Salinity influences disease-induced mortality of the oyster Crassostrea gigas and infectivity of the ostreid herpesvirus 1 (OsHV-1)

Mortality of young Pacific oysters Crassostrea gigas associated with the ostreid herpesvirus 1 (OsHV-1) is occurring worldwide. Here, we examined for the first time the effect of salinity on OsHV-1 transmission and disease-related mortality of C. gigas, as well as salinity-related effects on the pathogen itself. To obtain donors for OsHV-1 transmission, we transferred laboratory-raised oysters to an estuary during a disease outbreak and then back to the laboratory. Oysters that tested OsHV-1 positive were placed in seawater tanks (35‰, 21°C). Water from these tanks was used to infect naïve oysters in 2 experimental setups: (1) oysters acclimated or non-acclimated to a salinity of 10, 15, 25 and 35‰ and (2) oysters acclimated to a salinity of 25‰; the latter were exposed to OsHV-1 water diluted to a salinity of 10 or 25‰. The survival of oysters exposed to OsHV-1 water and acclimated to a salinity of 10‰ was >95%, compared to only 43 to 73% survival in oysters acclimated to higher salinities (Expt 1), reflecting differences in the levels of OsHV-1 DNA and viral gene expression (Expts 1 and 2). However, the survival of their non-acclimated counterparts was only 23% (Expt 2), and the levels of OsHV-1 DNA and the expression of 4 viral genes were low (Expt 1). Thus, OsHV-1 may not have been the ultimate cause of mortality in non-acclimated oysters weakened by a salinity shock. It appears that reducing disease risk by means of low salinity is unlikely in the field.

OsHV-1 countermeasures to the Pacific oyster’s anti-viral response

The host-pathogen interactions between the Pacific oyster (Crassostrea gigas) and Ostreid herpesvirus type 1 (OsHV-1) are poorly characterised. Herpesviruses are a group of large, DNA viruses that are known to encode gene products that subvert their host’s antiviral response. It is likely that OsHV-1 has also evolved similar strategies as its genome encodes genes with high homology to C. gigas inhibitors of apoptosis (IAPs) and an interferon-stimulated gene (termed CH25H). The first objective of this study was to simultaneously investigate the expression of C. gigas and OsHV-1 genes that share high sequence homology during an acute infection. Comparison of apoptosis-related genes revealed that components of the extrinsic apoptosis pathway (TNF) were induced in response to OsHV-1 infection, but we failed to observe evidence of apoptosis using a combination of biochemical and molecular assays. IAPs encoded by OsHV-1 were highly expressed during the acute stage of infection and may explain why we didn’t observe evidence of apoptosis. However, C. gigas must have an alternative mechanism to apoptosis for clearing OsHV-1 from infected gill cells as we observed a reduction in viral DNA between 27 and 54 h post-infection. The reduction of viral DNA in C. gigas gill cells occurred after the up-regulation of interferon-stimulated genes (viperin, PKR, ADAR). In a second objective, we manipulated the host’s anti-viral response by injecting C. gigas with a small dose of poly I:C at the time of OsHV-1 infection. This small dose of poly I:C was unable to induce transcription of known antiviral effectors (ISGs), but these oysters were still capable of inhibiting OsHV-1 replication. This result suggests dsRNA induces an anti-viral response that is additional to the IFN-like pathway.

Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections

Since 2008, massive mortality events of Pacific oysters (Crassostrea gigas) have been reported worldwide and these disease events are often associated with Ostreid herpesvirus type 1 (OsHV-1). Epidemiological field studies have also reported oyster age and other pathogens of the Vibrio genus are contributing factors to this syndrome. We undertook a controlled laboratory experiment to simultaneously investigate survival and immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Our data corroborates epidemiological studies that juveniles are more susceptible to OsHV-1, whereas adults are more susceptible to Vibrio. We measured the expression of 102 immune-genes by high-throughput RT-qPCR, which revealed oysters have different transcriptional responses to OsHV-1 and Vibrio. The transcriptional response in the early stages of OsHV-1 infection involved genes related to apoptosis and the interferon-pathway. Transcriptional response to Vibrio infection involved antimicrobial peptides, heat shock proteins and galectins. Interestingly, oysters in the later stages of OsHV-1 infection had a transcriptional response that resembled an antibacterial response, which is suggestive of the oyster's microbiome causing secondary infections (dysbiosis-driven pathology). This study provides molecular evidence that oysters can mount distinct immune response to viral and bacterial pathogens and these responses differ depending on the age of the host.