Isolation And Identification Of Variant Pseudorabies Virus And Synergistic Effect Of Immune Enhancing Agents In Swine Pseudorabies Inactive Vaccine

Pseudorabies virus can cause Swine pseudorabies which is one of the most serious infectious diseases in swine industry.At present,the prevention of pseudorabies mainly rely on vaccines,many European countries and the United States rely on biological purification and immunity to eradicate pseudorabies.In recent decades,pseudorabies virus attenuated vaccine was used to prevent and control this disease in our country,Bartha-K61 is the most widely usedand achieved good immune effect.However,since the end of 2011,China's pig farms immunized with PRV attenuated vaccine began to appear miscarriage,low birth,stillbirth,the production of pig respiratory symptoms,symptoms of nervous symptoms and even piglets death suspected of pseudorabies symptoms.In this study,a strain of PRV was isolated from a pig farm suspected of swine pseudorabies in Nanjing.The gE,TK and gB genes were sequenced and analyzed.pigs immunized by inactive vaccine with immunopotentiators and were challenged with with PRV epidemic strains,the immune characteristics were observed,this study laid the foundation for the evolution of PRV epidemic strains and the development of new PRV vaccines.1 Isolation and identification of variant pseudorabies virusIn 2014,we isolated a strain of virus from a pig farm which had vaccined with Bartha-K61.we identified gE,TK and gB by PCR,the results were all positive.gE,TK and gB gene of the strains named NJ-2014 were sequenced and analysed their phylogenetic tree,and compared their nucleotide and amino acid homology with reference sequence.The results showed that the gE gene of the strain was close to the Bartha strain,the gB gene had a close relationship with the SDWF1-China-2012 strain and TJ-China-2012 strain,the genetic relationship between TK gene and SDWF1-China-2012 were close.Comparing the homology of gE,TK and gB with the reference sequence,we found that the three genes had different degrees of variation,the nucleotide homology between gE and the reference sequence was 97.2%-99.6%,and the homology of amino acid was 95.0%-99.5%;The nucleotide homology between TK and the reference sequence was 99.1%-99.9%,the homology of amino acid was 98.7%-99.7%;The homology between gB and the reference sequence was 98.2%-99.7%,and the homology of amino acid was 96.6%-99.5%.Studies had shown that the epidemic strain of pseudorabies virus has changed in Nanjing pig farm.2 Immune protection of inactive Pseudorabies Vaccine with immunopotentiatorsIn this experiment,inactive vaccine was mixed with VA5 to evaluate a immunopotentiator(VA5)on immunity potency of inactive vaccine of Pseudorabies virus(PRV),.The comparison test of the immune effectiveness was conducted by using inactive vaccine of Bartha-K61 strain of PRV,inactive vaccine mixed with VA5 and conventional attenuated vaccine of Bartha-K61 strain.Blood samples were collected respectively at 14 and 35 days after the first immunization.We performed neutralization experiments and cytokine detection.Results showed that,compared with live and inactive vaccine of Bartha-K61 strain of PRV,the inactive vaccine with VA5 could induce higher neutralizing antibody against the heterologous strain of PRV and increase the expression levels of IL-2 and IFN-Y mRNA in porcine peripheral blood lymphocytes(PBMC).Neutralization test showed that VA5 could significantly increase the level of neutralizing antibody against the epidemic strain of PRV.After challenge on pigs,the inactivated vaccine with VA5 group had 100%survival rate,the weight of animal could grow stably,no fever happened,nasal cavity detoxification and tissue toxicity were the least.This experiment showed that VA5 could improve the humoral and cellular immunity of inactive PRV vaccine,the cytokine content of increases apparently,and provide a basis for paving the better immune effect of inactivated PRV vaccine.