鹅源class I类NDV生物学特性的研究及荧光定量RT-PCR检测方法的建立  

Biological characteristics of a class I newcastle disease virus isolated from goose and development of fluorescence quantitative RT-PCR assay

作　　者：李长宇 孙军峰 赵冉 王芳芳 韩宗玺[2] 刘胜旺[2] LI Chang-yu;SUN Jun-feng;ZHAO Ran;WANG Fang-fang;HAN Zong-xi;LIU Sheng-wang(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;Division of Avian Infectious Diseases,State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/禽呼吸道传染病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第9期923-930,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金团队项目(TD2021C001);蛋鸡产业技术体系岗位科学家项目(CARS-40)。

摘　　要：为了解鹅源class I类新城疫病毒(NDV)的生物学特性,本研究以2022年在安徽活禽市场的鹅中分离到的AH713/22株为研究对象,测定其全基因组序列,分析其基因组分子特征和遗传进化特性;将AH713/22株感染鸡胚,接种1日龄雏鸡脑内,分析其致病性;利用制备的单因子血清进行交叉血凝抑制试验,分析其抗原性;将AH713/22株56℃水浴不同时间后检测HA效价,分析其耐热特性。结果显示,AH713/22株基因组长15198 bp,具有典型的class I类NDV的基因组结构和分子特征,属于基因1.1.2亚型。F蛋白裂解位点为ERQERL,HN蛋白长度不同于已往报道的病毒株,含有618个氨基酸;鸡胚平均死亡时间(MDT)为134 h,1日龄雏鸡脑内接种致病指数(ICPI)为0.32,表明AH713/22属于弱毒株;抗原性检测结果显示AH713/22株与class II类基因II型NDV以及class I类代表性NDV之间存在不同程度的抗原性差异;耐热性试验显示AH713/22株在56℃热处理60 min后HA效价仍无显著变化,表明AH713/22株具有优良的热稳定性。进一步基于class I类基因1.1.2亚型NDV F基因的序列比对分析,针对其保守区域设计引物和探针,建立了荧光定量RT-PCR检测方法,结果显示该方法对质粒标准品的检测限为10^(2)拷贝数/μL,且能够特异性的检测class I类1.1.2亚型NDV,与其他常见的禽呼吸道病原核酸均无交叉反应。该方法用于临床样品检测结果与常规PCR检测结果一致。本研究系统鉴定了一株鹅源class I类NDV的遗传和分子生物学特性,加深了对我国class I类NDV遗传演化的认知,并建立了适用于我国优势基因型(1.1.2亚型)NDV的检测方法,为class I类NDV的监测和流行病学调查提供了技术支撑。In order to understand the biological characteristics of a goose origin class I Newcastle disease virus(NDV),named AH713/22,which was isolated from a goose in a live poultry market in Anhui in 2022.We determined whole genomic sequence of AH713/22 and analyzed its the genomic molecular characteristics and genetic evolution.Moreover,we assessed the pathogenicity,antigenicity and heat resistance of this strain.The results showed that the AH713/22 strain has a genomic length of 15198bp,exhibiting the typical genomic structure and molecular characteristics of class I NDV,and its belongs to genotype 1.1.2.The cleavage site of F protein was identified as ERQERL,and the length of HN protein was different from that of all previously reported NDVs,containing 618 amino acids.The mean death time(MDT)was 134 hours,and the intracerebral pathogenicity index in 1-day-old chicks(ICPI)was 0.32,classifying AH713/22 as an avirulent strain.We prepared the monofactor serum of AH713/22 and conducted a cross hemagglutination inhibition test,which indicated varying degrees of antigenic differences between AH713/22 and class II genotype II strains and representative strains of class I.In addition,the heat resistance test showed that the HA titer of AH713/22 remained unchanged after heat treatment at 56℃for 60 minutes,indicating that AH713/22 possesses excellent thermostability.Furthermore,based on the sequence comparison analysis of the 1.1.2 genotype class I NDV F gene,one pair of primers and probe target for the conserved sequence of F gene were designed,and a fluorescence quantitative RT-PCR assay was developed.This assay is capable of detecting at least 10^(2) copies/μL template and does not cross-react with other common avian respiratory pathogens.This study systematically identified the genetic and biological characteristics of a class I NDV isolated from a goose,enhancing our understanding of the genetic evolution of class I NDV in China.The established assay was suitable for the detection of dominant genotype(subtype 1.1.2)in C

关 键 词：新城疫病毒 鹅 分子特征 抗原性 热稳定性 荧光定量RT-PCR方法 

分 类 号：S852.65[农业科学—基础兽医学]