Abstract

BackgroundBovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures. Bovine enterovirus (BEV) has a broad host range with low virulence and is a good candidate as a viral vaccine vector. In this study, we explored new insertion sites for the expression of exogenous genes in BEV, and developed a recombinant infectious cDNA clone for BEV BJ101 strain expressing BVDV E0 protein.MethodsA recognition site for the viral proteinase 3Cpro was inserted in the GpBSK-BEV plasmid at the 2C/3A junction by overlapping PCR. Subsequently, the optimized full-length BVDV E0 gene was inserted to obtain the recombinant infectious plasmid GpBSK-BEV-E0. The rescued recombinant virus was obtained by transfection with linearized plasmid. Expression of BVDV E0 in the recombinant virus was confirmed by PCR, western blotting, and immunofluorescence analysis, and the genetic stability was tested in MDBK cells over 10 passages. We further tested the ability of the recombinant virus to induce an antibody response in mice infected with BVDV and immunized them with the recombinant virus and parental strain.ResultsThe rescued recombinant virus rBEV-E0 was identified and confirmed by western blot and indirect immunofluorescence. The sequencing results showed that the recombinant virus remained stable for 10 passages without genetic changes. There was also no significant difference in growth dynamics and plaque morphology between the recombinant virus and parental virus. Mice infected with both recombinant and parental viruses produced antibodies against BEV VP1, while the recombinant virus also induced antibodies against BVDV E0.ConclusionA new insertion site in the BEV vector can be used for the prevention and control of both BEV and BVDV, providing a useful tool for future research on the development of viral vector vaccines.

Highlights

  • Bovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures

  • Insertion of BVDV E0 in the Bovine enterovirus (BEV) genome After generating recombinant infectious BEV-E0 cDNA clones (GpBSK-BEV-E0) following the strategy outlined in Fig. 1, the polymerase chain reaction (PCR) and sequencing results (Table 3; sequencing results not shown) confirmed that the recombinant infectious clone contained the BVDV E0 gene at the correct position

  • To confirm that the rescued recombinant virus contained BVDV E0, the sequence across the insertion site of BVDV E0 was amplified by PCR (Fig. 3), and the sequencing results confirmed the existence of BVDV E0

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Summary

Introduction

Bovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures. Bovine enterovirus (BEV) has a broad host range with low virulence and is a good candidate as a viral vaccine vector. The optimized full-length BVDV E0 gene was inserted to obtain the recombinant infectious plasmid GpBSK-BEV-E0. The viral genome is a non-segmented single-stranded positivestranded RNA with a total length of about 7.5 kb. It can directly translate a polyprotein as mRNA and undergoes a series of degradation steps to produce four structural egg proteins (VP1–VP4) and seven non-structural proteins (2Apro, 2B, 2C, 3A, 3B, 3Cpro, and 3D), among which the viral protease 3Cpro recognizes and cleaves a characteristic amino acid sequence (ALPQG) within exposed and flexible structural domains [12, 13]

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