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Detection of Ostreid Herpesvirus 1 from adult Pacific Oysters <italic>Crassostrea gigas</italic> Cultured in Korea
Detection of Ostreid Herpesvirus 1 from adult Pacific Oysters Crassostrea gigas Cultured in Korea
Fisheries and aquatic sciences. 2013. Jun, 16(2): 131-135
Copyright ©2013, The Korean Society of Fisheries and Aquatic Science
This is an Open Access article distributed under the terms of the CreativeCommons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use,distribution, and reproduction in any medium, provided the original workis properly cited.
  • Received : April 04, 2013
  • Accepted : June 06, 2013
  • Published : June 30, 2013
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About the Authors
Bo Young Jee
Aquatic Life Disease Control Division, National Fisheries Research and Development Institute, Busan 619-902, Korea
Su Jin Lee
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Korea
Mi Young Cho
Aquatic Life Disease Control Division, National Fisheries Research and Development Institute, Busan 619-902, Korea
Soon Jeong Lee
Aquatic Life Disease Control Division, National Fisheries Research and Development Institute, Busan 619-902, Korea
Jin Woo Kim
Aquatic Life Disease Control Division, National Fisheries Research and Development Institute, Busan 619-902, Korea
Seung Hyuk Choi
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Korea
Hyun Do Jeong
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Korea
Ki Hong Kim
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Korea
khkim@pknu.ac.kr
Abstract
The presence of ostreid herpesvirus 1 (OsHV-1) and the percentage of viral DNA detected in Pacific oyster Crassostrea gigas adults were investigated monthly between May and November 2012 at three locations along the southern coast of Korea. Among 210 oysters examined by polymerase chain reaction (PCR) analysis, OsHV-1 DNA was detected in only one oyster collected in August. The low detection rate of OsHV-1 DNA was consistent with the lack of reported OsHV-1-associated disease in C. gigas cultured in Korea. The sequence of the present PCR product amplified with the C2/C6 primer pair was identical to that of OsHV-1 μVar except for one nucleotide, and the sequence amplified with Del36-37F2/Del36-37R showed a 605-bp deletion as in OsHV-1 μVar. Although these sequence data are insufficient to determine genotype, the results suggest that the herpesvirus detected was similar to OsHV-1 μVar. This is the first report on the presence of OsHV-1 in adult Pacific oysters cultured in Korea.
Keywords
Introduction
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.
Materials and Methods
- Oysters
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).
- PCR analysis
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
Oligonucleotides used in this study
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Oligonucleotides used in this study
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PCR analysis of ostreid herpesvirus 1 (OsHV-1) in the gill (G) and the mantle (M) of Crassostrea gigas collected from Tongyeong in August. Primary PCR was conducted with C13/C5 primer pair, and nested PCR was done with C2/C4 primer pair (right 353 bp). As a control, 18S ribosomal RNA gene was amplified with 18S-F/18S-R primer pair (left 537 bp). The figure is a part of the PCR results to show the positive band amplified with C2/C4 primer pair. MA, 1 kb ladder (Bioneer, Korea).
primary PCR. PCR products were analyzed on a 1% agarose gel containing Midori Green Advanced DNA stain (Nippon Genetics, Düren, 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.
Results
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. 3 A). A comparison of the present sequence with those recorded in other countries was identical except for two nucleotides ( Fig. 3 B).
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Multiple alignment of nucleotides sequence of the present C2/C6 PCR product with that of ostreid herpesvirus 1 (OsHV-1) reference (AY509253), OsHV-1 μVar (HQ842610.1), and OsHV-1 recorded from China (JN800132.1), and Japan (JN800133.1). Identical nucleotides are indicated by black shading. ★, indicates a different nucleotide of the present sample from OsHV-1 μVar.
Discussion
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.
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Multiple alignment of nucleotides sequence of the PCR product (Del36-37F and Del36-37R primers) with ostreid herpesvirus 1 (OsHV-1) reference (A), and with OsHV-1 recorded from New Zealand (JN800252.1), Ireland (JN800250.1), USA (JN800249.1) and France (JN800248.1) (B). Identical nucleotides are indicated by black shading.vv
Acknowledgements
This research was supported by the National Fisheries Research& Development Institute (NFRDI) (RP-2013-AQ-107)
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