Isolation and identification of a potential new bovine viral diarrhea virus genotype associated with severe outbreaks in yaks (Bos grunniens) in China

Figures

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  Figure 1. Phylogenetic analysis of BVDV isolates from yaks. (A) Maximum-likelihood tree inferred from the 5′ UTR region (200 bp). (B) Maximum-likelihood tree inferred from the Npro region (390 bp). Trees were constructed in MEGA using the best-fit nucleotide substitution model (selected in MEGA) and assessed with 1,000 bootstrap replicates. Isolates from this study are shown in bold. Bootstrap values ≥70% (1,000 replicates) are shown at the corresponding nodes. Scale bars indicate the number of nucleotide substitutions per site.

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  Figure 2. CPE and identification of BVDV isolates in MDBK cells. (A) Typical CPE observed in MDBK cells at 90 hpi, characterized by cell rounding, cytoplasmic granulation, cell detachment, and enlargement of intercellular spaces. (B) Uninfected control cells after 90 h of culture. (C) IFA of infected cells using a rabbit polyclonal anti-BVDV-1a (C24V) immune serum; widespread fluorescence indicates viral antigen expression. (D) TEM image showing pestivirus-like particles (~40–60 nm) in the cytoplasm; inset shows a higher-magnification view.

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  Figure 3. Phylogenetic relationships of the isolates with representative pestiviruses based on multiple genomic regions. (A) ML tree based on the CDS nucleotide sequences (GTR + G + I). (B) ML tree based on the 5′ UTR (280 bp) nucleotide sequences (K2 + G + I). (C) ML tree based on the Npro nucleotide sequences (GTR + G + I). (D) ML tree based on the E2 nucleotide sequences (GTR + G + I). (E) ML tree based on the conserved NS5B (RdRp) amino acid sequences (aa 3312–3837; LT + G + I). Bootstrap support values were estimated using 1,000 replicates. The numbers of reference sequences included for BVDV-1, BVDV-2, and BVDV-3 were 226, 69, and 28, respectively. Isolates obtained in this study are shown in bold. Only bootstrap values ≥70% are displayed at the corresponding nodes. Scale bars indicate the number of substitutions per site.

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  Figure 4. Homology modeling of the E2 protein three-dimensional structure using SWISS-MODEL with PDB ID: 4JNT as the template. Five major divergent regions are highlighted: A (aa 30–39), B (aa 85–93), C (aa 176–182), D (aa 224–228), and E (aa 246–251).