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 H9N2 Subtype Avian Influenza Virus and the Dynamics of Viral Shedding of Ducks

为研究一株鸭源H9N2亚型禽流感病毒(JN1株)全基因组的分子特征和遗传进化情况以及感染雏鸭后的排毒规律,对其生物学特性、全基因组序列以及遗传进化进行了分析,通过点眼滴鼻方式感染3周龄健康雏鸭,利用建立的实时荧光定量PCR(RT-PCR)检测鸭的排毒规律,并测定血清抗体效价。JNI株的鸡胚半数感染量为10^(-7.54)/0.2mL,鸡胚最小致死量的平均死亡时间为72h,静脉致病指数为0.266,抗原相关系数为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亚型禽流感病毒进化关系较近。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,饮食正常,精神良好,3~17d均有排毒,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。该H9N2亚型禽流感病毒属欧亚大陆分支,为低致病性禽流感,病毒可通过呼吸道和泄殖腔向外界排毒,排毒时间较长。

To study the genetic characteristics and dynamics of viral shedding of a duck-origin H9N2 subtype avian influenza separated from the laboratory, the biological characteristics, genome sequence and phylogenetiely were analyzed. Three weeks old ducklings were infected by intranasal inoculation, SYBR Green [ fluorescence quantitative RT-PCR anlysis of viral shedding. The results showed that the EIDs0 was 10-3s4/0.2 mL, the MDT was 72 h, 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, 64 and 65 amino acids. Viral RNA can be detected in cloacal and throat swabs from the third day to seventeenth day. The content of AIV in both eloaeal and throat swabs is higher on the third and thirteenth day. The results showed that duck-origin H9N2 subtype avian influenza belongs to Eurasia branch, and can be shed into the environment through throat and cloaca for a long time.