Isolation And Identification Of Non-cytopathic Bovine Viral Diarrhea-mucosal Disease Virus(BVDV)

In this study,12 samples of bovine lung disease randomly collected from a slaughterhouse in Hohhot,Inner Mongolia Autonomous Region were tested for BVDV by RT-PCR in 2017.One of the results was positive.After the positive disease was treated and MDBK cells were inoculated for virus isolation,blinded to the fifth passage and tested by RT-PCR.The result was positive and continued to the 11 th generation.RT-PCR was performed for each generation.In the assay,the isolates were cultured on monolayer MDBK cells without cytopathic effect.Physicochemical characteristics tests showed that the isolates were sensitive to ether,chloroform and temperature(56°C),which was in line with BVDV characteristics;the isolate H2017 was inoculated to grow into a single layer In the MDBK cells,the indirect immunofluorescence assay(IFA)was carried out,and the specific cells that were fluorescently stained were seen in the cells of the inoculated isolate H2017,and the fluorescent particles appeared in the cytoplasm;The isolated bovine viral diarrhea-mucosal disease virus(BVDV)is a non-cytopathic type(NCP).It was named H2017 strain.According to the published 5'uncoding region and E2 gene of bovine viral diarrhea-mucosa virus published in GenBank,specific primers were designed for gene cloning,sequence analysis and analysis.RT-PCR method was used to amplify 630 bp and 1067 bp,respectively.The target fragment was linked to the PMD-19 T vector to construct the recombinant plasmid PMD-19 T.The recombinant plasmid was transformed into E.coli DH5?,and the PCR-positive amplified liquid was sent to the bioengineering company for sequencing.The sequencing results were referenced to the BVDV in GenBank.The homology analysis and phylogenetic tree analysis of the strain showed that the homology of the isolates H2017 to the published LN-1 strain(accession number: KT896495)in GenBank was 96.5% and 93.6%,respectively.The strain belongs to the BVDV-1 type m gene subtype.The isolate H2017 provides a reference for understanding the geographical distribution,prevalence of BVDV,and further development of vaccines.