Final Report Summary - HERPISH (Herpes virus in Irish oysters and identification of resistant stocks)
The Pacific oyster, the most commercially important oyster in Europe, has suffered large mortalities that have had a significant economic impact in a number of European countries. Though due to a range of complex aetiologies one of the main factors associated with these mortalities has been the presence of a herpes virus. This virus has been particularly associated with larval and juvenile mortalities which were also demonstrated experimentally [1, 2, 3-9]. Ostreid herpesvirus-1 (OsHV-1) belongs to the genus Ostreavirus, family Malacoherpesviridae which is included in the order Herpesvirales [10]. OsHV-1μvar, a variant of OsHV-1 considered more virulent [11], was originally associated with C. gigas mortalities in France in 2008 and 2009 [12-13] and has subsequently been observed in Ireland, Italy, the Netherlands, the UK, New Zealand and South-East Australia [14].
Shellfish are hard to treat when exposed to disease due to the high population densities involved, the environment in which they are found and that immunisation is not a possibility due to their lack of an adaptive immune response. Treatment and control mechanisms tend to focus on long term solutions such as identification of resistant traits and breeding for the inclusion of these traits. In this study, a field and laboratory based approach were employed to assess the current status of Irish Pacific oyster populations to herpes virus infections and determine what resistance is developing.
Objectives of the Study
1. Investigate the relative resistance in populations of the Pacific oyster in Ireland to OsHV-1µvar.
2. Identification of genetic traits related to resistance.
3. Oysters with resistant traits will be used for small scale trials to test this resistance.
4. Recommendations will be made regarding traits that should be investigated in European populations of Pacific oyster for development of resistance.
Description of the work performed
The work performed for the last 2 years of duration of the project has consisted of:
1. A field survey to investigate the current status of a number of cultured oyster populations that have previously been a) infected with OsHV-1µvar and b) never been infected with OsHV-1µvar. The health status of these oysters was assessed using a range of methodologies to determine other factors that might be contributing to mortalities.
2. In a follow-up field trial several populations of oysters obtained from different sources were placed at several sites where a) OsHV-1µvar has been detected and b) where it has not been detected previously.
3. Survivors from field trial were returned to the lab and a laboratory transmission trial was carried out challenging them with OsHV-1µvar.
4. Conditioned oysters were used as broodstock. A range of crosses and families were produced and larvae were challenged with OsHV-1µvar in a laboratory transmission trial.
5. Genes that might be contributing to resistance were assessed using RNA-sequencing and quantification of expressed genes.
Description of main results and potential impact
The field survey of 1-year old oysters in different sites of the Irish coast revealed a very different incidence of the virus. The naïve site, never infected with OsHV-1µvar, remained uninfected during the survey even though the sea water temperature, critical factor for the development of the disease, reached very high values during the survey period. Oysters from one of the infected sites experienced significant mortalities and in the other infected site no abnormal mortalities were observed although the virus was detected by PCR in both locations. This might be related to intrinsic resistance due to the different genetic origins of the oysters. A significant relationship was found between the different parameters studied. Prevalence of infection and temperatures over 16ºC for at least 2 weeks were critical to trigger an event of mortality in 1-year old oysters. These conclusions might be of high interest in future recommendations for Pacific oyster producers.
In parallel, a field trial consisting of the transfer of one stock of spat to different infected sites was carried out. Spat from the naïve site remained uninfected. However, spat transferred to infected sites experienced higher mortalities than 1-year old oysters. The prevalence of infection was associated to mortality. Positive samples were sequenced to verify the variant affecting these oysters, all were infected with the µVar variant but a new genotype, differing from the previously described variant affecting Irish oysters, was found in 1-year old oysters in one of the infected sites.
Laboratory transmission trials succeeded in developing the disease in both spat and larvae. Several larvae families and spat with proven resistance during the field trial were experimentally infected and thereafter subjected to RNA-sequencing to identify the differently expressed genes after infection. Our results revealed the involvement of defence genes in oysters with less susceptibility to the virus. These results might contribute to the application of knowledge required for successful breeding programs. Indeed, the researcher has recently been awarded with another project to continue this investigation in collaboration with private companies to corroborate that these genes and the polymorphisms associated to them can be used as markers for selective breeding in the Pacific oyster.
[1] Renault et al., 1994. Rev Méd Vét. 145, 735–42; [2] Renault et al., 2000. Dis Aquat Org. 42, 173–183; [3] Le Deuff and Renault, 1999 . J Gen Vir, 80, 1317–1322 ; [4] Arzul et al., 2002. Vir Res, 84, 151-60; [5] Vásquez-Yeomans et al., 2004. J Shellfish Res, 23, 417-9; [6] Friedman et al., 2005. Dis. Aquat. Org. 63, 33–41; [7] Burge et al., 2006. Dis. Aquat. Org. 72, 31–43; [8] Burge et al., 2007. J Shellfish Res, 26, 163–172; [9] Schikorski et al., 2011. Vet Res, 42:27; [10] Davison et al., 2009. Arch Vir, 154, 171–7; [11] EFSA, 2010; EFSA Journal, 11, 1894; [12] Repamo, 2009; Bilan 2009 Repamo Report, 45p; [13] Segarra et al., 2010. Virus Res, 153, 92-9; [14] Dundon et al., 2011. Aquaculture, 314, 49-52.
Shellfish are hard to treat when exposed to disease due to the high population densities involved, the environment in which they are found and that immunisation is not a possibility due to their lack of an adaptive immune response. Treatment and control mechanisms tend to focus on long term solutions such as identification of resistant traits and breeding for the inclusion of these traits. In this study, a field and laboratory based approach were employed to assess the current status of Irish Pacific oyster populations to herpes virus infections and determine what resistance is developing.
Objectives of the Study
1. Investigate the relative resistance in populations of the Pacific oyster in Ireland to OsHV-1µvar.
2. Identification of genetic traits related to resistance.
3. Oysters with resistant traits will be used for small scale trials to test this resistance.
4. Recommendations will be made regarding traits that should be investigated in European populations of Pacific oyster for development of resistance.
Description of the work performed
The work performed for the last 2 years of duration of the project has consisted of:
1. A field survey to investigate the current status of a number of cultured oyster populations that have previously been a) infected with OsHV-1µvar and b) never been infected with OsHV-1µvar. The health status of these oysters was assessed using a range of methodologies to determine other factors that might be contributing to mortalities.
2. In a follow-up field trial several populations of oysters obtained from different sources were placed at several sites where a) OsHV-1µvar has been detected and b) where it has not been detected previously.
3. Survivors from field trial were returned to the lab and a laboratory transmission trial was carried out challenging them with OsHV-1µvar.
4. Conditioned oysters were used as broodstock. A range of crosses and families were produced and larvae were challenged with OsHV-1µvar in a laboratory transmission trial.
5. Genes that might be contributing to resistance were assessed using RNA-sequencing and quantification of expressed genes.
Description of main results and potential impact
The field survey of 1-year old oysters in different sites of the Irish coast revealed a very different incidence of the virus. The naïve site, never infected with OsHV-1µvar, remained uninfected during the survey even though the sea water temperature, critical factor for the development of the disease, reached very high values during the survey period. Oysters from one of the infected sites experienced significant mortalities and in the other infected site no abnormal mortalities were observed although the virus was detected by PCR in both locations. This might be related to intrinsic resistance due to the different genetic origins of the oysters. A significant relationship was found between the different parameters studied. Prevalence of infection and temperatures over 16ºC for at least 2 weeks were critical to trigger an event of mortality in 1-year old oysters. These conclusions might be of high interest in future recommendations for Pacific oyster producers.
In parallel, a field trial consisting of the transfer of one stock of spat to different infected sites was carried out. Spat from the naïve site remained uninfected. However, spat transferred to infected sites experienced higher mortalities than 1-year old oysters. The prevalence of infection was associated to mortality. Positive samples were sequenced to verify the variant affecting these oysters, all were infected with the µVar variant but a new genotype, differing from the previously described variant affecting Irish oysters, was found in 1-year old oysters in one of the infected sites.
Laboratory transmission trials succeeded in developing the disease in both spat and larvae. Several larvae families and spat with proven resistance during the field trial were experimentally infected and thereafter subjected to RNA-sequencing to identify the differently expressed genes after infection. Our results revealed the involvement of defence genes in oysters with less susceptibility to the virus. These results might contribute to the application of knowledge required for successful breeding programs. Indeed, the researcher has recently been awarded with another project to continue this investigation in collaboration with private companies to corroborate that these genes and the polymorphisms associated to them can be used as markers for selective breeding in the Pacific oyster.
[1] Renault et al., 1994. Rev Méd Vét. 145, 735–42; [2] Renault et al., 2000. Dis Aquat Org. 42, 173–183; [3] Le Deuff and Renault, 1999 . J Gen Vir, 80, 1317–1322 ; [4] Arzul et al., 2002. Vir Res, 84, 151-60; [5] Vásquez-Yeomans et al., 2004. J Shellfish Res, 23, 417-9; [6] Friedman et al., 2005. Dis. Aquat. Org. 63, 33–41; [7] Burge et al., 2006. Dis. Aquat. Org. 72, 31–43; [8] Burge et al., 2007. J Shellfish Res, 26, 163–172; [9] Schikorski et al., 2011. Vet Res, 42:27; [10] Davison et al., 2009. Arch Vir, 154, 171–7; [11] EFSA, 2010; EFSA Journal, 11, 1894; [12] Repamo, 2009; Bilan 2009 Repamo Report, 45p; [13] Segarra et al., 2010. Virus Res, 153, 92-9; [14] Dundon et al., 2011. Aquaculture, 314, 49-52.