Molecular Characteristics of Bovine Viral Diarrhea Virus Strains Isolated from Persistently Infected Cattle

Abstract

In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2-96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5'-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56-61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.

Keywords: BVDV-1b; bovine viral diarrhea virus; molecular characteristics; persistently infected cattle.

Figure A1

The genomic nucleotide identity between the new isolates and reference BVDV strains. The numbers from 1 to 27 represent the strains from TJ2109 to SD0803. The black cells represent the dividing line between percent identity and distance.

Figure 1

Nine BVDV isolates were isolated from serum samples of PI animals with mucosal disease. (A). Amplification signals with 255 bp were detected in all MDBK cells infected with the serum samples using RT-PCR. M. DNA Marker (D2000); 1–9. BVDV TJ2101–2109 infected cells; 10. Negative Control; 11. Positive Control. (B). The specific green signals were detected in infected cells with IFA. (C). Mock-infected cells were detected with IFA (400×). Cytopathic conditions of the NCP BVDV strain infected cells (D), CP BVDV strain infected cells (E), and mock-infected cells (F), were observed (200×).

Figure 2

Phylogenetic analysis of the new isolates with 31 BVDV reference strains based on the 5′-UTR nucleotide sequences. The 5′-UTR nucleotide sequences of the new isolates were aligned with 31 reference BVDV strains, and phylogenetic analysis was carried out by the neighbor-joining method using MEGA7 software. The strains marked with black triangles were the new isolates. The 5′-UTR nucleotide sequences of the reference BVDV strains were obtained from the GenBank data library.

Figure 3

Phylogenetic analysis of the new isolates with pestiviruses based on the full-length nucleotide sequences. The genomic sequences of the new isolates were generated and aligned with representative pestiviruses including BVDV-1, BVDV-2, BDV, and CSFV, in this study. The strains marked with black triangles were the new isolates. The full-length genome nucleotide sequences of the reference pestivirus strains were obtained from the GenBank data library.

Figure 4

Sequence alignment of the 5′-UTR of the new isolates and 13 reference strains. Some nucleotide deletions or mutations of these isolates are shadowed and described in detail in the text. * represent a number. For example, the * between 60 and 80 is 70.

Figure 5

Amino acid characteristics of E2 protein. (A) Amino acid sequence alignment of E2 proteins of the nine new BVDV isolates and 20 reference isolates. Amino acid deletions or mutations of these isolates were shadowed and described in detail in the text. (B) The linear B cell epitopes on E2 proteins of the 9 new isolates and 3 BVDV-1b reference strains were predicted using BepiPred-2.0. The linear B cell epitopes on the E2 proteins of the TJ2102, TJ2103 and TJ2107 strains were not shown with the consistency of the TJ2101 strain. The amino acids of the predicted linear B cell epitopes are highlighted with red letters and blue shading. (C) Tertiary structure on the E2 protein of the new isolates. The unique amino acid residues are marked in red. (D) Glycosylation sites analysis of the new BVDV isolates and reference strains. The unique glycosylation site is shadowed darker than the conserved ones. * represent a number. For example, the * between 60 and 80 is 70.

Figure 5

Amino acid characteristics of E2 protein. (A) Amino acid sequence alignment of E2 proteins of the nine new BVDV isolates and 20 reference isolates. Amino acid deletions or mutations of these isolates were shadowed and described in detail in the text. (B) The linear B cell epitopes on E2 proteins of the 9 new isolates and 3 BVDV-1b reference strains were predicted using BepiPred-2.0. The linear B cell epitopes on the E2 proteins of the TJ2102, TJ2103 and TJ2107 strains were not shown with the consistency of the TJ2101 strain. The amino acids of the predicted linear B cell epitopes are highlighted with red letters and blue shading. (C) Tertiary structure on the E2 protein of the new isolates. The unique amino acid residues are marked in red. (D) Glycosylation sites analysis of the new BVDV isolates and reference strains. The unique glycosylation site is shadowed darker than the conserved ones. * represent a number. For example, the * between 60 and 80 is 70.

Figure 6

Amino acid sequence alignment of E0 of the 9 new BVDV isolates and 13 reference strains. Seven linear epitopes of E0 were found and are less lightly shadowed than the amino acid mutations region. * represent a number. For example, the * between 60 and 80 is 70.

Figure 7

Recombination analysis on the genomes of the new BVDV isolates. Recombination analyses were conducted using RDP4 and SimPlot software. (A) Recombination breakpoints are marked with red lines, and the numbers beside the lines indicate the nucleotide position of the breakpoints relative to the genomic sequence of the recombinant virus. (B) Phylogenetic analyses of the recombination regions. Strains with red and blue letters were found to be major and minor parents of the recombinant strains.