Construction Of BVDV Reverse Genetic RNA Polymerase ? System And Study On The Mucosal Disease Model

Bovine viral diarrhea virus,which belongs to the genus Flaviridae plague virus,is the pathogen of bovine viral diarrhea.Because of its strong infectivity and lack of effective means of prevention and treatment,BVD has become a global infectious disease and caused huge economic losses to China and even countries in the global aquaculture industry.According to the biotype,BVDV is mainly divided into cytopathic type(Cp type)and non-cytopathic type(Ncp type).According to genotype,BVDV is mainly divided into BVDV1,BVDV2 and BVDV3(BVDV3 has not been confirmed by the International Committee on virus Classification).Previous studies have shown that persistent infection of animals is the main source of BVDV introduction into herds,and persistent infection of animals with the same genotype Cp BVDV may lead to mucosal disease.BVDV as a single-stranded positive-strand RNA virus,reverse genetic technology has become an important tool for the study of BVDV.In this study,RNA polymerase?system was used to perform reverse genetic operation on BVDV,and BVDV reverse genetic RNA polymerase?system was constructed to save virus rY2.BALB/c mice and New Zealand white rabbits were used to infect pregnant female animals with Ncp type BVDV during pregnancy.The persistent infection model of offspring was established.The offspring were reinfected with Cp type BVDV of the same or different genotypes to replicate different types of mucosal disease models,and to explore the molecular mechanism of BVDV leading to mucosal disease.1.Construction of BVDV reverse genetic RNA Polymerase?system and Rescue of rY2virusReferring to the existing rescue methods of plague virus,the full-length c DNA infectious clone of BVDV Y2 genome with RNA polymerase II rescue system was constructed by fusion PCR method with the vector of Y2 whole gene sequence preserved in laboratory as the template.The infectious clone p BR322-rY2,was transfected into MDBK cells to rescue virus rY2.Furthermore,the morphology,growth characteristics,pathogenicity and genetic stability of the recombinant virus were detected by transmission electron microscope,ELISA,immunofluorescence,monolayer cell peroxidase assay and RT-PCR.The results showed that the infectious cloning of each fragment of PCR of p BR322-rY2 was accurate,and the rY2,of the rescued virus was successfully obtained.The rY2 TCID50 of the recombinant virus was10^-5.25/0.1m L.The growth characteristics of the recombinant virus rY2 was similar to that of the parent virus Y2.2.Replication of mouse model of mucosal diseaseIn BALB/c mice,two genotypes of Cp type BVDV(JL22 and rY2)including recombinant virus rY2,and two genotypes of Ncp type BVDV(SY and SY4)were used to form a paired virus.Ncp type BVDV was used to infect pregnant mice,and the persistent infection model of offspring was established.The offspring were reinfected with Cp type BVDV of the same or different genotypes to establish different types of mucosal disease infection models.Through the analysis of pathological changes,changes of antibody levels,changes of immune cells and changes of cytokines,the feasibility of replication of mouse mucosal disease model was verified.The antigen and antibody levels of mother and offspring were detected by PCR and ELISA.The results showed that the persistent infection model of offspring with positive antigen and negative antibody was successfully constructed.Four groups of mucosal disease models of SY4+JL22,SY4+rY2,SY+rY2 and SY+JL22 were established by giving Cp type BVDV of the same genotype and different genotypes to the offspring of persistent infection model.The results showed that compared with the negative control group,the leucocyte and platelet levels of the four mucosal disease models were significantly decreased,and the lymphocytes showed a weakening trend,but there was significant difference only in the SY4+rY2 group.The surface antigens of spleen lymphocytes were detected by flow cytometry.The results showed that the levels of CD45⁺,CD19⁺,CD4⁺and MHC?decreased significantly in all groups,while the level of CD8⁺decreased significantly only in SY4+rY2 infection group,and there was no significant difference in costimulatory molecule CD80⁺/CD86⁺.The secretion levels of TNF-?,IL-1?and IFN-?in serum of mice in each group were detected by ELISA.The results showed that compared with the negative group,the levels of TNF-?and IFN-?in all intervention groups decreased,while the level of proinflammatory factor IL-1?increased significantly.Pathological results showed that the whole body of the SY4+rY2 intervention group had multiple organ diseases,and the digestive system was mostly seen in the mucosal layer of cells,and local lymphocyte infiltration.The results showed that the persistent infection model of BVDV could be established in mice,and the lethal mucosal disease could be induced in the offspring of mice reinfected with Cp type BVDV of the same genotype and different genotypes.The mechanism may be caused by immunosuppression and immune escape.3.Replication of rabbit model of mucosal diseaseNew Zealand white rabbits were used and SY4+rY2 intervention group was taken as the research object.The results showed that the body temperature of pregnant rabbits increased significantly after being infected with Ncp type BVDV SY4 strain,and it was continuously higher than that of the negative control group.The detection of antigen and antibody showed that pregnant rabbits were successfully infected with BVDV,and there were clinical symptoms of BVDV infection,such as slippery fetus,stillbirth and so on.After the detection of antigen and antibody,the persistent infection model with positive antigen and negative antibody was selected,and the Cp type BVDV rY2 strain was given to the newborn rabbit for the second challenge to replicate the model of mucosal disease.The results showed that Cp type BVDV rY2 alone in the control group could induce acute infection,resulting in transient increase of body temperature,significant decrease of white blood cells,lymphocytes and platelets,significant increase of T lymphocytes,significant increase of inflammatory factor IL-1?and decrease of IFN-?.On the other hand,the persistent infection model rabbits showed immunosuppressive states such as leukopenia and the decrease of T lymphocyte level.After the persistent infection model was challenged with Cp type BVDV rY2 for the second time,the body temperature of the newborn rabbits increased rapidly in the first 5 days and then stabilized,the levels of platelet and T lymphocytes decreased significantly,the ratio of CD8⁺T lymphocytes increased,the level of inflammatory factor IL-1?increased significantly,and the level of IFN-?decreased significantly.After continuous observation of the mucosal disease model induced by Cp type BVDV rY2 in the persistent infection model of young rabbits,it was found that the body temperature of the young rabbits increased temporarily about 2 weeks after the second challenge,and at this time,the young rabbits had symptoms such as loss of appetite or abstinence,mental malaise,rough coat,gradual emaciation and diarrhea,and the white blood cells,lymphocytes increased rapidly at this time.The level of inflammatory factor IL-1?increased significantly,while the level of IFN-?decreased significantly,and then the newborn rabbits began to die.The pathological results showed that there were pathological changes in multiple organs of the newborn rabbits at this time,and the immunohistochemical results showed that the virus was distributed all over the body.It is suggested that the persistent infection model of BVDV can also be established in rabbits,and the reinfection of Cp BVDV with different genotypes will lead to mucosal disease.To sum up,?in this study,the BVDV reverse genetic RNA polymerase?system was successfully constructed and the virus rY2 was rescued.?The persistent infection models of BALB/c mice and New Zealand white rabbits were successfully constructed,and it was clarified that reinfection with the same or different genotypes of Cp type BVDV in the persistent infection model may lead to mucosal disease.?It is preliminarily revealed that the mucosal disease induced by BVDV may be caused by immunosuppression and immune escape of the virus.