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.

Regulation of polymeric immunoglobulin receptor expression by reovirus.

Polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA and IgA-coated immune complexes from the lamina propria across epithelia and into secretions. The effect of reovirus infection on regulation of pIgR expression in the human intestinal epithelial cell line HT-29 was characterized in this report. Both replication-competent and UV-inactivated reovirus at m.o.i. equivalents of 1-100 p.f.u. per cell upregulated pIgR mRNA by 24 h post-infection and intracellular pIgR protein was increased at 48 h following exposure to UV-inactivated virus. Binding of virus to HT-29 cells was required, as pre-incubating virus with specific antiserum, but not non-immune serum, inhibited reovirus-mediated pIgR upregulation. Endosomal acidification leading to uncoating of virus is a required step for pIgR upregulation, as ammonium chloride or bafilomycin A1 pre-treatment inhibited virus-induced pIgR upregulation. Inhibition experiments using the calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal suggested that calpains are involved in reovirus-mediated pIgR upregulation. Upregulation of pIgR following virus infection appears to be an innate immune response against invading pathogens that could help the host clear infection effectively. Signalling induced by microbes and their products may serve to augment pIgR-mediated transcytosis of IgA, linking the innate and acquired immune responses to viruses.