Isolation Of Pseudorabies Virus,Construction Of Gene-deleted PRV Strain And Investigation Of Its Immune Efficacy

Pseudorabies(PR),or Aujeszky’s disease,is an infectious disease caused by pseudorabies virus(PRV),which can infect in cattle,sheep,pigs and other livestock and wild animals.Pigs are the main natural host of PRV.In general,PRVmainly infects pigs at various production phases,such as causing severe neurological diseases and high mortality in newborn piglets,as well as reproductive disorders in sows,and results in significant economic losses for the pork industry.At present,the control of PRV mainly depends on the Bartha-K61 vaccine,which can effectively control the disease.Since 2011,outbreaks of the new PR have occurred in many swine herds vaccinated with Bartha-K61 vaccine,indicating that Bartha-K61 vaccine cannot provide complete protection against current PRV and the new round of PR is very harmful to our pig industry.Therefore,it is urgent to develop an effective vaccine against the current epidemic mutant strains is urgent.A fluorescent quantitative PCR detection(qPCR)method capable of detecting PRV wild strains was successfully established.The qPCR could only specifically detect PRV.but not other pathogens,indicating that the method had good specificity;The minimum detection off-line of PRV is 37.0 copies/μL of the qPCR,indicating that the method had a good sensitivity;The repeatability results exhibited that the coefficient of variation within the batch of the standard template at different concentrations was 0.214%to 0.873%,and the coefficient of variation between batches was 0.964 to 1.233%,and the variation coefficients within and between batches are less than 2%,which demonstrated that the qPCR had the high repeatability and good stability.A total of 56 clinical samples collected from June 2018 to January 2020 in Henan Province from Henan Province were tested by the qPCR method,the positive rate of PRV was 17.86%(10/56),showing that PRV wild virus infection was still present in pig farms of Henan Province.This assay might provide technical support for the early diagnosis and epidemiological investigation of PRV.The PRV-positive pathogens were inoculated into ST cells,and two PRV strains were successfully isolated.The results of serum neutralization test and the targeting the PRV gE of qPCR displayed that the two PRV strains isolated in this test were wild strains The gE gene of the two PRV strains and one PRV positive strain were amplified,and analyzed the homology and the genetic evolution,The findings discovered that the three PRV wild strains in this experiment were all closely related to the domestic epidemic strains of PRV and belonged to the same branch,which implied that the three PRV wild strains were PRV epidemic strains of China.Subsequently,the proliferation and pathogenicity of PRV epidemic strains on ST cells were investigated.The outcomes revealed that the virulence of isolate JZ-1-2019 gradually increased at 4-24 hours after inoculation on ST cells,and reached the highest at 36 hours and then the virus titer began to decrease;the consequences of animal tests showed that the isolate JZ-1-2019 was highly lethal to mice,indicating that the epidemic strain ZJ-1-2019 was a PRV virulent strain.This test provides technical support for the detection method of PRV,and also provides a reference for the genetic variation of PRV.The rPRV NY-gE-/gI’/TK-/EGFP+ strain has been triumphantly constructed by the traditional homologous recombination method in our laboratory,but the traditional method of homologous recombination has low efficiency.Therefore,CRISPR/Cas9 method was used to knock out the large fragment of the foreign gene EGFP based on the rPRV NY-gE-/gI-/TK-/EGFP+mutant strain,generating the rPRV NY-gE-/gI-/TK-mutant strain without EGFP.Subsequently,the TCID50,one-step growth curve determination of the rPRV NY-gE-/gI-/TK-were explored,and the results showed that the knockout of EGFP did not significantly affect the virus infectivity,but the growth rate of the strain was slightly lower than that of the parental strain.Physical and chemical properties and genetic stability results displayed that the strains had similar physical and chemical characteristics to PRV wild strains,and the strains had good genetic.After rPRV NY-gE-/gI-/TK-and the rPRV NY-gE-/gI-virus solution stored in our laboratory were inactivated with formaldehyde,two virus solutions were respectively mixed with MONTANIDETM ISA 201 VG adjuvant and MONTANIDETM GEL PR adjuvant to make 4 inactivated preparations.4 inactivated preparations and DMEM culture medium were separately injected to 6-week-old female Kunming mice,and the immunogenicity of the 4 inactivated preparations was identified by ELISA,neutralization test and challenge protection test.The results manifested that the four inactivated preparations could effectively stimulate the mice to produce specific antibodies,and had certain resistance to the attack of PRV wild venom.Among them,The immune effect of the rPRV NY-gE-/gI-/TK--GEL inactivated mice was poor,while the other three groups were not significantly different,and the rPRV NY-gE-/gI-/TK--201 inactivated vaccine was safer due to the lack of the TK gene.This study will provide a theoretical foundation and scientific basis for the prevention and control of pseudorabies virus mutants.