Isolation And Characterization Of A Porcine Pseudorabies Virus Mutant And Detection Of Immunological Antibody

A pseudorabies virus strain was successfully isolated from the brain tissue of pigs in a pig farm in Henan Province.The strain named HN2012 was identified as a mutant strain by gE gene amplification and genetic evolution analysis.Meanwhile,the protective rate of gene knocking-out vaccines?gE^-and gE^-/gG^-?challenged with HN2012 was evaluated by antibody tests and protection test.1.Isolation,identification and genetic evolution analysis of pseudorabies virus mutant strainThe supernatant of tissue sample was inoculated onto BHK-21 cells and cultivated for 36 hours,the cells shown obvious lesions such as swelling,rounding,aggregation and syncytia.After three times cloning purification,the TCID₅₀ titer of virus increased to 10^-7.57/0.1 ml on BHK-21 cells.The virus was inoculated into experimental animals such as Kunming mice,rabbits,and piglets,and typical pseudorabies symptoms appeared.The comparison of gE amino acid sequence analysis showed that the homology of HN2012 with other strains was 94.82%-99.83%.among them,the homology with domestic isolates and foreign isolates were 97.07%-99.83%and 94.82%-96.03%,respectively.The homology of isolates After 2012 was 99.14%-99.83%.Variations ranging from 1aa to 33aa were shown between HN2012 sequence and other strains.The above results indicate that the domestically isolated PRV strains have been clearly divided into two subgroups:the subgroup of isolated strains before 2012 and the subgroups of isolated strains after 2012.This result provides a useful reference for selecting pig pseudorabies virus vaccine strains in the future.2.Vaccine efficacy testThe immunoprotective effects of three vaccines against HN2012 strain was evaluated by Kunming mice.According to the efficacy requirements of pig pseudorabies oil emulsion inactivated vaccine in Compilation of Quality Standards for Veterinary Biological Products?2011?,we found all the control groups died,which mean that the experimental results were reliable,and the protection rate of the experimental group after second vaccination was higher than 70%,indicating that the efficacy of the vaccine complied with the standard requirement.Three vaccines were tested and all of them shown good protective rates which were higher than 70%.The efficacy of gE-and gE-/gG-on target animals was tested according to the efficacy requirement for the inactivated vaccine against pseudorabies in the veterinary drug quality standard?2017 edition?.The experimental results showed that the serum neutralizing index of the two genetic engineering vaccines was higher than 316,and the results were judged to be qualified.Therefore,the efficacy tests of the two gene-deficient inactivated vaccines were in line with the national"veterinary drug quality standards"test standard.In summary,the two genetic engineering vaccines of the pseudorabies virus variant HN2012,especially gE-strain,have good immunoprotective effects which can provide positive data for the prevention and control of pseudorabies in China and the selection of vaccine strains.