The Isolation And Identification Of Genotype2of Bovine Viral Diarrhea Virus(BVDV-2)

Bovine viral diarrhea disease (BVD), characterized by fever, diarrhea, and acute/chronic form of mucosal disease, persistent infection and high abortion, is a worldwide distributed infectious disease of cattle caused by bovine viral diarrhea virus (BVDV). It is an important pathogen of cattle and also causes infection of sheep, pig, deer and other wild animals.BVDV has two genotype, BVDV-1and BVDV-2. BVDV-1which has involved in cattle industry for many years prevenced worldwide, whereas BVDV-2, first reported by research groups in Canadia and America in1980s, can cause the acute diseases which are characterized by thrombocytopenia, hemorrhages and high abortion and mortality rates. After the first BVDV strain isolated in China in1980, many places, more than twenty provinces, had reported the cases of infection of BVDV. BVDV-2was first isolated from cattles in China by our laboratory in2009. However, the information about BVDV-2is scant. Therefore, in this study, we focus on the following three parts:first, epidemiological survey of BVDV in China. Second, study about the characteristic of the isolates. Third, sequencing and genotyping of the isolates. The purpose of this study is to investigate the epidemic status of BVDV in China, which will be important for the development of vaccine.Study one:Altogether127samples, including spleen, lymph nodes, heart muscle, blood etc., were collected from different cattle farms in several province in China. After preparation, samples were detected by sandwich ELISA and RT-PCR. The viral RNA was extracted according to the manufacturer’s instructions and onlyBVDV-1, only BVDV-2, BVDV-1and BVDV-2directed primers were designed for RT-PCR. The results showed that eleven BVDV-2isolates were discovered and no BVDV-1isolate was discovered.Study two:we obtained11BVDV isolates from MDBK cells-inoculated positive blood samples, which couldn’t cause characteristic of CPE for serial passages of18times in MDBK cells. Indirect immunofluorescence assay demonstrated that all the11isolates-infected MDBK cells reacted with the monoclonal antibody specific to BVDV-2with fluoresce lights within the cytoplasms, and no reactivity with MAb specific to BVDV-1. By preparing the ultra-thin sections from the isolates-infected MDBK cells, there were virus-like particles about roughly size of60nm in diameter located in endoplasmic reticulums observed, the morphology of negative staining of phosphotungstic acid for the purified virions were round, with size of60nm in diameter under the transmission electronic microscope.Study three:primers were designed for the amplification of5’-UTR、Npro、E2and NS2-3genes. The PCR fragments were cloned into PMD18-T Vector and send to sequencing. The phylogenetic tree based on5’-UTR gene showed that the11isolates fall into BVDV-2. Comparation analysis of Npro、E2and NS2-3genes between the11isolates and other BVDV resulted that the nucleotide identity were93.1%～67.1%,92.2%～63.2%,91.7%～64.2%, respectively.