Development of an influenza D virus with an eight- or nine-segment genome

Influenza D virus (IDV) contains seven genome segments. The M and NS segments are regulated via splicing to express two proteins each (P42 and M1 from the M segment and NS1 and NS2 from the NS segment). Previously, we created an eight-segment IDV by separating the NS1 and NS2 genes into independent monocistronic segments. In this study, we designed another eight-segment IDV with two divided M segments, transcribing either P42 or M1 mRNA independently. Specifically, we constructed two plasmids for reverse genetics: one for viral RNA (vRNA) synthesis of the P42 segment with silent mutations at the splicing donor site and one for vRNA synthesis of the M1 segment with deletion of the intron in the M segment. We successfully created the virus via reverse genetics using these two segments and six other vRNA synthesis plasmids (PB2, PB1, P3, HEF, NP, and NS). Furthermore, we generated a nine-segment IDV including the P42, M1, NS1, and NS2 segments. These systems enable independent functional and interactive analyses of M and NS segment-encoded proteins for viral infectivity. Additionally, the eight- and nine-segment IDVs exhibited significantly reduced replication in cell culture compared with wild-type IDV, suggesting a strategy for attenuated live vaccine development.