Because EMCV infection is known to activate MDA5-mediated type I IFN signaling, we further determined whether MDA5 is required for DHX29-mediated antiviral immunity. Our investigation with MDA5 wild-type (WT) and deficient MEFs demonstrated the lack of IFN-β secretion in MDA5-deficient MEFs in response to EMCV regardless of DHX29 status (Fig 4C). In contrast, DHX29 KD in WT MEFs markedly decreased IFN-β release after EMCV infection, when compared with scrambled shRNA-transduced MEFs (Fig 4C). By contrast, we did not observe any difference in IFN-β production in DHX29 KD or MDA5-deficient MEFs in response to VSV infection (Fig 4C). Overall, our single and double knock down studies strongly indicate that DHX29-mediated antiviral response to EMCV is dependent upon MDA5. Subsequently, we pursued to determine whether RIG-I is required for DHX29-mediated antiviral immunity. We performed similar experiments with WT and RIG-I-deficient MEFs, and found no difference in IFN-β production between scrambled siRNA transfected WT and scrambled siRNA-transfected RIG-I-deficient MEFs or between DHX29-specific siRNA-transfected WT and DHX29-specific siRNA transfected RIG-I-deficient MEFs in response to EMCV infection (Fig 4D). However, we observed marked reduction in IFN-β production in both DHX29-specific siRNA-transfected (WT and RIG-I-deficient) MEFs, compared with scrambled siRNA transfected (WT and RIG-I-deficient) MEFs after EMCV infection (Fig 4D), suggesting that DHX29 KD, but not RIG-I deficiency, is responsible for the impairment of EMCV-induced IFN-β production. In contrast, there was no appreciable change in IFN-β production in response to VSV infection between DHX29 siRNA- and scrambled siRNA-transfected WT MEFs (Fig 4D). As expected, RIG-I-deficient MEFs failed to produce IFN-β after VSV infection regardless of DHX29 status (Fig 4D). Taken together, these results clearly suggest that both DHX29 and MDA5 are specifically required for EMCV-, but not VSV-, induced antiviral immune response in MEFs.