禽流感病毒WUDI-H9N2株的分离鉴定及 HA基因的分子特征研究

本研究于2011年8月从山东省某鸡场鸡群中分离一株病毒,通过对分离株进行病毒血凝试验及血凝抑制试验、RT-PCR、动物回归试验等方法证实已分离到禽流感病毒(avian influenza virus,AIV) H9N2,命名为WUDI-H9N2株。根据GenBank上发表的AIV H9病毒的HA基因序列,用Oligo 6.0生物学软件设计引物,对分离株WUDI-H9N2株HA基因进行扩增、测序及序列分析。将分离的AIV H9毒株与其他血清型代表参考毒株分别进行核苷酸和氨基酸系统发生进化关系分析。WUDI-H9N2株 HA基因全序列长为1708 bp,无核苷酸的插入和缺失,cDNA包含了1个完整的开放阅读框(ORF),编码区由1683 bp组成,共编码560个氨基酸。HA序列与国内参考毒株的核苷酸序列的同源性在92.9%~98.1%之间,其中与安徽分离株A/chicken/China/AH-10-012010(H9N2)同源性为98.1%。

Isolated and Identified of Avian Influenza Virus WUDI-H9N2 in China and Molecular Characteristics Research of HA Gene

An isolate of avian influenza virus,designated WUDI-H9N2,was isolated from Shandong area of China in Auguste 2011 and identified for pathogenicity to chicken embryo by hemagglutination test, hemagglutination inhibition test and RT-PCR. A pair of primers was designed to amplify the HA strain based on the reported gene sequences in GenBank by Oligo 6.0 and for WUDI-H9N2 HA gene amplifying and sequence analysis. The separated AIV H9 strain and other serotype were respectively analysed by nucleotide and amino acid phylogeny tree. The results of sequencing showed that the HA gene was 1708 bp in legth, no nucleotide insertion and deletion, the cDNA contains whole open reading frame(ORF) of HA gene was 1683 bp and encoded a 560 amino acid protein. Compared with reference strains, the nucleotide sequence similarity of HA was 92.9% to 98.1%. On the basis of phylogenetic analysis of HA gene, we concluded that the HA shared 98.1% similarity of structure protein encoding sequence with A/chicken/China/AH-10-012010(H9N2) strain.