Olivia Evans

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.

A SIMPLE CENTRIFUGATION METHOD FOR IMPROVING THE DETECTION OF OSTREID HERPESVIRUS-1 (OsHV-1) IN NATURAL SEAWATER SAMPLES WITH AN ASSESSMENT OF THE POTENTIAL FOR PARTICULATE ATTACHMENT

Ostreid herpesvirus-1 (OsHV-1) is responsible for massive mortality events in commercially farmed Pacific oysters (Crassostrea gigas) in Australia, New Zealand, Europe and the USA. Economic losses have been severe in many countries since 2008, associated with a strain known as OsHV-1μ-var. Despite intensive studies of the virus itself, there is almost no information on its detection in natural seawater, how it is spread over wide geographic distance in water or on how it is transmitted from oyster to oyster via seawater. The aim of the current work was to (1) assess and compare several centrifugation methods in order to detect OsHV-1 in natural seawater samples using real-time quantitative PCR, in such a way that large numbers of samples could be processed efficiently and (2) assess the potential for particulate attachment of OsHV-1 using filtration. Compared to testing unprocessed seawater samples, centrifugation of seawater at 1000×g for 20 min with testing of the pellet improved OsHV-1 detection rates by two fold. Results suggest that OsHV-1 may be attached to particles large enough to be pelleted at low g-force, as well as in the form of small particles, free virus or free viral DNA. Filtration of seawater using low protein binding filters could not be used to assess OsHV-1 particle attachment, due to interactions between particles, free virus or free viral DNA and the membranes.

Resistance of Black-lip learl oyster, Pinctada margaritifera, to infection by Ostreid herpes virus 1μvar under experimental challenge may be mediated by humoral antiviral activity

Ostreid herpesvirus 1 (OsHV-1) has induced mass mortalities of the larvae and spat of Pacific oysters, Crassostrea gigas, in Europe and, more recently, in Oceania. The production of pearls from the Black-lip pearl oyster, Pinctada margaritifera, represents the second largest source of income to the economies of French Polynesia and many Pacific Island nations that could be severely compromised in the event of a disease outbreak. Coincidentally with the occurrence of OsHV-1 in the southern hemisphere, C. gigas imported from New Zealand and France into French Polynesia tested positive for OsHV-1. Although interspecies viral transmission has been demonstrated, the transmissibility of OsHV-1 to P. margaritifera is unknown. We investigated the susceptibility of juvenile P. margaritifera to OsHV-1 μvar that were injected with tissue homogenates sourced from either naturally infected or healthy C. gigas. The infection challenge lasted 14 days post-injection (dpi) with sampling at 0, 1, 2, 3, 5, 7 and 14 days. Mortality rate, viral prevalence, and cellular immune responses in experimental animals were determined. Tissues were screened by light microscopy and TEM. Pacific oysters were also challenged and used as a positive control to validate the efficiency of OsHV-1 μvar infection. Viral particles and features such as marginated chromatin and highly electron dense nuclei were observed in C. gigas but not in P. margaritifera. Mortality rates and hemocyte immune parameters, including phagocytosis and respiratory burst, were similar between challenged and control P. margaritifera. Herpesvirus-inhibiting activity was demonstrated in the acellular fraction of the hemolymph from P. margaritifera, suggesting that the humoral immunity is critical in the defence against herpesvirus in pearl oysters. Overall, these results suggest that under the conditions of the experimental challenge, P. margaritifera was not sensitive to OsHV-1 μvar and was not an effective host/carrier. The nature and spectrum of activity of the humoral antiviral activity is worthy of further investigation.

Detection of ostreid herpesvirus 1 microvariant DNA in aquatic invertebrate species, sediment and other samples collected from the Georges River estuary, New South Wales, Australia

Ostreid herpesvirus 1 microvariants (OsHV-1) present a serious threat to the Australian Crassostrea gigas industry. Of great concern is the propensity for mortality due to the virus recurring each season in farmed oysters. However, the source of the virus in recurrent outbreaks remains unclear. Reference strain ostreid herpesvirus 1 (OsHV-1 ref) and other related variants have been detected in several aquatic invertebrate species other than C. gigas in Europe, Asia and the USA. The aim of this study was to confirm the presence or absence of OsHV-1 in a range of opportunistically sampled aquatic invertebrate species inhabiting specific locations within the Georges River estuary in New South Wales, Australia. OsHV-1 DNA was detected in samples of wild C. gigas, Saccostrea glomerata, Anadara trapezia, mussels (Mytilus spp., Trichomya hirsuta), whelks (Batillaria australis or Pyrazus ebeninus) and barnacles Balanus spp. collected from several sites between October 2012 and April 2013. Viral loads in non-ostreid species were consistently low, as was the prevalence of OsHV-1 DNA detection. Viral concentrations were highest in wild C. gigas and S. glomerata; the prevalence of detectable OsHV-1 DNA in these oysters reached approximately 68 and 43%, respectively, at least once during the study. These species may be important to the transmission and/or persistence of OsHV-1 in endemically infected Australian estuaries.

Detection of Ostreid herpesvirus-1 microvariants in healthy Crassostrea gigas following disease events and their possible role as reservoirs of infection

Ostreid herpesvirus-1 microvariants (OsHV-1) cause severe mortalities in farmed Crassostrea gigas in Europe, New Zealand and Australia. Outbreaks are seasonal, recurring in the warmer months of the year in endemic estuaries. The reference genotype and microvariant genotypes of OsHV-1 have been previously detected in the tissues of apparently healthy adult oysters naturally exposed to OsHV-1 in the field. However, the role of such oysters as reservoirs of infection for subsequent mortality outbreaks remains unclear. The aims of this study were: (1) to identify the optimal sample type to use for the detection of OsHV-1 DNA in apparently healthy C. gigas; and (2) to assess whether live C. gigas maintained on-farm after an OsHV-1 related mortality event remain infected and could act as a reservoir host for subsequent outbreaks. OsHV-1 DNA was detected in the hemolymph, gill, mantle, adductor muscle, gonad and digestive gland of apparently healthy adult oysters. The likelihood of detecting OsHV-1 DNA in hemolymph was equivalent to that in gill and mantle, but the odds of detecting OsHV-1 DNA in hemolymph and gill were more than 8 times that of adductor muscle. Gill had the highest viral loads. Compared to testing whole gill homogenates, testing snippets of the gill improved the detection of OsHV-1 DNA by about four fold. The prevalence of OsHV-1 in gill and mantle was highest after the first season of OsHV-1 exposure; it then declined to low or negligible levels in the same cohorts in subsequent seasons, despite repeated seasonal exposure in monitoring lasting up to 4years. The hemolymph of individually identified oysters was repeatedly sampled over 15months, and OsHV-1 prevalence declined over that time frame in the youngest cohort, which had been exposed to OsHV-1 for the first time at the start of that season. In contrast, the prevalence in two cohorts of older oysters, which had been exposed to OsHV-1 in prior seasons, was consistently low (<10%). Viral loads were <104 DNA copies per mg tissue or μL hemolymph, suggesting that OsHV-1 was not being maintained at or amplified to high quantities. Therefore, while OsHV-1 may persist within apparently healthy oysters that have survived an outbreak of disease, they may not be a major reservoir host for the virus for subsequent outbreaks. However, further investigation is required to ascertain whether OsHV-1 replication occurs in surviving oysters, and whether transmission from them to naive oysters and induction of clinical disease is possible.

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 outbreaks. The arrival of the disease in southeast Tasmania in January 2016 has put the entire

Comparison of Two External Tagging Methods Used for the Identification of Individual Adult Pacific Oysters, Crassostrea gigas

53M industry in Australia in jeopardy. The virus is amember of the FamilyMalacoherpesviridae, which includes several invertebrate herpesviruses. The OsHV-1 genome consists of 207439 base pairs, with organisation similar to that of mammalian herpesviruses. However, OsHV-1 contains two invertible unique regions (UL, 167.8kbp;US, 3.4 kbp) eachflankedby inverted repeats (TRL/IRL, 7.6 kbp; TRS/IRS, 9.8 kbp), with an additional unique sequence (X, 1.5 kbp) between IRL and IRS 4 . Unlike many herpesviruses which are host specific, OsHV-1 strains have been transmitted betweenmarine bivalve species 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 herpesvirus and abalone ganglioneuritis virus, it is part of adawning reality thatmarine herpesviruses are among the most virulent of pathogens. Finding solutions for industry requires more than laboratory-based research. In 2008, the microvariant genotype OsHV-1 mVar emerged as a dominant isolate against a background of prior endemic OsHV-1 strains in France, and has devastated the C. gigas industry there. Similar microvariant genotypes of OsHV-1 have since appeared throughout Europe, in New Zealand, and in Australia, with similar devastating impact (Figure 1). In 2010–11 when an emergency response was required in Australia, almost no information existed on the epidemiology of OsHV-1 infection, and the only management responses internationally that could be drawn from were to develop resistant oysters through selective breeding approaches. In Australia, a breeding program commenced immediately but it was complemented by research to identify mechanisms of viral transmission and environmental triggers, in order to develop husbandry recommendations to mitigate losses. Richard Whittington, Paul Hick, Olivia Evans, Ana Rubio, Navneet Dhand and Ika Paul-Pont Faculty of Veterinary Science, School of Life and Environmental Sciences, The University of Sydney, Camden, NSW 2570, Australia Hornsby Shire Council, Hornsby, NSW 1630, Australia CNRS, IUEM Technopôle Brest-Iroise, Rue Dumont d’Urville, 29280 Plouzané, France Corresponding author. Tel: +61 2 9351 1619, Email: richard.whittington@sydney.edu.au Under theMicroscope

Descriptive epidemiology of mass mortality due to Ostreid herpesvirus-1 (OsHV-1) in commercially farmed Pacific oysters (Crassostrea gigas) in the Hawkesbury River estuary, Australia

ABSTRACT Tagging methods used in bivalve research can be broadly categorized into two groups: internal and external. External tagging methods are more commonly used; however, the legibility of external tags tends to decline over time, and tag recovery rates are often low due to the abrasion or biofouling of shells, particularly during long-term studies. The aim of the current study was to compare two external identification methods placed on or in the shells of adult Crassostrea gigas: (1) a plasticlaminated glue-on tag and (2) novel use of a t-bar anchor tag inserted into the upper shell, to determine the optimal methodology to use in longitudinal studies of greater than 1-y duration. Over a 15-mo period, 100% of glue-on numbered tags were lost or became overgrown and could not be read, whereas 91.5% of t-bar anchor tags were retained and remained legible. The results of this study suggest that t-bar anchor tags are a better choice for long-term longitudinal studies of adult C. gigas in temperate marine conditions than plastic-laminated labels glued to the exterior of the shell. These findings may be applicable to other bivalves.