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Maryland Pathogen Research Institute and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Prince Georges County, MD 20742, USA



Present address: Aids Review Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD 20817, USA.





*

Author to whom correspondence should be addressed.



Abstract West Nile virus WNV is a positive-sense RNA arbovirus responsible for recent outbreaks of severe neurological disease within the US and Europe. Large-scale analyses of antiviral compounds that inhibit virus replication have been limited due to the lack of an adequate WN reporter virus. Previous attempts to insert a reporter into the 3’ untranslated region of WNV generated unstable viruses, suggesting that this region does not accommodate additional nucleotides. Here, we engineered two WNV infectious clones containing insertions at the Capsid C-Capsid Anchor CA junction of the viral polyprotein. Recombinant viruses containing a TAT1-67 or Gaussia Luciferase GLuc gene at this location were successfully recovered. However, rapid loss of most, if not all, of the reporter sequence occurred for both viruses, indicating that the reporter viruses were not stable. While the GLuc viruses predominantly reverted back to wild-type WNV length, the TAT viruses retained up to 75 additional nucleotides of the reporter sequence. These additional nucleotides were stable over at least five passages and did not significantly alter WNV fitness. Thus, the C-CA junction of WNV can tolerate additional nucleotides, though insertions are subject to certain constraints. View Full-Text

Keywords: West Nile virus; WNV; infectious clone; reporter; virus replication West Nile virus; WNV; infectious clone; reporter; virus replication





Autor: Rianna Vandergaast, Lisa I. Hoover, Kang Zheng and Brenda L. Fredericksen †,*

Fuente: http://mdpi.com/



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