一株无血凝性兔出血症病毒的分离鉴定及其衣壳蛋白VP60基因的克隆和序列分析

Isolation and Identification of a Non-haemagglutinating Strain of RHDV from China and Cloning and Sequence Analysis of VP60 Gene of the Strain

从临床病料中分离鉴定出1株无血凝性的致病性兔出血症病毒（RHDV），命名为Yaan-1。HA试验测得该毒株原代及经兔体传3代后的肝毒在25℃、37℃和4℃时均无血凝性；但在琼脂扩散试验和对流免疫电泳中均可见分离株与常规RHDV高免血清形成清晰的沉淀线；电镜观察可见30nm左右的病毒粒子。采用RT-PCR方法将Yaan-1株的衣壳蛋白VP60基因进行了克隆测序，序列分析表明VP60基因序列全长1740bp，编码579个氨基酸，测序结果提交GenBank（注册号为DQ069280），并与世界各国的常规RHDV分离株进行比较，结果表明核苷酸同源性为90．0％～98．0％，氨基酸同源性为94．1％～99．0％，氨基酸变异多发生在衣壳蛋白C、E区；同时对Yaan-1株的分子量、等电点、疏水性、跨膜区等分子结构特征进行了分析。本研究首次报道了无血凝性RHDV中国分离株，丰富了地方毒株资源，为明确我国地方株的分子流行病学，以及对VP60蛋白分子特征研究提供了新材料。

A variant strain of rabbit hemorrhagic disease virus which was named ＂Yaan-1＂ was isolated and identified in China. The virus lacked HA activity at 25,37 and 4 ℃ ,but gave positive results in agar diffusion reaction（ADR） and counter immunoelectrophoresis （CIE）. The capsid protein（VP60）of Yaan-1 strain was cloned by RT-PCR and sequenced. VP60 gene of Yaan-1 was 1 740 nt in size and encoding 579 aa. Alignment with other 14 sequences of RHDV isolates in the world showed that the homology were 90.0% -- 98.0% for nucleotide sequence, and 94.1% -- 99.0%for amino acid sequence respectively. The results indicated that the sequence of VP60 gene of different RHDV isolates, including non-haemagglutinating Yaan-1 strain, were highly homologous. The hydrophilicity plot, flexible regions, antigenic index of VP60 protein of Yaan-1 strain, etc. were also analyzed.