10株H9N2亚型禽流感病毒HA和NA基因的序列分析

HA and NA Gene Sequence Analysis of Ten Avian Influenza Virus Subtype H9N2

为了从分子水平上掌握我国H9亚型禽流感的变异情况和流行规律,本研究汇集近年来从我国部分省市养殖场分离的10株禽流感H9N2亚型毒株,采用RT-PCR技术对其HA和NA基因进行扩增、克隆和测序,并对所得全序列进行同源性和遗传进化分析。结果显示,本试验分离到的10株病毒的HA和NA基因变异程度不大,变异概率大;在遗传进化树中均属于欧亚分支中的类CBJ194亚分支,与CBJ194的HA基因核苷酸同源性在88.8%~95.8%,NA基因核苷酸同源性在93.4%~97.6%,可能与CBJ194由同一毒株进化而来。由于基因突变使ACHB101毒株的HA基因出现了新的糖基化位点,ACHN102毒株的保守受体结合位点发生变异。NA基因推导氨基酸序列中第63、64、65位有T、EI、3个氨基酸的缺失,导致61位糖基化位点的丢失。唾液酸吸附位点上有明显的突变。HA和NA基因的变异可能与频繁的疫苗免疫选择压力有关。

In order to explore the genetic mutations of the hemagglutinin gene and the neuraminidase gene and the law of molecular epidemiology of H9 subtype avian influenza in China,ten avian influenza virus subtype H9N2 were isolated from some poultry yards in Northern China,the whole HA and NA cDNA fragments of them were amplified by reverse transcription polymerase chain reaction,with primers specific to HA gene and NA gene.After cloning and sequencing,gene sequences were analyzed on homology and heredity evolution.The sequence analysis showed little degree of variation,the various types and the high rate of mutation.They belonged to a branch of the CBJ194-like in the phylogenetic tree.The nucleotide homology of the HA gene to the CBJ194 was from 88.8% to 95.8%.The nucleotide homology of the NA gene to the CBJ194 was from 93.4% to 97.6%,so they might come from the same ancestor AIV strain.Because of the gene mutation,the new potential glycosylation site appear in the HA gene of the ACHB101 and a stenoplastic receptor-binding site have changed in ACHN102.In the deduced amino acids sequence,the 63,64 and 65 sites lost T,E and I,which cause loss of the 61 glycosylation sites.Otherwise,there was conspicuous mutation in the acetylneuraminic acid adsorption sites.These findings may be fruitful for making further study of genetic mutations of AIV in the field and improving the control strategies.