Isolation And Identification Of Pseudorabies Virus And Construction Of A GI/gE Double Gene Deletion Strains

Pseudorabies ?PR?, also known as Aujeszky's disease, is an acute and high contagious disease of a variety of domestic and wild animals caused by Pseudorabies virus ?PRV?.which can characterize with fever, extreme itch ?except for pig? and encephalomyelitis. A wide range of mammals are able to be infected by PRV. but only pig is able to survive in an acute infection and represent the natural reservoir for the virus.the mode of transmission is vertical transmission, but also through the digestive tract, mutual contact and infection.At present, the world is widely popular, which brings huge economic losses to the pig industry in the world.Since 2011, many pig farms that had been vaccinated the sows with the Bartha k61 deleted vaccine have emerged a serious disease that appears to be pseudorabies symptoms, the sow usually display the reproductive disorders such as miscarriage, fetal death and mummy foetus,and the mortality rates of newly born piglets are very high. In this study, we detected the pathogen infection of the brain tissue samples from the dead piglets which had had been vaccinated, that had the symptoms of Pseudorabies from one pig farm of Sichuan province, named the isolated strain of PRV-XJ. The homogenated sample of brain tissue was subjected to infecting BHK-21cells,classical cytopathogenic effects of herpesvirus lesions appeared post infection 30 hours, such as cells aggregation, shrinking, rounding, net, the refractive index increased, flake off. After 5 rounds of plaque purification, a series of biological characterization and some biological characteristics were carried out. Tissue culture infectious dose(TCID₅₀) in BHK-21 cell line transfected with isolated virus was 10^7.67TCID₅₀/mL.It was proved that the virus could be neutralized by PRV positive serum, and the titer of the virus strain is 10^3.32TCID₅₀.This study were designed 5 pairs of primers for PCR amplification and sequence analysis of gE, gC, gB and gD genes. Results showed that all sequences belong to II genotype, while all of Europe and strains belong to genotype ?. which GE gene of other wild nucleotide homology between 92.3% to 99%. amino acid sequence homology between 94.2% to 98.4%. has a representative strain FA. TJ and EA strain homology were 96.7%,98.1% and 97.6%.Homology of nucleotide and amino acid PRV-XJ strain gC gene respectively were 92.3%?100% and 92.7%?100%, and China's early isolated strain EA, FA and the current separation of PRV strains were located in the II genotype. The gD gene of PRV-XJ strain of nucleotide and amino acid homology were 96%?100% and 96.3%?99.3%, the variation of multiple sites has the characteristics of type, GB protein is more conservative.The test strains PRV-XJ as parent strains, using PRV universal transfer vector pPI-2-EGFP. by homologous recombination. a double gene deletion virus was constructed successfully. In this experiment, the PRV-XJ strain was used as the parental strain, and the PRV was used as the universal transfer vector pPI-2-EGFP. A gI/gE-gene-deleted PRV mutant named rPRVXJ-delgl/gE-EGFP was generated using modified homologous recombination technology and confirmed by PCR, immunofluorescence assay ?IFA?.The results showed that the gE and gI genes were deleted as expected. The plaque and infinite dilution purification method to carry on the purification, and draw the growth curve. The results show that it has similar growth kinetics and between parental strain PRV-XJ line, between the two groups had no significant difference.The experiment was also carried out gene deletion virus rPRVXJ-delgl/gE-EGFP of piglets was safe and immunogenic inquiry. Our results showed that pigs immunized with different doses of immunization and different doses (10⁵? 10⁶?10⁸TCID₅₀) of rPRVXJ-delgl/gE-EGFP did not exhibit clinical signs and produced PRV specific antibodies. Immune after 7 days can be detected gB specific antibody, and intranasal and intramuscular injection compared the difference was not significant, immune after 7-28 days gE antibody were negative, could protect immune disease of pigs vaccinated, without showing clinical symptoms, but could not prevent wild virus antibody seroconversion and detoxification.This showed that the gene deleted virus rPRVXJ-delgI/gE-EGFP is very safe of piglets, can produce specific antibodies, can be used as a PRV gene deleted vaccine candidate strain.