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

Abstract

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

CLC Number:

S852.65

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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Isolation,Identification and Genetic Characterization of Equine Herpesvirus Type 8 from Donkey

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