhai, China Souchang River, Yangjiang, China Dafen River, Beihai, China Dafen River, Beihai, China Chengcun, China Guandu, Zhanjiang, China Guandu, Zhanjiang, China Shen Zhen, Bao An, China Shen Zhen, Bao An, China Podi, Beihai, China Dajiawa, Shandong/Tong'an Fujian, China Dajiawa, Shandong/Tong'an Fujian, China Zhanjiang, China Beihai, China Kahwa River, South Korea Sumjin River, South Korea Inchon, South Korea

7 Evidence for Latent Infections in Adults from Asian Oysters (indicates that quarantine is very important) Live C. hongkongensis* brought to the US from Beihai (southern China) in Fall 2006: Initial screening of a sub-sample = 0 % May 200 = 0 % June 200 = 3.6% August of 200 = 9.0% October 200 = 6.5% Sequence analysis indicated that all PCR fragments matched sequences that we found in other oysters directly from Asia: suggests not acquired after arrival at VIMS. *NOTE: Often confused with C. ariakensis and was imported to US east coast on several occasions as C. ariakensis

8 Horizontal Transmission to Larvae Uninfected C. ariakensis and C. hongkongensis larvae from the VIMS hatchery (Reece laboratory) and Uninfected C. gigas (Friedman laboratory) from Washington state All were exposed to homogenate from infected C. gigas from Tomales Bay, CA.

9 Larval survival curves (Similar for C. gigas, C. hongkongensis and C. ariakensis larvae exposed to infected C. gigas tissues) Percent Survival EXP EXP 2 Experiment Day Seawater Control Homogenate Control Homogenate Treatment Homogenate Control Homogenate Treatment DNA sequencing indicated that the California strain of the virus was transmitted to three species of Asian oyster larvae by exposure to infected tissue.

10 TEM analysis of larvae Image by P. Mason, VIMS 200 nm

11 DNA Sequencing (Reece Lab-Moss et al. 200 and Reece et al. unpublished) Many Different Genetic Strains of the Virus from Different Locations (Suggested potential differences in virulence and impacts to various hosts)

12 Summary Ostreid herpesvirus in Asian samples OsHV was detected in Asian oysters at 9 of 18 sites in China, Japan and Korea. There are many different genetic strains of virus that may demonstrate differences in virulence in different hosts. Should we be concerned about transport of infected hosts to different regions? Latent infections of virus can go undetected in adult broodstock for at least 6 months, indicating the importance of strict quarantine procedures for adults. Larvae of 3 Asian oyster species were infected by exposure to infected oyster tissue

2008:Emergence of another (more virulent) strain: OsHV-1 µvar First identified in association with a C. gigas mortality event in France in 2008 (Segarra et al.

13 2008:Emergence of another (more virulent) strain: OsHV-1 µvar First identified in association with a C. gigas mortality event in France in 2008 (Segarra et al. 2010), but has been found in archived samples from 2004/2005 (Martenot et al. 2012). Associated with numerous high mortality events around the world: France, New Zealand, Ireland, Netherlands, Spain, Italy.

14 Variant Sequencing (Renault et al. 2012) OsHV Japan, China, New Zealand OsHV µvar 3 different genome regions sequenced. Phylogenetic analysis based on composite sequences.

15 C fragment (ORF4) OsHV µvar Gulf of California (healthy oysters)- Grijalva-Chon et al. unpublished OsHV-1 China-acute viral necrosis virus (scallops) Histopathology and ISH indicate OsHV-like virus in Mexico (Vásquez-Yeomans et al. 2010, Cáceres- Martínez and Vásquez-Yeomans 2013)

Reece Lab OsHV Sequencing I34 8 1 2 1 1 NT 623 6 161 161 1 1 5 63 5 161 1613 4 I49 161 161 6 1 2 I49 4 I34 I34 I46 4 6 1 I33 I33 I34 2 4 1 I24 I24 5 6 I24 2 I24 1 I3 2 I3 I3 3 4 I2 6 CHCA06 CHCA06

C 2 305 4 CHCA06 6 305 4 QD24 1 QD18 QD18 QD18 3 4 5 QD18 QD18 1 2 QD18 6 1 1 215 215 215 215 215 8 12 34 5 215 6 100 92 56 100 84 100 88 92 8 100 92 A9 13 5 1 C24 C50 24 6 1 34 D13 56 13 D15 49 D25

8 2493 12 249 4 28 2566 34 CHCA06 256 81 305 2 CHCA06 305 5 3 D43 6 22 22 245 221 242 34 22 81 22 16 22 145 2213 12 3 22 81 136 22 1314 5 34 1 13 4 13 14 14 13 8 6 13 1311 145 41 2 13 119 13 8

16 Reece Lab OsHV Sequencing I NT I I49 4 I34 I34 I I33 I33 I I24 I I24 2 I24 1 I3 2 I3 I3 3 4 I2 6 CHCA06 CHCA CHCA CHCA CHCA CHCA CHCA CHCA USA and China CHCA06 CgCal1 CgCal3 F C24 C24 C 2 C 2 C C D25 1A9 4 CHCA06 C CHCA QD24 1 QD18 QD18 QD QD18 QD QD A C24 C D D15 49 D D2 4 5 D43 61 E E50 F QD QD QD QD C CHCA CHCA CHCA CHCA D CgCal CgCal CgCal9 66 CgCal CgCal CgCal8 59 CgCal CgCal CgCal 51 CgCal 49 4 CgCal CgCal CgCal CgCal6 38 CgCal CgCal5 F1 F CgCal3 30 CgCal CgCal c2 c CgCal2 CgCal fasta 11.fasta F1 F c 11.fasta c1 11.fasta F1 6 F c2 c5 11.fasta 11.fasta F c2 11.fasta F c 11.fasta F c1 11.fasta F2 4 F c6 c2 11.fasta 11.fasta F c3 11.fasta F2 F2 396 c4 11.fasta c3 11.fasta F c1 11.fasta F c1 11.fasta F c1 11.fasta F c2 11.fasta F3 5 T F3 F3 6 6 T 5 F3 8 F4 F4 2 1 T F4 3 11AMP F4 4 F14 F4 F4 6 5 F14 6 F14 F14 54 F4 F4 8 F14 F F 2 1 F F14 F13 18 F F 43 F13 F F 65 F13 F13 5 F F13 4 F13 3 F8 F 18 F13 2 F12 1 F8 F8 32 F12 8 F12 F8 F8 54 F12 6 F12 5 F8 F8 6 F12 F12 4 F12 32 F9 F9 F9 3 F F11 F11 1 F11 8 F11 6F11 5 F10 F9 F9 F9 28 F F F10 1 F10 86F10 3 µvar France and The Netherlands China F13 6 F10 1 Samples from USA France-OsHV and OsHV µvar China Japan The Netherlands-OsHV µvar (material from M. Engelsma) 3 regions have been sequenced A Fragment-unknown protein B Fragment-unknown protein ORF11-Ring finger protein Parsimony tree of ORF11 sequences

17 Hosts and Locations (OsHV-1, OsHV µvar and variants) Hosts C. gigas C. sikamea C. virginica C. ariakensis C. hongkongensis Ostrea edulis O. lurida O. angasi My8lus galloprovincialis Venerupis phillipinarum V. decussatus Chlamys farreri Pecten maximus Halio8s diversicolor supertexta Loca)ons USA Mexico France Ireland The Netherlands Spain England Italy Morroco New Zealand Australia China Japan Korea

18 Selection for Resistance to Juvenile Summer Mortality (USA West coast-friedman Lab) Can survival be improved with selected stocks? Oysters that survived 2000, 2001 and/or 2003 mortality events were collected from Tomales Bay, CA and spawned at the Bodega Marine Lab. (Origin: California and Washington stocks) Joe Newman

19 Percent Mortality Mortality & seed source: WA-1 WA-2 WA-3 OR-1 OR-2 Oyster stock Suggests that improved seed survival is possible. Survivors were used to spawn for the next year (Selected breeding). Similar observations in a recent selective breeding experiment ( ).

20 French C. gigas Selected for Resistance to Summer Mortality (Dégremont et al before µvar; Dégremont 2011-after µvar) Tested 3 C. gigas lines -Resistant (R), Susceptible (S), Control (C) (Note-Control oysters derived from broodstock at field deployment site) Selection for survival occurred , presumably before µvarassociated mortality events Field deployments of juveniles (~6 months old) in late summer 2009 Mortality and levels of 3 potential pathogens measured: OsHV-1/OsHV µvar Vibrio aestuarianus Vibrio splendidus

21 Results Dégremont 2011 Rapid (11-20 days) mortality in S and C batches (94% and 53%, respectively) Low mortality in R batch (5%) OsHV detected after only 3 days Rapid increase in all batches Levels dropped in R batch OsHV, but not Vibrio spp. levels correlated with mortality

22 Summary Many different genetic strains of Ostreid herpesvirus have been identified. Relative infection risks, as well geographic and host distributions need to be better characterized. Different strains may differ in virulence to different hosts. Surveys of hosts and locations would allow more accurate assessment of risks of transfer and inform management decisions. Host transfers/introductions should follow ICES protocols with disease screenings and certifications to help prevent transfer of pathogens such as the OsHV-1, OsHV µvar and variants to new locations: Even in endemic areas strain differences could be important For example, introduction of OsHV µvar could lead to massive mortality

23 Summary OsHV-1 or herpes-like virus has been found in Mexican C. gigas (Vásquez-Yeomans et al. 2010, Cáceres-Martínez and Vásquez-Yeomans 2013, Grijalva-Chon et al. unpublished, GenBank deposit-) Sequence results indicate that the US and Mexican viruses are more similar to OsHV-1 than to OsHV µvar. Selective breeding strategies for resistance to OsHV show promise. Mortality episodes associated with Ostreid herpesviruses are often impacted by other stressors such as temperature, phytoplankton blooms and infections by other pathogens (e.g. Vibrio spp.).

OsHV-1 μvar. Part II. Annual Meeting NRLs for mollusc diseases La Rochelle, March 2011 Sigrid Cabot, DG SANCO

OsHV-1 μvar. Part II. Annual Meeting NRLs for mollusc diseases La Rochelle, March 2011 Sigrid Cabot, DG SANCO OsHV-1 μvar Part II Annual Meeting NRLs for mollusc diseases La Rochelle, March 2011 Sigrid Cabot, DG SANCO sigrid.cabot@ec.europa.eu Background Overview of the presentation Measures adopted 2010 EFSA