Identification And Analysis Of Mutated Virulence Genes In LN/1301 Porcine Pseudorabies Virus

ObjectiveIsolation and identification of porcine pseudorabies virus strains in Liaoning. And analysis of its virulence genes genetic variation.Aims to identify the source of infection and pathogen mutation in Liaoning Province. It provides the basis and evidence for the targeted-diagnosis of establishing and method of controlling.MethodsFor the diagnosis of suspected cases by pathological examination and PCR testing.Vero cells were isolated and cultured. There are some mothods to identification of isolated viruse, for example electron microscopy, virus neutralization test, polymerase chain reaction, virus TCID50 assays and animal experiments. It analyzes of mutated virulence gene PRVg E and TK by PCR, cloning, sequencing and related bioanalysis software.ResultsSuspected cases of porcine pseudorabies by pathological examination and PCR detection diagnosed PR. The results showed that the main pathology observed necropsy lesions characterization was hemorrhage and necrotic spots of gray in meninges, lung, liver, kidney, spleen, lymph. There are some main histopathological changes including nonsuppurative encephalitis, interstitial pneumonia and hemorrhage and necrotic in organs above. Immunohistochemistry showed that the viruse was founded in a variety of organs, and consistent with the above-mentioned organ lesions occur. Collecting diagnosis brain tissue diseased lesions typical cells in porcine after seeded 16 hours. Observation under the electron microscope shows located vesicles were roughly circular, with envelope, mature virion size of 150~180nm,and effective and protective effect by PRV standard serum neutralization test. Virus titer was TCID50 for 10-6.565TCID50/m L. Pathogenicity tests in mice showed typical symptoms of pseudorabies and death, the brain tissue from died mice PRV gene was detected by PCR. Application of the PRV standard strain primers designed for isolated virus g E gene and TK gene was amplified by PCR, TA cloning and sequencing obtained target gene sequence. Through the above gene phylogenetic tree analysis indicates that the strains and reference strains homology abroad was more than 97%. Sequence analysis revealed the presence of the isolated gene PRVg E 995 point mutations by the C-A mutation, the 999 point mutations by T-G mutation, TK gene dose not mutation. Amino acid sequence alignment showed that there is a change in L-Q sites, leucine changes into glutamine, TK gene have not deletion and variation.Conclusions1.One case of porcine pseudorabies was confirmed by pathological observation combining with PCR testing. By isolating and culturing Vero cells, virus neutralization test, observation in electronic morphology and genetic testing, the porcine pseudorabies strain wild strains was success of the isolated in Liaoning Province, and then named PRVLN/1301.2. Major virulence genes g E and TK of pseudorabies virus Liaoning PRVLN/1301 was amplified successfully. By sequence and phylogenetic tree analysis showed that the strains and reference strains homology abroad in more than 97%.Description of the strain and other strains of the same source region, but there are some variation sites.