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Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein)

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Abstract

Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3+ T cells and CD14+ monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques.

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Acknowledgments

This work was supported by Agriculture and Food Research Initiative competitive grant number 2013-68004-20360 from the United States Department of Agriculture National Institute of Food and Agriculture (USDA-NIFA). The authors would like to thank Ms. Jennifer Strange and Mr. Greg Bauman, Flow Cytometry Core Facility, Department of Microbiology, Immunology and Molecular Genetics, College of Medicine, University of Kentucky (UK), for their assistance with flow cytometry. The UK Flow Cytometry & Cell Sorting core facility is supported in part by the Office of the Vice President for Research, the Markey Cancer Center and an NCI Center Core Support Grant (P30 CA177558) to the University of Kentucky Markey Cancer Center.

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Authors and Affiliations

  1. 108 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546-0099, USA

    Shankar P. Mondal, R. Frank Cook, R. Lakshman Chelvarajan, Pamela J. Henney, Peter J. Timoney & Udeni B. R. Balasuriya

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  1. Shankar P. Mondal
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  2. R. Frank Cook
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  3. R. Lakshman Chelvarajan
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  4. Pamela J. Henney
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  5. Peter J. Timoney
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  6. Udeni B. R. Balasuriya
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Correspondence to Udeni B. R. Balasuriya.

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Mondal, S.P., Cook, R.F., Chelvarajan, R.L. et al. Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein). Arch Virol 161, 821–832 (2016). https://doi.org/10.1007/s00705-015-2633-6

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  • DOI: https://doi.org/10.1007/s00705-015-2633-6

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