Identification Of Porcine Pseudorabies Virus Variant Strain HNJY And The Analysis Of Biological Characteristics

Pseudorabies(PR)is an acute and febrile infectious disease caused by Pseudorabies Virus(PRV)in a variety of livestock and wild animals with major clinical symptoms such as fever,itching,acute encephalomyelitis and reproductive disorders.However,since the end of 2011,large-scale farms that have been immunized with PRV vaccine have experienced a pseudorabies epidemic.In January 2018,an outbreak of suspected pseudorabies symptoms occurred in a large-scale pig pseudorabies immunized pig farm in Jiyuan City,Henan Province.This experiment was carried out by PCR to identify the suspected PR material and inoculated to PK-15.Pathogen isolation,plaque purification,virus titer determination,virus growth curve determination,and pathogenicity test of PRV strains in mice;PCR amplification of isolates gE,gB,gC,gD and TK genes,and The sequences were aligned and analyzed by establishing a genetic evolution tree.The PRV gE identification of the suspected PR infection from a pig farm in Jiyuan City,Henan Province was carried out to preliminarily determine the PRV wild-type infection;the disease material was ground,filtered and sterilized,and then inoculated onto PK-15 cells,inoculated 24-48h,PK-15 cells could be observed under the microscope,and the cells became round and detached,and the medium became turbid and other obvious lesions.After purification by plaque,a strain of PRV wild strain was obtained and named as HNJY;A wild strain of PRV was purified by plaque and named HNJY.The result of the PRV HNJY TCID50 strain was 10-8.7/0.1 mL.The results of the pathogenicity experiment in mice showed that after the HNJY virus was inoculated into the mice,the mice would develop itching symptoms and die.The LD50 result was 10-5.31 TCID50.The gE,gB,gC,gD and TK genes of HNJY strain were sequenced with reference strains at home and abroad,The results showed that the nucleotide and amino acid homology of gE,gB,gC and gD genes of HNJY strain were 99.2%-99.9%and 98.6%-100%,99.7%-100.0%and 99.2%-100.0%,respectively.96.9%-100%and 94.6%-100%,99.4%-99.9%and 99.3%-99.8%,And it was higher than that of European and American strains.gB and gC genes isolated after 2011.The homology of nucleotides and amino acids of domestic variant strains was 99.9%-100%and 99.8%-100%,99.8%-100.0%and 99.4%-100.0%,respectively,which was higher than that of domestic isolates and European and American strains before 2011.The homology of TK gene with all domestic and foreign strains of nucleotides and amino acids is as high as 99.4%-100%,TK gene is highly conserved.The results of amino acid evolution tree showed that the amino acid evolution tree of gE and gB gene was divided into two major branches:European and American strains and domestic strains,and the HNJY strain was in the same branch as the domestic strain,and the amino acid evolution tree of gC gene was obviously related to the isolation age of the strain.The strains were on the same small branch as the domestic mutants isolated after 2011,and the gD and TK genes were on the same branch as all the domestic mutants.The amino acid comparison of gD gene between HNJY strain and reference strain at home and abroad,the mutation from alanine to threonine at position 256 further indicated that the isolated strain HNJY belonged to an independent domestic variant strain.