Isolation And Identification Of PRV HuB20 And Construction Of Gene Deleted Strains

Pseudorabies（PR）is a virulent viral disease that can cause disease in a variety of mammals,including humans.PR is the most harmful to pigs.It can lead to severe neurological symptoms,death and breeding disorders of piglets,which greatly threatens the healthy development of pig industry.At the end of 2011,China experienced another outbreak of pseudorabies,and even pigs immunized with the classic pseudorabies Bartha K61 vaccine were not spared.Studies have shown that antibodies produced by classical PRV vaccine have a certain neutralization effect on the mutant strains,but the protective effect has been found to be limited in clinical application.In recent years,there have been reports on virulence and genome changes of PRV mutant strains.Therefore,it is of great importance for the subsequent prevention and control of PRV to continue the isolation,identification,biological characteristics,pathogenicity and genomics studies of PRV epidemic strains in pigs,especially those at high age.Therefore,this study conducted PRV isolation,identification and genetic evolution analysis on positive pathogen samples of pigs to be slaughtered in pig slaughterhouses in Hubei province and positive disease materials submitted for testing in pig farms,so as to understand the characteristics of PRV currently prevalent in China and lay a foundation for subsequent PRV research.At the same time,the whole genome of the PRV strains isolated by the research group was sequenced,including the strain information in NCBI Gen Bank to build the database PRVDB,and establish the research sharing platform of PRV strains.The biological characteristics,virulence test and bioinformatics analysis of the two isolated PRV variants were carried out.Then the candidate vaccine strain HuB20 was selected according to the pathogenicity,neutralization ability and genetic evolution characteristics of the strain,and it was used as the parent strain for ΔgGΔg I/g E gene deletion,which laid the foundation for the research and development of a novel PRV vaccine.Specific research work and progress are as follows:1.Detection of PRV Samples in Pig Slaughterhouse and Suspected Pathogen Detection in Pig FarmIn 2019-2022,a proportional sampling was conducted in four regions（East Hubei,North Hubei,West Hubei,Central Hubei）of Hubei Province,collecting 1795 blood samples and 2081 lung samples（lung and bronchial lymph nodes）from different scale slaughterhouses in 9 counties.PRV g E-ELISA and PCR methods were used to detect the infection antibodies in serum and pathogens in lung samples,respectively.The serological test results showed that 104 serum samples were positive,with an average positive detection rate of 5.79%（104/1795）.The pathogen test results showed that 82 lung samples were positive,with an average positive detection rate of 3.94%（82/2081）.At the same time,from 2019 to 2022,58 suspected samples were collected from large-scale pig farms in nine provinces and cities in China.Through PRV g E gene PCR detection,and the highest detection rate was in lung and lymph node samples.2.PRV Strain Isolation and Identification and Phylogeny AnalysisVirus isolation and identification were performed on the tissue samples positive for PRV pathogens in pig farms and slaughterhouses,and a total of 34 PRV strains that could be stably transmitted were isolated.The phylogeny analysis was conducted based on g B,g C and g E genes.The results showed that the 34 isolated strains were all gene type II variants,which were different from the early foreign isolates such as Bartha,NIA-3 and Becker,and were closest to the domestic isolates since 2012.Among them,the HBXG-4-2020（later renamed HuB20）strain was in an independent clade 2.2 genetic branch with the recombinant virus HuB1/CHN2017 reported earlier,suspected to be a recombinant strain of gene type I and gene type II viruses.3.Phenotypic Determination and Whole Genome Sequencing of Domestic PRVA total of 118 PRV isolated in this study and preserved by our research group from2012 to 2022 were determined by phenotypic analysis.The result showed that the TCID₅₀ of all strains on PK-15 cells was between 10^5.6910^7.63/ 0.1m L,and there was no significant difference in the virus titer of different years.The mouse LD₅₀ of the representative strain was between 10^3.4310^1.87 TCID₅₀.At the same time,58 PRV strains were sequenced by whole genome sequencing.The results showed that all sequencing libraries obtained more than 900 Mb of clean data,the Q30 value was greater than 90%,the whole genome length was between 171078-121071 bp,and the average GC content of the genome was between 70%⁷4%,which was in line with the basic characteristics of PRV whole genome.4.Construction of PRV Network DatabaseThe relevant information of 118 PRV stored in our research group and 47 PRV collected and sorted by NCBI were built through Apache HTTP server to establish the network interface of PRV network database PRVDB（http://vetinfo.hzau.edu.cn/PRV/）.The database integrates the functions of virus isolation background information collection,NCBI Gen Bank,Viral Zone and NCBI Pub Med,providing comprehensive information such as PRV strain information screening,visual epidemic map,genomics and latest research literature links,and data sharing can be realized through upload and download functions.5.Study of Variants HuB20 and He N21Two PRV strains HuB20 and He N21 were found through the epidemiological investigation,He N21 caused severe clinical symptoms in old pigs,and HuB20 showed suspected recombination events in the genome.The biological characteristics,animal virulence tests and bioinformatics characteristics of both were studied.The results of cell virulence test showed that the toxicity of HuB20 and He N21 to PK-15,Vero and SK-N-SH cells was similar to that of other PRV variants,but on PK-15 cells,He N21 had a higher virus titer and stronger plaque formation ability in the first to fifth generations compared to other PRV variants.The results of fixed virus neutralization test showed that HuB20 had the strongest neutralization ability against the epidemic strain.The results of animal virulence assessment in mice and fattening pigs showed that the infection of HuB20 and He N21 would lead to higher morbidity and mortality rates and more severe tissue pathology damage.Especially,the infection of He N21 caused severe respiratory and neuronal symptoms and death in 90-day-old fattening pigs.Both PRV strains showed high virus load on the main organs of infected mice and pigs.6.Construction of mutant PRV HuB20 ΔgGΔg I/g EBased on the above studies,the recombinant strain HuB20 with the strongest neutralization ability was selected as the candidate vaccine strain.The pc DNA3.1ΔgGΔg I/g E plasmid constructed was co-transfected with HuB20 complete genome into PK-15 cells,and multiple blank purification was performed.After PCR and sequencing verification,the purified HuB20ΔgGΔg I/g E strain was obtained.PCR and sequencing identification of the three-gene deletion strains of the fifth,tenth,fifteenth,twentieth and twenty-fifth generations showed that the deletion sites were stable and there was no recovery mutation.The plaque formation ability of the above generations was stable and significantly lower than that of the parent strain HuB20.The results of one-step growth curve showed that the proliferation rate of parent strain HuB20 was faster within12 h after inoculation,but the two strains could reach the same titer level after the virulence was stabilized(10^7.5 TCID₅₀).The HuB20ΔgGΔg I/g E strain prepared in this study was inactivated and emulsified to conduct high-dose safety tests and post-immunization challenge protection tests in mice.The results showed that no clinical symptoms were observed in mice after injection of 107 TCID₅₀ inactivated vaccine,and all survived.In the challenge protection experiment,the inactivated vaccine protected mice from the lethal attack of wild-type PRV and induced high levels of PRV g B antibodies and neutralizing antibodies.It showed that the prepared inactivated vaccine was nonpathogenic and effective for mice.