Detection Of Ostreid Herpesvirus Research Articles

The complex interactions of Ostreid herpesvirus 1, Vibrio bacteria, environment and host factors in mass mortality outbreaks of Crassostrea gigas

AbstractFrom the first detection in the Eastern oysters (Crassostrea virginica) in 1972, herpes‐like viruses/herpesviruses have been identified in at least 20 bivalve species and have caused massive mortalities in six cultured species of bivalves. Among them, the Pacific oyster (Crassostrea gigas) is most threatened by massive mortalities associated with detection of Ostreid herpesvirus 1 (OsHV‐1) and its variants. These mortality events have been reported in more than 12 countries, and OsHV‐1 itself has been reported in 15 countries worldwide. Traditionally, the genotype OsHV‐1 (or OsHV‐1 Var) is the main contributor to bivalve mortalities, but the emergence of variants (e.g. OsHV‐1 μVar and related ones, acute viral necrosis virus—AVNV, OsHV‐1‐SB) is now responsible for new mortality outbreaks in Pacific oysters and other bivalve species. In addition to OsHV‐1, Vibrio bacteria and other abiotic and host factors also significantly contribute to mortality outbreaks. To this end, the current contribution aims to synthesize the body of literature on our current knowledge of these mass mortality events to provide insights into the role of each risk factor and complex interactions in the disease process. Specifically, this review encompasses our current knowledge and critical gaps. Important suggestions for future investigation are also presented.

Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

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.

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 from adult Pacific Oysters Crassostrea gigas Cultured in Korea

The presence of ostreid herpesvirus 1 (OsHV-1) and the percentage of viral DNA detected in Pacific oyster Crassostrea gigas adults were investigated monthly between May and November 2012 at three locations along the southern coast of Korea. Among 210 oysters examined by polymerase chain reaction (PCR) analysis, OsHV-1 DNA was detected in only one oyster collected in August. The low detection rate of OsHV-1 DNA was consistent with the lack of reported OsHV-1-associated disease in C. gigas cultured in Korea. The sequence of the present PCR product amplified with the C2/C6 primer pair was identical to that of OsHV-1 μVar except for one nucleotide, and the sequence amplified with Del36-37F2/Del36-37R showed a 605-bp deletion as in OsHV-1 μVar. Although these sequence data are insufficient to determine genotype, the results suggest that the herpesvirus detected was similar to OsHV-1 μVar. This is the first report on the presence of OsHV-1 in adult Pacific oysters cultured in Korea.

Detection of Ostreid Herpesvirus 1 (OsHV-1) DNA in seawater by PCR: influence of water parameters in bioassays

Since 1991, herpesvirus infections have been reported among larvae and juveniles of various bivalves. Most of the studies focused on detection of viral infections of economically important species. However, the persistence of bivalve herpesviruses in the marine environment is poorly documented. The present study concerns the role of seawater parameters in Ostreid Herpesvirus 1 (OsHV-1) detection by polymerase chain reaction (PCR). Viral DNA extracted from purified particles or virions present in infected oyster larvae were detected by PCR after storage in different media at different temperatures. The lowest detection threshold was found using distilled water or Tris EDTA buffer. In seawater, the threshold was higher. The use of sterile media permitted detection of viral DNA stored over a longer period. Storage temperature also had a significant influence on detection, with lower temperatures promoting DNA detection over a longer period. In summary, water parameters such as temperature influenced detection of OsHV-1 DNA by PCR. However, the PCR technique may also be successfully applied to samples in natural seawater. Indeed, the PCR technique permitted detection of naked viral DNA at 100 ng l(-1) in seawater in bioassays.