鹅呼肠孤病毒GRV1株分离鉴定及其σC基因特征性分析

Isolation and characterization of minor core protein σC from goose reovirus GRV1

从患运动障碍的雏鹅肝脏和脾脏分离到一株鹅源呼肠孤病毒,该病毒经琼脂扩试验可与番鸭呼肠孤病毒(MuscovyDuckReovirus,MDRV)的抗血清发生交叉反应,推测其为呼肠孤病毒,命名为GRV1。参考GenBank禽呼肠孤病毒(AvianReovirus,ARV)和番鸭呼肠孤病毒小外壳蛋白(minorcoreprotein)σC蛋白基因序列设计合成了一对引物,病毒RNA经RT-PCR扩增,产物为810bp,与预期的目的片断大小一致,测序结果表明σC基因在280～1089区间是一个开放性阅读框架,编码269个氨基酸的蛋白,GC含量为49.88%,等电点为6.497,分子量为29.4Ku。核苷酸序列经DNAStar(6.0)软件分析,与法国番鸭呼肠孤病毒89026株核苷酸同源率为93.0%,与鸡呼肠孤病毒σC基因同源率仅为21%～25%,同源性分析表明鹅呼肠孤病毒与番鸭呼肠孤病毒可能来自同一祖先,建议将鹅呼肠孤病毒连同番鸭呼肠孤病毒归属为正呼肠孤病毒属第二个亚群中不同于禽和内尔森贝海湾呼肠病毒的独立基因群。由蛋白质分析软件Anthepro5.0和MultiCoil软件分析表明,抗原区多位于N末端,没有跨膜区,σC为三股螺旋结构,这是我国第一次在鹅体内分离到呼肠孤病毒,同时也是第一次在数据库中提供σC的序列。

Goose reovirus named GRVI isolated from the liver and spleen of goose with moving difficulty disease. The virus could cross react with the serum of reference strain S14 muscovy duck reoviruses by agarose gel precipitation test. Minor core protein σC-encoding gene of GRVI was amplified by RT-PCR with the primers designed by the reported avian and muscovy duck reoviruses. The PCR products were cloned to the pMD18-T vector and identified by enzyme cutting analysis. The sequence results showed that σC-encoding gene is 810 bp. It contains one open reading frame between 280 to 1089 that encodes a protein of 269 amino acids with a molecular mass of 29.4 Ku. Isolectric point is 6.497, Based on MultiCoil analysis, GRV1 σC protein should contain three-stranded coiled-coil structure. Nucleotide analysis by the DNASTAR（6.0） software showed that GRV1σC-encoding gene is highly related to the muscovy duck reovirus 89026, with 93.0 % homology, and 21% to 25 % identity to avian reoviruses. The homologous of σC of GRV1 to the muscovy and avian suggested that both GRV1 and MDRV should segregated NBV and avian reoviruses into one specified genogroup among second subgroup of orthoreovirus genus. This study isolated the reovirus from geese and demonstrated that geese reoviruses GRV1 is highly related to that of muscovy duck reovirus and give one conclusion that GRV and MDRV might come from an unknown common progenitor.