Ostreid Herpesvirus-1 Microvariant Research Articles

Rapid detection of Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus (AngHV-1) in Japanese eel (Anguilla japonica) using a qLAMP assay

Two DNA viruses, Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus 1 (AngHV-1), cause serious diseases in eels that can negatively affect the economy of eel aquaculture industries in South Korea. Efficient virus detection is crucial for disease management; however, the practical application of current methodologies is challenging, in part owing to extensive time and specialized equipment requirements. The aim of this study was to develop a rapid real-time quantitative loop-mediated isothermal amplification (qLAMP) technique for the detection of JEECV and AngHV-1. Six different primers were designed to target eight different regions of the large T-antigen gene for JEECV detection, and the DNA polymerase catalytic subunit gene for AngHV-1 detection. Both sets of primers worked optimally at 65 °C. The qLAMP assay exhibited a minimum detection limit of 2.39 × 104 copies/μL for JEECV and 2.37 × 104 copies/μL for AngHV-1. Moreover, the primers demonstrated excellent specificity, showing no reaction with two major viruses, namely ostreid herpesvirus-1 microvariant (OsHV-1 μvar) and red sea bream iridovirus (RSIV). In addition, JEECV primers did not react with AngHV-1, and vice versa. Based on the standard curve and coefficient of determination values, both viruses could be quantified using this method. The qLAMP assay was validated with 104 clinical samples, including 72 infected and 32 healthy eels, and the assay findings were compared with previously reported duplex qPCR results that reported high sensitivity and specificity. The qLAMP results concorded well with the previous results. Evaluation of the threshold time (Tt) of the qLAMP results revealed that the samples could be analyzed in less than 15 min. The clinical diagnostic sensitivity (dSe) and diagnostic specificity (dSp) were assessed based on the confusion matrix; dSe and dSp for JEECV were 100% and 94.1%, respectively, and dSe and dSp for AngHV-1 were 96.8% and 77.8%, respectively. The Kappa values, which were used to represent the level of agreement between qLAMP and duplex qPCR results, for JEECV and AngHV-1 were 0.956 and 0.755, respectively, indicating that the qLAMP assay is a promising detection method for those viruses. Thus, the qLAMP assay developed herein could be applied as a rapid and sensitive tool to diagnose diseases caused by JEECV and AngHV-1 in the eel aquaculture industry.

Development of a duplex qPCR assay for the detection of coinfection in eels by Japanese eel endothelial cell-infecting virus and Anguillid herpesvirus-1

Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus 1 (AngHV-1) are virulent pathogens in eel aquaculture farms in the Republic of Korea and coexist in the same host. To date, no diagnostic method has been developed that detects them simultaneously. We developed a duplex real-time polymerase chain reaction (duplex qPCR) assay to detect both JEECV and AngHV-1 simultaneously in a single reaction. Two sets of specific primers and TaqMan probes were designed to target the large T-antigen gene of JEECV and the DNA polymerase catalytic subunit gene of AngHV-1. The assay was optimized by changing the primer and probe concentrations and the annealing temperature of the reaction. The duplex qPCR could specifically amplify the specific genes of the two viruses with high efficiency and coefficient of determination. The assay could detect both viruses at a limit of detection of 8 fg/μL. The diagnostic performance of the developed assay was determined by analyzing 82 clinical samples (72 infected and 10 healthy) and comparing the results with those of conventional PCR and SYBR Green-based qPCR; the results correlated well with each other. The duplex qPCR showed 100% and 98.6% diagnostic sensitivity for JEECV and AngHV-1, respectively. The diagnostic specificity of this assay was validated using two potential viral strains, red sea bream iridovirus and Ostreid herpesvirus-1 microvariant, reported in the aquaculture industry. In contrast, the methods already developed including conventional PCR and qPCR cannot detect both JEECV and AngHV-1 at the same time as they should perform in separate reactions. Thus, the duplex qPCR developed herein is a sensitive and specific diagnostic tool for the simultaneous detection of JEECV and AngHV-1 and has subsequent applications in the health management of eels.

The first detection of a novel OsHV-1 microvariant in San Diego, California, USA

The spread, emergence, and adaptation of pathogens causing marine disease has been problematic to fisheries and aquaculture industries for the last several decades creating the need for strategic management and biosecurity practices. The Pacific oyster (Crassostrea gigas), a highly productive species globally, has been a target of disease and mortality caused by a viral pathogen, the Ostreid herpesvirus 1 (OsHV-1) and its microvariants (OsHV-1 µvars). During routine surveillance to establish health history at a shellfish aquaculture nursery system in San Diego, California, the presence of OsHV-1 in Pacific oyster juveniles was detected. Quantification of OsHV-1 in tissues of oysters revealed OsHV-1 viral loads > 106 copies/mg. We characterized and identified the OsHV-1 variant by sequencing of ORFs 4 (C2/C6) and 43 (IA1/IA2), which demonstrated that this variant is a novel OsHV-1 microvariant: OsHV-1 µvar SD. A pilot transmission study indicates that OsHV-1 µvar SD is infectious with high viral loads ~ 7.57 × 106 copies/mg detected in dead individuals. The detection of OsHV-1 µvar SD in a large port mirrors previous studies conducted in Australia where aquaculture farms and feral populations near port locations may be at a higher risk of OsHV-1 emergence. Further research is needed to understand the impacts of OsHV-1 µvar SD, such as transmission studies focusing on potential vectors and characterization of virulence as compared to other OsHV-1 µvars. To increase biosecurity of the global aquaculture industry, active and passive surveillance may be necessary to reduce spread of pathogens and make appropriate management decisions.

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.

CgMyD88s Serves as an Innate Immune System Plug During Ostreid Herpesvirus 1 Infection in the Pacific Oyster (Crassostrea gigas).

Ostreid herpesvirus-1 microvariant (OsHV-1 μVar) is considered a major infectious microbe that can reduce the survival of natural or cultured oysters in summer. Because they lack an adaptive immune system, oysters are dependent on their innate immune systems to fight pathogens. The duplication and functional divergence of innate immune genes in the oyster have been studied, but the contribution of molecular mechanisms underlying innate immunity remains to be defined. Here, we identified the interacting proteins associated with Crassostrea gigas Toll-like receptors (CgTLR) using a yeast two-hybrid (Y2H) screening system. A total of eight proteins were identified that could interact with CgTLR. Three of these appeared at least four times in the screening and were related to MyD88. Two genes encoding these MyD88-like proteins, CgMyD88-1 and CgMyD88-2, possessed typical death and TIR domains. The third gene encoding an MyD88-like protein possessed only a TIR domain, and we named it CgMyD88s. CgMyD88s interacted only with CgTLR, but not CgMyD88-1 or CgMyD88-2. Both CgMyD88-1 and CgMyD88-2 mRNAs were upregulated after OsHV-1 μVar infection, whereas the expression of CgMyD88s decreased. When overexpressed in HEK293T cells, CgMyD88-1 and CgMyD88-2 activated an NF-κB reporter, whereas CgMyD88s impaired activation induced by CgMyD88-1 or CgMyD88-2. Intriguingly, the silencing of CgMyD88s using double-stranded RNA (dsRNA)-mediated RNA interference increased the expression of CgMyD88-1 and CgMyD88-2. Taken together, our results revealed that CgMyD88-1, CgMyD88-2, and CgMyD88s may all participate in the TLR-mediated innate immune pathway and that CgMyD88s served as a plug to avoid oysters from excessive inflammatory response during OsHV-1 μVar infections.

First comparison of French and Australian OsHV-1 µvars by bath exposure.

Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.

First evaluation of resistance to both a California OsHV-1 variant and a French OsHV-1 microvariant in Pacific oysters

BackgroundVariants of the Ostreid herpesvirus 1 (OsHV-1) cause high losses of Pacific oysters globally, including in Tomales Bay, California, USA. A suite of new variants, the OsHV-1 microvariants (μvars), cause very high mortalities of Pacific oysters in major oyster-growing regions outside of the United States. There are currently no known Pacific oysters in the United States that are resistant to OsHV-1 as resistance has yet to be evaluated in these oysters. As part of an effort to begin genetic selection for resistance to OsHV-1, 71 families from the Molluscan Broodstock Program, a US West Coast Pacific oyster breeding program, were screened for survival after exposure to OsHV-1 in Tomales Bay. They were also tested in a quarantine laboratory in France where they were exposed to a French OsHV-1 microvariant using a plate assay, with survival recorded from three to seven days post-infection.ResultsSignificant heritability for survival were found for all time points in the plate assay and in the survival phenotype from a single mortality count in Tomales Bay. Genetic correlations between survival against the French OsHV-1 μvar in the plate assay and the Tomales Bay variant in the field trait were weak or non-significant.ConclusionsFuture breeding efforts will seek to validate the potential of genetic improvement for survival to OsHV-1 through selection using the Molluscan Broodstock Program oysters. The lack of a strong correlation in survival between OsHV-1 variants under this study’s exposure conditions may require independent selection pressure for survival to each variant in order to make simultaneous genetic gains in resistance.

Variability in the Composition of Pacific Oyster Microbiomes Across Oyster Families Exhibiting Different Levels of Susceptibility to OsHV-1 μvar Disease.

Oyster diseases are a major impediment to the profitability and growth of the oyster aquaculture industry. In recent years, geographically widespread outbreaks of disease caused by ostreid herpesvirus-1 microvariant (OsHV-1 μvar) have led to mass mortalities among Crassostrea gigas, the Pacific Oyster. Attempts to minimize the impact of this disease have been largely focused on breeding programs, and although these have shown some success in producing oyster families with reduced mortality, the mechanism(s) behind this protection is poorly understood. One possible factor is modification of the C. gigas microbiome. To explore how breeding for resistance to OsHV-1 μvar affects the oyster microbiome, we used 16S rRNA amplicon sequencing to characterize the bacterial communities associated with 35 C. gigas families, incorporating oysters with different levels of susceptibility to OsHV-1 μvar disease. The microbiomes of disease-susceptible families were significantly different to the microbiomes of disease-resistant families. OTUs assigned to the Photobacterium, Vibrio, Aliivibrio, Streptococcus, and Roseovarius genera were associated with low disease resistance. In partial support of this finding, qPCR identified a statistically significant increase of Vibrio-specific 16S rRNA gene copies in the low disease resistance families, possibly indicative of a reduced host immune response to these pathogens. In addition to these results, examination of the core microbiome revealed that each family possessed a small core community, with OTUs assigned to the Winogradskyella genus and the Bradyrhizobiaceae family consistent members across most disease-resistant families. This study examines patterns in the microbiome of oyster families exhibiting differing levels of OsHV-1 μvar disease resistance and reveals some key bacterial taxa that may provide a protective or detrimental role in OsHV-1 μvar disease outbreaks.

Transmission of Ostreid herpesvirus-1 microvariant in seawater: Detection of viral DNA in seawater, filter retentates, filter membranes and sentinel Crassostrea gigas spat in upwellers

To understand the transmission of Ostreid herpesvirus-1 microvariant(s) (OsHV-1) in seawater, it is necessary to understand the temporal pattern of occurrence of the virus in seawater in relation to demonstrated infection events in oysters. The aims of the current study were: (1) to investigate the temporal sequence of detection of OsHV-1 in seawater and Crassostrea gigas spat in an upweller system, (2) to assess whether filtration or ageing of seawater reduced the detection of OsHV-1 in seawater and spat, and (3) to assess whether retentate or membranes from filters that were installed to enable continuous sampling of particles in seawater could be used for OsHV-1 surveillance and to investigate OsHV-1 particulate attachment. This study is the first to detect OsHV-1 DNA in 5μm retentates and membranes used to continuously filter natural seawater. OsHV-1 DNA was detected in seawater before it was detected in oysters in 65.4% of the 26 detection events examined across 6 trials in an OsHV-1 endemic estuary. The time of sampling (morning or afternoon) did not affect the detection of OsHV-1 DNA in seawater. Seawater treatment by filtration or ageing did not reduce the frequency of detection of OsHV-1 DNA in seawater. In contrast, the odds of detecting OsHV-1 DNA in the tissues of oysters kept in aged seawater and some filtered seawater treatments were reduced (odds ratios: 0.16–0.36) in comparison to the control. Although OsHV-1 DNA was detected in the retentate from 5μm filtration, it is unlikely these samples will be useful for investigating OsHV-1 particle attachment due to low viral signals. Similarly, OsHV-1 DNA was detected on the 5μm filter membranes, however further investigation is required to ascertain their usefulness in assessing OsHV-1 particle attachment. Despite this and with further development, 5μm filter retentates and filter membranes may prove to be useful samples for the surveillance of OsHV-1 in seawater.

Distribution of Ostreid herpesvirus-1 (OsHV-1) microvariant in seawater in a recirculating aquaculture system

Ostreid herpesvirus-1 (OsHV-1) microvariant presents a significant threat to the commercial production of Crassostrea gigas worldwide. Investigating the transmission of the virus in the estuarine environment where oysters are grown has proven to be difficult due to the complexity of the ecosystem. The aims of this trial were to (1) investigate the distribution of OsHV-1 in seawater over time in a recirculation system during an experimental disease outbreak; (2) assess the effects of the biofiltration and ultraviolet light components of the recirculation system on the detection of OsHV-1; and (3) assess the potential for OsHV-1 to associate with particles in the artificial seawater matrix. The source of virus was experimentally infected donor and cohabitating recipient oysters. Both the presence of infected oysters and the biofilter and UV light source significantly affected the detection rate of OsHV-1 in seawater. It was detected more frequently after high dose exposure (n=6 donors per tank) compared with low dose exposure (n=2 donors per tank), and when the biofilter and UV were disconnected from the treatment tanks. The odds of detecting OsHV-1 in the treatment tanks, which were periodically disconnected from the biofilter and UV, were 2.56 that of the system sumps which were always connected to the biofilter and UV. It may not be possible to remove OsHV-1 completely from the seawater of a recirculation system during a disease event using biofiltration and UV irradiation alone. The presence of food did not significantly affect the detection rate of OsHV-1 in seawater. The pellet produced by low speed centrifugation of seawater had ~2 to 5 fold greater odds of being positive for OsHV-1 by qPCR, in comparison to unprocessed seawater and the supernatant fraction. These results are consistent with the hypothesis that OsHV-1 may be attached to particles, which in this model may include algal feed, oyster faeces, fragments of oyster tissues or microorganisms introduced to the system with the oysters, but confirmation of this is required. Statement of relevanceIt is important to understand the nature and persistence of OsHV-1 microvariant within seawater in order to undertake aquaculture in hatchery environments, where water reticulation, recirculation, biofiltration, UV and other water treatments may be used. Understanding the distribution and mode of transmission of OsHV-1 within seawater has proven difficult. Experimental studies in controlled environments are important to furthering this understanding.

Pacific oyster mortality syndrome: a marine herpesvirus active in Australia

Genotypes of Ostreid herpesvirus 1 (OsHV-1) known as microvariants cause the disease Pacific oyster mortality syndrome (POMS). Since its appearance in NSW in 2010, OsHV-1 microvariant has prevented the farming of Pacific oysters (Crassostrea gigas) in the affected estuaries near Sydney, following the initial massive outbreaks1,2. The arrival of the disease in southeast Tasmania in January 2016 has put the entire $53M industry in Australia in jeopardy3. The virus is a member of the Family Malacoherpesviridae4, which includes several invertebrate herpesviruses. The OsHV-1 genome consists of 207 439 base pairs, with organisation similar to that of mammalian herpesviruses. However, OsHV-1 contains two invertible unique regions (UL, 167.8 kbp; US, 3.4 kbp) each flanked by inverted repeats (TRL/IRL, 7.6 kbp; TRS/IRS, 9.8 kbp), with an additional unique sequence (X, 1.5 kbp) between IRL and IRS4. Unlike many herpesviruses which are host specific, OsHV-1 strains have been transmitted between marine bivalve species5 and the virus is transmitted indirectly. The virus may have relatively prolonged survival in the environment, has extremely high infection and case fatality rates, and latency is unproven. Along with pilchard herpesvirus6–8 and abalone ganglioneuritis virus9,10, it is part of a dawning reality that marine herpesviruses are among the most virulent of pathogens. Finding solutions for industry requires more than laboratory-based research.

Experimental infections of Pacific oyster Crassostrea gigas using the Australian ostreid herpesvirus-1 (OsHV-1) µVar strain.

In Australia, the spread of the ostreid herpesvirus-1 microvariant (OsHV-1 µVar) threatens the Pacific oyster industry. There is an urgent need to develop an experimental infection model in order to study the pathogenesis of the virus under controlled laboratory conditions. The present study constitutes the first attempt to use archived frozen oysters as a source of inoculum, based on the Australian OsHV-1 µVar strain. Experiments were conducted to test (1) virus infectivity, (2) the dose-response relationship for OsHV-1, and (3) the best conditions in which to store infective viral inoculum. Intramuscular injection of a viral inoculum consistently led to an onset of mortality 48 h post-injection and a final cumulative mortality exceeding 90%, in association with high viral loads (1 × 105 to 3 × 107 copies of virus mg-1) in dead individuals. For the first time, an infective inoculum was produced from frozen oysters (tissues stored at -80°C for 6 mo). Storage of purified viral inoculum at +4°C for 3 mo provided similar results to use of fresh inoculum, whereas storage at -20°C, -80°C and room temperature was detrimental to infectivity. A dose-response relationship for OsHV-1 was identified but further research is recommended to determine the most appropriate viral concentration for development of infection models that would be used for different purposes. Overall, this work highlights the best practices and potential issues that may occur in the development of a reproducible and transferable infection model for studying the pathogenicity of the Australian OsHV-1 strain in Crassostrea gigas under experimental conditions.

Further observations on the influence of husbandry practices on OsHV-1 μVar mortality in Pacific oysters Crassostrea gigas: Age, cultivation structures and growing height

Abstract Management of mass mortality events associated with Ostreid herpesvirus-1 microvariant (OsHV-1 μVar) is vital for aquaculture of Crassostrea gigas . As a consequence, the understanding of transmission mechanisms and risk factors enabling husbandry solutions to be developed constitutes an international research priority. In this context, a longitudinal intervention study was set up in Woolooware Bay, Australia, during summer in 2012–2013. The primary objective was to improve the external validity of a study completed in 2011–2012 that demonstrated mortalities in adult oysters but not spat could be halved by reducing their immersion time in an inter-tidal tray cultivation system (by elevating growing height by 300 mm). It is unknown whether an even higher growing height (+ 600 mm) could further improve survival or benefit the survival of spat. Consequently Pacific oysters of different ages (9.5 and 17.5 month) were placed in inter-tidal and sub-tidal rearing structures (trays and baskets) at three different sites in the bay. Mortality, growth, infection prevalence and viral load were monitored for 8 months (October–May). As in the previous summer, the disease was not continuously active over time in Woolooware Bay; mortalities were observed mainly in November 2012 and March 2013. The mortality was highly correlated with the prevalence of OsHV-1, which was in turn highly correlated with viral load, confirming the strong relationship between the virus and oyster mortality. The protective effect of high growing height on adult oysters was confirmed, with a final cumulative mortality below 40%, associated with significantly lower infection prevalence. Even though infection prevalence was significantly lower in baskets than in trays considering all ages and all sites, the final cumulative mortalities for spat were high (> 60%) regardless of the height and the type of cultivation structure; other mitigation strategies need to be developed for young oysters. The disease showed temporal and spatial variability suggesting that substantial variations in exposure to the virus occurred within and between years.

Identification and characterisation of an ostreid herpesvirus-1 microvariant (OsHV-1 µ-var) in Crassostrea gigas (Pacific oysters) in Australia

Between November 2010 and January 2011, triploid Crassostrea gigas (Pacific oysters) cultivated in the Georges River, New South Wales, experienced >95% mortality. Mortalities also occurred in wild diploid C. gigas in the Georges River and shortly thereafter in the adjacent Parramatta River estuary upstream from Sydney Harbour. Neighbouring Saccostrea glomerata (Sydney rock oysters) did not experience mortalities in either estuary. Surviving oysters were collected to investigate the cause of mortalities. Histologically all oysters displayed significant pathology, and molecular testing revealed a high prevalence of ostreid herpesvirus-1 (OsHV-1). Quantitative PCR indicated that many C. gigas were carrying a high viral load at the time of sampling, while the load in S. glomerata was significantly lower (p < 0.001). Subsequent in situ hybridisation experiments confirmed the presence of a herpesvirus in C. gigas but not S. glomerata tissues, suggesting that S. glomerata is not susceptible to infection with OsHV-1. Naïve sentinel triploid C. gigas placed in the Georges River estuary in January 2011 quickly became infected and experienced nearly 100% mortality within 2 wk of exposure, indicating the persistence of the virus in the environment. Phylogenetic analysis of sequences derived from the C2/C6 region of the virus revealed that the Australian strain of OsHV-1 belongs to the microvariant (µ-var) cluster, which has been associated with severe mortalities in C. gigas in other countries since 2008. Environmental data revealed that the Woolooware Bay outbreaks occurred during a time of considerable environmental disturbance, with increased water temperatures, heavy rainfall, a toxic phytoplankton bloom and the presence of a pathogenic Vibrio sp. all potentially contributing to oyster stress. This is the first confirmed report of OsHV-1 µ-var related C. gigas mortalities in Australia.