Isolation And Identification Of Swine Pseudorabies Virus Guizhou Strain And It's GB/gE/TK Gene Molecular Feature Analysis

Pseudorabies?PR?is an acute infectious disease of pigs caused by Pseudorabies virus?PRV?.Sows are mainly found to have reproductive disorders in the clinic,producing stillbirth or mummified Sows fetuses;boar semen is with virus;piglets develop neurological symptoms until they die.In recent 30 years,the vaccine for gene deleted pseudorabies virus has been widely used in China,and the disease has been effectively controlled.However,at the end of 2011,there was an outbreak of pseudorabies in China again.In some places,epidemic strains were mutated and the pig industry in China suffered huge economic losses.The purification of porcine pseudorabies has been listed as The National Medium and Long Term Plan for Animal Disease Prevention and Control?2012-2020 years?.In this study,we collected samples of piglets suspected of being infected with pseudorabies and identified it as pseudorabies virus after infection with swine pseudorabies virus by PCR.We successfully isolated PRV by cell culture,electron microscopic observation of morphological structure,TCID₅₀0 assay,and susceptible animal inoculation experiments.Guizhou strains were cloned and gene molecular characteristics were analyzed for the gB/gE/TK gene of the isolated strains,in order to provide a theoretical basis for the purification of pseudorabies virus in Guizhou Province.1.Isolation and identification of pseudorabies virus T4 Guizhou strainBy collecting the piglet's sample of suspected porcine pseudorabies,the virus was isolated and identified after related treatment,and the morphological structure of cell culture,electron microscope observation,TCID₅₀0 assay,susceptible animal inoculation experiment,and the homology analysis of viral nucleic acid sequences were performed.Identification,the results showed,when Vero cells were inoculated into the diseased cells,they passed to the 4th generation and they had a significant cytopathic effect?CPE?60 h after the virus was inoculated.When they passed to the 6th generation,stable CPE appeared;electron microscopic observations revealed that the isolated strain had the common morphological characteristics of herpes virus.Transmission electron microscopy showed that the virus particles were elliptical or round,and the nucleus was a lattice-like inclusion body with a diameter of approximately 120 to 150 nm.Intracytoplasmic and nucleus enveloped mature virus particles with a diameter of 150-180 nm;after the sixth passage of the virus was inoculated into the rabbits,two rabbits in the experimental group showed obvious symptoms of pruritus 52 hours after the virus inoculation and eventually died;the result of PCR product sequencing was compared with swine pseudorabies virus and it was confirmed that the virus was PRV.In this experiment,a pseudorabies virus was successfully isolated and named as PRV-T4 isolate.The TCID₅₀0 was measured to be 10^-6.24/100?L.Lower thanGuizhou-DY strain TCID₅₀(10^-9.71/100?L)?Hao Fei et al.,2013?and Zeng Zhiyong et al.?2010?isolated the GZ-Z1 strain TCID₅₀(10^-7.26/100?L)?Zeng Zhiyong et al.,2011?.The results of the separation showed that there was a PRV wild-type infection on the farm.2.Cloning and Sequencing of gB/gE/TK Gene of Isolated PRV-T4 StrainIn this study,the isolated PRV-T4 stain were selected as the research object.Three pairs of specific primers were designed and the whole genes of gB,gE and TK genes were amplified by LA-Taq enzyme respectively.The reaction conditions were as follows:94?pre denaturation for 5 min,94?denaturation for 1 min,62??gB gene?/55??gE/TK gene?annealing for 1 min,72?extension for 45s,for 35 cycles,and finally 72?extension for 10min,4?,heat preservation.At the end of the reaction,5?L of PCR product was taken for1%agarose gel electrophoresis.The remaining 45?L of the product was gel-purified,cloned into pMD19-T vector,and transformed into DH5?competent cells.Positive colonies identified by colony PCR were enriched and cultured in LB liquid medium containing Amp,and plasmids were extracted.BamH I/EcoR I double enzyme digestion was performed on the recombinant plasmids.Electrophoretic detection gave two large and small ones that were in line with expectations.In the band,the positive plasmid was sent to Beijing Liuhe Huada for sequencing,the sequencing results showed that the gB gene was 2 751 bp,the gE gene was 1737 bp,and the TK gene was 938 bp,which was in agreement with the expected results,indicating successful cloning,the successfully constructed recombinant plasmids were named:pMD19-T4-gB,pMD19-T4-gE,and pMD19-T4-TK.This study laid the foundation for further research on the gB/gE/TK gene of PRV-T4 isolates.3.Bioinformatics analysis of gB/gE/TK gene of PRV-T4 isolateThe gB/gE/TK gene sequences of the PRV-T4 isolates that had been successfully cloned and sequenced were analyzed for nucleotide and protein sequences using biological software.The results showed that compared with the domestic Barth,La and Fa strains of the PRV-T4strain isolated in this experiment,the gB gene-encoded amino acid sequence analysis showed that the PRV-T4 isolate isolated in this experiment was in gB.There are no amino acid changes in the amino acid encoded by the gene at positions 395,562,and 739;the amino acid sequence analysis of the gE gene encoded by the domestic classical strain Ea strain,Yangsan strain,and GDSH strain showed that the aspartic acid was inserted into the 48th,496th,and498th positions of the amino acid sequence encoded by the gE gene?Asp D?.In contrast to the domestic classic strains Bartha,La,Fa,and MinA,the PRV-T4 strain isolated from this experiment did not occur at amino acid positions 16,129,215,and 284 of the TK gene.Related mutations.The G+C content of gB/gE/TK gene in the isolated PRV-T4 strain was71.03%,74.09%,and 74.40%,respectively,both of which were abundant in secondary structure,and the hydrophilic amino acids were evenly distributed throughout the entire peptide chain.The difference in the number of hydrophobic amino acids is not obvious and has a strong hydrophilicity.The results showed that the PRV strain isolated in this experiment was a variant strain.It is speculated that due to the rapid development of the pig-raising industry in Guizhou Province in recent years,trans-regional introduction led to an increase in herd mobility.This study has certain guiding significance for the prevention,control and purification of PR in our province.