H5N1亚型和H3N2亚型禽流感病毒广西株NS基因的克隆和序列分析

Cloning and sequence analysis of NS gene of H5N1 and H3N2 avian influenza virus strains from Guangxi,China

目的对3株分离自广西的禽流感病毒的NS基因进行序列分析,试图从分子水平分析和了解广西地区禽流感病毒NS基因的变异特点和进化规律。方法根据GenBank上登录的已知禽流感病毒的NS基因全序列设计引物,对3株2004-2005年分离自广西的禽流感病毒株A/DK/GX/1566/04(H3N2)、A/Goose/GX/737/05(H5N1)、A/Goose/GX/2775/05(H5N1)的cDNA进行PCR扩增NS基因,并将其克隆到pMD-18T载体上,分别获得全长为855bp、834bp、837bp的NS基因全序列。结果2株H5N1亚型禽流感病毒广西株间NS基因序列的同源性为95.3%,而2株H5N1亚型禽流感病毒株与H3N2亚型禽流感病毒株之间NS基因序列的同源性为94.0%～94.1%。H5N1亚型禽流感病毒广西株的NS基因在第238-253位核苷酸处均发生了15个核苷酸缺失,与我国广东、香港、云南、湖南、湖北及东南亚等不同地区的毒株比较,NS基因的核苷酸同源性为70.2%～97.6%。在NS基因系统进化树中,3株禽流感病毒广西株都属于A亚群,但处在不同的分支上,其中A/DK/GX/1566/04(H3N2)与广东、香港2001-2003年分离株处于同一分支;A/Goose/GX/737/05(H5N1)、A/Goose/GX/2775/05(H5N1)与我国华南地区以及韩国、越南、泰国等亚洲东南部国家的2003年以后的分离株有共同起源。结论3株2004-2005年分离自广西的禽流感病毒株NS基因之间的同源性均高于94%,都属于A亚群。

A pair of primers specific to NS gene of HSN1 subtype and H3N2 subtype avian influenza virus isolates from Nanning poultry market between 2004 and 2005 was designed on the basis of the NS gene sequence from GenBank. The cDNA of the NS gene was amplified by RT PCR and cloned into pMD18 T vector, and nucleotide sequences of the NS gene of the three isolates were 855 bp,834 bp and 837bp in length respectively. The sequence analysis showed that the nucleotide homologies between the two isolates of HSN1 subtype was 95.3 % ,however,those between HSN1 subtype and H3N2 subtype were from 94.0% to 94.1%. The NS gene of the HSN1 isolates had 15 nucleotide deletions,which was typical of highly pathogenic HSN1 viruses isolated in various countries from 2000. Comparison with the strains from Guangdong, HongKong, YuNNan, Hunan, Hubei and South Asia indicated that the nucleotide homology were from 70. 5 % to 97 %. Phylogenetic analysis showed that three isolates were situated in sublineages A, but they formed different distinct little sublineages, A/ DK/GX/ 1566/04（H3N2） showed closer relationship with virus islaoted from Guangdong and HongKong from 2001 to 2003. A/Goose/ GX/ 737/05（H5N1） and A/ Goose/GX/ 2775/05 （H5N1） had high aligment located in the same lineage together with virus isolated from southeast region of China and some areas of Asia including Korea, Vietnam and Thailand after 2003.