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. 2012 May;86(10):5942–5947. doi: 10.1128/JVI.06534-11

Analysis of Clinical Ostreid Herpesvirus 1 (Malacoherpesviridae) Specimens by Sequencing Amplified Fragments from Three Virus Genome Areas

Tristan Renault a,✉, Pierrick Moreau a, Nicole Faury a, Jean-François Pepin a, Amélie Segarra a, Stephen Webb b
PMCID: PMC3347280  PMID: 22419803

Abstract

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 “specimens” collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.

TEXT

Ostreid herpesvirus 1 (OsHV-1) has been classified within the Malacoherpesviridae family (6, 7, 12). Although OsHV-1 variants have already been reported (2, 3, 14, 19, 20), more work is needed to gauge the range of OsHV-1 polymorphisms. Since 2008, massive mortality outbreaks among Pacific oysters (Crassostrea gigas) have been reported in Europe (8, 21) and have been associated with a virus genotype labeled μVar (22). In addition, mortality outbreaks were reported recently in New Zealand and Australia (15, 16) in association with a virus identified as OsHV-1 μVar. Moreover, the acute viral necrosis virus (AVNV), a herpesvirus that has close affinities to OsHV-1 and that infects Chinese cultured scallops (Chlamys farreria), has been recently sequenced (GenBank accession no. GQ153938). Comparative genomic analysis of AVNV and OsHV-1 suggests that AVNV is a variant of OsHV-1 (R. Weicheng, personal communication).

Seventy-two samples of Pacific oysters collected from 1993 to 2010 and covering different stages of development (larval, spat, and adult) (Table 1) were selected. Most of the samples (63) were collected in France during episodes of mortality and were stored frozen at −20°C. Nine samples came from elsewhere (Ireland, China, Japan, the United States, and New Zealand) and included three paraffin-embedded archival specimens (Table 1). Nucleic acid extraction was performed by using a QIAamp DNA minikit (Qiagen) according to the manufacturer's handbook (17). For frozen tissues, 60 to 200 mg of larvae or 20 to 60 mg of mantle from juveniles and adults was used. For paraffin-embedded specimens (spat collected in 2005 during mortality events in New Zealand), five sections (each 30 μm in thickness) were cut from each histology block (1, 4, 23).

Table 1.

List of isolation codes of DNA extracted from C. gigas samples, geographical origins, years of sampling, stages of development, and GenBank accession numbers

Isolate code Geographical origin of isolate Yr of sampling Development stage or age of isolate source GenBank accession no. for sequence obtained with indicated primer pair
C2 and C6 Del 36-37F2 and Del 36-37R IA1 and IA2
1993/002 France 1993 Larval JN80065 JN800134
1993/004 France 1993 Larval JN80066 JN800135
1993/012 France 1993 <1 yr JN80067 JN800201 JN800136
1994/005 France 1994 <1 yr JN80068 JN800202 JN800137
1994/006 France 1994 <1 yr JN80069 JN800203 JN800138
1994/011 France 1994 <1 yr JN80070 JN800204 JN800139
1994/012 France 1994 Larval JN80071 JN800205 JN800140
1995/020 France 1995 Larval JN80072 JN800205 JN800141
1995/023 France 1995 Larval JN80073 JN800206 JN800142
1995/027 France 1995 Larval JN80074 JN800207 JN800143
2003/001 France 2003 <1 yr JN80075 JN800208 JN800144
2003/003 France 2003 <1 yr JN80076 JN800209 JN800145
2003/006 France 2003 <1 yr JN80077 JN800210 JN800146
2003/009 France 2003 <1 yr JN80078 JN800211 JN800147
2003/012 France 2003 <1 yr JN80079 JN800212 JN800148
2003/013 France 2003 <1 yr JN80080 JN800213 JN800149
2005/001 France 2005 <1 yr JN80081 JN800214 JN800150
2005/005 France 2005 <1 yr JN80082 JN800215 JN800151
2005/008 France 2005 Larval JN80083 JN800216 JN800152
2005/012 France 2005 <1 yr JN80084 JN800217 JN800153
2006/002 France 2006 Larval JN80085 JN800218 JN800154
2006/003 France 2006 Larval JN80086 JN800219 JN800155
2006/005 France 2006 Larval JN80087 JN800220 JN800156
2006/009 France 2006 <1 yr JN80088 JN800157
2006/013 France 2006 <1 yr JN80089 JN800158
2006/018 France 2006 <1 yr JN80090 JN800159
2007/004 France 2007 Larval JN80091 JN800221 JN800160
2007/012 France 2007 Adult JN80092 JN800222 JN800161
2007/025 France 2007 <1 yr JN80093 JN800223 JN800162
2007/026 France 2007 <1 yr JN80094 JN800163
2007/028 France 2007 <1 yr JN80095 JN800164
2007/029 France 2007 <1 yr JN80096 JN800165
2007/030 France 2007 <1 yr JN80097 JN800224 JN800166
2007/034 France 2007 1-2 yr JN80098 JN800225 JN800167
2007/035 France 2007 <1 yr JN80099 JN800226 JN800168
2008/017 France 2008 <1 yr JN800100 JN800227 JN800169
2008/019 France 2008 1-2 yr JN800101 JN800170
2008/021 France 2008 Larval JN800102 JN800171
2008/023 France 2008 Adult JN800103 JN800172
2008/025 France 2008 Adult JN800104 JN800173
2008/030 France 2008 Adult JN800105 JN800174
2008/039 France 2008 Larval JN800106 JN800228 JN800175
2008/045 France 2008 1-2 yr JN800107 JN800229 JN800176
2008/050 France 2008 <1 yr JN800108 JN800230 JN800177
2008/055 France 2008 <1 yr JN800109 JN800231 JN800178
2008/059 France 2008 <1 yr JN800110 JN800232 JN800179
2008/073 France 2008 1-2 yr JN800111 JN800180
2008/079 France 2008 <1 yr JN800112 JN800233 JN800181
2008/083 France 2008 <1 yr JN800113 JN800234 JN800182
2008/092 France 2008 <1 yr JN800114 JN800235 JN800183
2009/002 France 2009 <1 yr JN800115 JN800236 JN800184
2009/021 France 2009 <1 yr JN800116 JN800237 JN800185
2009/022 France 2009 <1 yr JN800117 JN800238 JN800186
2009/027 France 2009 <1 yr JN800118 JN800239 JN800187
2009/035 France 2009 <1 yr JN800119 JN800240 JN800188
2010/002 France 2010 <1 yr JN800120 JN800241 JN800189
2010/008 France 2010 <1 yr JN800121 JN800242 JN800190
2010/012 France 2010 <1 yr JN800122 JN800243 JN800191
2010/013 France 2010 <1 yr JN800123 JN800244 JN800192
2010/021 France 2010 <1 yr JN800124 JN800245 JN800193
2010/023 France 2010 <1 yr JN800125 JN800246 JN800194
2010/026 France 2010 <1 yr JN800126 JN800247 JN800195
2010/028 France 2010 <1 yr JN800127 JN800248 JN800196
2002/E50 China 2002 JN800132 JN800253
2007/07-CB2 USA (California) 2007 JN800128 JN800249 JN800197
2010/158-144 Japan 2010 <1 yr JN800133 JN800254
2009/Ireland Ireland 2009 JN800129 JN800250 JN800198
2010/01 New Zealand 2010 <1 yr JN800130 JN800251 JN800199
2010/02 New Zealand 2010 Larval JN800131 JN800252 JN800200
2005/2Ea New Zealand 2005 <1 yr
2005/2Ha New Zealand 2005 <1 yr
2005/BN1Ca New Zealand 2005 <1 yr
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a

DNA extracted from histological blocks.

PCR assays were performed using 3 extant primer pairs targeting 3 virus genome regions: primer pair C2 and C6 (open reading frame 4 [ORF4]) (18), primer pair IA2 and IA1 (ORF42 and -43) (22), and primer pair Del 36-37F2 (5′-ATACGATGCGTCGGTAGAGC-3′) and Del 36-37R (5′-CGAGAACCCCATTCCTGTAA-3′) (ORF35, -36, -37, and -38) (Fig. 1). All tested samples yielded amplicons of the expected sizes with primer pair C2 and C6 (709 bp) and primer pair IA1 and IA2 (607 bp) except for one specimen, with that exception perhaps due to limiting template conditions (Fig. 2A). With primer pair Del 36-37F2 and Del 36-37R, the DNA samples each gave 1 of 3 different patterns: a PCR product of the expected size (989 bp), a PCR product of 384 bp, or no amplification (Fig. 2A). Twenty-eight French samples collected from 1993 to 2008 yielded 989-bp amplicons and might be interpreted as representing the reference type (accession no. AY509253), a virus isolated from French Pacific oyster larvae in 1995 (6). A large deletion (605 bp) was reported for all samples identified as representing variant OsHV-1 μVar (7 French samples collected in 2008, 13 French samples collected in 2009 and 2010, and a sample collected in Ireland in 2009) and also for samples collected in China, the United States, Japan, and New Zealand. Finally, a third group of virus specimens (from French oysters collected in 1993 and from 2003 to 2008) was defined based on the absence of amplification. This lack of amplification was not related to the absence of virus DNA, as amplicons were obtained from the same samples with primer pair C2 and C6 and primer pair IA1 and IA2 (Fig. 2A). The 605-bp deletion reported for OsHV-1 μVar and related specimens entirely covered both ORF36 and ORF37 and a part of ORF38 (Fig. 1). This deletion of 2 genes and the modification of a third might be more than coincidental with respect to the apparently increased virulence of OsHV-1 μVar. ORF38 encodes a RING finger protein. The RING finger domain of ICP0 and of homologs from alphaherpesviruses is required for the activation of quiescent genomes (5, 9, 10, 11, 13). Modifications of the RING finger protein encoded by ORF38 might affect its activities and influence OsHV-1 virulence.

Fig 1.

Fig 1

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Scale diagram of ORF35, -36, -37, and -38 areas for the OsHV-1 reference type (A) and virus specimens presenting a 605-bp deletion area (B). The vertical lines indicate the limits of the different ORFs. The deleted sequence is located between the dashed arrows: ORF36 and -37 are totally missing, as is a part of ORF38.

Fig 2.

Fig 2

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(A) PCR products (obtained using primer pair Del 36-37F2 and Del 36-37R, primer pair C2 and C6, and primer pair IA1 and IA2) from selected virus specimens subjected to electrophoresis on 1.5% agarose. Lanes: M, Small Marker (Eurogentec); 1, 1993/012/France; 2, 1994/005/France; 3, 1995/020/France; 4, 2005/012/France; 5, 2006/005/France; 6, 2007/004/France; 7, 2008/020/France; 8, 1993/002/France; 9, 2005/005/France; 10, 2006/009/France; 11, 2006/013/France; 12, 2007/028/France; 13, 2008/019/France; 14, 2008/073/France; 15, 2008/011/France; 16, 2010/001/New Zealand; 17, 2010/158-144/Japan; 18, 2009/002/France; 19, 2009/022/France; 20, 2010/002/France; 21, 2010/021/France; N, negative control. (B) PCR analysis of the DNA samples extracted from histological blocks (Pacific oysters collected in 2005 in New Zealand during a mortality outbreak). Lanes: M, Small Marker (Eurogentec); 1, 2005/2E/New Zealand; 2, 2005/2H/New Zealand; 3, 1995/020/France (OsHV-1 reference); 4, 2008/055/France (OsHV-1 μVar); N, negative control.

For DNA samples extracted from paraffin-embedded specimens, PCR analyses were carried out using primer pair C9 (5′-GAGGGAAATTTGCGAGAGAA-3′) and C10 (5′-ATCACCGGCAGACGTAGG-3′), primer pair CF (5′-CCCCGGGGAAAAAGTATAAA-3′) and CR (5′-GTGATGGCTTTGGTCAAGGT-3′), and primer pair Del 36-37F2 and Del 36-37R (see above). Primer pair C9 and C10 and primer pair CF and CR targeted ORF4. Two histological blocks from the 3 analyzed samples yielded amplicons with the 3 primer pairs used (Fig. 2B). With primer pair CF and CR, the 2 samples yielded a 157-bp amplicon similar in size to that obtained for OsHV-1 μVar (Fig. 2B). The specimen considered the reference type (1995/020/France) gave a 173-bp product. With primer pair Del 36-37F2 and Del 36-37R, a 384-bp amplicon was obtained for the 2 samples from New Zealand (Fig. 2B), although specimen 1995/020/France (the reference type) yielded a 989-bp product. The obtained PCR product sizes suggest that variant OsHV-1 μVar or a related virus was present in Pacific oysters collected in New Zealand in 2005. With primer pair C9 and C10, moreover, PCR products from a paraffin-embedded specimen appeared identical in sequence to OsHV-1 μVar and presented a single deletion in comparison with the sequence of OsHV-1 (accession no. AY509253) (data not shown).

PCR products were purified by kit (Amicon Ultra centrifugal filter; Millipore) (0.5 ml; 30 K) according to the supplied protocol and were then directly sequenced. Samples were loaded into an ABI Prism 3130 XL-Avant Genetic Analyzer. Phylogenetic analyses were performed on sequence concatenations of the 3 genome areas (1,426 positions) by the maximum-likelihood method with the MEGA5 program (24).

For IA1 and IA2, amplicon sequences (ORF42 and -43) were compared to the OsHV-1 sequence (accession no. AY509253) by the use of the ClustalW program. Two mutations were observed that differentiated samples into 3 groups, the first one containing samples presenting 100% sequence identity with OsHV-1 (accession no. AY509253; reference type), the second group containing samples presenting 100% sequence identity with OsHV-1 μVar (22), and the third group consisting of both samples collected in New Zealand in 2010 and presenting only one of the mutations characterizing OsHV-1 μVar, representing an A deletion (22).

With primer pair Del 36-37F2 and Del 36-37R, amplicons from samples collected in France in 2009 and 2010, some of the samples collected in 2008, and samples collected in China, the United States, Japan, and New Zealand demonstrated a 605-bp deletion. Although amplicons obtained from samples collected in France from 1993 to 2007 and some of the samples collected in 2008 did not demonstrate the 605-bp deletion, a few point mutations were reported that differentiated the samples into 3 groups, one of these groups presenting 100% sequence identity with the reference type (accession no. AY509253).

Finally, with primer pair C2 and C6, the obtained amplicons demonstrated the highest polymorphism, with 82 positions of a 460-nucleotide sequence showing mutations defining at least 19 virus groups. All the sequences of the French samples collected in 2009 and 2010 showed 100% identity with the OsHV-1 μVar sequence (accession no. HQ842610) except for 2 virus specimens (2010/02/France and 2010/12/France). Although most of the French samples collected before 2007 were similar to the reference type (accession no. AY509253), some of them demonstrated mutations in comparison with the reference sequence. Thus, polymorphisms for several microsatellite zones with variable numbers of repetitions were observed: 5 to 9 A, 5 to 6 G, 4 to 5 C, and 4 to 13 CTA, with 3 repetitions for AVNV, 4 for OsHV-1 μVar, and 8 for the reference type (data not shown). For this microsatellite zone, some virus specimens collected in France also showed 9, 11, or 13 repetitions.

Phylogenetic analysis allowed identification of 2 main groups from 54 virus specimens (Fig. 3). The first group contained French specimens collected from 1994 to 2008, including the reference type (accession no. AY509253). This group also integrated samples collected in the United States and in China and AVNV samples (Fig. 3). The second main group was composed of French specimens collected from 2008 to 2010 and an isolate collected in Ireland in 2009. The sequence of OsHV-1 μVar deposited in GenBank (accession no. HQ842610) was included in this group. This group also integrated samples from Japan and New Zealand (Fig. 3). Although the C2-C6 fragment sequence for these specimens (Japan and New Zealand) was similar to the OsHV-1 μVar sequence (accession no. HQ842610), they differed from HQ842610 by two shared mutations (two As replaced by two Gs).

Fig 3.

Fig 3

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Phylogenetic tree generated by the maximum-likelihood method. Bootstrap values were obtained from 1,000 resampled data sets. The analysis involved 54 nucleotide sequences (from concatenated PCR products obtained with primer pair C2 and C6, primer pair Del 36-37F2 and Del 36-37R, and primer pair IA1 and IA2). There were a total of 1,426 positions in the final data set. OsHV-1 (reference type) and AVNV were also included for phylogenetic analysis.

Several French samples collected from 1993 to 2008 demonstrated 100% sequence identity with the reference type and as such could be identified as OsHV-1 (6). Other samples collected in France from 2003 to 2008 showed some differences from the reference type. Although they appeared closely related to this virus type (accession no. AY509253) with respect to ORF4, these samples did not yield amplicons when primer pair Del 36-37F2 and Del 36-37R was used. These results suggest that different OsHV-1 variants coexisted in France before 2008. Variant OsHV-1 μVar was not detected in French samples collected before 2008 in the present study. These results are in accordance with those reported by Segarra et al. (22), who concluded that OsHV-1 μVar was not detected in archival samples and that, in Europe, OsHV-1 μVar was an emerging genotype. Phylogenetic analysis suggested also that, although the reference type and OsHV-1 μVar share an ancestor (Fig. 3), OsHV-1 μVar is not directly derived from the reference type. Moreover, 3 different types were detected in 2008 in France: specimens identical to the reference type, specimens related to the reference type (closely related with respect to ORF4 but not amplified by primer pair Del 36-37F2 and Del 36-37R), and specimens identical to OsHV-1 μVar. Finally, 2 French virus specimens collected in 1993 showed high homology to variant OsHV-1 Var (3). This variant was reported in 1997 during a mortality outbreak affecting both larval Pacific oysters and larval Manila clams (Ruditapes philippinarum) in a commercial hatchery (19, 20). Both French samples from 1993 correspond to C. gigas larvae collected in a commercial hatchery. It is thus possible that the intensive farming conditions under which different bivalve species are kept at the same time in close proximity might promote interspecies transmission (2, 3).

The sample collected in California demonstrated the 605-bp deletion observed in the area of ORF35, -36, -37, and -38 for OsHV-1 μVar. However, it did not present the mutations characterizing this variant within ORF4 and it appeared identical to OsHV-1 in the area of ORF42 and -43. AVNV grouped with the isolate collected in California based on sequence data obtained in the present study (Fig. 3). Although AVNV presents variations in coding and noncoding regions in comparison to OsHV-1, these results suggest strongly that AVNV is an OsHV-1 variant.

ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups. However, areas of ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in defining different genotypes.

Nucleotide sequence accession numbers.

Partial PCR product sequences from virus specimens were submitted to GenBank under accession numbers JN80065 to JN800254 (Table 1).

ACKNOWLEDGMENTS

We thank C. Friedman, C. Burge, K. S. Reece, and D. Cheslett for providing DNA samples from the United States, China, and Ireland.

Footnotes

Published ahead of print 14 March 2012

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