Production of Alexa Fluor 488-labeled reovirus and characterization of target cell binding, competence, and immunogenicity of labeled virions.
Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time of flight analysis indicated that Alexa Fluor 488 labeled the outer capsid proteins of reovirus. Labeled virus bound to murine L929 fibroblasts as determined by flow cytometry and fluorescence microscopy, and the specificity of binding were demonstrated by competitive inhibition with non-labeled virus. Labeled reovirus induced apoptosis and cytopathic effect in infected L929 cells. Mice infected with labeled virus mounted robust serum antibody and CD8(+) T-cell responses, indicating that labeled virus retained immunogenicity in vivo. These results indicate that Alexa Fluor 488-labeled virus provides a powerful new tool to analyze reovirus infection in vitro and in vivo.
Fecek
Ronald J
RJ
Department of Microbiology, Immunology, and Cell Biology, Robert C. Byrd Health Sciences Center of West Virginia University, P.O. Box 9177, Morgantown, WV 26506-9177, USA.
Busch
Ryan
R
Lin
Hong
H
Pal
Kasturi
K
Cunningham
Cynthia A
CA
Cuff
Christopher F
CF
D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008297:Male; D012089:Mammalian orthoreovirus 3 / Q000737:chemistry* / Q000276:immunology / Q000472:pathogenicity; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D012088:Reoviridae Infections / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods*; D013388:Succinimides / Q000737:chemistry*; D014771:Virion / Q000737:chemistry*
C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D036022:Capsid Proteins; D005456:Fluorescent Dyes; D013388:Succinimides