Abstract
Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.
Highlights
IFN-a and IFN-b are structurally related cytokines of the type I interferon family which mediate an early innate immune response to viral infections
Generation of the vesicular stomatitis virus (VSV)*DG(Luc) replicon Previously, a VSV replicon vector was generated by replacing the glycoprotein G gene of VSV with the hemagglutinin (HA) gene of an H7N1 influenza virus and inserting an extra transcription unit into the HA-L intergenic junction to drive expression of a modified green fluorescent protein (GFP) gene [28]
Several mammalian and avian cells were found to be susceptible to infection with the virus replicon particles (VRP) in line with previous observations on the very broad cell tropism of VSV [29]
Summary
IFN-a and IFN-b are structurally related cytokines of the type I interferon family which mediate an early innate immune response to viral infections. IFN-a and IFN-b act by binding to a common, ubiquitously expressed IFN-a/b receptor resulting in the activation of the JAK/STAT signal transduction pathway and transcription of ‘‘IFN-induced genes’’ [1,2]. Several of these genes encode for proteins with strong antiviral activity, i.e. Mx protein, protein kinase R, and 29-59oligo(A) synthetase [3]. More important is the paracrine action of type I IFN, which induces an antiviral state in previously uninfected cells, thereby blocking virus dissemination in the organism. An issue of increasing importance is the determination of neutralizing antibodies that are induced in some patients following recombinant IFN therapy [10]
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