Pathogenicity Study Of Two Yak BVDV1 Strains In Mice And Preliminary Preparation Of Inactivated BVDV1 Vaccine

Bovine viral diarrhea virus?BVDV?is a positive-stranded,envelope-encapsulated RNA virus belonging to the genus Pestivirus of the Flaviviridae family.BVDV can cause persistent infection and immunosuppression after entering the body,resulting in decreased milk production,reduced fertility,slow growth and secondary infection of other pathogens,It has caused huge economic losses to the cattle industry and other aquaculture industries in various countries around the world.In recent years,the BVDV infection in yaks has become increasingly serious in the Qinghai-Tibet Plateau,causing certain economic losses to the plateau animal husbandry.Vaccination is the main measure to prevent BVDV infection.In this study,BALB/c mice were injected intraperitoneally by two cytopathic yak-derived BVDV strains SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015 isolated in our laboratory,aiming at analyzing the pathogenicity of bovine viral diarrhea virus?BVDV?in mice.The inactivated vaccine was prepared to study the antibody growth and decline after immunizing BALB/c mice or cows.The results obtained are as follows:1.Pathogenicity study of two yak BVDV1 strains in BALB/c miceTwo yak BVDV1 strains of cytopathic SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015 were successfully isolated and identified in our previous study.This study aims at analyzing the pathogenicity of BVDV in mice,BALB/c mice aged 6-8 weeks were selected as experimental animals and inoculated by intraperitoneal injection.The Oregon C24V standard strain was used as a positive control.The blood,feces and tissues of the mice were collected on the 5^th,7^th,and 10^thh day after inoculation,and the pathogenicity of the virus-infected mice were studied by blood routine,histopathology,and the real-time RT-PCR.The results exhibited that BALB/c mice infected with 2 yak-derived BVDV1 strains showed a certain clinical symptoms,including rough coat,reduced feed intake,aggregated heap,mental depression and diarrhea.The kidney,liver and lung hemorrhages were observed at autopsy,and the spleen margin was cyanotic with mild enlargement,and the intestinal contents were yellow and watery,The clinical symptoms of the mice in the SMU-Z6/1a/SC/2016 group were more severe than those in the SMU-DJ2/1d/QH/2015 group.The lymphocyte,leukocyte,and platelet contents of the challenge group decreased significantly?p<0.05?.The results of the real-time RT-PCR showed that two BVDV1 could be detected in different tissues of mice,and the highest load of virus in the lungs was 1.47×10⁷ copies/?L?SMU-Z6/1a/SC/2016?and 3.30×10⁶ copies/?L?SMU-DJ2/1d/QH/2015?.HE staining showed that there were inflammatory cell infiltration,necrosis and shedding of intestinal villus epithelial cells in the liver of the challenge group,and it also showed that there were increased spleen endocytosis and reactive hyperplasia of spleen,alveolar atrophy and hemorrhage and other pathological changes.Compared with the OregonC24V group,the lesion of SMU-Z6/1a/SC/2016 group were the most serious,and the the lesion of SMU-DJ2/1d/QH/2015 group was lighter.The above results showed that BALB/c mice was successfully infected by two BVDV1 strains,and caused certain clinical symptoms and different degrees of pathological damage to tissues.2.Preparation and immune evaluation of two yak BVDV1 inactivated vaccinesIn this study,SMU-Z6/1a/SC/2016 and SMU-DJ2/1d/QH/2015 strains were used as vaccine candidates to prepared inactivated vaccine with MONTANIDE ISA 201VG adjuvant.BALB/c mice aged 6-8 weeks were selected as experimental animals,and different doses of BVDV1 inactivated vaccine were inoculated by multiple subcutaneous injections in the back.Antibody neutralization assay was used to study the antibody growth and decline after immunizing BALB/c mice.After immunizing,the results showed that the neutralizing antibody titering in BALB/c mice reached the highest on the 5^th day after the second immunization with the inactivated vaccine prepared by the two isolates,SMU-Z6/1a/SC/2016 with the maximum of 1:177,SMU-DJ2/1d/QH/2015 with the maximum of 1:89.The above results indicated that the neutralizing antibodies in mice with the SMU-Z6/1a/SC/2016 vaccine have higher titers compared to the SMU-DJ2/1d/QH/2015 vaccine.Furthermore the antibody cross-neutralization protection test was performed by using the SMU-DJ2/1d/QH/2015 strain with the serum of the SMU-Z6/1a/SC/2016 inactivated vaccine,with the highest antibody titer of 1:177,but the antibody cross-neutralization protection test was performed by using the SMU-Z6/1a/SC/2016 strain with the serum of the SMU-DJ2/1d/QH/2015 inactivated vaccine,with the only antibody titer of 1:22,it was lower than the SMU-Z6/1a/SC/2016 inactivated vaccine.This result showed that the neutralizing antibody prepared by the SMU-Z6/1a/SC/2016 strain could neutralize the SMU-DJ2/1d/QH/2015 strain in mice.Furthermore,we also studied the growth and decline of antibodies with SMU-Z6/1a/SC/2016 inactivated vaccines in cows.The three cows aged from 6-8months were selected as experimental animals which were inoculated with smus-z6/1a/SC/2016 inactivated vaccine through neck muscle,and commercial bovine viral diarrhea/mucosal disease and infectious rhinobronchitis inactivated vaccine was used as a positive control.The antibody titers were detected by neutralization test and ELISA methods.After immunizing,the results showed that the neutralizing antibody titer in cows reached the highest on the 7^th day after the second immunization,with the maximum of 1:1413,but commercial inactivated vaccine with the maximum of 1:178.The result showed that the neutralization antibody in cows immunized with SMU-Z6/1a/SC/2016 inactivated vaccine was significantly higher than that of the commercial vaccine group?p<0.05?.The antibody cross-neutralization protection test was performed by using the SMU-DJ2/1d/QH/2015 strain with the serum of the SMU-Z6/1a/SC/2016 inactivated vaccine,with the highest antibody titer of 1:708,but the antibody cross-neutralization protection test was performed by using the SMU-DJ2/1d/QH/2015 strain with the serum of the commercial inactivated vaccine,with the highest antibody titer 1:178,it was significantly lower than the SMU-Z6/1a/SC/2016 inactivated vaccine?p<0.05?.This result showed that the neutralizing antibody prepared by the SMU-Z6/1a/SC/2016 strain could neutralize the SMU-DJ2/1d/QH/2015 strain.In addition,the ELISA based on E2 recombinant protein was developed to detect the BVDV antibody in immunized cows,The results showed that the antibody level of the SMU-Z6/1a/SC/2016 inactivated vaccine group?Cut-off value 3.252?was significantly higher than that of the commercial inactivated vaccine group?Cut-off value 1.920??p<0.05?.The above results indicated that the inactivated vaccine prepared with the SMU-Z6/1a/SC/2016 strain could stimulate high levels of neutralizing antibodies in BALB/c mice and cows,therefore,Therefore,our study provides some data to develop the BVDV inactivated vaccine and to screen the vaccine candidate strains in China.