Abstract
Eighty calf blood samples were collected from different areas of China and were examined. Sixteen non cytopathic isolates of bovine viral diarrhea virus genotype 2 (BVDV-2) were isolated by specific BVDV-2-directed RT-PCR and sandwich ELISA. The results were verified by the indirect immunofluorescence assay and the ultra-thin sections. Genes of 5'-UTR, E2 and Npro from all the 16 field isolates were sequenced. The sequence identities at the nucleotide and amino acid levels were higher than 99.0%. The phylogenetic analysis revealed that the BVDV-2 USA strain, AzSpin, had the highest sequence homology with each of the Chinese isolates. Key words: Bovine viral diarrhea virus genotype 2 (BVDV-2), isolation and characterization, phylogenetic analysis.
Highlights
Bovine viral diarrhea virus (BVDV) is an enveloped positive-stranded RNA virus in the genus Pestivirus of the family Flaviviridae (Collett et al, 1988; Meyers et al, 1989; Ridpath and Bolin, 1995; Becher et al, 1997)
The 16 Madin Darby bovine kidney (MDBK) cells-infected BVDV isolates were isolated from the positive blood samples respectively, which reacted with the mouse-originated polyclonal antibody against recombination E2 glycoprotein from bovine viral diarrhea virus genotype 2 (BVDV-2) and monoclonal antibody (MAbBz-53 ) specific to BVDV-2 with fluoresce lights within the cytoplasms in the indirect immunofluorescence assay, and no reactivity with MAb D89 specific to BVDV-1
By preparing the ultra-thin sections from the isolates-infected MDBK cells (Wang et al, 2000), there were virus-like particles, roughly about the size of 60 nm in diameter located in endoplasmic reticulums observed (Figure 1B) and the morphology of negative staining of phosphotungstic acid for the purified virions were round, enveloped, with size of 60 nm in diameter under the transmission electronic microscope (TEM) (Figure 1A)
Summary
Bovine viral diarrhea virus (BVDV) is an enveloped positive-stranded RNA virus in the genus Pestivirus of the family Flaviviridae (Collett et al, 1988; Meyers et al, 1989; Ridpath and Bolin, 1995; Becher et al, 1997). Sixteen non cytopathic isolates of bovine viral diarrhea virus genotype 2 (BVDV-2) were isolated by specific BVDV-2directed RT-PCR and sandwich enzyme-linked immunosorbent assay (ELISA). Genes of 5'-UTR, E2 and Npro from all the 16 field isolates were sequenced.
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