Since Farley et al. (1972) first described a herpes-type virus in the Eastern oyster Crassostrea virginica, herpes or herpeslike viruses have been reported worldwide from various bivalves including oysters, clams, and scallops (Renault and Novoa, 2004). Minson et al. (2000) named a virus isolated from infected Pacific oyster Crassostrea gigas larvae as ostreid herpesvirus 1 (OsHV-1), and Davison et al. (2005) reported the full genome sequence of OsHV-1 (GenBank accession no. AY509253). Initially, two OsHV-1 genotypes, OsHV-1 reference and OsHV-1 var, were reported in France (Arzul et al., 2001), but additional genotypes such as OsHV-1 microvariant (μVar) (Segarra et al., 2010) and OsHV-1 μVar Δ9 (Martenot et al., 2011) have been newly added. OsHV-1 has been detected in C. gigas spat mortalities in France since 1993. However, severe mortality outbreaks in Pacific oyster spat and juveniles have been reported in different member states of the European Union including France and Ireland since 2008, and OsHV-1 μVar has been proposed as the possible culprit (Segarra et al., 2010; Schikorski et al., 2011a, 2011b).

Korea is among the top three countries in the world for production of Pacific oysters. However, as oyster farms are concentrated on the southern coastal area of Korea, an outbreak of highly infectious disease at a farm can bring highly disastrous results in the Korean oyster aquaculture industry. Although Moss et al. (2007) reported the presence of molluscan herpesvirus from Crassostrea ariakensis collected from Korea, no reports have been published on the presence of OsHV-1 in Korean Pacific oyster. The purpose of this study was to investigate the presence and prevalence of OsHV-1 in adult Pacific oysters in Korea.

Crassostrea gigas adults (1-2 years old) were collected monthly between May 2012 and November 2012 from oyster farms located at Tongyeong, Goseong, and Geoje on the southern coast of Korea. Immediately after the oysters arrived in the laboratory each month, 30 oysters (10 oysters from each region) were sampled, and gill and mantle tissues were excised to analyze for the presence of OsHV-1 by polymerase chain reaction (PCR).

The excised gill and mantle were lysed by adding 500 μL of lysis buffer containing Proteinase K and incubated in a shaking water bath at 60℃ for 3 h to extract total DNA. Then, 250 μL of 6 M NaCl was added to each tube, followed by vigorous shaking of the tubes 20 times and a 10-min incubation on ice. The supernatants were recovered by centrifugation at 10,000 g for 10 min, mixed with 650 μl isopropanol, and centrifuged at 10,000 g for 10 min. The resulted pellet was further air-dried for 5 min and eluted with RNAse-free water.

The primers used are listed in Table 1. Primary PCR reactions were conducted with primer pairs of C13/C5 (open reading frame [ORF]4), C13/C6 (ORF4), and Del-F/Del-R (ORF35, 36, 37, and 38), and amplification conditions were one cycle of 3 min at 95℃ (initial denaturation), 30 cycles of 30 s at 95℃, 30 s at 55℃, and 30 s at 72℃, followed by 7 min at 72℃. Primer pairs used for nested PCR were C2/C4, C2/C6, and Del36-37F/Del36-37R, respectively, and 0.2 μl of each primary PCR product was used as the template. The PCR conditions for the nested PCR were the same as those for the

primary PCR. PCR products were analyzed on a 1% agarose gel containing Midori Green Advanced DNA stain (Nippon Genetics, Duren, Germany) for visualization, purified using a gel purification kit (Nucleogen, Seoul, Korea), subcloned into pGEM-T Easy Vector (Promega, Madison, WI, USA), and sequenced. Multiple sequence alignments were generated using the CLUSTALW 1.8 program.

In total, 210 Pacific oysters were PCR-analyzed to detect OsHV-1 DNA. The band corresponding to the partial 18S ribosomal RNA gene was detected from all analyzed specimens (Fig. 1). No amplified band was identified in any of the samples by primary PCR with the primer pairs for amplifying OsHV-1 DNA. However, nested PCR with the C2/C4 primers amplified a band of the expected size (352 bp) from only one oyster (Fig. 1), which was collected from Tongyeong in August; thus, the percentage of OsHV-1 DNA detection in C. gigas adults was less than 1% (1/210).

The sequence of the C2/C6 PCR product of the positive sample showed deletion of 13 consecutive nucleotides, and another 11 nucleotides were sporadically changed or deleted when compared to the OsHV-1 reference sequence (Fig. 2). Only one G (Fig. 2) was deleted in the present sample compared to that in the OsHV-1 μVar sequence.

Nested PCR with the Del36-37F/Del36-37R primers amplified a band of expected size (384 bp) that represented a deletion of the middle region of the gene corresponding to 605 nucleotides in the OsHV-1 reference sequence (Fig. 3A). A comparison of the present sequence with those recorded in other countries was identical except for two nucleotides (Fig. 3B).

We report for the first time the presence of OsHV-1 from Pacific oyster adults in Korea. Although OsHV-1 has been associated with mass mortalities of Pacific oyster larvae or spat, adult oysters asymptomatically carrying the virus could have the potential to transmit OsHV-1 to C. gigas larvae or other bivalve species.

According to the results of Moss et al. (2007), C. ariakensis collected from three regions in Korea showed a prevalence of molluscan herpesvirus of up to 40%, whereas C. gigas was completely negative for the herpesvirus (all sampled oysters from Korea were considered adults based on the description). Similarly, the percentage of OsHV-1 DNA that we detected in Pacific oyster adults was very low (<1%). This result was extremely low compared to the OsHV-1 occurrence in adult Pacific oyster in France, where prevalence is above 70% (Arzul et al., 2002; Martenot et al., 2011). The low detection rate of OsHV-1 DNA observed in the present study is consistent with the lack of reported OsHV-1-associated disease in C. gigas cultured in Korea.

One of the main differences in the nucleotide sequence of OsHV-1 μVar from that of OsHV-1 reference is a partial deletion of the microsatellite zone of the region amplified with the primer pair C2 and C6 (Segarra et al., 2010). The deleted nucleic acids included three consecutively repeated “CTA,” followed by a “CTGA.” The C2/C6 sequence of the present OsHV-1 showed the same deletion and was identical with that of OsHV-1 μVar except for one nucleotide, whereas the previously reported OsHV-1 from China and Japan showed four and three different nucleotides, respectively, compared to that of OsHV-1 μVar. Renault et al. (2012) reported that all specimens identified as OsHV-1 μVar show a large deletion (605 bp) of the amplicon with the primer pair Del36-37F2 and Del 36-37R, and the present virus specimen also had the same deletion. Considering the full genome size of OsHV-1, the present sequence data are insufficient to determine the genotype; however, our results suggest that the herpesvirus detected in this study was similar to OsHV-1 μVar.