上海市三株H9N2鸭禽流感病毒全基因遗传进化分析

为了解上海市鸭群中H9N2亚型禽流感病毒（Avian influenza virus,AIV）的遗传变异特征,以及与疫苗株A/Chicken/Shan dong/6/1996和A/Chicken/Shanghai/F/1998之间的遗传距离,对2007年和2009年分离自上海市鸭气管和泄殖腔样品采用荧光RT-PCR检测,将H9亚型禽流感病毒核酸阳性样品处理后,经鸡胚尿囊腔接种分离病毒,HI进一步确定血凝素（haemagglutin,HA）亚型,随后进行了全基因测序,并结合GenBank中的相关序列进行遗传进化分析。结果表明：3株分离毒株为H9N2亚型鸭禽流感病毒,HA蛋白裂解位点的氨基酸组成为PARSSRGLF,符合低致病性禽流感病毒特征,均属于经典的H9N2 Ck/Bei群系;NA基因均属于Y280系;NP、PA基因和A/Goose/Guangdong/1/1996（H5亚型）归为一群;PB2和M基因属于Qa/HK/G1/97系;NS基因仍为Ck/Bei系;2007年的分离株和2009年的分离株在PB1基因上分属不同亚群。3株病毒的HA1基因与疫苗株A/Chicken/Shandong/6/1996和A/Chicken/Shanghai/F/1998之间的遗传距离均大于7%。由此可见,3株鸭H9N2亚型毒株可能是由不同禽流感病毒基因亚群间发生自然重排的产物,现有疫苗对分离株的保护性需要进一步评估。

PHYLOGENETIC ANALYSIS OF THREE H9N2 SUBTYPE AVIAN INFLUENZA VIRUS ISOLATES FROM DUCKS IN SHANGHAI

To explore genetic variation of field isolates of H9N2 subtype Avian influenza virus （AIV） and their genetic relationship with the vaccine strains （A/ChiCken/Shandong/6/1996 and A/Chicken/Shanghai/F/1998）, oropharyngeal and cloacal samples were collected from ducks in Shanghai in 2007 and 2009. These samples were tested for H9 subtype AIV in Real-time RT-PCR and then positive samples were inoculated into 10-day-old SPF chicken eggs for virus isolation. Three AIV isolates were obtained, one from the sample collected in 2007 and two from the samples collected in 2009. These three isolates were further identified in HI test. As a result, three isolates were all AIV H9N2 Subtype. Subsequently, cDNAs of eight gene segments of these isolates were amplified in RT-PCR and sequenced for phylogenetical analysis as compared with the relevant sequences from GenBank. All three isolates were grouped into low pathogenic AIV as the cleavage site of their haemagglutinin （HA） was PARSSRGLF. Sequencing results showed that the three isolates belonged to the Ck/Bei sublineage based on HA and NS genes or to the same clade with A/Duck/Y280/1997 based on their NA genes. Additional phylogenetic analysis of their NP and PA genes revealed that they were in the same evolutionary branch with A/Goose/Guangdong/1/1996 （H5 subtype） while PB2 and M genes belonged to Qa/HK/G1/97 lineage. However, the genetic origin of PB1 gene from the isolate of 2007 was different from two isolates of 2009. The genetic distances between these three isolates and the vaccine viruses （A/Chicken/ Shandong/6/1996 and A/Chicken/Shanghai/F/1998） were more than 7%. The results from the present study suggested that these three duck H9N2. subtype isolates might have emerged through natural gene reassortment among AIVs with different sublineages.