Rescue and characterization of a recombinant HY12 bovine enterovirus carrying a foreign HA epitope in the 3A nonstructural protein
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Full-length infectious cDNA clones for recombinant HY12 bovine enteroviruses designated as rHY12-3A-2-HA, rHY12-3A-3-HA, and rHY12-3A-9-HA were constructed by the insertion of an epitope from influenza virus hemagglutinin (HA) at the N-terminus of the HY12-encoded 3A protein at amino acid positions 2, 3, and 9. The recombinant HY12 viruses expressing the HA epitope were rescued and characterized using immunoperoxidase monolayer assay, western blotting, and electron microscopy. The three rescued recombinant marker viruses showed similar characteristics, such as TCID50 titer, plaque size, and growth properties, to those of parental rHY12 virus. Comparative analysis of the nucleotide sequences demonstrated the three recombinant marker viruses remained stable for 15 passages with no genetic changes. The recombinant viruses remained viable in various permissive cell lines, including BHK-21, Vero, and PK15 cells, suggesting that the insertion of the HA epitope tag had no effect on virus infectivity. Mice infected with the recombinant marker viruses and the parental virus produced anti-HY12-virus antibodies, while the recombinant marker viruses also produced anti-HA-epitope-tag antibodies. Taken together, these results demonstrate that HY12 viruses containing genetic markers may be useful tools for future investigations of the mechanisms of viral pathogenesis and virus replication, as well as for vaccine development.
We thank Dr. Yanjin Zhang at the University of Maryland for providing us a gift of the pCI-T7 plasmid. We also thank Dr. Encheng Sun, Dr. Liyan Cao at the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, for technical assistance and helpful suggestions.
This work was supported by grants from the National Natural Science Foundation of China (31572531), National Key Research and Development Programs (2017YFD0500104, 2016YFD0500904, and 2017YFD0500903) and the Natural Science Foundation of Heilongjiang Province in China (C2015064).
Compliance with ethical standards
Conflicts of interest
The authors declare that there are no conflicts of interest.
The handling of mice and the procedures used for this study were done following a standard protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (approval no JLU-20150226), in strict compliance with the requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
- 1.Barya MA, Moll T, Mattson DE (1967) Antigenic analysis of bovine enteroviruses through studies of the kinetics of neutralization. Am J Vet Res 28:1283–1294Google Scholar
- 3.Datta U, Dasgupta A (1994) Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro. J Virol 68:4468–4477Google Scholar
- 4.Dunne HW, Huang CM, Lin WJ (1974) Bovine enteroviruses in the calf: an attempt at serologic, biologic, and pathologic classification. J Am Vet Med Assoc 164:290–294Google Scholar
- 8.Giachetti C, Hwang SS, Semler BL (1992) cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication. J Virol 66:6045–6057Google Scholar
- 10.Han Q, Williams WB, Saunders KO, Seaton KE, Wiehe KJ, Vandergrift N, Von Holle TA, Trama AM, Parks RJ, Luo K, Gurley TC, Kepler TB, Marshall DJ, Montefiori DC, Sutherland LL, Alam MS, Whitesides JF, Bowman CM, Permar SR, Graham BS, Mascola JR, Seed PC, Van Rompay KKA, Tomaras GD, Moody MA, Haynes BF (2017) HIV DNA-adenovirus multiclade envelope vaccine induces gp41 Antibody immunodominance in rhesus macaques. J Virol 91(21):e00923–17CrossRefGoogle Scholar
- 14.Kunin CM, Minuse E (1958) The isolation in tissue culture, chick embryo and suckling mice of filtrable agents from healthy dairy cattle. J Immunol 80:1–11Google Scholar
- 20.Lu HH, Li X, Cuconati A, Wimmer E (1995) Analysis of picornavirus 2A(pro) proteins: separation of proteinase from translation and replication functions. J Virol 69:7445–7452Google Scholar
- 22.Moll T, Davis AD (1959) Isolation and characterization of cytopathogenic enteroviruses from cattle with respiratory disease. Am J Vet Res 20:27–32Google Scholar
- 24.Paul AV (2002) Possible unifying mechanism of picornavirus genome replication. In: Semler BL (ed) In Molecular Biology of PicornaViruses. ASM Press, Washington, DC, pp 227–246Google Scholar
- 34.Tsuchiaka S, Rahpaya SS, Otomaru K, Aoki H, Kishimoto M, Naoi Y, Omatsu T, Sano K, Okazaki-Terashima S, Katayama Y, Oba M, Nagai M, Mizutani T (2017) Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein. BMC Microbiol 17:18CrossRefGoogle Scholar