中国大菱鲆虹彩病毒主要衣壳蛋白基因的PCR扩增及序列分析

应用同源PCR技术，从被一种球状病毒感染的患病大菱鲆（Scophthalmus maximus）脾脏和肾脏组织中扩增出了一段长度为620bp的DNA片断。序列测定和Blast分析表明，该DNA片断与鱼类虹彩病毒主要衣壳蛋白（MCP）C末端编码区的DNA序列高度相似，由此证实感染养殖大菱鲆的这种球状病毒为一种鱼类虹彩病毒，暂命名为大菱鲆红体病虹彩病毒（TRBIV）。多序列比对和分析发现，TRBIV MCP C末端的205个氨基酸序列与GenBank中20种虹彩病毒相应序列的相似性分别为99．47％（韩国大菱鲆虹彩病毒）、97％～98％（待指定病毒属的7种病毒），以及50％以下（蛙病毒属、淋巴囊肿病毒属、虹彩病毒属的12种病毒），由此绘制出了包含TRBIV在内的21种虹彩病毒的系统发育树。研究结果表明，感染中国养殖大菱鲆的TRBIV属于虹彩病毒科待指定病毒属，位于该属ISKNV亚群和RSIV亚群之间，是该病毒属的一个新成员。

PCR amplification and sequence analysis of major capsid protein gene of turbot reddish body iridovirus (TRBIV)

Turbot,Scophthalmus maximus, has become an important marine aquaculture species in North China.Recently,more and more epizootic diseases of farmed turbot in China occurred because of high density stocking and improper management.In a disease survey of farmed turbot,a reddish body syndrome'(RBS) in both juveniles and adults was found.Further researches showed that an iridovirus-like agent was the causative pathogen of RBS that afflicts farmed turbot in China.To determine the taxonomic position of the virus,a genomic fragment of the virus was amplified and sequenced by PCR and DNA cloning techniques.Briefly,spleens and kidneys were removed from diseased turbots and DNA was extracted.A pair of PCR primers corresponding to the consensus sequence of piscine iridovirus genome was applied and a 620 bp of virus-specific DNA fragment was amplified successfully.After amplification,the PCR product was cloned into pUCm-T vector and sequenced by DNA cloning techniques.The determined nucleotide sequence was deposited into GenBank and the accession number is AY590687.1(AAT01301.1).Blast analysis showed that the sequence is highly identical to the major capsid protein(MCP) gene of piscine iridovirus.The result showed that the virus was a new piscine iridovirus and subsequently named as turbot reddish body syndrome iridovirus'(TRBIV).Further analysis revealed that the sequence encoded 205 amino acid residues which were corresponding to the C-terminal of TRBIV MCP.Alignment of the deduced amino acid sequence of TRBIV MCP C-terminal and those from other 20 iridoviruses in GenBank showed that they had a degree of identity from 41% to 99%.Calculation of the match ratio showed that the deduced sequence of the TRBIV MCP C-terminal shared 99% amino acid identity with that of turbot iridovirus in Korea,97%-98% identity with red seabream iridovirus(RSIV) and infectious spleen and kidney necrosis virus(ISKNV),below 50% identity with those viruses belonging to genus Ranavirus,Lymphocystivirus and Iridovirus.Based on the alignment,a phylogenetic tree of iridoviruses was yielded.The phylogenetic tree indicated that TRBIV was more closely linked to RSIV or ISKNV than to lymphocystis disease virus 1 or frog virus 3.TRBIV should be a new member of genus unassigned,family Iridoviridae and located between the subgroup of RSIV and subgroup of ISKNV in this genus.