Study On Pathogenicity Of Pseudorabies Virus Variant Strain And Immune Efficacy Of The Live Vaccine With Genes Deletion

Porcine pseudorabies virus(PRV)is an important pathogen that endangers the pigs industry,which causing fever and encephalomyelitis in many kinds of domestic animals and some wild animals,such as pigs,sheep,cattle and cats.In recent years,a PRV variant strain broke out in China,having high virulence and variant antigenicity.But there is no commercial live vaccine to effectively control this disease.In this study,by the serological detection of anti-gE antibody,it was proved that PRV spread wildly in Shandong province.And a live genes-deleted vaccine from the variant PRV strain ZJ01 could provide good cross immune protection against classical PRV LA strain challenge.And the immune protective efficacy of the vaccine were also measured in mice and rabbits,which laid foundation for the development of a new vaccine of pseudorabies.At the same time,by foot pad inoculation of PRV in mice,it was clarified that the inflammatory reaction caused by PRV strain was the main reason of death in mice.The main contents of this study are as following:1.Epidemiological investigation of the serum antibody against PRV gE in pigs in Shandong province in 2018In order to know the virus infection in Shandong province,we used PRV-gE-ELISA antibody test kit to detect the anti-gE antibody in 2240 serum samples from 99 pig farms in this province in 2018.The results showed that the positive rate of PRV-gE antibody in those pig farms was 82.83%(82/99),and the positive rate of serum samples was 61.74%(1383/2240).The highest positive rate was in spring,and the lowest in winter.The positive rate of serum in lactating sows was the highest,96.00%,followed by boars and reserve sows,which indicated that PRV infection rate was high in this area.It is necessary to formulate scientific and reasonable immunization procedures and strengthen the comprehensive prevention and control of this disease.2.The cross protective efficacy of PRV variant strain live vaccine with deletion of gI/gE/TK genesIn this study,28-30 day old piglets were inoculated with ZJ01ΔgI/gE/TK from pseudorabies virus variant strain,and challenged with ZJ01 variant strain and LA traditional strain,respectively.The results showed that the piglets had no significant clinical symptom within 21 days after vaccination and shed no vaccine virus in the noses.And the piglets produced neutralizing antibody against LA and ZJ01 strain with no significant difference in titer at 21 days post vaccination.The protective effect of the vaccine was 100%.The levels of PRV ZJ01 gE gene in brain and lung tissues were significantly lower than that in control group.In addition,the piglets was also able to resist the attack of LA virulent strain,without clinical symptoms and pathological changes.The levels of PRV LA gE gene in brain and lung tissue were significantly lower than that in the control group.It indicated that the vaccine had a good cross protective effect on traditional virulent strain,which being helpful to develop a vaccine to control this disease.3.The alternative method of testing the efficacy of PRV variant strain live vaccine with deletion of gI/gE/TK genesIn this study,mice and rabbits were inoculated with ZJ01ΔgI/gE/TK in different doses,and injected subcutaneously with PRV ZJ01 strain(106.0TCID50/mL)at 21 days post vaccination.The mice experiment results showed that the mice inoculated with the vaccine at the doses of 103.0,104.0,105.0,106.0TCID50/mL,had no obvious clinical abnormality and pathological change within 21 days after vaccination,and produced neutralizing antibody against ZJ01 strain.After challenged with ZJ01 strain at 21 days post vaccination,they did not have clinical symptoms and pathological changes.The levels of PRV gene in brains and lungs tissues were significantly lower than those in the control group.And the levels of the antibody and protective effect in 106.0TCID50/mL group were significantly higher than those in 103.0-105.0TCID50/mL groups.The rabbits experiment results showed that the rabbits inoculated with the vaccine at the doses of 104.0,105.0,106.0TCID50/mL subcutaneously,also had no obvious clinical symptom within 21 days after vaccination,produced neutralizing antibody against PRV,and resisted the attack of ZJ01 strain,with a protection rate of 100%.The levels of PRV in the in brain and lung tissue were significantly lower than that in the control group.The results showed that mice and rabbits could be used as the evaluation model of immune efficacy of this vaccine.4.The relationship between pathogenesis and inflammatory responses in mice infected with PRV through foot padPRV is an important pathogen that causes serious diseases in many kinds of domestic animals and wild animals,such as pigs,cattle,sheep,etc.in the world.The virulence of different strains is quite different,and the pathogenic mechanism is not full clear.In order to clarify whether the neuron infection of PRV can cause cytokine storm to cause the clinical morbidity and death of infected animals,C57BL6 mice of 5-7 weeks old were inoculated with PRV ZJ01,LA,ZJO1ΔAgI/gE/TK and Bartha-K61 strains by foot pad inoculation.The results showed that the foot of the mice inoculated with ZJ01 and LA showed severe itching and dying state.The levels of IL-6,G-CSF,MCP-1 and GRO-1 in the plasma of mice infected with ZJ01 and LA were significantly higher than those of ZJO1ΔgI/gE/TK and Bartha-K61.The levels of IL-6 and G-CSF in spinal cord,DRGs,heart and liver also increased.The levels of PRV in the organs and tissues of mice was detected by fluorescence quantitative PCR.The results showed that the virus was positive in the foot pad and nervous system of mice in ZJ01 and LA virulent groups,but negative in other groups.At the same time,the plasma CRP level of the mice in ZJ01 and LA groups increased significantly,indicating that the infection was an acute inflammatory reaction.The results showed that PRV ZJ01 and LA virulent strains infection caused acute inflammatory reaction and death in mice.PRV attenuated strains infection did not cause specific and lethal inflammatory reaction.It indicates that inflammation is closely related to death in mice caused by PRV virulent strains.Systemic inflammatory reaction induced by PRV was the main cause of severe pruritus and acute death in mice.