Influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range and a broad geographical distribution. Recent IDV outbreaks in swine along with serological and genetic evidence of IDV infection in humans have raised concerns regarding the zoonotic potential of this virus. To better study IDV at the molecular level, a reverse-genetics system (RGS) is urgently needed, but to date, no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven-plasmidbased system and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV minigenome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein (RNP) complex, resulting in either attenuated or replicationincompetent viruses. Finally, by using the minigenome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3= conserved noncoding region in IDV and influenza C virus (ICV) resulted in the inefficient crossrecognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a minigenome replication assay and a robust reversegenetics system that can be used to further study replication, tropism, and pathogenesis of IDV. [ABSTRACT FROM AUTHOR]