Molecular Epidemiology of Bovine Enteroviruses and Genome Characterization of Two Novel Bovine Enterovirus Strains in Guangxi, China

FIG 1. Prevalence and phylogenetic analysis of BEV in Guangxi Province, China. (A) Positivity rates and geographic distribution of BEV in Guangxi Province. (B) Phylogenetic analysis of global BEV strains based on the 5′-UTR gene.

FIG 2. Cytopathogenic effect (CPE), plaque purification, and growth curve analysis in MDBK cells. (A) CPE in MDBK cells infected with strain GXNN2106. Mock-infected and virus-infected MDBK cells were observed at 36 h postinfection (hpi). (B) MDBK cells without virus inoculation (original magnification, ×20). (C) Plaque morphology of strain BEV-GXGL2215 on MDBK cells. (D) Plaque morphology of the mock MDBK cells. (E) Growth kinetics of the eight isolated strains. At different times postinfection, the cell supernatants were collected, and the virus titers were determined as the 50% tissue culture infective dose (TCID50) values. The results represented here are the means of three independent experiments.

FIG 3. Recombinant protein and IFA analysis. (A) Analysis of expression of the purified BEV-GXNN2106 VP2 by SDS-PAGE. (B) Western blot analysis of expressed recombinant BEV-GXNN2106 VP2 showing reactivity with horseradish peroxidase-conjugated anti-His tag monoclonal antibody. (C) IFA analysis of the VP2 protein expression was conducted in MDBK cells. The virus-infected cells were fixed and stained using an anti-buffalo BEV-VP2 PcAb and goat anti-rabbit IgG H+L (green). The cell nuclei were visualized by DAPI (4′,6-diamidino-2-phenylindole staining) (blue). IFA, indirect immunofluorescence assay.

FIG 4. Phylogenetic trees based on the full-length amino acid sequences of the polyprotein (A), P1 (B), and VP1 (C) genes in the virulent strains isolated (black dots).

FIG 5. Recombination within the genomes of the virulent strains GXYL2213 (A), GXNN2204 (B), and GXGL2215 (C). Recombination analysis performed using RDP5.