Herpes-Type Virus Disease of Oysters

Category

Category 1 (Not Reported in Canada)

Common, generally accepted names of the organism or disease agent

Herpes-type virus disease. In Australia, the acronym POMS (Pacific Oyster Mortality Syndrome) was created to refer to mass mortalities due to Ostreid Herpesvirus type 1 (OsHV-1) (Paul-Pont et al. 2013a).

Scientific name or taxonomic affiliation

Herpes-type virus or herpes-like viruses. Ostreid Herpesvirus type 1 (OsHV-1) from Crassostrea gigas in France has been described and represents a major class of herpesviruses that is different from those in vertebrates (Davison et al. 2005, Renault 2008a). Like other herpesviruses, OsHV-1 is a DNA virus. It was placed in the Order Herpesvirales, Family Malacoherpesviridae and Genus Ostreavirus(Davison 2010, Renault 2011). The apparent lack of host specificity in bivalves and loss of several gene functions in OsHV-1 prompted speculation that this virus may have resulted from interspecies transmission in the context of introduction and intensive culture of non-native bivalve species (Arzul et al. 2001a, b; ICES 2004). It is not known if all herpes-like viruses reported from various species of oysters and other bivalves are the same or different species of herpesvirus. However, variations among DNA sequences of OsHV-1 have been reported (Arzul et al. 2001c, Friedman et al. 2005, Moss et al. 2007, Barbosa Solomieu et al. 2015, Martenot et al. 2015a, Bai et al. 2015, Morrissey et al. 2015) including the variant OsHV-1 μVar described by Segarra et al. (2010) from several locations in France. This variant apparently spread rapidly around the world (Martenot 2013). Between 2008 and 2010, Martenot et al. (2011) characterized the genotype (variants or OsHV-1 reference) of 300 positive samples from C. gigas collected along the French, Jersey, and Irish coasts and reported that the majority of samples (257) were characterized as OsHV-1 μVar. The genotype OsHV-1 reference was never detected and a novel genotype named OsHV-1 μVar Δ9 was found in five samples from Normandy, France (Martenot et al. 2011). Subsequently, Martenot et al. (2012, 2013, 2015a), Renault et al. (2012), Lynch et al. (2012) and Morrissey et al. (2015) reported additional microvariants (virus type) of OsHV-1 from France, Ireland and other locations around the world including China (Bai et al. 2015). In addition to other species of bivalves (see below), herpes-like viruses have been reported from abalone.

Because some of the herpes-like viruses reported in various species of oysters (and other bivalves) have not been specifically identified, the reports were grouped according to main host/geographic region and alphabetically listed below. Each reported geographic location and host species was consistently assigned the same letter code with the classical OsHV-1 listed in position a). The remainder of the topics (i.e., impact on host, diagnostic techniques, and methods of control) focus on OsHV-1.

Geographic distribution

  1. Oyster larvae in hatcheries in New Zealand (Hine et al. 1992) and hatchery-reared oyster larvae and juveniles (including spat) in France (Nicolas et al. 1992). OsHV-1, OsHV-1 μVar, and/or variant genotypes closely related to the OsHV-1 μVar were also detected in juvenile C. gigas (often associated with high mortality events) farmed: in Italy (Dundon et al. 2011, Domeneghetti et al. 2014), on the Mediterranean and Atlantic coasts of Spain (Roque et al. 2012, Aranguren et al. 2012), in the Netherlands (Gittenberger et al. 2016), in Jersey (Martenot et al. 2011), in Ireland (Peeler et al. 2012, Lynch et al. 2012, Clegg et al. 2013, Morrissey et al. 2015) and Great Britain (Barbosa Solomieu et al. 2015), in New South Wales, Australia (Paul-Pont et al. 2013a, b, 2014; Jenkins et al. 2013), in Korea (Hwang et al. 2013) and in samples obtained from China, Japan and New Zealand (Renault et al. 2012, Shimahara et al. 2012, Bai et al. 2015). In Japan, Shimahara et al. (2012) found variable types of OsHV-1 but their nucleotide sequences were not identical to those of OsHV-1 μVar. A closely related virus was detected in juvenile C. gigas in California, USA (Friedman et al. 2005; Burge et al. 2006, 2011; Burge 2010) and in the eroded gills of adult C. gigas along the northwestern Pacific coast of Baja California, Mexico (Vásquez-Yeomans et al. 2004, 2010). Grijalva-Chon et al. (2013) detected a new OsHV-1 DNA strain using molecular assays in healthy, juvenile and adult, C. gigas from the Gulf of California, Mexico without visual confirmation of infection (for example: histology, transmission electron microscopy, and/or  in-situ hybridization were not conducted). Morphological structures (observed by histology and transmission electron microscopy) similar to those of the herpesvirus-like agent were detected in C. gigas from Alaska, USA (Meyers et al. 2009). In Portugal, OsHV-1 μVar was detected in adult Crassostrea angulate experiencing a 47 to 59% mortality outbreak (Batista et al. 2015).
  2. Coastal waters of Maine and New York, USA (Farley et al. 1972).
  3. Various locations in Europe including: France (Comps and Cochennec 1993, Renault et al. 2000a, Renault and Arzul 2001); the United Kingdom (Davison et al. 2005); and in juvenile oysters from various stocks grown experimentally in Galicia, Spain (da Silva et al. 2008).
  4. Tasmania and Western Australia (Hine and Thorne 1997).
  5. New Zealand (Hine et al. 1992, 1998).
  6. Asia including Japan, South Korea and China (Moss et al. 2007, Shimahara et al. 2012, Bai et al. 2015).

Host species

  1. Crassostrea gigas (Renault 2006). Ostreid Herpesvirus type 1 (OsHV-1) from C. gigas larvae in France is infective to larvae of other species of oyster (Crassostrea angulata, Crassostrea rivularis and Ostrea edulis) and clam larvae (Ruditapes decussatus and Venerupis (=Ruditapes) philippinarum) under experimental conditions (Arzul et al. 2001a, b) and in bivalve hatcheries (Renault and Arzul 2001, Renault et al. 2001). This virus has also been detected: in asymptomatic adult C. gigas in France (Arzul et al. 2002); in healthy spat and juvenile C. gigas, Crassostrea ariakensis and Crassostrea sikamea in Japan (Shimahara et al. 2012);and in apparently healthy young adult C. gigas, C. angulata and their F1 hybrids in Portugal (Batista et al. 2014). Subsequently, OsHV-1 μVar was detected in dying adult C. angulata in Portugal (Batista et al. 2015). Saulnier et al. (2011) demonstrated OsHV-1 viral DNA in mussels, barnacles and seawater during mortality events in cultured juvenile C. gigas. A variant of OsHV-1 has also been detected in larval French scallops (Pecten maximus) experiencing sporadic high mortalities (Arzul et al. 2001c).  In New South Wales, Australia, low levels of OsHV-1 with no associated mortality were detected by quantitative PCR but not by in situ hybridisation in Saccostrea glomerata growing adjacent to C. gigas that were experiencing high mortalities (greater than 95%) and had heavy loads of OsHV-1 (Jenkins et al. 2013). The herpesvirus-like agent detected in farmed C. gigas from Alaska, USA was also detected in wild clams (Protothaca staminea) and scallops (Crassadoma gigantea) in Alaska (Meyers et al. 2009). Burge et al. (2011) detected related oyster herpesvirus (OsHV) DNA in asymptomatic C. gigas, Crassostrea sikamea, Crassostrea virginica, O. edulis, Mytilus galloprovincialis, and V. philippinarum in northern California, USA. Tan et al. (2015) determined that under the conditions of the experimental challenge, the black-lip pearl oyster Pinctada margaritifera was not sensitive to OsHV-1 μVar and was not an effective host/carrier.
  2. Crassostrea virginica(Farley et al. 1972).
  3. Ostrea edulis (Comps and Cochennec 1993, da Silva et al. 2008).
  4. Ostrea angasi(Hine and Thorne 1997).
  5. Larvae of C. gigas (Hine et al. 1992) and Ostrea (=Tiostrea) chilensis(Hine et al. 1998) from hatcheries in New Zealand near Auckland and Wellington, respectively. A subsequent survey of 885 molluscs (9 species of bivalves and 1 species of gastropod) from various locations around New Zealand did not detect OsHV-1 (Webb et al. 2007).
  6. Crassostrea ariakensis, C. gigas, C. sikamea, and Crassostrea hongkongensis (Moss et al. 2007, Shimahara et al. 2012). In China, Bai et al. (2015) detected two clades of OsHV-1 associated with abnormal mortalities of the scallop, Chlamys farrier and the clam, Scapharca broughtonii.

Impact on the host

A herpes-like virus was associated with high mortalities (80 - 90%) among C. gigas juveniles (spat) in France (Renault et al. 1994a, b). The disease quickly spread among C. gigas larvae in commercial hatcheries in France, indicating a short productive cycle, with cumulative mortalities approaching 100% within a few days. Between 1998 and 2002, herpes-like viruses were reported from an increasing number of larval bivalve species from around the world, especially in Europe and are now thought to be ubiquitous with associated substantial mortalities in hatcheries (Renault et al. 2000, 2001; ICES 2004). Arzul et al. (2001a, b) have shown that the Ostreid Herpesvirus type 1 (OsHV-1) from C. gigas can infect several bivalve species. Pathology may be related to poor husbandry such as elevated temperatures and crowding and is most prevalent during the summer periods.

OsHV-1, as well as other pathogens, environmental factors (such as elevated temperatures, physiological stress associated with gonadal maturation, aquaculture practices, water quality, predators, phytoplankton blooms, etc.) and genetic factors, have been associated with the syndrome known as ‘summer mortality' which severely affects the aquaculture production of C. gigas in many countries including Japan; Pacific Northwest, USA; Australia; Ireland; and France (Sauvage et al. 2010, Carnegie 2011, Lynch et al. 2012, Fleury and Huvet 2012, Pernet et al. 2014). The term "summer mortality" was typically associated with prolonged losses that affect older, reproductively mature animals (Burge 2010). Various studies have investigated the role of OsHV-1 and variants as causative agents in C. gigas summer mortalities and have found this virus to be particularly associated with larval and juvenile mortalities (Comps and Cochennec 1993, Renault et al. 1994a, b, 2000a; LeDeuff and Renault 1999; Friedman et al. 2005; Burge et al. 2006, 2007; Lynch et al. 2012; Peeler et al. 2012; Degremont and Benabdelmouna 2014; Pernet et al. 2014). For example, in France, Oden et al. (2011) detected high concentrations of OsHV-1 (5×107 DNA copies per 50 mg of dying juvenile C. gigas tissue as measured using a molecular assay) that were associated with summer mortality occurrence. Dégremont (2011) also reported an association between summer mortality and OsHV-1 in Marennes-Oléron Bay, France.

Since 2008, massive mortality outbreaks were reported in several C. gigas oyster farms in France, Ireland, the United Kingdom and Australia. The outbreaks were attributed to adverse environmental conditions (especially a rise in water temperatures (+0.5°C/day, > 4 days) and/or other factors such as toxic phytoplankton blooms or the presence of a pathogenic Vibrio sp., etc.) associated with a particular variant of OsHV-1, called OsHV-1 μVar (Renault 2011, Saulnier et al. 2011, Jenkins et al. 2013, Domeneghetti et al. 2014, Barbosa Solomieu et al. 2015). In 2010, a panel of the European Food Safety Authority suggested that OsHV-1 infection is a necessary cause but may not be a sufficient cause of the severe mortality events of 2008 and 2009 in cultured juvenile C. gigas in Europe, and the strain OsHV-1 μVar seemed to be dominant (EFSA (AHAW) 2010). Although the role of other pathogenic agents such as Vibrio spp. had not yet been fully resolved (EFSA (AHAW) 2010, Barbosa Solomieu et al. 2015), Saulnier et al. (2011) and Dégremont (2011) indicated that bacteria belonging to the Vibrio splendidus group and Vibrio aestuarianus were frequently detected in the living spat but the intensity of bacteria in the tissues did not significantly increase during periods of increased mortality. Petton et al. (2015) found no correlation between Vibrio spp. concentration and OsHV-1 load (DNA concentration determined by molecular assays) in co-infected animals. Also, the quantity of viral DNA was a predictor of mortality, but in the absence of bacteria, a high load of herpesvirus was not sufficient to induce the full expression of the disease (Petton et al. (2015). Selection of C. gigas with resistance to OsHV-1 seemed to provide resistance to V. aestuarianus at the juvenile stage of oyster development but did not benefit adult oysters (Azema et al. 2015c). However, it is important to note that mortality related to V. aestuarianus tends to increase with oyster size and age (Azema et al. 2015a, Moreau et al. 2015b).

In Italy in 2010, OsHV-1 and the microvariant (OsHV-1 μVar) were detected in juvenile C. gigas during grow-out despite the absence of clinical and pathological signs normally associated with the presence of this variant (Dundon et al. 2011). However, in France, OsHV-1 viral DNA detection was negligible except during mortality events and its prevalence and intensity in C. gigas tissues increased rapidly just before and during mortality outbreaks (Saulnier et al. 2011, Dégremont 2011). Garcia et al. (2011) also suspected that OsHV-1 was associated with oyster mortalities but indicated that subsequent work, particularly epidemiological surveys, would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas.

Several epidemiological studies conducted since 2011 have revealed various factors associated with disease caused by OsHV-1. Dégremont (2013) and Whittington et al. (2015b) determined that mortalities can be attributed to OsHV-1 alone because the pattern of mortalities and the presence of a high viral load of OsHV-1 in moribund juvenile C. gigas sampled during peak mortality were observed in field studies that began with the absence of detection of OsHV-1 in oysters at deployment. During the Dégremont (2013) study, all mortalities occurred when seawater temperature was above 16 °C, which was termed the ‘risk' period. For all deployments made during the risk period, mortality was observed within two weeks post-deployment and most episodes lasted over a week. For deployments made outside of the risk period, mortality occurred as soon as the next risk period began (Dégremont 2013). Renault et al. (2014a) detected a similar correlation between juvenile C. gigas mortality associated with OsHV-1 and increases in water temperature. During investigations into the interactive effects of temperature and disease exposure on physiological parameters of juvenile C. gigas, Tamayo et al. (2014) suggested that although energetic reserves were being diminished in infected oysters, their metabolic activities remained similar to that of healthy animals. In Australia, Paul-Pont et al. (2014) found that although seawater temperatures were consistently above 24 °C (±3 °C) during the month preceding mass mortalities, high temperatures did not necessarily lead to mortality events when the virus was present. The incubation period for mass mortality was less than 4 days, however subclinical OsHV-1 infection was detected three months prior to the first signs of mortality in the index case site (first location affected), which suggests that low viral loads of OsHV-1 are insufficient to induce the disease (Paul-Pont et al. 2014). Rapidity of onset and virulence of OsHV-1 in naïve C. gigas spat was also reported in New Zealand waters (Keeling et al. 2014).

Experimental field studies by Dégremont (2011) and Jenkins et al. (2013) also found that the infection in juvenile C. gigas can occur suddenly. No OsHV-1 was detected in seed at deployment, but all juvenile oysters tested 7 days post-deployment were infected and the peak of the viral load was observed 11 days post-deployment (Dégremont 2011). Jenkins et al. (2013) reported that the sentinel triploid C. gigas (60 mm shell height) quickly became infected and experienced nearly 100% mortality within 2 weeks of exposure. Similar results were obtained in the laboratory by Schikorski et al. (2011b) who demonstrated that healthy one-year old C. gigas (about 40 mm in length), after 2 days of cohabitation with oysters experimentally infected by injection, develop the disease with mortality rates increasing gradually for the first eight days and reached a maximum of about 50 % by Day 8. At 6 hours after cohabitation exposure, haemocytes contained more viral DNA than the gills, mantle, adductor muscle and digestive gland (Schikorski et al. 2011b).

Various other factors that seem to affect the possibilities of infection and outcome of the disease have been reported. Lassudrie et al. (2015) indicated that exposure to the harmful dinoflagellate Alexandrium catenella modified the host–pathogen interaction by reducing prevalence of OsHV-1 μVar infection in C. gigas spat. In addition, oysters challenged with OsHV-1 μVar and possibly other pathogens from the environment accumulated smaller amounts of Paralytic Shellfish Toxins than unchallenged oysters. The authors suggested three possible mechanisms for these results: (i) possible direct interactions between A. catenella and herpesvirus (or associated pathogens) could reduce viral transmission and algal availability for oyster consumption; (ii) oyster feeding behavior or digestive functions may have been altered, thus decreasing both uptake of viral particles and consumption or digestion of toxic algae and consequent toxin accumulation; (iii) immuno-activation by A. catenella could enhance defense efficiency against OsHV-1 μVar infection (Lassudrie et al. 2015). Moreau et al. (2015a) determined that pesticides at realistic concentration exerted adverse effects on C. gigas and caused an increased susceptibility to the OsHV-1 infection in experimental conditions. Other laboratory studies indicated that infectious OsHV-1 μVar stability in the sea water was modulated by temperature and elevated water temperatures (25 °C for over 33 hours) were not favorable for virus survival (Martenot et al. 2015b). In other controlled laboratory experiments, Schikorski et al. (2011a) demonstrated that filtered (0.22 μm) tissue homogenates prepared from naturally OsHV-1 μVar infected spat collected on the French coast during mortality outbreaks in 2008 induced mortalities by intramuscular injection into healthy juvenile C. gigas (about 40 mm in length and 5 grams wet weight). However, the same tissue homogenates exposed to an ultraviolet (UV) treatment did not induce any mortality suggesting that juvenile oyster mortalities caused by OsHV-1 require the presence of a UV sensitive agent (Schikorski et al. 2011a).

Adult C. gigas that appear normal (healthy) can be infected with OsHV-1 (Arzul and Renault 2001, Arzul et al. 2002, Batista et al. 2014). Olicard et al. (2005a, b) reported antiviral activity (to Herpes simplex virus type 1 (HSV-1) replicating in African green monkey kidney (Vero) cell monolayers and to two fish RNA viruses replicating in fish cell lines) in the fresh filtered haemolymph from adult C. gigas and seasonal variation in the haemolymph antiviral activity was detected. Green and Montagnani (2013) demonstrated that juvenile C. gigas (mean weight of 3.8 grams) can recognise double-strand RNA to initiate an innate immune response that inhibits viral infection with OsHV-1 μVar. Green et al. (2014) provided evidence that cavortin, a multifunctional protein involved in immunity, was associated with this antiviral activity in C. gigas. Allam and Raftos (2015) reviewed the subject of antiviral activity (immunity) in bivalves. The resistance to mortality caused by OsHV-1 increased with both age and size of C. gigas suggesting a maturation of the immune system against the virus. In field conditions, the relationship between mortality and size was stronger than the relationship between mortality and age (Dégremont 2013). In Australia, mortalities associated with OsHV-1 were recorded in all age classes but were greater in spat and juveniles than in adults (Paul-Pont et al. 2014). Segarra et al. (2014a) suggested that apoptosis could be a main mechanism involved in disease resistance in adult C. gigas. Moreau et al. (2015b) indicted that the autophagy pathway was present and functional in C. gigas less than one year old and played an important role in protecting animals from infections.

Horizontal transmission (between individuals in a population) seems to be the usual mechanism of spread of OsHV-1. For example, Degremont and Benabdelmouna(2014) demonstrated that OsHV-1 was horizontally transmitted from the wild-caught C. gigas spat to the hatchery-produced C. gigas. Paul-Pont et al. (2013b) determined that the distribution of OsHV-1 associated mortalities was spatially clustered, highly variable and clearly dependent on the age of oysters and their position in the water column with hydro-dynamics, physical disturbances, host density/distribution, and variations of environmental parameters thought to influence the mechanism of disease transmission. They hypothesized that OsHV-1 may be carried through water by particles, possibly by planktonic organisms, which may also explain the patchy distribution of mortalities (Paul-Pont et al. 2013b).

Vertical transmission of OsHV-1 (from adults to larvae) has been implicated for asymptomatic oysters (C. gigas by Le Deuff et al., (1996) and Arzul et al. (2002) and in O. edulis by da Silva et al. (2008)) and scallops (P. maximus by Arzul et al. (2001c)). In both the adult oysters and scallops, the connective tissue of the gonad was among the regions that contained infected cells (Arzul et al. 2001c, 2002). Barbosa-Solomieu et al. (2005) detected OsHv-1 in three successive generations of C. gigas including 2 day-old larvae.Crosses involving an OsHV-1 infected male and a non-infected female resulted in hatching and larval survival rates statistically lower than those observed in the other types of crosses suggesting that OsHV-1 infected females may transmit to their offspring some kind of protection or resistance against viral infection (Barbosa-Solomieu et al.2005). Pepin et al. (2008) confirmed the high genetic basis underlying the variance of resistance of C. gigas to OsHV-1. Renault et al. (2011) identified virus-induced genes in C. gigas haemocytes that were challenged with OsHV-1.

Meyers (1981) reported an apparent herpesvirus infection, with no associated disease, in one of 243 adult C. virginica from the south shore of Long Island, New York. Hine and Thorne (1997) detected intranuclear inclusions associated with herpes-like viruses in haemocytes of 23% of the adult O. angasi population that was experiencing low level mortalities associated with Bonamia sp. but with no apparent mortalities attributable to the viral infection. Jenkins et al. (2013) suggested that Saccostrea glomerata  is not susceptible to infection with OsHV-1 because mortalities were not evident and  molecular assays (quantitative PCR and in situ hybridisation) were negligible for the virus while neighbouring C. gigas were experiencing high mortalities (greater than 95%) and had high loads of OsHV-1. In T. chilensis larvae and spat, the interstitial cells, mantle and digestive tract epithelial cells were infected with a herpesvirus and infection appeared to be associated with about 95% mortality over 3 to 4 days at 16 to 18°C among experimentally exposed veligers (Hine et al. 1998).

Diagnostic techniques

The virus is easier to detect in moribund animals than in apparently healthy animals. Also, young stages of oysters including larvae, spat and juveniles, seem to be more susceptible to the infection (World Organisation for Animal Health, OIE 2014).

Gross Observations: Pale digestive gland in spat and older oysters. Infected larvae stop feeding, swimming and exhibit velar lesions (less extended velum and detached parts of the velum are observed free in the water). Usually significant mortalities occur six days after spawning with peak mortalities (80 to 100%) between day 8 and 12 often during the summer time.

Wet Mounts: Larval velum cells are hypertrophied and detached from the tissue. Schikorski et al. (2011a) developed a protocol for the maintenance of infective OsHV-1 virus which involved the preparation of filtered (0.22 μm) tissue homogenates stored at 4°C for one month. They indicated that this preparation could be useful for studies concerning the transmission and the development of OsHV-1 infection.

Histology: Presumptive diagnosis can be made on the histological observations of intranuclear inclusion bodies that are eosinophilic, Feulgen positive and referred to as Cowdry type A nuclear inclusions, abnormal chromatin pattern (usually emarginated) and hypertrophied (enlarged) nuclei in various cells of oysters (including interstitial cells, connective tissue cells or fibroblasts and epithelium) as tabulated by Hine et al. (1998). Cowdry type A nuclear inclusions were not observed in OsHV infected juvenile C. gigas in California (Burge et al. 2006). The main histological change in juvenile oysters is the presence of abnormal nuclei (enlarged nuclei associated with marginated chromatin in fibroblast-like cells and highly condensed nuclear chromatin in ovoid cells interpreted as haemocytes) throughout the connective tissue especially in the mantle, labial palps, gills and digestive gland. In juveniles, infections were usually focal where fibroblastic-like cells exhibit abnormal cytoplasmic basophilia (Renault 2008b). Cellular abnormalities are not associated with massive haemocytic accumulations (Renault and Novoa 2004). Burge et al. (2007) reported that microscopic changes in connective tissue and digestive tubules are consistent with herpesvirus infections in juvenile C. gigas from the west coast of the USA including: diffuse to multifocal peritubular (pertibular) haemocyte infiltration, diapedesis, dilation of the digestive tubules, nuclear hypertrophy, and chromatin margination.

Electron Microscopy: Herpes-like virogenesis begins in the nucleus of infected cells where capsids and nucleocapsids appear. The virions pass into the cytoplasm and are released at the cell surface or by cytolysis. Because bivalve viruses have not been cultured, a confirmatory diagnosis is based on the following transmission electron microscopic features in infected cells from bivalves. Intranuclear inclusion bodies consisting of icosahedral (6- and 5-sided particles) virions 70-120 nm in diameter, a single coat and, occasionally, a dense nucleoid (or toroidal core). In C. gigas larvae, viral particles (capsid size of 72-97 nm) occur in the nuclei and occasionally in the cytoplasm of connective tissue cells of the velum, adductor muscle tissue and digestive gland (Renault 2011, Schikorski et al. 2011a, Hwang et al. 2013). Extracellular enveloped virons measure between 100 to 180 nm in diameter (Renault 2006, 2008b). The C. gigas viruses are most similar to the Betaherpesvirinae according to Hine et al. (1992) but are antigenically cross-reactive with channel catfish virus, which belongs to the Alphaherpesvirinae according to Le Deuff et al. (1995). Intranuclear particles 80 nm in diameter and enveloped cytoplasmic particles 160 to 180 nm in diameter occurred in infiltrating cells with enlarged nuclei (tentatively identified as haemocytes and fibroblasts) in the digestive gland of O. edulis (Comps and Cochennec 1993). A tailed envelope extensions (250-350 nm) has been reported on cytoplasmic mature viral particles found in some of the oyster species but not others. Hine et al. (1998), Renault and Novoa (2004) and Davison et al. (2005) presented morphological comparison of herpes-like viruses described from various species of oysters and comparison to other herpesviruses. Renault (2008b) indicated that larvae exhibit generalized infections with velar and mantle lesions, whereas focal infections usually occur in juveniles.

In scallops, features of OsHV-1 replication were reported from intranuclear and intracytoplasmic locations and were associated with various cellular lesions, including host cell lysis. Replication involved two classes of nucleocapsid; one had an electron-dense core and corresponds to DNA-containing capsids, and the other lacked the core. In addition, enveloped capsids (virions) were observed in perinuclear spaces, cytoplasmic vesicles, and extracellular locations (Arzul et al. 2001c).

Immunological Assay: An immunochemistry analysis using polyclonal antibodies was conducted on 7 µm tissue sections as described by Arzul et al. (2002).

DNA Probes: Details on molecular procedures are available from the World Organisation for Animal Health (OIE 2014). DNA has been extracted from virions purified from fresh, heavily infected C. gigas larvae (Le Deuff and Renault 1999, Renault 2006) and the total genome of the virus (207439 base pairs) was completely sequenced (GenBank number AY509253). Sequence data demonstrate that the Ostreid Herpesvirus type 1 (OsHV-1) is not closely related to herpesviruses from vertebrate hosts (including fish) (Renault and Novoa 2004, Davison et al. 2005, Renault 2008a, Davison 2010). The OsHV-1 genes are unspliced whereas those in herpesviruses of mammals and birds contain one interon and fish and amphibians contain two interons (Renault 2008b). Polymerase Chain Reaction (PCR) and in situ hybridisation (ISH) procedures have been developed for the detection of herpes-like viruses of oysters in France (Renault and Lipart 1998; Renault et al. 2000b; Renault and Arzul 2001; Arzul et al. 2002; Lipart and Renault 2002; Renault and Novoa 2004; Renault 2008a, b; Pepin et al. 2008; Martenot et al. 2010; Corbeil et al. 2015; Segarra et al. 2016) and in New Zealand (Webb et al. 2007). Garcia et al. (2011) suggested two measures for improving the molecular detection of OsVH-1 during oyster mortality outbreaks: (1) collecting animals preferentially less than one week after mortality events and (2) using OsHV-1 PCR on both live and moribund oysters. Lynch et al. (2013) cautioned that tissue selection, sample storage, DNA extraction method used, subsequent storage of DNA, and selection of primers used in the assay, can impact on PCR success or failure.

Renault et al. (2004) developed an internal standard for OsHV-1 detection by PCR using a competitive PCR method in order to detect PCR inhibitors in oyster tissues, to validate sample preparation methods for PCR analysis and to quantify OsHV-1 DNA. Vigneron et al. (2004) indicated that PCR could be used to successfully amplify OsHV-1 DNA in samples of natural seawater water. Barbosa-Solomieu et al. (2004) adapted existing DNA extraction protocols and designed specific primers targeting small fragments (less than 200 bp) in order to detect OsHV- 1 in fixed paraffin-embedded archival samples using PCR and ISH. Batista et al. (2005) developed a simple and rapid method of DNA extraction from oyster larvae for the detection of OsHV-1 by PCR. A critical review of the above procedures was published (Batista et al. 2007) and the European Food Safety Authority published a document that tabulated PCR detection methods (EFSA (AHAW) 2010). Pepin et al. (2008, and see Pepin 2013) developed a real-time PCR (quantitative PCR (qPCR)) assay for rapid, sensitive and quantitative detection of OsHV-1. Subsequently, this procedure was used by researchers to detect OsHV-1 in oysters under various experimental conditions in order to understand the epizootiology (Sauvage et al. 2009, Schikorski et al. 2011b, Clegg et al. 2013, Dégremont 2013, Renault et al. 2014a, Petton et al. 2015, Segarra et al. 2016). Ren et al. (2010) developed a loop-mediated isothermal amplification (LAMP) assay for rapid, specific and sensitive detection of OsHV-1 DNA.

Martenot et al. (2010) described an alternative real-time PCR protocol based on TaqMan® chemistry, which they claimed was an improvement on the reference protocol of Pepin et al. (2008) in terms of sensitivity, specificity and rapidity (<3 h). This assay was used by several researchers including: Oden et al. (2011) to determine a viral load threshold for the use of oyster farmers in evaluating mortality risk during the different stages of handling C. gigas; Petton et al. (2015) to quantify OsHV-1 in haemolymph samples; Gittenberger et al. (2016) to detect OsHV-1 μVar in invasive C. gigas from wild beds in the Wadden Sea, Netherlands where mixed age classes hamper the detection of mortality among juvenile oysters; and Paul-Pont et al. (2013a) and Whittington et al. (2015a, b) to examine the influence of husbandry practices on OsHV-1 associated mortality of C. gigas in New South Wales, Australia. Evans et al. (2014), Keeling et al. (2014) and Hick et al. (2016) used variations of the Martenot et al. (2010) procedures to assay for OsHV-1 in natural seawater samples, in naïve C. gigas spat relocated to an OsHV-1 PCR-positive area, and for assessment of OsHV-1 stability after various disinfection assays, respectively. Burge et al. (2011) developed a SYBR® Green qPCR based on the A-region of the OsHV-1 genome. Various other primers (probes) and PCR assay variations have been used to detect OsHV-1 and its variants (Renault et al. 2012, Jenkins et al. 2013, Lynch et al. 2013, Martenot et al. 2013)

Sequencing of the PCR products obtained by amplification with primers C2-C6 (described by Renault and Arzul 2001) can be used to identify the OsHV-1 μVar strain definitively (Segarra et al., 2010; Martenot et al. 2011; Lynch et al. 2012, 2013; Jenkins et al. 2013). The European Union Commission published a regulation that described a PCR assay using another primer pair (the CF-CR primers) that could be used to distinguish OsHV-1 and the variant OsHV-1 μVar based on the different mobility of the amplified products in agarose gel (EU Commission Regulation 175/2010). However, Aranguren et al. (2012) indicated that differentiation based on this assay was not conclusive and could lead to an erroneous interpretation, especially when co-infection of two genotypes occurred in the same sample, because there is only a difference of 16 bp between the two strains (i.e., the primers CF/CR, amplified 157 bp of OsHV-1 μVar and 173 bp of OsHV-1). Thus, they proposed a specific polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) assay using the MfeI restriction enzyme to identify OsHV-1 μVar and distinguish it from the reference OsHV-1 genotype (Aranguren et al. 2012). Renault et al. (2014b) developed a genotyping method to characterise clinical OsHV-1 specimens by targeting a particular microsatellite locus located in the ORF4 area which allowed accurate discrimination to detect OsHV-1 polymorphism. Batista et al. (2015) suggested that a non-coding region (NC1/NC2 region) located between ORFs 49 and 50 may be a highly polymorphic region and hence of particular interest for molecular epidemiological studies. Segarra et al. (2014b) suggested that an OsHV-1-specific reverse transcriptase real time PCR targeting 39 viral genes may be a new tool for diagnosis and could be used to complement viral DNA detection in order to monitor viral replication thereby assessing the viral cycle and infection status. Nevertheless, the PCR assay chosen for surveillance must be validated for all components of the assay and as indicated by Lynch et al. (2013), further validation of a reliable and sensitive PCR method should include an interlaboratory comparison using a range of diagnostic techniques and tissue types.

In situ hybridization (ISH) indicated that OsHV-1 occurred in the connective tissues of C. gigas spat as identified previously using light and transmission electron microscopy as well as in the visceral ganglion of the oyster nervous system (Renault and Lipart 1998, Lipart and Renault 2002). Infected organs included gills, labial palps, mantle, digestive gland, heart, adductor muscle, and gonads (Lipart and Renault 2002, Batista et al. 2014, Segarra et al. 2016). Segarra et al. (2016) also used ISH to detect RNA of OsHV-1 in tissues of C. gigas spat. Application of PCR, ISH, and immunochemistry suggested that herpes-like virus occurred in a high prevalence (>75%) of apparently normal C. gigas adults and the presence of viral DNA and viral proteins in the gonad supported the hypothesis of vertical transmission from adults to larvae (Arzul and Renault 2001, Arzul et al. 2002). However, the detection of viral DNA in parental oysters did not systematically correspond to infection or result in successful transmission to the progeny although the infection status of the parents appeared to influence infection and survival rates of the progeny (Renault and Novoa 2004).

Methods of control

The European Food Safety Authority panel on Animal Health and Welfare determined that risk factors associated with the severe mortality events in cultured juvenile C. gigas in the summers of 2008 and 2009 in the main European producing countries included an increase or a sudden change in the temperature, and husbandry practices such as introduction of non-certified possibly infected spat, movements and mixing of populations and age groups. The panel concluded that it is not safe to transfer oysters older than 18 months from affected areas to areas not affected. They recommended that to promote and preserve a high health status and in particular to prevent and/or control "increased mortality", measures are urgently needed to improve the general level of biosecurity in the oyster aquaculture industry in Europe. Furthermore, to minimize the risk of subsequent transfer of infectious agents from hatcheries and wild-caught spat, there is a need to establish the health status of oyster spat at source. An assessment of the health status should include results of regular batch laboratory testing (at least in regards to OsHV-1 reference strain and OsHV-1 μVar) and epidemiological assessment. Improved diagnostic methods should be developed and clear criteria for viral strain differentiation taking in account genotype and epidemiological criteria are necessary (EFSA (AHAW) 2010).  Oden et al. (2011) concluded that molecular monitoring of viral load should be a particularly useful tool for preventing mortality by enabling oyster farmers to evaluate risks before batch transfers or before setting oysters out in the sea. They established that a viral load threshold of 4.4×105 copies of OsVH-1 DNA in 50 mg of C. gigas tissue appeared to be useful to oyster farmers for this purpose (Oden et al. 2011). Lynch et al. (2013) confirmed that rapid, reliable, and highly sensitive diagnostic techniques are necessary for the effective control and management of disease in aquaculture. Normand et al. (2014) discussed the use of detection methods in relation to early rearing practices and disease control strategies. In Australia, the management of OsHV-1 mainly involves active surveillance, rigorous biosecurity protocols and mollusc breeding programs targeting production of resistant animals (Paul-Pont et al. 2013a). Pande et al. (2015) proposed a survey design methodology that used hydrodynamically-modelled epidemiological units (based on groups of animals sharing a similar risk) that could help inform management decisions.

Moving oysters into cooler waters may reduce the pathogenicity of the virus. The case in Maine, U.S.A. was reported following transfer of oysters to thermal effluent from a power station, for grow-out. Some cases in New Zealand, France and California occurred in mid-summer at a time of elevated water temperatures. Le Deuff et al. (1996) reported 80 to 90 % mortality in C. gigas larvae reared at 25 to 26°C but not in larvae reared at 22 to 23°C. However, morbidity and mortality can occur below temperatures at which viral replication is completed and latent infections will persist. Using laboratory studies, Hick et al. (2016) determined that OsHV-1 remained infectious in seawater for 2 days at 20 °C and in wet or dry, non-living C. gigas tissues for at least 7 days at 20 °C. Also using laboratory studies, Pernet et al. (2015) found that OsHV-1 persists in C. gigas even at low temperature (10 and 13 °C) and was reactivated during subsequent thermal elevation to 21 °C. Thus, low temperature treatments did not improve overall survival of oyster seed infected with OsHV-1 suggesting that moving infected oysters to a cooler area would only delays mortality and may increase the risk of infection in neighbouring stocks when rising temperatures become permissive for viral replication (Pernet et al. 2015). In T. chilensis, the opposite appeared to occur because the virus was observed to replicate at ambient temperatures (16° to 18°C) but not at higher temperatures (Hine et al. 1998). Results of experimental transmission studies using infected C. gigas larvae indicate that the virus remained infectious after several months of storage at -20°C (Le Deuff et al. 1994).

For areas in which the virus is not enzootic, the most sensible management is to prevent its introduction with contaminated broodstock, larvae or seed (juveniles for grow-out). In Enzootic areas, mortality related to OsHV-1 in hatchery-produced oysters can be avoided using ponds or tanks because these oysters can be protected from exposure to OsHV-1, (Degremont and Benabdelmouna 2014). Results of experiments conducted by Whittington et al. (2015a) supported the hypothesis that OsHV-1 is carried on particles rather than being uniformly distributed in water. Thus, the removal of the putative particulate vector of OsHV-1 from seawater using aging/sedimentation of water or filtration to 5 μm enabled C. gigas spat to survive despite the presence of OsHV-1 μVar in the water supply (Whittington et al. 2015a). Although the results of Evans et al. (2016) were consistent with the hypothesis that OsHV-1 may be attached to particles, they determined that it may not be possible to remove OsHV-1 completely from the seawater of a recirculation system during a disease event using biofiltration and ultra-violet light (40W—65LMP/3900LPH at 30mJ/cm2 (90% UVT) 40 mm connections) irradiation alone.  Hick et al. (2016) indicated that OsHV-1 was inactivated by: commercial multi purpose disinfectants used according to label directions (Virkon-S, Dupont; quaternary ammonium preparation, Livingston); sodium hydroxide (20 g/L, 10 min), iodine (0.1%, 5 min) and formalin (10% v/v, 30 min); and physical measures including heating to 50 °C for 5 min and exposure to a high dose of ultraviolet light (greater than 1000 mW/cm2 at 254 nm at a distance of 15 cm from 2× Sankyo Denki G15T8 15 W germicidal lamps rated at 4.9 W output each, in the UVC range for 10 min in a white plastic reagent reservoir). Sodium hypochlorite (50 ppm available chlorine for 15 min) inactivated OsHV-1 in relatively clean seawater, but this treatment was not effective after addition of protein (10% v/v foetal bovine serum) and a concentration of 200 ppm available chlorine for 15 min did not inactivate OsHV-1 in oyster tissue.

Clegg et al. (2013) considered a range of risk factors relating to farm management but none were found significantly related to mortalities associated with OsVH-1 μVar. However, Dégremont (2013) and Azema et al. (2015b) indicated that larger juvenile C. gigas in a cohort always tended to be more resistant to OsHV-1 than smaller ones. Prudent management strategies for oyster growers such as deploying juveniles at a site favouring the growth of oysters after the threat of exposure to OsHV-1 has passed (i.e. at the end of the risk period when seawater temperatures return to less than 16 °C), and by using cultural practices favouring high growth and/or a site for which the risk period is short due to the seawater temperature could potentially offer viable solutions for disease control (Dégremont 2013). In Australia, Whittington et al. (2015b) confirmed that cumulative mortality for adult C. gigas attributed to OsHV-1 can be reduced by cultivation at a high height in the inter-tidal zone during the summer risk period for OsHV-1 infection. However, the final cumulative mortalities for C. gigas spat were high regardless of the tidal height indicating that other mitigation strategies need to be developed for young oysters (Whittington et al. 2015b). Abollo Rodrígues and Villalba García (2013) published, on the internet, a document that describes the disease and recommended methods of control.

Based on the high genetic basis underlying the variance of resistance of C. gigas to the prevalence and intensity of infection and resulting mortality, Sauvage et al. (2009, 2010) and Dégremont et al. (2015a, b, 2016) suggested that there might be a possibility to improve resistance to OsHV-1 by selective breeding. Segarra et al. (2014c) and Azema et al. (2015a, b, c) confirmed that susceptibility to OsHV-1 infection has a significant genetic component resulting in divergent responses of C. gigas families in terms of mortality. Dégremont (2011, 2013) reported that juvenile (6 month old) C. gigas selected for resistance to "summer mortality" (mainly attributed to OsHV-1 during the study) had a significant resistance to mortality (only 5% mortality) when compared to a control batch (53% mortality) and a batch descended from one family selected to be susceptible to the summer mortality phenomenon (94% mortality). Fleury and Huvet (2012) also reported high heritability for resistance to "summer mortality" in C. gigas and developed lines of oysters resistant to this disease. Apparently, triploidy in C. gigas confers neither advantage nor disadvantage in survival after challenge with OsHV-1 and resistance to OsHV-1 was not substantially altered by triploidization (Dégremont et al. 2015b, 2016). The genes induced in C. gigas haemocytes challenged with OsHV-1 that were identified by Renault et al. (2011) may serve as markers of interest in breeding programs to obtain selected oysters presenting OsHV-1 resistance. Green et al. (2014) suggested that cavortin, a multifunctional protein involved in immunity might be useful for marker-assisted selection of disease resistant oysters. Dégremont et al. (2015c) indicated that after four generations C. gigas subjected to mass selection under field conditions for their higher resistance to OsHV-1 infection showed higher growth (58.4 mm – 19.4 g) than controls (51.4 mm – 15.2 g), and had significantly improved yield (13.3 kg for the selected oysters over 1.2 kg for the controls).

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Citation Information

Bower, S.M. (2016): Synopsis of Infectious Diseases and Parasites of Commercially Exploited Shellfish: Herpes-Type Virus Disease of Oysters.

Date last revised: August 21, 2016

Comments to Susan Bower

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