鹅圆环病毒浙江永康株全基因组的克隆及序列分析

Cloning and analysis of the complete genome of a Goose circovirus from Yongkang Zhejiang

为研究水禽流感大规模爆发的机理,进行了水禽流感病例中并发病原,特别是免疫抑制性病原的检测研究。根据已发表的鹅圆环病毒(Goosecircovirus,GoCV)序列,设计了一对检测引物,对浙江永康禽流感病死鹅样品进行PCR扩增,获得与预期552bp大小相符的DNA片段,经测序确认为GoCV特异序列,推测样品中存在GoCV。根据测定的序列进一步设计反向扩增引物,经扩增、测序、拼接后获得GoCV全长基因组序列。基因组序列分析表明,浙江永康株GoCV_yk01全长1821bp,具有圆环病毒共同的与病毒复制相关的茎环结构和Rep蛋白保守基序等特征,它与德国、中国台湾发表的序列在全基因组水平有91%～93%的同源性,在Rep和外壳蛋白的氨基酸水平有94%～97%的同源性。应用ClustalW方法作进化树分析显示,GoCV_yk01序列与德国株及中国台湾株均不在同一分支。圆环病毒可以感染淋巴细胞等增殖快的细胞,引起免疫抑制,从而造成其他病原的并发和继发感染,怀疑GoCV可能在2004年初永康爆发的鹅流感中起到了一定的协同作用。该GoCV_yk01是中国内地首次检测确认并测定全基因组序列的鹅圆环病毒。

To investigate the mechanism of avian influenza outbreak wildly in water fowls, the co-pathogens, especially that caused immuno-depression were studied. A pair of degenerated primers, which amplified a fragment of 552bp in length, was designed and synthesized based on the published goose and other Circovirus sequences. The specific PCR product was amplified from the goose sample of Yongkang avian influenza case of Zhejiang Province. The fragment was then sequenced and the result showed the existence of the GoCV. The opposite part of the genome was further amplified using inverse primers designed based on the 552bp sequence obtained and the 1821bp full length genomic sequence of GoCV-yk01 was compiled using Seqman program of DNAStar. Sequence analysis showed that the GoCV-yk01 possessed common features of circovirus included potential replication associated stem-loop structure and the Rep protein motifs. Homology analysis showed that the sequence of GoCV-yk01 had 91% -93 % similarity to that of Taiwan and Germany strains. Phylogenetic analysis with ClustalW, however, showed that the GoCV-yk01 was on a different branch away from Taiwan and Germany strains. Circovirus usually causes immuno-depression and results in secondary infection, as it infects rapid growing cells as lymphocyte. It is speculated that the infection of GoCV may be a part of the reason of the avian influenza outbreak of the Yongkang case. The GoCV-yk01 is the first GoCV strain reported in mainland of China.