A previously undescribed ostreid herpesvirus 1 (OsHV-1) genotype detected in the Pacific oyster, Crassostrea gigas, in Ireland.

Parasitology.  2012 Oct; 139(12):1526-32.

Lynch SA1,  Carlsson J2,  O’ Reilly A1,  Cotter E1,   Culloty SC1

1Aquaculture & Fisheries Development Centre, School of Biological, Earth & Environmental Sciences, University College Cork, The Cooperage, Distillery Fields, North Mall, Cork, Ireland.

2School of Biology and Environmental Science, Science Centre-West, University College Dublin, Belfield, Dublin 4, Ireland.

Abstract

Significant mortalities of the Pacific oyster, Crassostrea gigas, have been reported worldwide since the 1950’s.  The impact these reoccurring mortality events have had on the C. gigas industry has highlighted the necessity to determine the factors that may be causing these mortalities.  This study investigated the possible role of ostreid herpesvirus (OsHV-1) in C. gigas mortalities over two successive summers at two study areas in Ireland. A single sample of adult C. gigas, which had been experiencing mortalities at one of the sites was screened.  Successive cohorts of C. gigas spat obtained from a hatchery outside Ireland was relayed to both sites in 2003 and in 2004. Spat were screened each year prior to relaying. Samples were collected every two weeks and mortality counts were recorded and observed at both sites. Polymerase chain Chain reaction Reaction (PCR) and subsequent sequencing indicated that a previously undocumented variant genotype of OsHV-1 was present in the single cohort of adult C. gigas and in seed and juveniles at both sites, in both years.  Analysis suggests that the Irish OsHV-1 μvar variant genotype is closely related to and could be derived from OsHV-1 μvar, first described in France in 2008.

Sharon Lynch-f1

Figure 1.  Pacific oysters in PVC mesh bags on trestles at Irish cultivation site.

 

Supplement:

The Pacific oyster, originally from Japan, has become the oyster of choice for cultivation in many parts of the world due to its rapid growth and its wide-ranging tolerance to environmental conditions. Intentional introductions of this oyster species have occurred worldwide, including Ireland, to replace native oyster stocks significantly depleted by over-fishing or disease, or to create a new industry where none existed before. Accidental introductions have occurred through the movement of oyster larvae in ships’ ballast water or by adults being attached to the hulls of ships. In 2000, the Pacific oyster accounted for 98% of the world’s cultured oyster production and by 2003 global production had expanded to 4.3 million tonnes with a worth of USD 3.69 billion, more than any other fish or shellfish species.

Ostreid herpesvirus 1 (OsHV-1) belongs to the genus Ostreavirus, family Malacoherpesviridae which is included in the order Herpesvirales.  Viruses belonging to the order Herpesvirales are relatively large double-stranded DNA (dsDNA) viruses and can cause latent and/or lytic re-occurring infections.  OsHV-1 μvar is a variant of OsHV-1 and is considered to be a more virulent strain and was initially associated with significant Pacific oyster summer mortalities in France in 2008 and in 2009 and has subsequently been observed in Ireland, Italy, the Netherlands, the UK and south east Australia. Other variants have been observed in a wide range of bivalves including oysters, scallops, clams and abalone.

Several molecular based tools, including standard PCR, are currently used to detect OsHV-1 and variant infections. The PCR primers designed to amplify in the ORF 4 (C2/C6) have been used to amplify DNA yielding mutations or polymorphisms (detected by DNA Sanger sequencing) in the variants compared to the characterized OsHV-1 (1). This PCR detects oyster herpes DNA in region C, which encodes for two proteins of unknown function, and is routinely used for the screening of OsHV-1 and variants.

The main objective of this study was to investigate the role of ostreid herpesvirus 1 in summer mortalities of Pacific oyster adult, seed and juveniles and was part of a larger study that looked at a range of biological and physical factors contributing to oyster mortalities along the coast of Ireland. A sample of market sized C. gigas (24 months old, 60+g) from an oyster farm on the south east coast of Ireland was provided by fishermen from stock that had been experiencing mortality in May 2003. In June 2003, a single cohort of hatchery-reared OsHV-1 uninfected C. gigas spat (shell length ~ 15mm) was obtained from a hatchery outside of Ireland and was relayed to the oyster farm experiencing adult mortalities and an additional oyster farm in one of the most productive areas of operation in Ireland, both located on the south east coast of Ireland.  The oyster spat were held under the same conditions as the oyster growers own stock at both farms and were sampled at two week intervals from July to October 2003.  The trial was repeated in 2004 using a second cohort of spat imported from the same hatchery.

PCR was carried out on all oyster samples, extracting DNA from a range of tissue types and using different extraction methods.  A lower percentage of seed and juveniles were observed to be infected compared to the adult oysters. DNA from the PCR amplified products were isolated and sent for sequencing to confirm the identity of what was being detected.  Sequencing confirmed that the DNA sequences obtained from the adult, seed and juvenile oysters at the two Irish farms were identical.  In the BLASTn analysis, the Irish sequences were similar to OsHV-1 (GenBank accession number AY509253). Further, sequences were aligned and genetic variation was detected in the C region in the Irish OsHV-1 μvar sequences compared to all previously published sequences.  The Irish sequence was almost identical to the OsHV-1 µvar strain identified in France and now epizootic in Europe, however, the differences consisted of one nucleotide deletion (adenine/thymine) and one nucleotide insertion (guanine/cytosine) over the entire 360 bp DNA sequence indicating a previously undescribed variant genotype at the Irish sites.  In the Maximum Likelihood tree (Figure 2), OsHV-1 μvar and the Irish OsHV-1 μvar variant genotype are closely related.

Sharon Lynch-f2

Figure 2. Maximum Likelihood tree showing the phylogentic (Kimura 2 parameter model distances) relationship among four OsHV-1 sequence variants,  using all available variability.  Sequences are coded as follows; GenBank, (GenBank accession number Y509253, Davison et al., 2005); Arzul, (Arzul et al., 2001a), b; Segarra, (Segarra et al., 2010); and Irish variant genotype.

 

The importance of this study. A previously undescribed Irish OsHV-1 μvar variant genotype was detected at two Irish C. gigas cultivation sites and was associated with on-going mortalities.

The phylogenetic relationship among the four variants of OsHV-1 shows that OsHV-1 µvar and the Irish variant genotype are most similar to each other. The additional indels observed in the Irish variant genotype that are not present in OsHV-1 μvar suggests that it may be derived from OsHV-1 μvar or alternatively, OsHV-1 μvar is derived from the Irish variant, although that would demand that the two indels observed in the Irish variant are ancestral for the clade containing OsHV-1 μvar and the Irish variant, and have consequently been lost in OsHV-1 μvar.  It is not clear if the Irish variant differs from OsHV-1 μvar in virulence, tissue tropism etc. and it is obvious that more research is needed to confirm any biologically significant differences between the variant genotypes.  Nevertheless the close resemblance to OsHV-1 μvar in oysters sampled prior to 2008 in France may indicate that OsHV-1 µvar has been present for longer than thought, but had escaped detection until 2008, which has important implications for the management and control of this virus.

References

1.         Renault T, Le Deuff RM, Lipart C, Delsert C 2000 Development of a PCR procedure for the detection of a herpes-like virus infecting oysters in France. Journal of Virology Methods 88: 41–50

Sharon Lynch-f3Contact:

Sharon Lynch, Ph.D.

Research fellow

AFDC, School of BEES,

University College Cork, Cork, Ireland.

s.lynch@ucc.ie

http://publish.ucc.ie/researchprofiles/D026/slynch

 

 

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