野生鸟类新城疫病毒分离株的生物学特性和系统发育分析

Molecular characterization and phylogenetic analysis of Newcastle disease virus isolates from healthy wild birds

为了掌握野鸟新城疫病毒(NDV)的流行情况,2016年对山东东营黄河口湿地野鸟NDV进行了流行病学调查,共采集了352份健康野鸟的泄殖腔拭子,在鸡胚上进行病毒的分离鉴定,然后对分离株进行了生物学特性和遗传进化分析。结果显示,共分离到9株NDV,其中8个毒株MDT和ICPI分别为>96h和≤0.5,为低毒力株;1个毒株MDT和ICPI分别为79h和1.62,为中等毒力株。对F基因测序及序列分析显示9个分离株F蛋白裂解位点处氨基酸序列为^(112)E-R-QE-R-L^(117)或^(112)G-R-Q-G-R-L^(117),具有典型的弱毒株特征。F基因系统发育分析将9个分离株分为ClassⅠ和ClassⅡ两类,其中6个分离株属于ClassⅠ,3株属于ClassⅡ,进一步分析显示6株ClassⅠ毒株中2株属于基因1.2型,4株属于基因1.1.1型,3株ClassⅡ毒株中1株属于基因Ⅰ型,2株属于基因Ⅱ型。结果表明,野鸟携带NDV非常普遍且基因型较广泛,Ⅰ类NDV仍是野鸟中的优势基因型。分离到2株与疫苗株LaSota同源的毒株,其中虽然1个分离株F蛋白裂解位点氨基酸基序与疫苗株LaSota一致,但生物学特性显示为中等毒力株,该毒株可能是在疫苗的选择压力下进化而来,对环境及家禽存在潜在的威胁。本研究为进一步了解野鸟携带NDV与家禽NDV传播、暴发之间的联系提供了依据。

In order to understand the epidemiology of Newcastle disease virus in wild birds,352 cloacal swabs of healthy wild birds were collected from wild birds in the Yellow River Estuary Wetland of Dongying,Shandong Province in 2016.The virus was isolated and identified on chicken embryos,and the biological characteristics and genetic evolution of the isolates were analyzed.Results showed that nine Newcastle disease virus strains were isolated,among them,eight strains were lentogenic with MDT and ICPI>96 h and≤0.5,respectively,and one strain was mesogenic with MDT and ICPI of 79 h and 1.62,respectively.Sequence analysis of the F gene indicated that the amino acid sequences at the cleavage site of F protein of nine isolates were ^(112)E-R-Q-E-R-L^(117)or ^(112)G-R-Q-G-R-L^(117),which had typical characteristics of lentogenic strains.Phylogenetic analysis of F gene showed that nine isolates were divided into classⅠand classⅡ,of which six belonged to classⅠand three belonged to classⅡ.Further analysis showed that among the six classⅠisolates,two belonged to genotype 1.2,four belonged to genotype 1.1.1.Among the three strains of ClassⅡvirus,one belongs to genotypeⅠand the other two belong to genotypeⅡ.The results indicated that NDV carried by wild birds was very common and the genotypes were diverse.ClassⅠNDV was still the dominant genotype in wild birds.Two isolates were homologous to vaccine strain LaSota,and one of them was a mesogenic strain despite its amino acid motif of F protein cleavage site was consistent with LaSota,it may have evolved under the pressure of vaccine selection,which may pose a potential threat to the environment and poultry.This study provides a scientific basis for further understanding the relationship between Newcastle disease virus carried by wild birds and the spread and outbreak of Newcastle disease virus in poultry.