Rescue and characterization of a recombinant HY12 bovine enterovirus carrying a foreign HA epitope in the 3A nonstructural protein

  • Dan Liu
  • Changming Liu
  • Xing Liu
  • Xin Li
  • Liping Huang
  • Junying Hu
  • Yanwu Wei
  • Hongzhen Zhu
  • Qun Zhang
  • Xinping WangEmail author
Original Article


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.


Author contributions

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.

Ethical approval

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.


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Dan Liu
    • 1
  • Changming Liu
    • 2
  • Xing Liu
    • 3
  • Xin Li
    • 1
  • Liping Huang
    • 2
  • Junying Hu
    • 1
  • Yanwu Wei
    • 2
  • Hongzhen Zhu
    • 2
  • Qun Zhang
    • 1
  • Xinping Wang
    • 1
    Email author
  1. 1.Key Laboratory for Zoonoses Research, Ministry of Education, College of Veterinary MedicineJilin UniversityChangchunChina
  2. 2.State Key Laboratory of Veterinary Biotechnology, Division of Swine Infectious Diseases, Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbinChina
  3. 3.Inactivated Vaccine Production Workshop Comprehensive GroupHarbin Weike Biotechnology Development CompanyHarbinChina

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