Isolation,Identification and Mouse Pathogenicity Analysis of a Strain of Bovine Viral Diarrhea Virus from Imported Fetal Bovine Serum
[Objective]The experiment aims to isolate,identify and analyze the genetic evolution of Bovine viral diarrhea virus(BVDV)in imported fetal bovine serum.[Method]The presence of BVDV nucleic acid contamination in the imported fetal bovine serum purchased by the laboratory was detected by RT-PCR,the contaminated serum was inoculated with MDBK cells,and the cells were subcultured to the 7th passage,and the cultures of each generation of cells were amplified by BVDV 5'-UTR,and the PCR amplification product was ligated with the pMD19-T vector and sequenced for genetic evolution analysis.Indirect immunofluorescence assay(IFA)was used to detect the antigen and virus titer of the isolates.BALB/c mice were inoculated with the isolates,and orbital blood was collected for blood routine detection and RT-PCR detection.[Result]The results showed that the virus strain failed to cause cytopathy during MDBK cell culture,and the RT-PCR amplification of cell culture was positive.Analysis based on the similarity of the 5'-UTR sequence indicated that the isolated strain had 99.6％similarity in its 5'-UTR sequence to the BVDV UDEC-COY7-17(OL860952.1)strain,both belonged to the BVDV-1e subtype.IFA result detected specific green fluorescent signals in the cytoplasm of MDBK cells infected with the isolated strain,while no signals were observed in the control group.The 50％tissue culture infectious dose(TCID50)was determined to be 10-4.7/0.1 mL.Blood routine tests conducted on BALB/c mice on the 7th day after immunization revealed extremely significantly lower counts of white blood cells(WBC)and lymphocytes(LYM)compared with control group(P＜0.01).Total RNA extracted from the blood was subjected to 5'-UTR RT-PCR testing,which yielded positive results with an amplified product size of 298 bp,consistent with expectations.[Conclusion]A NCP BVDV-1e subtype strain was isolated in this study.It was proved that there was BVDV antigen contamination in imported fetal bovine serum,and also provided experimental materials for further study of the related molecular mechanisms of BVDV.
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