Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein).
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.
Publication Date: 2015-12-28 PubMed ID: 26711457DOI: 10.1007/s00705-015-2633-6Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Animal Models
- Biotechnology
- Cell Culture
- Disease control
- Disease Diagnosis
- Disease Management
- Disease Prevention
- Disease Surveillance
- Disease Treatment
- Equine Diseases
- Equine Health
- Equine Viral Arteritis
- In Vitro Research
- In Vivo
- Infectious Disease
- Molecular biology
- Vaccine development
- Veterinary Research
- Virology
- Virus
Summary
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This research discusses the creation and examination of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus (EAV), coded with mCherry, a red fluorescent protein. This study highlights the value of using advances in nucleic acid synthesis technology as a research tool for identifying how a virus infects different cell types and how these viral proteins attach and enter various cell subpopulations.
Creation and Examination of a Synthetic Infectious cDNA Clone of EAV
- The researchers designed and synthesised an infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV, which was also encoded with mCherry, a red fluorescent protein. This was done using advances in in silico design and de novo nucleic acid synthesis technology.
- The resulting progeny virions (EAV sVBSmCherry) were then characterized in vitro based on their ability to infect cells, their replicative capacity, and the stability of the mCherry coding sequences after repeated passage in cell culture.
Use of mCherry Sequence as a Research Tool
- The study found that the mCherry sequence remained relatively stable during sequential cell culture passage, suggesting that the EAV-sVBSmCherry-derived virus could serve as a valuable research tool.
- This tool could be used to identify which cell types are prone to infection by the virus (host-cell tropism) and to characterize the proteins in the virus that facilitate its attachment and entry into various subpopulations of peripheral blood mononuclear cells.
Advantages of Nucleic Acid Synthesis Technology
- The study also shows that using the latest methods in nucleic acid synthesis allows for the creation of complex viral genomes, such as those found in arteriviruses, without resorting to the complex techniques traditionally used in molecular cloning.
- This technology makes the process of designing viral vectors more efficient and less risk-ridden, bypassing the difficulties and risks associated with conventional lab techniques.
Cite This Article
APA
Mondal SP, Cook RF, Chelvarajan RL, Henney PJ, Timoney PJ, Balasuriya UB.
(2015).
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(4), 821-832.
https://doi.org/10.1007/s00705-015-2633-6 Publication
Researcher Affiliations
- 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.
- 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.
- 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.
- 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.
- 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.
- 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. ubalasuriya@uky.edu.
MeSH Terms
- Animals
- Antibodies, Monoclonal
- Antigens, Viral
- Cell Line
- Cloning, Molecular
- Cricetinae
- DNA, Complementary / genetics
- Equartevirus / genetics
- Equartevirus / pathogenicity
- Flow Cytometry
- Gene Expression Regulation, Viral / physiology
- Horses
- Luminescent Proteins / genetics
- Luminescent Proteins / metabolism
- Microscopy, Fluorescence
- Rabbits
- Virulence
- Red Fluorescent Protein
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