猪伪狂犬病病毒分离鉴定及gE和gC基因遗传进化分析

doi:10.16431/j.cnki.1671-7236.2025.05.030

摘要/Abstract

摘要： 【目的】探究伪狂犬病病毒(PRV)野毒株的变异情况及流行趋势，为PRV新型疫苗的研究提供数据参考。【方法】采集河北省保定市规模化养殖场经Bartha-K61疫苗免疫猪中疑似感染PRV的病料组织，通过PCR方式进行病原检测，将仅PRV阳性的组织滤液接种PK-15细胞进行病毒分离与纯化，通过间接免疫荧光试验和电镜观察对分离到的病毒进行鉴定，测定分离毒株的病毒滴度并绘制生长曲线，对分离毒株进行血清中和试验、动物致病性研究及gE、gC基因序列分析。【结果】分离株在PK-15细胞中产生典型的拉丝、圆缩、聚集和脱落等病变。纯化后的病毒传代培养至F10代，经PCR隔代检测均能扩增出大小约为742 bp的目的条带，将分离株命名为BD-HB-2024。间接免疫荧光试验鉴定可见绿色荧光标记的细胞；透射电镜下观察到病毒颗粒直径约为150 nm，呈圆球状，符合PRV典型粒子特征。依照Reed-Muench法测定该毒株的半数组织培养感染剂量(TCID₅₀)为10^5.55/0.1 mL。血清中和试验发现，抗Bartha-K61株的血清对该分离株的中和抗体效价为1∶11.22，而抗变异株血清的中和抗体效价为1∶89.13。该毒株对小鼠有一定的致病性，F10代病毒对小鼠的半数致死量(LD₅₀)为10^-2.5 TCID₅₀/0.1 mL。通过与国内PRV变异毒株gE、gC基因序列比对发现，核苷酸序列相似性分别为99.8%～99.9%和99.8%～100%。遗传进化树分析表明，该分离毒株与经典毒株SC、Italy2014和Kaplan等相似性较低，亲缘关系较远；与国内近几年分离到的变异株HNB、HLJ8、JM和SX1911等相似性较高，亲缘关系较近且位于同一进化分支，比对氨基酸序列后发现符合国内变异株的典型变化。【结论】本试验证实了该养殖场存在PRV的感染，并成功分离到1株PRV变异毒株，该研究为后续新型疫苗的研发和免疫防控方案的制备提供了参考依据。

关键词: 伪狂犬病病毒(PRV); 变异株; 分离鉴定; 遗传进化

Abstract: 【Objective】 The purpose of the trial was to explore the variation and epidemic trend of Pseudorabies virus (PRV) wild strains,and provide data reference for researching new vaccines against PRV.【Method】 In this study,samples from pigs suspected to be infected with PRV which immunized with Bartha-K61 vaccine were collected from large-scale farms in Baoding city,Hebei province.Pathogen detection was performed by PCR.The filtrate of tissues with only PRV positive was inoculated with PK-15 cells for virus isolation and purification.The isolated virus was identified by indirect immunofluorescence assay and electron microscope observation.The virus titer of the isolated strain was determined and the growth curve was drawn.Serum neutralization test,animal pathogenicity study and gE and gC gene sequence analysis were performed.【Result】 The results showed that the isolate produced typical lesions such as wiredrawing,rounding,aggregation and shedding in PK-15 cells.The purified virus was subcultured to F10 generation,and the target band of about 742 bp could be amplified by PCR alternate generation detection.The isolate was named BD-HB-2024.The green fluorescent labeled cells were identified by indirect immunofluorescence assay.Transmission electron microscopy showed that the virus particles were about 150 nm in diameter and were spherical,which was consistent with the typical particle characteristics of PRV.According to Reed-Muench method,the TCID₅₀ of the strain was 10^5.55/0.1 mL.Serum neutralization test showed that the neutralizing antibody titer of the serum against Bartha-K61 strain was 1∶11.22,while the neutralizing antibody titer of the serum against the variant strain was 1∶89.13.The strain had certained pathogenicity to mice,and the median lethal dose (LD₅₀) of F10 generation virus to mice was 10^-2.5 TCID₅₀/0.1 mL.The nucleotide sequence similarity was 99.8%-99.9% and 99.8%-100% compared with the gE and gC gene sequences of domestic PRV reference strains,respectively.Genetic evolutionary tree analysis showed that the isolated strain had low similarity with classical strains such as SC,Italiy2014 and Kaplan,and was far related.It had high similarity with the variant strains HNB,HLJ8,JM and SX1911 isolated in China in recent years.It was closely related and located in the same evolutionary branch.After comparing the amino acid sequence,it was found that it conformd to the typical changes of domestic variant strains.【Conclusion】 This test confirmed the existence of PRV infection in the farm,and a PRV variant strain was successfully isolated.This study provided reference for the subsequent research and development of new vaccines and the preparation of immune prevention and control programs.

Key words: Pseudorabies virus (PRV); mutant strain; isolation and identification; genetic evolution

中图分类号:

S852.65⁺1

武月, 石兴亚, 张帅, 赵云环, 郭立明, 左玉柱, 范京惠. 猪伪狂犬病病毒分离鉴定及gE和gC基因遗传进化分析[J]. 中国畜牧兽医, 2025, 52(5): 2266-2277.

WU Yue, SHI Xingya, ZHANG Shuai, ZHAO Yunhuan, GUO Liming, ZUO Yuzhu, FAN Jinghui. Isolation and Identification of Porcine Pseudorabies Virus and Genetic Evolution Analysis of gE and gC Genes[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2266-2277.

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