The goal of the proposed research is to understand the basis for alphavirus induced disease. In the previous grant period we deduced a consensus sequence for the prototype alphavirus, Sindbis (SB), eliminating mutations for cell culture-adaptation and attenuation of virulence in animals. The consensus sequence was reconstituted as a cDNA clone, TR339, to provide a stable genotype representing a native isolate. When injected subcutaneously into neonatal mice, TR339 causes an acute, fatal disease. Extensive replication in extraneural tissues is coincident with surprisingly high levels of the proinflammatory cytokines IFNalphabeta, IFNgamma, TNFalpha and IL-6 in a systemic inflammatory response. Attenuation of the virus infection, either with increasing host age or by introduction of attenuating mutations, is associated with more restricted virus replication, reduced cytokine induction, and the onset of classical encephalitis. TR339 and all other non-neuroadapted SB strains are avirulent in adult mice. However, in adult animals lacking a functional receptor for IFNalphabeta (IFNalphabetaR-/- mice), TR339 infection was fatal and was characterized by high proinflammatory cytokine levels. In normal adult mice, infection of macrophages and dendritic cells is suppressed by the IFNalphabeta response, whereas these cells are fully permissive in IFNalphabetaR-/- adult mice and may have been directly or indirectly responsible for the proinflammatory cytokine cascade. The Specific Aims of this proposal are: 1) to characterize the earliest events in SB infection, 2) to determine the mechanism whereby SB infection induces a systemic inflammatory response, 3) to characterize the effects of IFNalphabeta and IFNgamma on SB infection, and 4) to examine physiological parameters which influence the efficacy of the IFN response and which may contribute to fatal infection of neonatal animals and resistance of adults. These experiments will address fundamental issues in viral pathogenesis using the SB-mouse model, taking advantage of the extensive genetic characterization of this virus, the availability of a genomic clone representative of a native isolate, and expression systems which will allow the identification of the first cells infected in vivo.