Isolation And Identification Of Swine Pseudorabies Epidemic Virus Strains From South China And Preliminary Study On Inactivated Vaccine Of GE Gene Deletion In YC Strain

Pseudorabies in pigs is a highly contagious disease caused by swine pseudorabies virus,which can cause miscarriage and piglet death,and bring huge losses to the pig industry.Porcine pseudorabies virus belongs to herpesvirus A subfamily of herpes zoster virus type.The attenuated gene vaccine is the main method to prevent and control the disease.This study was to conduct an epidemiological survey of swine pseudorabies virus in herds in South China to understand its prevalence in swine herds.At the same time,isolate the pseudorabies virus epidemic strains and screen them for high titer proliferation in the cells.Virus strains,to prepare safe,efficient inactivated vaccines,provide an effective means for the prevention and control of the disease.This study collected clinical samples(brain,tonsils,lymph nodes,and fetal clothing)from 99 farms in four provinces in southern China from 2013 to 2014,and used the established PCR detection method to detect the collected clinical samples.The results showed that the average positive rate of pig farms was 29.3%,indicating that the pseudorabies were widely prevalent among pigs in South China,and the infection rate was high,which provided basis for timely mastering the epidemic variation of wild virus PRV in South China.Then the positive samples detected by PCR were inoculated into MARC-145 cells for virus isolation and identification.Six strains of pseudorabies virus were successfully isolated by sequencing and named YC,ST,FL,BE,SY and YQ.The gE gene sequences of six strains were amplified and sequenced.The results showed that the gE gene was 1740 bp in length.The gE gene of six strains was further analyzed by phylogenetic evolution and sequence alignment.The results showed that: The amino acid homology of the gE gene between strains was 98.1% to 99.8%.All isolates are located on the same branch,indicating that the evolutionary relationship between the isolates is closer,but with the earlier European and American strains are on different branches.The pathogenicity test of YC strains in pigs showed that the body temperature of the pigs could be increased on the second day after the challenge;all the pigs in the challenge group died on the 6th day after the challenge,indicating that the YC strain has a high pig's exposure.Disease,necropsy pathological changes include lung bleeding,brain congestion and hemorrhage,liver white necrosis,tonsil purulent lesions.In order to prepare the inactivated vaccine of the pseudorabies gE gene deletion,we selected the YC strain as a vaccine candidate strain by comparison with the amino acid sequence of the prevalent strain,and knocked out the gE gene for the preparation of a gene deletion inactivated Vaccine.The results showed that the prepared gE gene deletion inactivated vaccine had stable physical properties and met the relevant requirements for inspecting inactivated vaccines.The immune challenge protection experiment showed that the gE gene inactivated vaccine prepared in this study could protect pigs only twice after inactivated vaccine,which provided an effective means for the prevention and control of swine pseudorabies.