1株赛鸽新城疫强毒株的致病特性及分子特征分析

Pathogenicity Analysis and Molecular Characterization of Newcastle Disease Virus Isolated from Racing Pigeon

为了解赛鸽群中新城疫流行毒株的分子遗传特征及变异情况,从河北省疑似赛鸽新城疫病例中采集病料,常规处理后接种SPF鸡胚,经血凝试验(HA)和血凝抑制试验(HI)鉴定病毒,并对分离毒株进行致病性试验、毒力测定,采用RT-PCR方法扩增F基因,并与其他不同种属新城疫毒株进行遗传进化发育分析和氨基酸序列同源性分析。结果显示,从病料中分离到1株新城疫病毒(命名为HB株),致病性试验产生与发病鸽相似的临床症状和剖检变化;其鸡胚平均死亡时间(MDT)、1日龄雏鸡脑内致病指数(ICPI)和6周龄雏鸡静脉致病指数(IVPI)分别为52.8 h、1.78和2.59,表明HB株新城疫病毒为强毒株;HB株F基因碱基序列包含1个大小为1 662 bp的完整开放阅读框,基因分型结果显示,HB株属于基因型Ⅵ型。根据遗传进化发育关系和同源性分析结果发现,HB株与pi/CH/LGD/110208、dove/Italy/2736/00、Q-GB 506/97等毒株核苷酸序列相似性和氨基酸同源性较高,尤其与pigeon/Qinghai/1344/2017毒株亲缘关系最近,分别达98.45%和99.5%;与目前使用的传统新城疫疫苗毒株LaSota、B1、V4、Mukteswar株等遗传关系较远;F蛋白裂解位点氨基酸序列为112 R-R-Q-K-R-F 117,符合强毒株特征。以上结果表明,HB株为强毒株且与目前广泛使用的新城疫疫苗毒株遗传关系较远。

In order to understand the molecular genetic characteristics and variation of Newcastle disease epidemic strains in racing pigeon population,in this experiment,the disease material was collected from the suspected pigeon Newcastle disease case in Hebei Province.After routine treatment,the SPF chicken embryo was inoculated.The chicken embryo allantoic fluid was collected and identified by Hemagglutination and Hemagglutination inhibition test as Newcastle disease virus,and pathogenicity tests,virulence assays were conducted.The sequences of F gene were amplified by RT-PCR and genetic evolution and amino acid sequence homology with other different species of Newcastle disease virus strains were analyzed.The results showed that a Newcastle disease virus(named HB strain)was isolated from the disease material.The pathogenicity test of HB strain presented similar clinical symptoms and necropsy changes with those found in naturally diseased pigeons.The mean death time,intracerebral pathogenicity index and intravenous pathogenicity index of HB strain were 52.8 h,1.78,and 2.59,which indicated that the HB strain was a virulent strain.F gene of HB strain contained a complete open reading frame(ORF)of 1 662 bp.The genotyping results showed that the HB strain belonged to theⅥgenotype.Genetic relationship of HB strain showed that it was closed to pi/CH/LGD/110208,dove/Italy/2736/00,Q-GB 506/97 based on F gene.Especially with the pigeon/Qinghai/1344/2017 strain,the closest relativities of nucleotide and amino acid sequences were 98.45%and 99.5%.However,it had far genetic relationship with traditional chicken Newcastle disease vaccine strains of B1,LaSota,V4 and Mukteswar.The F protein cleavage site amino acid sequence of HB strain was 112 R-R-Q-K-R-F 117,which was consistent with the characteristics of virulent strains.The results indicate that the HB strain is a virulent strain and has a large genetic difference from the current widely used Newcastle disease vaccine strains.