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