Isolation And Identification Of Porcine Pseudorabies Virus Epidemic Strain And Its Immunogenicity Analysis

Pseudorabies is a kind of acute,strong and highly contact infectious disease of most mammals(including many domestic animals and wild animals)and some birds.It can infect the nervous system.After infection,almost all animals will have symptoms such as fever,pruritus,encephalomyelitis and clinical symptoms of death.Pig is the natural host of the disease and the only viable host after infection.Pseudorabies is one of the major infectious diseases that affect the global pig breeding industry.Therefore,countries all over the world attach great importance to this disease,and have launched and implemented the plan of eradicating Pseudorabies,and have achieved periodic victories,such as the United States,Germany,Sweden,Denmark,the United Kingdom and other countries,and have realized the purification of Pseudorabies in domestic pigs.Since 2011,there has been a new outbreak of PRV in the world,especially in China.Since then,many PRV variants have been isolated and identified.Heilongjiang Province is a big pig raising Province,and there is also the prevalence of Pseudorabies in some areas.The isolation of Pseudorabies virus in Heilongjiang Province is helpful to understand the prevalence of the virus,research and development of vaccines prepared with the epidemic strains and provide help for the prevention and control of the disease.In this study,submaxillary lymph nodes,spleen and other tissues of piglets with suspected pseudorabies abortion were collected from a pig farm which had been immunized with Pseudorabies Vaccine in Harbin suburb of Heilongjiang Province.Then the virus was isolated by inoculating Vero cells,and the virus was identified by PCR,serum neutralization test,direct immunofluorescence and negative staining electron microscopy.The pathogenicity and growth characteristics of the virus were further studied.the gene of g E was cloned,sequenced and sequenced.The immunogenicity and antigenic difference of the isolate were also analyzed in this study.In addition,the inactivated vaccine prepared by the isolated strain was preliminarily applied.The results showed that after inoculating Vero cells,typical Pseudorabies virus cytopathy appeared,such as round shrink,round shrink,round shrink and round shrink The results of PCR showed that PRV was positive,the virus could be neutralized by Pseudorabies virus positive serum,the direct fluorescence reaction of cells inoculated with PRV had positive signal and electron microscope observation,all of which proved that the virus was PRV,and the isolate was named hrb-2018.After inoculated with rabbits,mice and weaned piglets,all the infected animals died All of them showed typical clinical symptoms of pseudorabies,indicating that the isolate had strong Pathogenicity;the one-step growth curve of the virus showed that the highest virus content of the isolate was higher than that of the classic strain,and the time when the virus content reached the peak was shorter than that of the classic strain,indicating that the isolate had stronger cell adaptability.The molecular characteristics of the isolate were understood by GE gene.The results showed that the ORF of g E gene was 1737 bp in length and encoded 578 amino acids.The homology of ORF with reference strain was 98.3% ? 99.7%,and the homology of ORF with reference strain was 97.7% ? 99.3%.The genetic evolution tree constructed shows that the isolates are in a small evolutionary branch with the closest genetic relationship with the domestic strains isolated after2011;they are in a larger evolutionary branch with the domestic more classic isolates such as ea strain,and their genetic relationship is relatively close;they belong to genotype II.Compared with foreign isolates,such as Becker strain,they are in two different large evolutionary branches and have a far genetic relationship,indicating that the isolates were indeed mutants.Furthermore,the amino acids pushed by g E gene were analyzed.One D-aspartic acid was inserted at position 48 and492 respectively,and the change of V-I at position 488 was consistent with the molecular biological characteristics of PRV variant after 2011.The results showed that the vaccine prepared by the isolate could produce a good neutralizing antibody level after immunizing weaned piglets.After the virus attack,the pigs in the immune group had no other clinical symptoms except one transient temperature rise,while all the uninjured pigs had mental depression and anorexia The results of antigen difference analysis showed that there were significant differences in neutralizing antibody level and protection rate of different strains.The preliminary application effect of the vaccine was good.The above results show that the virus isolated from the collected materials is pseudorabies virus,which has strong pathogenicity and cell adaptability.It is a strain that has become popular in China since 2011.It has good immunogenicity,and has antigen differences with the widely used vaccine strains in China.The preliminary application of the inactivated vaccine has good effect,It is also to provide valuable strains for further study of its pathogenesis,screening of vaccine strains Molecular epidemiology and pathogen diagnosis.