2012年上海地区发病鸡群H9N2亚型禽流感病毒的变异分析

GENETIC VARIATION OF H9N2 SUBTYPE AVIAN INFLUENZA VIRUS ISOLATES FROM DISEASED CHICKEN FLOCKS OF SHANGHAI IN 2012

为了了解2012年分离自上海发病鸡群的H9N2亚型禽流感病毒(Avian influenza virus,AIS)的遗传变异特征,采集发病鸡群中病死鸡气管、肺、脑和肾脏等内脏,经实验室诊断为H9亚型禽流感病毒核酸阳性。将阳性样品处理后,经鸡胚尿囊腔接种分离病毒,HI进一步确定HA亚型,通过RT-PCR方法分别扩增了3个分离株的8个基因片段并进行了序列分析。3株分离株均为H9N2亚型禽流感病毒,与以往从上海地区健康鸡群中分离的4株H9N2毒株相比,本研究中的3株分离株由于HA基因220位的氨基酸发生了变异,缺失了HA上218～220位的一个潜在的糖基化位点,并且HA基因受体结合位点的左缘235位发生了变异。从HA基因抗原位点的分析表明,与疫苗株Ck/SD/6/96相比,这3株H9N2亚型禽流感病毒7个抗原位点中已有4个发生了改变。遗传发生关系表明这3株分离株均属于Ck/Bei/1/94系,与08年的分离株Ck/SH/Y1/08 8个基因片段所属基因型相同。2012年上海地区发病鸡群中分离的上述3株H9N2禽流感病毒基因序列已发生变异,是否导致其生物学特性发生改变以及目前使用的疫苗能否给禽群提供足够的保护力还需要作进一步的研究。

To explore genetic variation of H9N2 subtype Avian influenza viruses(AIVs) isolated from three diseased chicken flocks in Shanghai,trachea,lung,brain and kidney samples from diseased chicks were collected and tested for H9 subtype AIV.All samples were positive and then three isolates were inoculated into 10-day-old SPF chicken eggs.Re-isolated viruses were further identified as H9 subtype in HI test.The cDNAs of eight gene segments were amplified in RT-PCR,and sequence analysis confirmed that they belonged to H9N2 subtype.As compared with previous 4 H9N2 AIV isolates from healthy chicken flocks in Shanghai,these 3 isolates obtained in 2012 had a deletion of potential glycosylation site due to a single nucleotide mutation at aa 220 in HA gene.Mutation also occurred at aa 235 on the left edge of receptor binding site in HA gene.Analysis of antigenic sites in HA gene illustrated that 4 out of 7 antigenic sites mutated as compared with the vaccine strain Ck/SD/6/96.Phylogenetic analysis of HA gene showed that these 3 H9 viruses were included in the lineage represented by Ck/Bei/1/94.These results indicated that H9N2 subtype AIV isolates from diseased chicken flocks of experienced genetic variation.Further study is needed to find out whether biological characters of these mutated viruses have changed and the currently used vaccines can provide effective protection in chickens.