产蛋鸡传染性支气管炎病毒的分离鉴定及其遗传变异和血清型分析

Isolation and identification of an infectious bronchitis virus from laying hens and analysis of its genetic variability and serotype

【目的】明确产蛋鸡源传染性支气管炎病毒(IBV)分离株(GX-YL170808)的结构基因及抗原变异情况,为广西种鸡场的传染性支气管炎(IB)防控提供科学依据。【方法】通过鸡胚尿囊液血凝试验、鸡胚侏儒化试验及3'端非编码区(3'-UTR)测序对分离株进行鉴定;采用RT-PCR扩增S1、E、M和N基因,以MegAlign和MEGA 6.0分别进行核苷酸序列相似性及系统发育进化树分析,运用RDP4和SimPlot对S1、E、M和N基因进行重组分析,利用NetNGlyc 1.0和NetOGlyc 4.0进行糖基化位点预测分析,并通过中和试验进行血清型鉴定。【结果】分离株的鸡胚尿囊液血凝试验呈阴性,鸡胚盲传5代后出现侏儒胚典型病变,其3'-UTR序列与IBV的3'-UTR序列相似性为99.13%;综合病鸡临床症状及其病理变化可确定该病毒为IBV,命名为GX-YL170808。GX-YL170808分离株S1、E、M和N基因的开放阅读框(ORF)长度分别为1620、327、675和1230 bp,对应编码540、109、225和410个氨基酸残基,与参考株的核苷酸序列相似性分别为60.4%~96.5%、81.8%~97.2%、85.6%~93.5%和85.7%~92.0%;GX-YL170808分离株的S1、M和N基因属于LX4型,而E基因属于LDT3型;4个结构基因内部均无重组区域;除S1基因同时具有N-糖基化和O-糖基化位点外,E、M和N基因均只有N-糖基化位点;S1蛋白裂解位点为HRRRR,与参考株LX4的裂解位点相同。GX-YL170808分离株属于血清4型,不同于常用的疫苗株H120(血清3型)和4/91(血清5型),也不同于广西主要侵害雏鸡的IBV优势血清型。【结论】产蛋鸡源IBV分离株并非疫苗株,其基因型和血清型均已发生变异,且该毒株的血清型不同于广西地区侵害雏鸡的优势血清型,提示了广西地区IB防控的严峻性及新型多价疫苗研发的紧迫性。

【Objective】The objective was to identify the structural genes and antigenic variation of infectious bronchitis virus(IBV)isolate GX-YL170808 from laying hens,and to provide scientific basis for the prevention and control of infectious bronchitis(IB)in breeder farms in Guangxi.【Method】The virus was identified by hemagglutinin test,chicken embryo dwarf test and gene sequence analysis of the 3'untranslated region(3'-UTR).The S1,E,M and N genes were amplified by RT-PCR,and the nucleotide sequence similarity and phylogenetic tree analysis were carried out by MegAlign and MEGA 6.0;the recombined analysis of S1,E,M and N genes were conducted by RDP4 and SimPlot;the glycosylation sites prediction analysis were carried out with NetNGlyc 1.0 and NetOGlyc 4.0,and serotype identification was carried out by neutralization test.【Result】The results showed that the hemagglutination test was negative and dwarf embryo appeared typical lesions after five generations;the similarity between isolate GX-YL170808 and reference IBV strains in 3'-UTR was 99.13%.According to the clinical symptoms and pathological changes of the chickens,the virus was determined as IBV,named GX-YL170808.The open reading frame(ORF)length of S1,E,M and N genes of GX-YL170808 isolate were 1620,327,675 and 1230 bp,corresponding to 540,109,225 and 410 amino acid residues.The nucleotide sequence similarity between S1,E,M and N genes of GX-YL170808 isolate and reference strain were 60.4%-96.5%,81.8%-97.2%,85.6%-93.5%and 85.7%-92.0%.The S1,M and N genes belonged to LX4 type,while E gene belonged to LDT3 type.There was no recombination region in the S1,E,M and N structural genes.The E,M and N genes had only N-glycosylation sites except S1 gene had both N and O-glycosylation sites,and the S1 protein cleavage site of the isolate was HRRRR,which was the same as the reference strain LX4.The GX-YL170808 isolate belonged to serotype 4,which was different from those of vaccine strains H120(serotype 3)and 4/91(serotype 5),and also different from the dominant serotype of IBV which mainly affected the chicks in Guangxi.【Conclusion】The IBV isolate GX-YL170808 from laying hens is not vaccine strain,and its genotype and serotype have mutated,and the serotype of this strain is different from the dominant serotype which affects the chicks in Guangxi,suggesting that the severity of IBV prevention and control in Guangxi and the urgency of new multivalent vaccine development.