IFREMER, Laboratoire de Génétique et Pathologie, 17390 La Tremblade, France1
MRC Virology Unit, Church Street, Glasgow G11 5JR, UK2
Author for correspondence: Tristan Renault. Fax +33 5 46 36 37 51. e-mail trenault{at}ifremer.fr
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Sporadic virus infections of C. gigas larvae and juveniles have been observed in France each summer since 1991 (Renault, 1998 ). In the case of larvae, symptoms typically appear 45 days after fertilization. Larvae reduce their feeding and swimming activities and sediment from the water (Le Deuff et al., 1994
). Substantial mortality occurs by day 6, reaching 100% by days 812 (Renault et al., 1994a
, 1995
). A causative role for the virus in larval mortality is supported by transmission experiments (Le Deuff et al., 1994
). In the case of juveniles, high mortality levels (8090%) occur sporadically among farmed spat at months 312 (Renault et al., 1994b
). Prior to death, no gross physiological signs are detectable. The main histological changes consist of enlarged and abnormally shaped nuclei and abnormal chromatin patterns throughout the connective tissues (Renault et al., 1994b
, 1995
). Mortality or morbidity has not been reported in adult oysters in France.
As histological lesions are not specific to herpesvirus infection and no bivalve cell line is available to facilitate virus culture, infections are routinely diagnosed by detection of virus particles by transmission electron microscopy (TEM). The size, structure and sequence of the genome support the hypothesis that the virus that infects C. gigas larvae is a member of the Herpesviridae (Le Deuff & Renault, 1999 ; A. J. Davison, unpublished data), now termed oyster herpesvirus or ostreid herpesvirus-1 (OsHV1; Minson et al., 2000
). Specific PCR tools have been developed to enable rapid diagnosis of OsHV1 in large numbers of samples (Renault & Lipart, 1998
; Renault et al., 2000a
).
Herpesviruses have been detected in several bivalve species in different parts of the world, but it is not known whether these agents represent different viruses, as might be anticipated from the fact that vertebrate herpesviruses are invariably associated closely with individual host species. In order to answer this question, samples of larvae from different bivalve species obtained from different locations were analysed by PCR, restriction endonuclease digestion of PCR products and DNA sequencing.
Animals were obtained at 126 days after fertilization from three hatcheries on the Atlantic and Channel coasts of France; in Normandy, Vendée (370 km south of Normandy) and Charente-Maritime (530 km south of Normandy). Samples were collected during the period 19951999, in some cases from broods that presented abnormally high mortality, and were stored at -20 °C. Thirty infected samples were selected for analysis (Table 1). Twenty consisted of C. gigas larvae that had sedimented before sampling. Three of these samples (1012) and the three samples of R. philippinarum larvae (1315) originated from the same hatchery and were obtained during an episode of high mortality. Four samples consisted of O. edulis larvae that did not exhibit mortality (13 and 29). Lastly, three samples (46) consisted of R. decussatus larvae presenting high mortality.
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The primers A3/A4 and B1/B2 amplified fragments of the sizes predicted for OsHV1 from all larval samples and from reference DNA (summarized in Table 1). In contrast, the primers C1/C6 amplified the predicted fragment from only 24 samples. The six negative samples originated from two bivalve species obtained from the same hatchery (1012 and 1315) and were shown to contain herpesvirus particles by TEM (Table 1
). Further investigations were undertaken on these samples, with the primer pairs C2/C4, C2/C6 and C1/C4 (Fig. 2a
). No product was obtained with samples amplified with C1/C4. Products were obtained with C2/C6 and C2/C4, but were aberrant in being about 180 bp smaller than those obtained with reference DNA, at 530 and 170 bp, respectively. The negative controls did not yield PCR products in any experiment, indicating that laboratory contamination had not occurred.
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C2/C4 and C2/C6 products from one of the aberrant C. gigas samples (sample 11), one of the aberrant R. philippinarum samples (sample 14) and reference DNA were cloned into plasmids and sequenced. Three independent plasmids were sequenced for each product in order to rule out errors induced by PCR amplification. As expected, the reference sequence was identical to the appropriate part of the genome sequence. The sequence of the aberrant fragment from C. gigas was identical to that from R. philippinarum, but differed from that of the reference (Fig. 1d). The differences included several single nucleotide substitutions, insertions and deletions and, more notably, a deletion of 200 bp near the C2 sequence, accompanied by an insertion of 27 bp. These differences would be expected to disrupt the functions of the two proteins encoded by the C region. The provenance of the 27 bp insert is unknown; it is not present in the entire reference virus genome. It is possible that primer C2 functioned as a result of fortuitous hybridization to a non-specific sequence located nearer to the C4 and C6 primer recognition sites in the aberrant genome than the cognate sequence in the OsHV1 genome. Further assays with combinations of six additional primers up to 2·8 kbp upstream from the deletion end-point failed to yield products from the aberrant samples, indicating that a region of the inverted repeat of at least 2·8 kbp is absent from the virus genome. PCR products of the expected sizes were obtained when reference DNA was amplified with these primers. The upstream end-point of the deletion was not determined. Sequencing was also carried out for 380 bp at each end of the 1001 bp PCR product from the A region and for 332 bp in the B region. The reference sequence was identical to the appropriate part of the genome sequence in both regions. Samples 11 and 14 were identical to each other in regions A and B, region B being identical to the reference sequence and region A differing by a single synonymous nucleotide substitution.
The results of this study indicate that four bivalve species belonging to three genera were infected with OsHV1 and that infection of more than one species was not confined to a single hatchery. Infection with the variant of OsHV1 was detected in two bivalve species in a single hatchery during one episode of mortality. The variant did not persist to later episodes, however, as infected samples taken from the same hatchery at subsequent dates yielded PCR products characteristic of reference DNA (data not shown). We conclude that OsHV1 (and the variant) may be transmitted from one species of bivalve to another, and therefore that the natural host of OsHV1 must be considered a matter of uncertainty.
In the natural setting, vertebrate herpesviruses are invariably associated with a single host species. Moreover, the implication that the majority of these viruses have evolved with their hosts over long periods of time finds strong support from molecular phylogenetic studies (McGeoch & Cook, 1994 ; McGeoch et al., 1995
). Exceptionally, transmission can occur from one species to another in the context of farms or zoos. Examples include infection of humans with the simian herpesvirus B virus (Whitley, 1996
) and of cattle, sheep, dogs and cats with the porcine herpesvirus pseudorabies virus (Gustafson, 1981
). Interspecies transmission may also have played a limited part in herpesvirus evolution (McGeoch et al., 1995
). It is possible that bivalve herpesviruses, like vertebrate herpesviruses, are confined to single host species in nature, but that intensive farming conditions, under which different bivalve species are kept in large numbers in unnaturally close proximity, promote transmission to new host species. It is also possible that OsHV1 is itself a mutant of a virus infecting a single bivalve species that has gained the ability to cross species boundaries.
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References |
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Received 5 October 2000;
accepted 5 December 2000.