Apoptosis is a pathological hallmark of encephalitis and myocarditis caused by reovirus in newborn mice. In cell culture models, the antiviral transcription factor interferon regulatory factor 3 (IRF-3) enhances reovirus-induced apoptosis following activation via retinoic acid inducible gene I and interferon promoterstimulating factor 1. To determine the role of IRF-3 in reovirus disease, we infected newborn IRF-3 ؉/؉ and IRF-3 ؊/؊ mice perorally with mildly virulent strain type 1 Lang (T1L) and fully virulent strain type 3 SA؉ (T3SA؉) and monitored infected animals for survival. Both wild-type and IRF-3 ؊/؊ mice succumbed with equivalent frequencies to infection with T3SA؉. However, the absence of IRF-3 was associated with significantly decreased survival rates following infection with T1L. The two virus strains achieved similar peak titers in IRF-3 ؉/؉ and IRF-3 ؊/؊ mice in the intestine, brain, heart, liver, and spleen. However, by day 12 postinoculation, titers in all organs examined were 10-to 100-fold higher in IRF-3 ؊/؊ mice than those in wild-type mice. Increased titers were associated with marked pathological changes in all organs examined, especially in the heart, where absence of IRF-3 resulted in severe myocarditis. Cellular and humoral immune responses were equivalent in wild-type and IRF-3 ؊/؊ animals, suggesting that IRF-3 functions independently of the adaptive immune response to enhance reovirus clearance. Thus, IRF-3 serves to facilitate virus clearance and prevent tissue injury in response to reovirus infection.

The interferon-β (IFN-β) response is critical for protection against viral myocarditis in several mouse models, and IFN-α or -β treatment is beneficial against human viral myocarditis. The IFN-β response in cardiac myocytes and cardiac fibroblasts forms an integrated network for organ protection, however the different IFN-α subtypes have not been studied in cardiac cells. We developed a quantitative RT-PCR assay that distinguishes between thirteen highly conserved IFN-α subtypes, and found that reovirus T3D induces five IFN-α subtypes in primary cardiac myocyte and fibroblast cultures: IFN-α1, -α2, -α4, -α5, and -α8/6. Murine IFN-α1, -α2, -α4, or -α5 treatment induced IRF7 and ISG56 and inhibited reovirus T3D replication in both cell types. This first investigation of IFN-α subtypes in cardiac cells for any virus demonstrates that IFN-α is induced in cardiac cells, that it is both subtype- and cell type-specific, and that it is likely important in the antiviral cardiac response.

Viruses frequently infect the heart but clinical myocarditis is rare, suggesting that the cardiac antiviral response is uniquely effective. Indeed, the Type I interferon (IFN) response is cardiac cell typespecific and provides one integrated network of protection for the heart. Here, a proteomic approach was used to identify additional proteins that may be involved in the cardiac antiviral response. Reovirus-induced murine myocarditis reflects direct viral damage to cardiac cells, and offers an excellent system for study. Primary cultures of murine cardiac myocytes were infected with myocarditic or non-myocarditic reovirus strains, and whole cell lysates were compared by twodimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption / ionization -time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry. Results were quantitative and reproducible, and demonstrated that whole proteome changes clustered according to viral pathogenic phenotype. Moreover, the data suggest that the heat shock protein Hsp25 is modulated differentially by myocarditic and non-myocarditic reoviruses and may play a role in the cardiac antiviral response. Members of seven virus families modulate Hsp25 or Hsp27 expression in a variety of cell types, suggesting that Hsp25 participation in the antiviral response may be widespread. However, results here provide the first evidence for a virus-induced decrease in Hsp25/27, and suggest that viruses may have evolved a mechanism to subvert this protective response, as they have for IFN.