Ostreid Herpesvirus 1 Variants Research Articles

Pathogenic and genomic characterization of Ostreid herpesvirus 1 associated with mass mortalities of blood cockle (Anadara kagoshimensis)

Ostreid herpesvirus 1 (OsHV-1) is an infectious viral disease of bivalve mollusks with a wide geographic distribution across five continents. Since its initial identification in 1991, the disease has been identified as the cause of mortality in more than ten species of bivalve mollusks. The transmission of OsHV-1 is thought to be facilitated by water. In 2019 and 2021, mass mortalities of blood cockle (Anadara kagoshimensis) hatchery broodstock associated with OsHV-1 infection were reported. The presence of OsHV-1 DNA was confirmed in all hatchery samples with levels exceeding 106 copies per milligram of DNA. OsHV-1 was not detected in the wild populations from which the broodstock was collected, indicating that the virus was introduced into the hatchery with seawater from the open ocean. The results of the histopathological examination indicated that tissue lesions and associated hemocyte infiltration were most prevalent in the connective tissues of the digestive tract, mantle, gills and foot of the diseased A. kagoshimensis. In situ hybridization (ISH) signals of OsHV-1 DNA were frequently observed in infiltrated hemocytes, putative fibroblasts, and on occasion, in muscle cells with weak staining. Transmission electron microscopy analysis demonstrated the presence of herpes-like viral particles within the nucleus of infected cells. The genomes of the OsHV-1 variants from cases 2019 and 2021 were successfully resolved through direct high-throughput sequencing of infected tissues. A phylogenetic analysis of the complete genome sequences of 32 OsHV-1 variants revealed that the segregation of the virus was primarily structured by host species and geographic origin, with some exceptions. We propose that the sympatric distribution of different host species and the potential occurrence of cross-species transmission of OsHV-1 are responsible for the inconsistency between the phylogenetic segregation and their host species. This is the first report on the pathogenic and genomic characterization of OsHV-1 infecting A. kagoshimensis. The epidemiological and phylogenetic analyses offer new insights into the evolution of the virus and provide valuable information for the prevention and control of the disease.

Herpesvirus de los ostreidos tipo 1 en la almeja mano de león Nodipecten subnodosus de Baja California Sur, México

This study is the first report of the presence of Ostreid herpes virus 1 (OsHV-1) in the scallop Nodipecten subnodosus from Baja California Sur, Mexico; collected from 2010 to 2012.The retrospective study was conducted to evaluate whether OsHV-1 was implicated in the population decline of 2010. The virus was detected by PCR, and viral particles were quantified by qPCR. The OsHV-1 detection was conducted by PCR amplification of three different regions of the genome used to identify OsHV-1 variants. A phylogenetic analysis indicated the phylogenetic position of N. subnodusus OsHV-1 among OsHV-1 samples previously collected in different countries and bivalves. Phylogeny analyses indicated differences between OsHV-1 of N. subnodosus and OsHV-1 μVar. Similarity analyses showed differences between the OsHV-1 of Crassostrea gigas and the OsHV-1 of N. subnodosus from Mexico, however the sequence was identical to that of OsHV-1 from France 1993. This study contributes to better understand the genetic diversity of OsHV-1 and to identify potential host of the virus or species involved in the viral cycle.

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

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

Unraveling concordant and varying responses of oyster species to Ostreid Herpesvirus 1 variants

The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (μVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 μVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-μVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) μVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both μVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS μVar (~22%) and higher mortality when challenged with the French μVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French μVar (~22%) and higher mortality with the AUS μVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA μVar (69% correlation) but not with that of the AUS μVar, which also lacked correlation with the FRA μVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.

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.

Long-range PCR and high-throughput sequencing of Ostreid herpesvirus 1 indicate high genetic diversity and complex evolution process

Ostreid herpesvirus 1 (OsHV-1) is an important pathogen associated with mass mortalities of cultivated marine mollusks worldwide. Since no cell line allows OsHV-1 replication in vitro, it is difficult to isolate enough high-purity viral DNA for High-Throughput Sequencing (HTS). We developed an efficient approach for the enrichment of OsHV-1 DNA for HTS with long-range PCR. Twenty-three primer pairs were designed to cover 99.3% of the reference genome, and their performances were examined on ten OsHV-1 infected samples. Amplicon mixtures from six successfully amplified samples were sequenced with Illumina platform, and one of them (ZK0118) was also sequenced with the PacBio platform. PacBio reads were assembled into 2 scaffolds compared to 9−68 scaffolds assembled from the Illumina reads. Genomic comparison confirmed high genetic diversity among OsHV-1 variants. Phylogenetic analysis revealed that OsHV-1 evolution was mainly impacted by its host species rather than spatial segregation.

Experimental infection of European flat oyster Ostrea edulis with ostreid herpesvirus 1 microvar (OsHV-1 μvar): Mortality, viral load and detection of viral transcripts by in situ hybridization

Ostreid herpesvirus 1 (OsHV-1) infections have been reported in several bivalve species. Mortality of Pacific oyster Crassostrea gigas spat has increased considerably in Europe since 2008 linked to the spread of a variant of OsHV-1 called μvar. In the present study we demonstrated that O. edulis juveniles can be infected by OsHV-1μvar when administered as an intramuscular injection. Mortality in the oysters injected with OsHV-1μvar was first detected 4 days after injection and reached 25% mortality at day 10. Moreover, the high viral load observed and the detection of viral transcripts by in situ hybridization in several tissues of dying oysters suggested that OsHV-1μvar was the cause of mortality in the O. edulis juveniles. This is therefore the first study to provide evidence about the pathogenicity of OsHV-1μvar in a species that does not belong to the Crassostrea genus. Additionally, we present a novel method to detect OsHV-1 transcripts in infected individuals’ using in situ hybridization.

Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas

The ostreid herpesvirus 1 (OsHV-1) and variants were implicated in mass mortality affecting the young Pacific cupped oysters, Crassostrea gigas, in European countries and those around the world. From 2008 onwards, oyster mortality had greatly increased on the French coast and was associated with the detection of a new OsHV-1 variant, entitled OsHV-1 μVar. The OsHV-1 μVar is predominant in oysters; however, other OsHV-1 variants have been detected in samples collected during mortality periods or collected out of mortality periods in France, Ireland, Spain, Portugal, Italy, Mexico, United States, South Korea, Australia, and New Zealand. A retrospective study conducted on 1047 OsHV-1 specimens sampled mainly in France between 2009 and 2012, revealed 17 undescribed OsHV-1 variants found in 65 oyster samples. These specimens presented point mutations situated downstream and upstream from the microsatellite area in the C region (ORF 4/5) which were different from the OsHV-1 reference and the OsHV-1 μVar. In the present work, investigation was performed to further characterize these OsHV-1 specimens by sequencing two habitually targeted regions to study genetic polymorphism of the virus: ORF 41/42 and ORF 35–38. An OsHV-1 variant detected in six oyster samples, contained a nucleotide substitution in the C region which impacted the amino acid sequence and might modify the function of the unknown protein encoding by ORF 4. For the ORF 41/42 region, only two specimens presented a synonymous mutation in comparison with the OsHV-1 μVar. All specimens contained the same deletion with the OsHV-1 μVar in ORF 35–38. Then, a phylogenetic analysis based on the C region was performed to investigate the distribution of undescribed specimens among 21 OsHV-1 DNA sequences notified in GenBank and collected from different countries (France, Japan, New Zealand, China, Ireland, and United States) between 1995 and 2012. All analyzed samples and the OsHV-1 μVar were placed in the same group, excepted for a Japan specimen.Our results contribute to improve the description of the genetic diversity of the OsHV-1 and the C region (ORF 4/5) appears to be a better target than ORF 42/42 and 35–38 to distinguish variants between themselves.

Complete genome sequence of Ostreid herpesvirus-1 associated with mortalities of Scapharca broughtonii broodstocks.

BackgroundOstreid herpesvirus-1 (OsHV-1) is the major bivalve pathogen associated with severe mortality events in a wide host range. In the early summer of 2012 and 2013, mass mortalities of blood clam (Scapharca broughtonii) broodstocks associated with a newly described variant of OsHV-1 (OsHV-1-SB) were reported.MethodsIn this study, the complete genome sequence of the newly described variant was determined through the primer walking approach, and compared with those of the other two OsHV-1 variants.ResultsOsHV-1-SB genome was found to contain 199, 354 bp nucleotides with 38.5 % G/C content, which is highly similar to those of acute viral necrosis virus (AVNV) and OsHV-1 reference type. A total of 123 open reading frames (ORFs) putatively encoding functional proteins were identified; eight of which were duplicated in the major repeat elements of the genome. The genomic organization of OsHV-1-SB could be represented as TRL-UL-IRL-IRS-US-TRS, which is different from that of OsHV-1 reference type and AVNV due to the deletion of a unique region (X, 1.5Kb) between IRL and IRS. The DNA sequence of OsHV-1-SB is 95.2 % and 97.3 % identical to that of OsHV-1 reference type and AVNV respectively. On the basis of nucleotide sequences of 32 ORFs in OsHV-1-SB and the other nine OsHV-1 variants, results from phylogenetic analysis also demonstrated that OsHV-1-SB is most closely related to AVNV.ConclusionsThe determination of the genome of OsHV-1 with distinguished epidemiological features will aid in our better understanding of OsHV-1 diversity, and facilitate further research on the origin, evolution, and epidemiology of the virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0334-0) contains supplementary material, which is available to authorized users.

Emerging and endemic types of Ostreid herpesvirus 1 were detected in bivalves in China

Viral infection caused by Ostreid herpesvirus 1 (OsHV-1) is one of the proximate causes of mass mortalities of cultivated bivalves around the world. The emergence and spread of different variants of OsHV-1 accompanied by different epidemiological characteristics have been reported frequently in different countries around the world. In this paper, we present a study of the detection of OsHV-1 DNA and their variations from 1599 samples over 18 species collected in 27 aquaculture sites and two food markets during 2001–2013 in China. All of the samples were examined by a nested PCR assay targeting the C2/C6 fragment of OsHV-1 followed by sequencing. Our results showed 338 individuals (21.1%) of seven species sampled from 14 (14/27=51.9%) sites and the two food markets were positive for viral DNA. Sequencing of 289 PCR products revealed 24 virus types. No shared virus type was found among different countries with 47 types (23 in Japan, 16 in France, 2 in South Korea and 1 in each country of Australia, USA, Ireland, New Zealand, Mexico and China) identified in previous studies. As previously reported, two main phylogenetic groups were identified by phylogenetic analysis based on the 71 virus types; within which 6 separate clades were identified.Our results also demonstrated that two clades were associated with abnormal mortalities of the scallop, Chlamys farrier and the calm, Scapharca broughtonii in China. These findings indicated that cultivated bivalves may face potential threats from OsHV-1 types found in our study.

Genotyping of a microsatellite locus to differentiate clinical Ostreid herpesvirus 1 specimens

Ostreid herpesvirus 1 (OsHV-1) is a DNA virus belonging to the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 has been associated with mortality outbreaks in different bivalve species including the Pacific cupped oyster, Crassostrea gigas. Since 2008, massive mortality events have been reported among C. gigas in Europe in relation to the detection of a variant of OsHV-1, called μVar. Since 2009, this variant has been mainly detected in France. These results raise questions about the emergence and the virulence of this variant. The search for association between specific virus genetic markers and clinical symptoms is of great interest and the characterization of the genetic variability of OsHV-1 specimens is an area of growing interest. Determination of nucleotide sequences of PCR-amplified virus DNA fragments has already been used to characterize OsHV-1 specimens and virus variants have thus been described. However, the virus DNA sequencing approach is time-consuming in the high-scale format. Identification and genotyping of highly polymorphic microsatellite loci appear as a suitable approach. The main objective of the present study was the development of a genotyping method in order to characterise clinical OsHV-1 specimens by targeting a particular microsatellite locus located in the ORF4 area. Genotyping results were compared to sequences already available. An excellent correlation was found between the detected genotypes and the corresponding sequences showing that the genotyping approach allowed an accuraté discrimination between virus specimens.

Genome exploration of six variants of the Ostreid Herpesvirus 1 and characterization of large deletion in OsHV-1μVar specimens

The genetic polymorphism of the Ostreid Herpesvirus 1 (OsHV-1) has generally been investigated in three areas: ORFs 4/5, ORFs 42/43, and ORFs 35 to 38. The present study, however, focuses on 40 ORFs, representing 30% of the OsHV-1 genome, encoding four categories of putative proteins: 4 ORFs encoding putative inhibitor of apoptosis proteins; 17 ORFs encoding membrane proteins; 10 ORFs encoding secreted proteins; and 9 ORFs encoding RING finger proteins. The potential role of these proteins in major steps of the life cycle of the OsHV-1 motivated their selection. Seven specimens have been selected in accordance with their nucleotide variations in the C region (area located between the end of the ORF4 and the beginning of ORF 5): 3 OsHV-1μVar specimens, 2 OsHV-1μVar Δ9, one specimen of OsHV-1μVar Δ15, and one OsHV-1 specimen (reference control) close to the reference genome to validate PCRs. The OsHV-1μVar is mainly characterized by a deletion of 12 consecutive nucleotides followed by a deletion of one adenine in a microsatellite area located in the C region. A representation of nucleotide modifications between the different specimens was performed by building evolutionary trees with respect to the category of ORFs. This phylogenetic analysis revealed two groups: the first one corresponded to the reference control and the reference genome AY509253, and the second one included the 6 OsHV-1 variants. These results suggest that the two main groups come from the same common ancestor, and that the divergence between the reference OsHV-1 and its variants occurred quite far back in time. Moreover, consequences of nucleotide variations in the amino acid sequences, especially the change of the N glycoslyation sites, were investigated. Herein is the first report of four important deletions in these OsHV-1μVar variants: a deletion of 1385bp in ORF 11; a deletion of 599bp in ORF 48; a deletion of 3549bp in ORFs 61 to 64; and a deletion of 712bp in ORF 114. The size of the deletions differed between OsHV-1μVar specimens, OsHV-1μVar Δ9 specimens, and the OsHV-1μVar Δ15 specimen. These zones seem to correspond to special points of gene rearrangements for producing new proteins. Further investigation necessary proves to link such nucleotide modifications with consequences of protein functions in the OsHV-1 life cycle.

Complete genome sequence of acute viral necrosis virus associated with massive mortality outbreaks in the Chinese scallop, Chlamys farreri

BackgroundAcute viral necrosis virus (AVNV) is the causative agent of a serious disease resulting in high mortality in cultured Chinese scallops, Chlamys farreri. We have sequenced and analyzed the complete genome of AVNV.ResultsThe AVNV genome is a linear, double-stranded DNA molecule of 210,993 bp with a nucleotide composition of 38.5% G + C. A total of 123 open reading frames were predicted to encode functional proteins, ranging from 41 to 1,878 amino acid residues. The DNA sequence of AVNV is 97% identical to that of ostreid herpesvirus 1 (OsHV-1), and the amino acid sequences of the encoded proteins of these two viruses are 94-100% identical. The genomic organization of AVNV is similar to that of OsHV-1, and consists of two unique regions (170.4 kb and 3.4 kb, respectively), each flanked by two inverted repeats (7.6 kb and 10.2 kb, respectively), with a third unique region (1.5 kb) situated between the two internal repeats.ConclusionsOur results indicate that AVNV is a variant of OsHV-1. The AVNV genome sequence provides information useful for understanding the evolution and divergence of OsHV-1 in marine molluscs.

Detection of the OsHV-1 μVar in the Pacific oyster Crassostrea gigas before 2008 in France and description of two new microvariants of the Ostreid Herpesvirus 1 (OsHV-1)

Since the summer of 2008, the mortality of young Pacific oysters Crassotrea gigas has been greatly increased on the French coast and may be linked to a microvariant of the Ostreid Herpesvirus 1 (OsHV-1), named OsHV-1 μVar. This variant is mainly characterized by a deletion of 12 consecutive nucleotides followed by a deletion of one adenine in a microsatellite area located in the C region. The present study is the continuity of a previous study that was conducted on oysters collected from July 2008 to June 2010 (Martenot et al., 2011). The purpose of the current survey is to determine the genotype of oyster samples collected from July 2010 to August 2011 and samples taken before 2008. The OsHV-1 μVar was detected in 98% of characterized samples and the two new forms of microvariants were found in the remaining 2%. The first genotype has 15bp successive deletions and the second genotype is similar to the OsHV-1 reference with an insertion of three nucleotides. The OsHV-1 μVar Δ9 previously described in the literature was detected in a sample originating in Brittany. These observations reinforce the possibility of the emergence of different OsHV-1 variants.

Detection of different variants of Ostreid Herpesvirus 1 in the Pacific oyster, Crassostrea gigas between 2008 and 2010

Since summer 2008, high mortality rates of young Pacific oysters Crassostrea gigas have been recorded in association with the detection of the Ostreid Herpesvirus 1 (OsHV-1). A new variant called μVar has been recently described, characterized mainly by 12 consecutive deletions followed by one deletion of an adenine in the C region. The purpose of this study is to characterize the genotype (variants or OsHV-1 reference) of 300 positive samples of C. gigas analyzed between July 2008 and July 2010 collected along the French, Jersey, and Irish coasts. Samples were quantified by TaqMan® PCR, amplified with conventional PCR, targeting the area of the deletion, and then sequenced. Eighty-seven percent of the samples were characterized and the OsHV-1 μVar was detected in 257 oyster samples. The genotype OsHV-1 reference was never detected during the 25 months of the present survey. Thirty-eight samples could not be determined and the majority of them had a low viral load. A novel genotype containing only 9 consecutive deletions named OsHV-1 μVar Δ9 was found in 5 samples. These observations indicate the emergence of different OsHV-1 variants.

Les virus infectant les mollusques marins: un exemple d'actualité, les herpèsvirus

Bien que des épisodes de mortalité associés à la détection de virus aient été rapportés chez différentes espèces de mollusques, peu d’informations sont aujourd’hui disponibles concernant l’affiliation exacte de ces virus et leur position taxinomique. Parmi les virus infectant les coquillages, les herpèsvirus ont fait l’objet d’une attention particulière du fait de leurs impacts économiques et écologiques sur les animaux en élevage et sauvages. Des recherches par des approches de biologie moléculaire ont été menées en France sur le virus infectant les huîtres creuses, Crassostrea gigas. Sur la base des données moléculaires et ultra-structurales, ce virus a été classé, sous le nom d’<i>ostreid herpesvirus</i> 1 (OsHV-1), comme première espèce virale de la famille Malacoherpesviridae. Depuis 2008, des épisodes de surmortalité ont été rapportés dans différentes régions de production des huîtres creuses C. gigas, en France, en Irlande et au Royaume Uni. Ils ont été attribués à des conditions environnementales défavorables associées à la découverte d’un variant génotypique viral particulier, appelé OsHV-1 μVar.

French scallops: a new host for ostreid herpesvirus-1.

Sporadic high mortalities were reported among larval French scallops (Pecten maximus). Electron microscopy of moribund larvae revealed particles with the characteristics of a herpesvirus in association with cellular lesions. PCR and DNA sequencing showed that the virus is a variant of ostreid herpesvirus-1 that has already been described in clams and oysters. This is the first description of a herpesvirus infection of a scallop species. The virus was transmitted successfully from an extract of infected scallop larvae to uninfected scallop or oyster (Crassostrea gigas) larvae, demonstrating that it is able to infect both species. Detection of viral DNA in asymptomatic adult scallops by in situ hybridisation indicates that the herpesvirus may have been transmitted from adults to larvae. It is notable that, unlike most herpesviruses, this virus has a wide host range reflected by its ability to infect several species of marine bivalve.