一株鸭源禽流感病毒（H9N2）全基因序列分析及其排毒规律的研究

Sequence Analysis of A Strain H9N2 Subtype Avian Influenza Virus and the Dynamics of Viral Shedding of Ducks

关键词:  H9N2亚型AIV、生物学特性、抗原相关性、序列分析、排毒

Key words:  H9N2 subtype avian influenza virus  biological characteristics  cross-hemagglutinin-inhibition(HI) assay  sequence analysis  shedding

摘要:

为了研究实验室分离的一株鸭源H9N2亚型AIV（JN1株）全基因组的分子特征和遗传进化情况以及其在鸭体内的排毒规律，方法：对其进行了生物学特性分析、HI交叉抑制实验、全基因组测序以及遗传进化分析，通过点眼滴鼻感染3周龄健康雏鸭，利用建立的实时荧光定量（RT-PCR）检测鸭的排毒规律，并测定血清HI抗体效价。结果：该毒株的鸡胚半数感染量EID50为10-7.54/0.2mL，鸡胚最小致死量的平均死亡时间MDT为72h，静脉致病指数IVPI为0.266，抗原相关系数R=0.47。系统发育分析表明，JN1株的HA与CK/HK/G9/97和DK/HK/Y280/97在同一分支上，NA、M、NP、PA、PB1、PB2基因与SH/F/98同源性较高，NS基因则与H5N1亚型AIV进化关系较近。HA的裂解位点为PSRSSR↓GL，存在6个潜在糖基化位点，在234位的氨基酸残基为L，NA基因颈部存在缺失。雏鸭在感染该病毒后，3~17天均由排毒，咽拭子和泄殖腔棉拭子排毒规律基本一致，分别在第3天和第13天出现排毒高峰，咽拭子比泄殖腔棉拭子排毒量高。结论：该H9N2亚型AIV属欧亚大陆分支，生物特性分析为低致病性禽流感，在实验室条件下，对鸭的致病力较弱，病毒可通过呼吸道和泄殖腔向外界排毒，排毒时间较长。

Abstract:

To analysis the genetic characteristics and dynamics of viral shedding of a strain of duck-origin H9N2 subtype avian influenza separated from the laboratory,The biological characteristics and a cross-hemagglutinin-inhibition(HI) assay were analyzed，the full genome was amplified,and then sequenced and photogenically analyzed.Three weeks old ducklings were infected by intranasal inoculation,SYBR Green Ⅰ fluorescence quantitative RT-PCR analysis of viral shedding.The results showed that the EID50 was 10-7.54 / 0.2mL, the MDT was 72h, the IVPI was 0.266, and the HI correlation indices (R) exceeded 0.47.HA gene was located on the same branch with CK/HK/G9/97 and DK/HK/Y280/97,NA,M,NP,PA,PB1,PB2 genes had higher homology with SH/F/98, the cleavage site of HA gene was PSRSSR↓GL,6 potential glycosylation were found on HA gene,the 234 site amino acid was L.The neuraminidase gene had deletions at 63,64and 65 amino acids.Viral RNA can detected in cloaca and throat swabs from the third day to seventeenth day.the content of AIV in both cloaca and throat swabs is higher on the third and thirteenth day.the throat swabs were higher than cloaca swabs.The results showed that duck-origin H9N2 subtype avian influenza belongs to Eurasia branch, experimental infection showed low pathogenicity to ducks, and can be shed into the environment through throat and cloaca for a long time and can invade all major organs.