| Since the 1990s, the outbreak of ostreid herpesvirus-1 (OsHV-1) has brought great losses to the French oyster industry. Besides, OsHV-1 has also caused great mortalities in other European countries and and New Zealand. The host of OsHV-1 is not limited to the Pacific oyster(Crassostrea gigas), OsHV-1 has also been detected in the Manila clam(Ruditapes philippinarum), the King scallops(Pecten maximus) and other bivalve mollusc. These OsHV-1 strains showed some kind of differences in the sequence. After 2008, a new genotype OsHV-1 μvar has replaced OsHV-1 and became the major pathogenic type. In China, a herpes-like virus, Acute Viral Necrosis Virus (AVNV) has also caused great mortalities in Zhikong scallops(Chlamysfarreri) since 1990s which were mainly cultured along the northern coast ofChina. According to the similarities of AVNV in the whole sequence, epidemiology, morphology by comparison with OsHV-1, AVNV was considered to be a variant of OsHV-1.Blood clam(Scapharca broughtonii) is one ofthe economic shellfish farmed in northern China. Since 2012, blood clam has suffered great mortality and the symptoms were similar to that occurred in the dying scallop caused by AVNV including the mantle atrophy and slow response to stimulation. OsHV-1 was detected in moribund blood clam by electron microscopy and PCR detection. However, this strain rendered some differences in the detection region and was named OsHV-1 CDSB2012 strain, OsHV-1-SB in short.To study the changes in OsHV-1 at the molecular level, this experiment aims at sequencing the OsHV-1-SB genome. PCR primers were designed according to the sequenced (A3/A4, C2/C6 and Gp3/Gp4) nucleotide sequences and published OsHV-1 genome sequence (GenBank No. AY509253). By using PCR amplification and molecular cloning technique, a series of cloning OsHV-1-SB sequences were obtained and the ends of which were overlapped. After splicing the entire genome of OsHV-1-SB (GenBank No.:KP412538) was determined. OsHV-1-SB genome is 199,354 bp in length with GC content of 38.5%. It contains two larger terminal inverted repeats (1～7638 and 178,052-185,689; 187,200～197,411 and 200,782～210,993) which is type D of herpesvirus structure. OsHV-1-SB genome contains 123 open reading frames (ORF) and eight repeated ORFs resulting in a total of 131 ORFs. The size of amino acid residues encoded by these ORFs range from 71 to 1878. The functions of the proteins which have identities in GenBank are involved in viral DN A replication, nucleic acid metabolism as well as virus-host interaction. Twenty large insertions and deletions (more than 63bp) are present in OsHV-1-SB by comparison with OsHV-1 and AVNV. ORF48, ORF50,IRL in ORF5/ORF4, ORF115, IRs of ORF117, TRS in ORF117,IRs of ORF122, ORF123 are deleted and three new open reading frames (ORF 125/126/127) occur in OsHV-1-SB due to the sequence inserton.Most ORFs of OsHV-1-SB share the similarity of 96%-100%to counterparts in AVNV and OsHV-1 ORF except for ORF70 (82%) and ORF114 (43%) which are caused by frameshifts. The number ofORFs in OsHV-1-SB shared the same size and orientation to counterparts in OsHV-1 and AVNV are 96 and 94, respectively. Among these ORFs 11 ones (ORFs 2,13,30,35,36,52,74,81,91,96 and 109) are highly conserved that share 100% identity.10 ORFs vary in length due to the mutations in OsHV-1-SB. The change of seven ORFs (ORFs 3,4,9,12,37,70,120) are caused by single base indel (insertion/deletion) or two consecutive bases change. ORFs 21,68, 106 present differences ofconsecutive three nucleotides repeats.Phytogenetic analysis with 32 ORFs’sequences of ten OsHV-1 strains can cluster them into two main branches, OsHV-1 and OsHV-lμVar type. OsHV-1-SB, AVNV and OsHV-1 locate in the same branch, but based on the differences of the hosts, the geographical distribution, OsHV-1-SB and AVNV can be divided into a new genotype. Thus the ten OsHV-1 strains are divided into three genotypes that are European OsHV-1 genotype, OsHV-1μVar genotype and the Chinese AVNV genotype.In order to determine the epidemic of OsHV-1 in China, bivalve samples were collected since June 2013 in six Chinese coastal provinces and detected by conventional PCR method. The infection rate of Chinese scallop has fallen while increased in Patinopecten yessoensis and Crassostrea hongkongensis. Accumulation of these data will provide basis for the rapid diagnosis, molecular epidemiology exploration and monitoring ofOsHV-1. |
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