一株蛋鸡新城疫病毒的分离鉴定及全基因组序列分析

Identification and Complete Genomic Analysis of a Newcastle Disease Virus from a Laying Hen

为研究云南新城疫病毒(NDV)分子流行特性和致病性,对云南省1个蛋鸡场的组织样品进行病原分离,通过血凝、血凝抑制试验和RT-PCR检测分离毒株,证明为NDV。通过病毒最小致死量(MLD)、鸡胚半数感染量(EID 50)、鸡胚平均死亡时间(MDT)、脑内接种致病指数(ICPI)和静脉接种致病指数(IVPI)测定病毒毒力,结果EID 50为10-4.4,MLD为10-4,MDT为97.5 h,ICPI为0,IVPI为0,显示分离株为弱毒株。高通量测序及遗传进化分析发现,分离毒株基因组全长为15198 bp,从3′端到5′端有6个结构基因依次为NP-P-M-F-HN-L,各基因之间有1~48个核苷酸间隔。全基因组、F和HN基因遗传进化分析证明分离毒株属于ClassⅠ分支,F基因ORF全长为1662 bp,编码553个氨基酸,F蛋白裂解位点氨基酸序列为112-ERQERL-117,符合NDV弱毒裂解位点特征,HN基因ORF全长为1851 bp,编码616个氨基酸,在HN基因保守区域234-NRKSCS-239位氨基酸序列没有发生变异。F蛋白有6个糖基化位点比较保守,HN蛋白有6个潜在的糖基化位点,但与LaSota相比存在缺失的和多出的糖基化位点。F蛋白半胱氨酸残基分布在76、199、338、347、362、370、394、399、401、424、514、523位氨基酸,F蛋白中性抗原位点在72、75、79和170位氨基酸发生了变异,HN蛋白受体结合部位和NA活性位点氨基酸为174R、401E、416R、526Y,未发生变异。本研究首次报道了云南省蛋鸡新城疫ClassⅠ毒株的全基因序列特征,为新城疫病毒ClassⅠ毒株研究奠定了基础。

In order to study the molecular epidemic characteristics and pathogenicity of Newcastle disease virus(NDV)in Yunnan province,the pathogen was isolated from the tissue samples of an laying hens farm in Yunnan province.The isolates were proved to be NDV by hemagglutination,hemagglutination inhibition test and RT-PCR detection.The virulence of the virus was determined by the minimal lethal dose of(MLD),50%egg infectious does of(EID 50),the mean death time of(MDT),the pathigenicity index of(ICPI),and the pathogenicity index(IVPI)of intravenous inoculation.The results showed that the EID 50 was 10-4.4,MLD was 10-4,MDT was 97.5 h,ICPI was 0 and ICPI was 0,which showed that the isolates were attenuated strains.High throughput sequencing and genetic evolution analysis showed that the genomic length of the isolated strain was 15198 bp,and there were 6 structural genes from 3′end to 5′end,which were 1-48 nucleotides interval between NP-P-M-F-HN-L genes.The genetic evolution analysis of the whole genome,F gene and HN gene showed that the isolated strain belonged to ClassⅠbranch.The full length ORF of F gene was 1662 bp,encoding 553 amino acids,and the amino acid sequence of F protein cracking site was 112-ERQERL-117,which consistent with the characteristics of NDV weak virus cleavage site.The ORF of HN gene was 1851 bp,encoding 616 amino acids.There was no amino acid sequence variation at 234-NRKSCS-239 site in the conservative region of HN gene.There were six glycosylation sites in F protein and six potential glycosylation sites in HN protein,but there were missing and more glycosylation sites compared with LaSota.The cysteine residues of F protein were 76,199,338,347,362,370,394,399,401,424,514,523 amino acids,the neutral antigen sites of F protein were mutated at 72,75,79 and 170 amino acids,the amino acids of HN protein receptor binding site and NA active site were 174R,401E,416R,526Y,no mutation occurred.In this study,the whole gene sequence characteristics of Newcastle disease ClassⅠstrain in laying hens in Yunnan Province were reported for the first time,which laid a foundation for the study of Newcastle disease virus ClassⅠstrain.