驴源马疱疹病毒8型的分离鉴定与生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.08.036

摘要/Abstract

摘要： 本研究旨在分离1株驴源马疱疹病毒8型（Equine herpesvirus type 8，EHV-8）毒株并分析其遗传特征。从山东省聊城地区某驴场采集病驴肺脏组织，经PCR方法鉴定EHV-8的感染情况；对EHV-8感染阳性的肺组织进行研磨，经反复冻融后接种于兔肾细胞（RK-13）细胞中，盲传3代，待出现细胞病变（CPE）时收集细胞，并通过PCR、间接免疫荧光试验、透射电镜等技术对EHV-8进行鉴定。利用PCR扩增获得分离株的ORF70全基因组序列，并进行生物信息学分析。研究结果显示，经PCR鉴定获得EHV-8感染阳性的肺脏组织，病料接种易感细胞RK-13后出现典型CPE，分别收集前3代的细胞培养物，经PCR扩增，均获得与预期大小一致的ORF70基因片段，将分离获得的EHV-8毒株命名为SDLC66，序列上传GenBank，获得登录号：MW816102。经测序和序列比对发现，SDLC66毒株与AHV-3毒株（GenBank登录号：U24184.1）ORF70基因的相似性为99%，与国内的EHV-8 wh毒株（GenBank登录号：JQ343919.1）、国外EHV-8/IR/2015/40（GenBank登录号：MF431614.1）、EHV-8/IR/2003/19（GenBank登录号：MF431611.1）参考毒株的相似性最高，均为99.8%。经遗传进化树分析发现，SDLC66与EHV-8（EHV-8 wh、EHV-8/IR/2015/40和EHV-8/IR/2003/19株）在一个小分支上，亲缘关系最近；与EHV-1型参考毒株（Hong Kong/57/1984、United Kingdom/32/1982、Oxfordshire/206/2013株）序列来源于同一大分支，亲缘关系较近，与EHV-4毒株（91c1和TH20p株）亲缘关系较远。间接免疫荧光试验可见与病毒蛋白特异结合的红色荧光信号；透射电镜观察可见直径约110 nm的圆形病毒粒子，并且核衣壳外有一层亮晕，亮晕外有一层囊膜。说明本试验成功分离得到1株驴源EHV-8毒株，为进一步研究其致病性和致病机制奠定基础。

关键词: 驴; 疱疹病毒8型; ORF70基因; 遗传变异

Abstract: The aim of the study was to isolate Equine herpesvirus type 8 (EHV-8) from donkeys and analyze genetic characteristics.The lung tissues of diseased donkeys were collected from a donkey farm in Liaocheng area of Shandong province.The infection of EHV-8 was identified by PCR.The lung tissues with positive EHV-8 infection were ground and inoculated into RK-13 cells after repeated freezing and thawing 3 times.The cells were collected when cytopathic effects (CPE) appeared and identified by PCR, indirect immunofluorescence test and transmission electron microscope.The whole genome sequence of ORF70 was amplified by PCR and analyzed by bioinformatics.The results showed that the lung tissues with positive EHV-8 infection were identified by PCR, and typical CPE appeared after inoculating the susceptible cells RK-13.The first three generations of cell cultures were collected, and the ORF70 gene with the expected size was obtained by PCR amplification.The EHV-8 strain was named SDLC66, and the sequence was uploaded to GenBank, and the accession No.was MW816102.Sequencing and sequence alignment showed that the similarity of ORF70 gene between SDLC66 strain and AHV-3 strain (GenBank accession No.:U24184.1) was 99%, and the similarity of ORF70 gene between SDLC66 strain and domestic EHV-8 wh strain (GenBank accession No.:JQ343919.1), foreign EHV-8/IR/2015/40 (GenBank accession No.:MF431614.1) and EHV-8/IR/2003/19 (GenBank accession No.:mf431611.1) reference strain was the highest (99.8%).Phylogenetic tree analysis showed that SDLC66 and EHV-8 (EHV-8 wh, EHV-8/IR/2015/40 and EHV-8/IR/2003/19) were closely related in a small branch.It was closely related to EHV-1 reference strains (Hong Kong/57/1984, United Kingdom/32/1982, Oxford/206/2013), but far from EHV-4 strains (91c1 and TH20p).Indirect immunofluorescence assay showed a red fluorescent signal specifically binding to viral protein.The round virus particles with diameter of about 110 nm were observed by transmission electron microscope, and there was a halo outside the nucleocapsid, and a capsule outside the halo.These data indicated that EHV-8 strain from donkey was isolated, which laid the foundation for the study of pathogenicity and mechanism of EHV-8.

Key words: donkey; Equine herpesviruses type 8; ORF70 gene; genetic variation

中图分类号:

S852.65

王彤彤, 刘梦媛, 张敬文, 郗灿坤, 贾淑楠, 于跃, 刘文强, 任慧英, 王长法, 李亮亮. 驴源马疱疹病毒8型的分离鉴定与生物信息学分析[J]. 中国畜牧兽医, 2021, 48(8): 3030-3037.

WANG Tongtong, LIU Mengyuan, ZHANG Jingwen, XI Cankun, JIA Shunan, YU Yue, LIU Wenqiang, REN Huiying, WANG Changfa, LI Liangliang. Isolation,Identification and Genetic Characterization of Equine Herpesvirus Type 8 from Donkey[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 3030-3037.

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