Establishment Of Taq Man Fluorescence Quantitative PCR Assay And Challenge Model Of Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus(BVDV)is one of the major pathogens causing bovine respiratory disease syndrome(BRDC),to which cattle of all ages are susceptible and which is widely spread worldwide.BVDV infection can present with a variety of intestinal and respiratory symptoms,subclinical infection may be accompanied by secondary disease,reproductive disease in susceptible breeding heifers,and vertical transmission leading to persistent infection(PI)in output In cattle,BVDV infection may be subclinical or secondary to infection and develop into a serious and fatal disease.PI cattle are the main hosts of BVDV and can excrete large amounts of the virus in urine,feces,excreta,milk and semen,posing a serious risk to the cattle industry,and early screening of PI cattle is a key factor in the prevention and control of BVDV.In this study,we designed specific primers and probes for the 5’UTR of BVDV,amplified the BVDV 5’UTR,then constructed positive recombinant plasmids to establish a standard curve,and established a Taq Man real-time fluorescence quantitative PCR assay for BVDV after verification of specificity,sensitivity and compliance.The results showed that the established real-time fluorescence PCR method had good linearity with R~2=0.998at positive standard plasmid copy number of 1×10~4～1×10~8copies/μL,and the minimum detection limit of the method was 10 copies/μL.It can specifically detect BVDV and has no cross-reactivity with bovine coronavirus(BCV),bovine rotavirus(BRV),bovine parainfluenza virus type 3(BPIV-3)and bovine respiratory syncytial virus(BRSV).The compliance rate was 97.5%when compared with commercial kits.The BVDV attack test was also performed to establish the BVDV attack model.The results showed that all three calves in the attack group showed elevated body temperature and exceeded 40℃;and clinical signs of BVDV such as mental depression,increased ocular discharge and diarrhea were apparent,and the clinical score was significantly higher than that of the control group.The nasal swabs collected were subjected to fluorescence quantitative PCR to detect the detoxification,which started on the 5th and 6th day and lasted for 7 days,and reached the peak of detoxification on the 9th and 10th day,with the highest detoxification amount reaching 1380.38 copies/μL.The anticoagulated whole blood was collected for leukocyte count,and the results showed that the leukocyte level in the attack group decreased significantly,with the leukocyte decrease rate ranging from 25.3%to 38.8%,and the average The average decrease rate was 30.5%.The results of histopathological sections showed that the ileum of the attack group showed typical intestinal mucosal epithelial cell necrosis and detachment,congestion and hemorrhage;the liver and lung both showed foci of inflammatory cell infiltration.In conclusion,the Taq Man real-time fluorescence quantitative PCR assay established in this study provided technical support for the early and rapid diagnosis of BVDV,and the BVDV attack model was beneficial for the subsequent vaccine evaluation and pathogenesis research.