Attenuation Of The Pseudo-rabies Virus Double Gene Deletion Strain And Evaluation Of Its Immunological Efficacy

Pseudorabies(PR)is an acute porcine infectious disease caused by Pseudorabies virus(PRV;ADV),which can infect pigs and cause a series of clinical symptoms of fever,respiratory system and nervous system dysfunction.PRV belongs to the herpesvirus family and the genome is double-stranded linear DNA.The host range of PRV is wide and pigs are the only animals that can survive infection,but PRV can seriously affect the growth status of infected pigs.Currently,vaccination is the main way of prevention and control,among which the live attenuated vaccine with PRV gene deletion is the most widely used and the effect is enviable.Since 2011,however,many pig farms in China that have been vaccinated with Bartha-K61 have occurred new epidemics and spread to pig farms in most parts of the country,causing huge economic losses to the development of China's pig industry.The study found that the new outbreak was caused by the PRV variant and the Bartha-K61 vaccine can not provide complete protection.Therefore,Chinese pig industry is pressing a safer and more effective PRV vaccine.In this laboratory,based on the BAC constructed by the PRV AH02LA strain,a pseudorabies double gene deletion strain PRVATK&gE-AH02 was constructed by homologous recombination and named as PRV LA 1206 strain.Our laboratory studies have shown that PRV LA 1206 strain is safe for weaned pigs but pathogenic for 1-day-old piglets.This experiment aims to reduce the pathogenicity of PRV LA 1206 strain by passage,and to study its biological characteristics,safety and immunoprotective efficacy,and strive to become a safe and effective vaccine for preventing and controlling PRV mutant strains.In this experiment,based on the constructed pseudorabies virus gE/TK double gene deletion strain PRV LA 1206,the highest titer was 107.25TCID50/0.1mL,and 0.1 MOI virus was inoculated into chicken embryo fibroblasts.The virus was cultured and passed down to the 40th generation.The 17th generation virus was labeled as PRV LA 1206 F17,the 40th generation virus was labeled as PRV LA 1206 F40,and the DNA was extracted for PCR to identify the deletion of gE and TK genes in the passage.The results showed that the deletion of gE and TK loci were in the passage process.After specificity and purity tests,it was confirmed that there were no foreign virus,bacteria,fungi and mycoplasma contamination in the transgenic strains PRV LA 1206 F17 and F40.The growth curves of PRV LA1206,passage strains PRV LA1206 F17 and PRV LA1206 F40 were measured.The results showed that the growth trends of the three were similar on CEF cells,and the highest titers were close,but the highest titer of PRV LA 1206 F40 was relatively lowest.The safety of PRV LA1206 F40 was tested.PRV LA1206 F40 and DM nutrient solution were intramusculally injected into 1-day-old PRV negative piglets,respectively.No clinical symptoms and temperature changes were observed after inoculation for 14 consecutive days,and no lesions were found in the organs and tissues of the experimental pigs after cesarean section,suggesting that PRV LA1206 F40 is safe for 1-day-old piglets.The immune efficacy challenge test of PRV LA1206 F40 was carried out.The Bartha-K61 attenuated live vaccine was selected as the vaccine control group.The 30-day-old PRV-negative piglets were intramuscularly injected into the neck.No clinical symptoms and temperature changes were detected within 14 days after immunization.PRV LA 1206 F40 can produce higher PRV neutralizing antibodies in the test pigs;after the 28th day of immunization,the PRV variant virulent strain AH02LA was challenged by the nose.After the challenge,the pigs had no body temperature reaction and clinical symptoms,and the gE antibodies all turned positive.PRV LA1206 F40 provides complete protection for the test pigs,while the test pigs of the Bartha-K61 attenuated live vaccine immunization group do not provide complete protection and do not prevent detoxification.The above results indicate that PRV LA 1206 F40 can provide complete protection against PRV epidemic strains,and the serum antibodies produced by the immunization can be distinguished from the wild-type antibodies,and the immune effect is significantly better than the Bartha-K61 attenuated live vaccine.It provides an important basis for the development of a new vaccine for the prevention of pseudorabies in China.