Diagnosis, Isolation And Identification Of Porcine Pseudorabies Virus And Sequence Analysis Of Its GB, GC And GE Genes

Pseudorabies?PR?is a porcine infectious disease caused by pseudorabies virus?PRV?,which is characterized by neural symptoms in the piglets and miscarriage in pregnant sows.The disease has caused huge economic losses in pig industry.Since the 1980s,most pig herds in China have been inoculated with vaccines,so PR had been effectively controlled.However,since 2012,pseudorabies has occurred again in large-scale swine farms in various regions of China.Our laboratory collected relevant materials from suspected pigs with pseudorabies in a pig farm in Baoji City,Shanxi Province.In this study,we isolated virus from the collected material,plaque purified the virus,observed virus particles by transmission electron microscope,finished PRV infection TCID₅₀0 determination,the rabbit infection test and nucleotide sequence analysized gB,gC and gE genes on this isolated strain.We got the following results.?1?An epidemic of pseudorabies in a pig farm in Baoji City in August 2017 was determined by observing the clinical symptoms and pathological necropsy of pigs suspected of being infected with pseudorabies,and then identifying by laboratory PCR of PRV gB gene.?2?PK-15 cells infection with the relevant materials and appeared obvious cytopathic effect?CPE?when the cells infected six times in a row.Some cells appeared rounded and scattered likelesion at 24 h.Cells appeared rounded and aggregated at 48 h.Cells are broken and dead at 72 h.After 48 hours virus infected of cells,we observd pellucid plaques with apposite size and number.When the purified virus was amplified by transmission electron microscopy at a magnification of 70,000 times,the typical morphology of PRV virus particles was observed,showing many fibers were on the surface of the capsule and a solid spherical shape with a diameter of about 130 to 150 nm.The isolated virus was further confirmed to be a pseudorabies virus and named SX17 strain.?3?The virus titer in SX17 is 10^-11.4237TCID₅₀/100?L.Transparent rabbit infection test results show that SX17 strain can cause the symptoms of pseudorabies in rabbits,what is wilting food,elevated body temperature,some rabbits appear scratching phenomenon,stroke symptoms within 48 hours after onset and successively death.Pathological necropsy results showed that all three rabbits in the poisoning group showed typical PRV lesions,cardiac congestion,hepatomegaly,severe peripheral hemorrhage,and extensive congestion in the lung;splenic congestion was evident,and bleeding was found in the brain.?4?The nucleotide sequence comparison of the gB gene of SX17 strain showed that the homology of the genotype of the PRV strain was high?99.4%⁹9.7%?,and the genetic phylogenetic tree showed that most of the domestic PRV strains belong to the different branch;gC The nucleotide sequences of the genes showed relatively little difference?98.3⁹9.9%?.The phylogenetic tree showed that the PRV epidemic strains in each region of the SX17 strain should belong to the same branch;the nucleotide sequence of the gE gene showed a certain difference?98.9⁹9.4%?.Some parts of the population showed mutations or deletions,and the genetic evolutionary tree showed that PRV epidemic strains in each region of SX17 strain do not belong to the same branch and have shown certain regional characteristics.