| 1. RT-PCR method established to detect and distinguish BVDV-1 and BVDV-2Based on the GenBank data of the 5,untranslated regions (5`UTR) known in the GenBank from BVDV for both BVDV-1 and BVDV-2 isolates, Three pairs of primers were purposely designed to amplify specifically 5`UTR fragments from only BVDV-1, or only BVDV-2, and both BVDV-1 and BVDV-2, respectively. Based the above each pair of specific primers for the reverse-transcription polymerase chain reaction (RT-PCR), reference strains NADL (BVDV-1) and 890 (BVDV-2) were differentiated. The diseased tissue and organs (spleen, mesentery lymph node, heart,cannon born medulla,et.al) from clinical cases of cattle probably involved in BVDV-2 infection were detected by the RT-PCR established above using the specific primers for BVDV-2, the results showed that 8 of 18 (44%) samples were BVDV-2 related infection.2. Isolation and identification of BVDV-2The BVDV-2 positive samples by RT-PCR from three regions of XinJiang, QinHai, ShanDong were chose and inoculated into the MDBK cells for the virus isolation, respectively. After 5～8 blind passages in the cells. BVDV-2 infected MDBK were detected by RT-PCR, 3 field isolates of BVDV-2 from the three regions described above was isolated, named XJ-04 isolate, QH-92isolate, SD-06 isolate respectively. Under the light microscope, isolates XJ-04 and SD-06 can produce cell pathogenic effect (CPE) including many vacuolus, cells gradually losing its capability of adherence, eventually becoming mesh leading to CPE. But isolate QH-92 has no visible CPE in MDBK cell lines. Indirect immunofluorescence assay with the specific fluorescence recognized in the cytoplasm showed 3 field isolates of BVDV-2 reacted with the anti-mouse IgG of isolate 890 (BVDV-2 genotype) based-E2 fusion protein. Under the electron microscope many virus-like particles with envelope, round, around 60 nm in diameter were observed in the ultra-thin section of the endoplasmic reticulum from the MDBK cells infected 3 field isolates of BVDV-2. Same virions were found by electron microscope using negative staining of phosphotungstic acid, which were purified from the above infected cells through the procedure of the combination of series of differential centrifugation with ultracentrifugation of 20 % sucrose cushion.3. Clone and sequence analysis of both glycoprotein E2 and non-structural protein NS2-3 genes from 3 field isolates of BVDV-2E2 and NS2-3 gene of BVDV-2 field isolate(sXJ-04,SD-06,QH-92)with the expected size of 1116bp (for E2 gene) and 1527bp (for NS 2-3 gene) was amplified by RT- PCR, respectively, then the amplified genes were cloned into Pmd19-T vectors and further sequenced. Compared with reference sequence from GenBank data, the homology of the E2 nucleotide sequences were between 80.9%～92.1%, the homology of the deduced amino acid of E2 amino acid sequences was 83.1～93.5%; The homology of the NS2-3 nucleotide sequences was 89.7%～91.8%, and the homology of the deduced amino acid of NS2-3 amino acid sequences was 99.0%. Based on the phylogenetic analysis, 3 field isolates of BVDV-2 have the closest relationship of genetic distance with North American reference strain (p11Q,890,New York,93,1373,p24515).This is the first report involved in BVDV-2 infected isolation and identification in China. The RT-PCR method established can be used to detect and distinguish BVDV-2.
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