Isolation And Identificaton Of Porcine Pesudorabies Virus And Established Real-time PCR Assay For Application

Swine pesudorabies virus(PRV)belongs to herpesvirus genus of herpesvirus family.PRV viron shows the ball shape appearance with 150-180 nanometer diameter.External of PRV nucleocapsid are envelop formed by lipid bilyer.PRV can infect many livestocks while pig is the majar host and reservoir.Pseudorabies(PR)is caused by PRV in pigs,and lead to heavy economic losses in all major swine-producing countries.The characterization of PR is abortion,stillbirth and mummified pigs in pregnant swine.PR characterization in mature pigs is respiratory disorders.Newborn Piglet infected with PRV show neurological signs and ataxia,and suffer from approximately 100%death rate.In china,a great many swine farms have increased the incidence of Swine Pseudorabies seine 2011,and suffered great economic losses.We perform this study to further acquire the genetic variation and the incidence of PRV.A strain of pseudorabies virus(PRV)was isolated from piglet in Jiangsu.The gB gene of PRV was amplified and sequenced to analyzed genetic evolution and antigenic analysis of this stain.Real-time fluorescent quantitative PCR(real-time PCR)method for detecting gB gene was established to detect pseudorabies,and the relationship between PRV gene copies and viral titer was acquired.Experiment 1.Phylogeny and antigenic analysis on gB gene of PRV NJ-guoyao strain isolatedIn this study,a strain of pseudorabies virus(PRV)was isolated from lung tissue of piglet in Jiangsu province,and named as NJ-guoyao strain.The homogenated sample of lung tissue was subjected to infecting BHK-21 cells,classical cytopathogenic effects of herpesvirus lesions appeared post infection 24 hours.We further identified the pathogen as PRV using PCR methods to amplify the corresponding DNA segment of PRV gB and gE gene.Phylogeny analysis of gB gene indicated that this strain was belong to a classical strain.The homology of PRV gB gene was higher than 99%between NJ-guoyao and classical strain,NIA3,Becker,and Kaplan strain.Antigenic analysis of gB protein showed that transmembrane region could be located at 255-276(22)amino acid.There were 5 potential glycosylation sites which located at 37,147,294,341 and 365 amino acid residues of gB protein,and the 294 amino acid residue was the highest possibility of glycosylation.These peptides at 10-30,320-340 and 350-367 of gB protein may be located on the surface of the protein,and could be the main part of antigen-antibody reaction reaction.Isolation of PRV NJ-guoyao strain provides materials for the study of vaccines and diagnostic method.Experiment 2.Application and establishment of Real-time fluorescent quantitative PCR method for Detecting gB gene of PRVIn order to establish a real-time PCR method for detecting gB gene of PRV,three pair of primers was designed according to the acquired gB gene sequence of PRV.The real-time PCR method was analyzed with by quantitive concentration of plasmids containing PRV gB gene.A standard curve was achieved,and the result showed that this method had good repeatability,good specificity,and did not reat with PCV2,PPV,CSFV,JEV and PRRSV.The sensitive of this method was 1×103 copies/μL,and the sensitive of conventional PCR was 1×105 copies/μL.20 samples were detected by real-time PCR,and this method could increase detection rate compared with the conventional PCR assay.The establishment of real-time PCR for detecting PRV gB gene provided an effective method for differential diagnosis and quantitative analysis of PRV.Experiment 3.The relationship between PRV gene copies and viral titer detected by Real-time PCR and CPEIn order to acquire the relationship between gene copies of PRV and viral titer,PRV titer was first detected with cytopathic effect in BHK-21 cells.The second,the gB gene copies of different PRV titer was detected by real-time PCR of PRV-gB.In the end,The relationship of viral titer and gene copies of different PRV dilution was acquired by regression analysis,linear regression equation is y = 0.978x + 4.216,y = lg(copies/mL),x=lg(TCID50/mL),and R2(correlation coefficient)=0.9993 showed good correlation.Real-time fluorescence quantitative PCR of PRV-gB become possible for detecting PRV virus titer.Swine pesudorabies virus(PRV)belongs to herpesvirus genus of herpesvirus family.