Reduction in Virulence over Time in Ostreid herpesvirus 1 (OsHV-1) Microvariants between 2011 and 2015 in Australia

Microvariant genotypes of Ostreid herpesvirus 1 (OsHV-1) are associated with mass mortality events of Pacific oysters in many countries. The OsHV-1 microvariant (µVar) emerged in France 2008 and caused significant economic losses as it became endemic and displaced the previously dominant OsHV-1 reference genotype. Recently, considerable genotypic variation has been described for OsHV-1 microvariants, however, less is known about variation in viral phenotype. This study used an in vivo laboratory infection model to assess differences in total cumulative mortality, peak viral load, transmissibility, and dose-response for three OsHV-1 isolates obtained between 2011 and 2015 from endemic waterways in Australia. This followed field observations of apparent reductions in the severity of mass mortalities over this time. Significantly higher hazard of death and cumulative mortality were observed for an isolate obtained in 2011 compared to isolates from 2014–2015. In keeping with other studies, the hazard of death was higher in oysters challenged by injection compared to challenge by cohabitation and the mortality was higher when the initial dose was 1 × 104 OsHV-1 DNA copies per oyster injection compared to 1 × 102 DNA copies. There was no difference in the quantity of OsHV-1 DNA at time of death that could be related to isolate or dose, suggesting similar pathogenetic processes in the individual oysters that succumbed to end-stage disease. While the isolates examined in this study were biased towards pathogenic types of OsHV-1, as they were collected during disease outbreaks, the variation in virulence that was observed, when combined with prior data on subclinical infections, suggests that surveillance for low virulence genotypes of OsHV-1 would be rewarding. This may lead to new approaches to disease management which utilize controlled exposure to attenuated strains of OsHV-1.

Phylogeography of infectious disease: genetic diversity and connectivity of the Ostreid herpesvirus 1 population in France

The genetic diversity of viral populations is a key to understanding ther phylogeographic and dissemination history of viruses, but studying the diversity of whole genomes from natural populations remains a challenge. Molecular ecology approaches are commonly used for RNA viruses harboring small genomes, but have only rarely been applied to DNA viruses with large genomes. Here, we used the Pacific oyster mortality syndrome (POMS, a disease that affects oyster farms around the world) as a model to study the genetic diversity of its causative agent, the Ostreid herpesvirus 1 (OsHV-1) in the three main French oyster-farming areas. Using ultra-deep sequencing on individual moribund oysters and new bioinformatics methodology, we de novo assembled 21 OsHV-1 genomes. Combining whole-genome comparisons with phylogenetic analysis and quantification of major and minor variants, we assessed the connectivity of OsHV-1 viral populations between the three oyster-farming areas. Our results suggest that the Marennes-Oleron Bay represents the main source of OsHV-1 diversity, from where the virus has dispersed to other farming areas, a scenario consistent with current practices of oyster transfers in France. Here, we demonstrate that molecular ecology approaches can be applied to large-genome viruses to determine the extent of their genetic diversity and better understand the spread of viral populations in natural environments.

Immunomodulatory and Antiviral Effects of Macroalgae Sulphated Polysaccharides: Case Studies Extend Knowledge on Their Importance in Enhancing Shellfish Health, and the Control of a Global Viral Pathogen Ostreid Herpesvirus-1 microVar

Macroalgae are the primary source of non-animal sulphated polysaccharides (SPs) in the marine environment with fucoidans derived from brown algae (Phaeophyta) and carrageenans from red algae (Rhodophyta). Much research has been carried out on SP effects on Asian shrimp species (genera Penaeus and Metapenaeus) but their effect on commercially important bivalve mollusc species is limited and in Pacific oyster Crassostrea gigas is unknown. Knowledge of their impact on bivalve pathogens and Palaemon shrimp is unknown. The objectives of this study were to assess the effects of Fucus vesiculosus (Phaeophyta), Mastocarpus stellatus (Rhodophyta) and algal derivatives (fucoidan and κ-carrageenan) on C. gigas performance, and on ostreid herpesvirus-1 microvar (OsHV-1 μVar) and bacteria Vibrio spp. development. Both pathogens have been associated with significant oyster mortalities and economic losses globally. The effects of sulphated galactan from Gracilaria fisheri (Rhodophyta) on European common prawn Palaemon serratus, an important fishery species, was also assessed. Findings indicate a rapid and prolonged increase in total blood cell count, lysozyme (enzyme that destroys pathogens), and a difference in the ratio of blood cell types in treated individuals compared to their control counterparts. A significantly lower OsHV-1 μVar prevalence was observed in treated oysters and κ-carrageenan was found to suppress viral replication (loads), while OsHV-1 μVar was not detected in the fucoidan treated oysters from Day 8 of the 26-day trial. No antibacterial effect was observed however, the oysters did not succumb to vibriosis. These findings contribute further knowledge to macroalgae sulphated polysaccharide biotherapeutic properties, their twofold effect on animal health and viral suppression.

Differential Mortality and High Viral Load in Naive Pacific Oyster Families Exposed to OsHV-1 Suggests Tolerance Rather than Resistance to Infection

Pacific oysters, Crassostrea gigas, are one of the most productive aquaculture species in the world. However, they are threatened by the spread of Ostreid herpesvirus-1 (OsHV-1) and its microvariants (collectively “µvars”), which cause mass mortalities in all life stages of Pacific oysters globally. Breeding programs have been successful in reducing mortality due to OsHV-1 variants following viral outbreaks; however, an OsHV-1-resistant oyster line does not yet exist in the United States (US), and it is unknown how OsHV-1 µvars will affect US oyster populations compared to the current variant, which is similar to the OsHV-1 reference, found in Tomales Bay, CA. The goals of this study were to investigate the resistance of C. gigas juveniles produced by the Molluscan Broodstock Program (MBP) to three variants of OsHV-1: a California reference OsHV-1, an Australian µvar, and a French µvar. This is the first study to directly compare OsHV-1 µvars to a non-µvar. The survival probability of oysters exposed to the French (FRA) or Australian (AUS) µvar was significantly lower (43% and 71%, respectively) than to the reference variant and controls (96%). No oyster family demonstrated resistance to all three OsHV-1 variants, and many surviving oysters contained high copy numbers of viral DNA (mean ~3.53 × 108). These results indicate that the introduction of OsHV-1 µvars could have substantial effects on US Pacific oyster aquaculture if truly resistant lines are not achieved, and highlight the need to consider resistance to infection in addition to survival as traits in breeding programs to reduce the risk of the spread of OsHV-1 variants.

Host plasticity supports spread of an aquaculture introduced virus to an ecosystem engineer

Background The common cockle Cerastoderma edule plays an important ecological role in the marine ecosystem both as an infaunal engineer (reef forming and bioturbation) and a food source for protected bird species in its European range. Cockle beds are found in close proximity to aquaculture and fisheries operations, which can be “hot spots” for infectious agents including viruses and bacteria. Ostreid herpesvirus-1 microVar (OsHV-1 μVar) has spread to many Pacific oyster Crassostrea gigas culture sites globally, where it has been associated with significant mortalities in this cultured bivalve. Knowledge on the impact of the virus on the wider ecosystem, is limited. As the likelihood of released virus dispersing into the wider aquatic ecosystem is high, the plasticity of the virus and the susceptibility of C. edule to act as hosts or carriers is unknown. Methods In this study, wild C. edule were sampled biweekly at two C. gigas culture sites over a four-month period during the summer when OsHV-1 μVar prevalence is at its highest in oysters. C. edule were screened for the virus molecularly (PCR, qPCR and Sanger sequencing) and visually (in situ hybridisation (ISH)). The cockle’s ability to act as a carrier and transmit OsHV-1 μVar to the oyster host at a temperature of 14 ℃, when the virus is considered to be dormant until water temperatures exceed 16 ℃, was also assessed in laboratory transmission trials. Results The results demonstrated that OsHV-1 μVar was detected in all C. edule size/age cohorts, at both culture sites. In the laboratory, viral transmission was effected from cockles to naïve oysters for the first time, five days post-exposure. The laboratory study also demonstrated that OsHV-1 μVar was active and was successfully transmitted from the C. edule at lower temperatures. Conclusions This study demonstrates that OsHV-1 μVar has the plasticity to infect the keystone species C. edule and highlights the possible trophic transmission of the virus from cockles to their mobile top predators. This scenario would have important implications, as a greater geographical range expansion of this significant pathogen via migratory bird species may have an impact on other species that reside in bird habitats most of which are special areas of conservation.