Regulatory Effect And Mechanism Of NLRP3 Inflammasome In Anti-CP BVDV Infection

Bovine Viral Diarrhea Virus(BVDV)is a single-stranded positive-stranded RNA virus,which belongs to the family Flaviviridae and the genus Pestivirus.Studies have shown that BVDV acutely infected cattle show leukopenia and immunosuppression.The NLRP3 inflammasome is an important member of the host’s innate immune system and participates in host defense responses against multiple pathogens,recruiting and activating cysteine protease-1(Caspase-1),which mediates the secretion of the inflammatory cytokine IL-1β/IL-18 and induces an immune response.Morales-Aguilar A et al.found that BVDV infection of bovine monocyte-macrophages can mediate IL-1β release by dependent Caspase-1 pathway,and activation of this pathway affects BVDV replication,suggesting that the NLRP3 inflammasome may be involved in the development of BVDV.To this end,the study used BVDV to infect mouse and bovine peritoneal macrophages as a research model and NLRP3 inflammasome as the research object to deeply explore the activation law of NLRP3 inflammasome by BVDV infection and its effect on BVDV replication.This study could provide a theoretical basis for establishing effective immune prevention and control measures for BVDV infection.First,to determine the correlation between acute BVDV infection and NLRP3 inflammasome activation,SPF BALB/c mice aged 4～6 weeks were selected for infection with CP BVDV(NADL strain).The results showed that BVDV infection can cause pathological changes in the spleen and duodenum of mice,and virus particles were detected in the duodenum,spleen and blood at different time points,and the viral load reached peak at 7th day.,which demonstrate the success of BVDV infection mouse model.In addition,the study found that the m RNA and protein expression levels of NLRP3,Caspase-1 and IL-1β in the duodenum of mice at the early stage of BVDV infection(1 d,2 d,and 4 d)was increased,with the most significant change at 1 d.However,the expression of NLRP3 inflammasome related proteins was decreased in the later stages of BVDV infection(7 d),and the significantly decrease on the 7th day.These studies suggest that the NLRP3 inflammasome may be involved in the pathogenesis of BVDV.Secondly,in order to clarify the role of NLRP3 inflammasome in BVDV-infected mice,the NLRP3 inflammasome inhibitor MCC950 and the agonist Nigericin were treated respectively,and further verified by NLRP3 gene-deficient mice.The results showed that protein expression of both Caspase-1 and IL-1 was significantly suppressed and both inhibition and knockout of the NLRP3 gene in vivo significantly increased the BVDV load in the duodenum,spleen and blood of mice on 1th day of BVDV infection.Indicating that the NLRP3 inflammasome was activated at the early stage of BVDV infection and inhibited its activation promoting the replication of the virus.Given that NLRP3 inflammasome was inhibited on day 7th day of BVDV infection,treating mice with Nigericin,and found that Nigericin significantly reduced the load of BVDV in the duodenum,spleen and blood,indicating that the NLRP3 inflammasome is in the BVDV infection is inhibited in the later stage of infection,and its activation has anti-BVDV infection effect.Finally,in order to further clarify the effect of BVDV infection on the activation of NLRP3 inflammasome in bovine peritoneal macrophages and the regulatory role of NLRP3 inflammasome on BVDV replication.The cell samples with different multiplicity of infection and different infection time of BVDV were collected respectively.The IFA results showed that,4MOI BVDV infection for 24 h,the activation of NLRP3 is the most obvious.To further explore the effect of NLRP3 inflammasome on BVDV replication,MCC950 was used to treat cells,and the results showed that MCC950 could significantly promote BVDV replication,which was consistent with the results of in vivo experiments in mice.Taken together,our results suggest that the NLRP3 inflammasome is activated in mouse and bovine peritoneal macrophages at the early stage of BVDV infection and promotes IL-1βexpression dependent on the Caspase-1 pathway.Inhibition of NLRP3 inflammasome activation promotes BVDV replication.This study provides a rationale for the mechanism of BVDV antagonism against host innate immunity.