Study On The Pathogenicity And Genomic Characterization Of A Pseudorabies Virus Strains FJ-2012

Pseudorabies(PR)caused by Pseudorabies virus(PRV)is a major infectious disease in the pig breeding industry.Since PR epidemics in pig farms all over the world,it had caused great economical loss.In late 2011,PR outbreaked in gE-deleted-vaccinated farms again,the disease rapidly spread throughout China.In order to understand the pathogenicity and genomic characterization of novel epidemic PRV,we had successfully isolated and identified a wild-type PRV that was collected from a suspected pseudorabies farm in Fujian Province.Then we analyzed the virulence of it by a series of animal infection experiments,sequenced the complete genome sequences and analyzed the bioinformatics analysis.1 The isolation and identification of PRV FJ-2012In this research,we first identified the pathological sample collected from a suspected pseudorabies farm and confirmed the disease was caused by PRV.Then we inoculated PK-15 cells with a homogenized tissue supernatant,and did some works of viral identification tests.The result showed that the elassical CPE of herpesvirus lesions appeared after infection and the typical herpesvirus particles structural morphology was visible observed in the electron microscope test.The infected PK-15 cells showed positive fluorescence with antibodies against PRV in IFA,and the fluorescence intensity of isolate is high than classical strains PRV Min-A.The titer of this strains that had purified 6 times propagated in PK-15 cells was amount to 10-9.40/0.1mL.Then the new isolate PRV was named as FJ-2012 strains.These results indicated that the farm existed PRV infection and the reason of death was caused by PRV.2 The pathogenicity of PRV FJ-2012To further explore the pathogenicity of PRV FJ-2012,three kinds of animal models of rabbits,B ABL/c mice and serological negative of PRV pigs were used in this research.And the classical strains PRV Min-A was used as the reference to compare the differences between two isolate strains by artificial infection test and histological analysis.The rabbits showed typical clinical symptoms and temperature was up to 42.2? after infection 40 hours.The median lethal dose(LD50)of PRV FJ-2012 was 102 69 TCID50 which lower than that in PRV Min-A(103.87 TCID50).Moreover,the pigs in PRV FJ-2012 strains group was observed with high mortality,high temperature,significant clinical symptoms and necropsy lesions compared with PRV Min-A group.The histological analysis in PRV FJ-2012 group showed strong pathological changes.These results prompted that the pathogenicity of PRV FJ-2012 was more enhanced than PRV Min-A.3 Genomic characterization of PRV FJ-2012To further understand the genomic characterization of PRV FJ-2012,the complete genome sequences was sequenced with Pacific Biosciences RS ? sequencer,using single-molecule long-read sequencing technology.Then we used bioinformatics software to analyze the differences between PRV FJ-2012 with other strains that downloaded at GenBank.The sequences analysis indicated that PRV FJ-2012 showed high variation with other strains.The main genes sequences of immune,virulence and immune evasion showed insertions,deletions and substitutions in different degrees.Furthermore,analysis of hydrophilicity,antigenic index and surface probability of the gB protein show that there are three differences with classic vaccine strains(Ea?Bartha),some amino acid residues differences of gE protein between isolates and other reference strains,and great variations between vaccine strains and novel Chinese strains.Result above revealed the genomic characterization and potential epidemic source of pseudorabies viru strains FJ-2012.This study provided an effective reference for the genetic evolution of PRV and laid a scientific foundation for the development of new vaccines.