The Development of a Screening Tool for a Reovirus T1L Mutant

Interferons (IFNs) are cytokines that provide innate protection for essentially all cells in response to viral infections 1 . Viruses can induce IFN-β, which is then secreted and binds to other cells to induce antiviral gene expression and protection in those neighboring cells. Induction of IFN-β is particularly important in protection of cardiac myocytes against reovirus-induced myocarditis (inflammation of the heart) in mice, which provides a good model for viral myocarditis in humans. In general, nonmyocarditic reoviruses, such as T3D, induce IFN-β well while myocarditic reoviruses, such as T1L, induce IFN-β poorly 2 . T3D is highly sensitive to the antiviral effects of IFN-β, in contrast to myocarditic reovirus strains which are less sensitive to the effects of IFN-β. We hypothesized that a novel selection procedure can be developed to select T1L mutants that induce IFN. These T1L mutants could be used in the future to identify phenotypes that vary concomitantly with increased induction of IFN. In order to select T1L mutants that induce IFN, we employed vesicular stomatitis virus (VSV), which is highly sensitive to the antiviral effects of IFN-β 3 . The overall strategy is to use T3D, a virus known to induce IFN, in varying dilutions to see whether limited reovirus infection is sufficient to protect against a subsequent challenge with VSV. After finding a cell culture well that was not killed by VSV, and thus protected by reovirus, 0.1% NP-40 would be used to lyse the cells and inactivate the enveloped VSV while leaving the nonenveloped reovirus intact for isolation and further amplification. After optimizing the assay using reovirus T3D, the assay would be used to select T1L mutants for further study of parameters of viral infection that induce IFN. INTRODUCTION Interferons (IFNs) are cytokines made by essentially all cells in response to viral infections in the body. In general, IFNs inhibit viral replication within neighboring cells. IFN-β is controlled by multiple regulatory factors in the cell. Viruses can induce IFN-β, which is then secreted and binds to other cells to induce antiviral gene expression. This process protects adjacent cells by stopping the spread of virus. Viruses in the Reoviridae (Respiratory Enteric Orphan virus) family are not enveloped and have a genome that consists of 10 segments. The two reovirus strains of interest are Type 1 Lang (T1L) and Type 3 Dearing (T3D), which differ in all 10 genomic segments. It is known that nonmyocarditic reoviruses, such as T3D, induce IFN-β well while myocarditic reoviruses, like T1L, induce IFN-β poorly. T3D is highly sensitive to the antiviral effects of IFN-β, which is unusual when compared to myocarditic reovirus strains which are less sensitive to the effects of