Defective interfering particles (DIPs) replicate at the expense of co-infecting, fully infectious homologous virus. Typically, they contain a highly deleted form of the viral genome. Utilizing single-cell analysis, we here report the discovery of a yet unknown DIP type, derived from influenza A viruses (IAV), termed OP7 virus. Instead of deletions, the genomic viral RNA (vRNA) of segment 7 (S7) carried 37 point mutations compared to the reference sequence, affecting promotor regions, encoded proteins and genome packaging signals. Co-infection experiments demonstrated strong interference of OP7 virus with IAV replication, manifested by a dramatic decrease in infectivity of released virions. Moreover, an over-proportional quantity of S7 in relation to other genome segments was observed, both intracellularly and in the released virus population. Concurrently, OP7 virions lacked a large fraction of other vRNA segments, which appears to constitute its defect in virus replication. OP7 virus might serve as a promising candidate for antiviral therapy. Furthermore, this novel form of DIP may also be present in other IAV preparations.IMPORTANCE Defective-interfering particles (DIPs) typically contain a highly deleted form of the viral genome, rendering them defective in virus replication. Yet, upon complementation through co-infection with fully infectious standard virus (STV), interference with the viral life cycle can be observed, leading to a suppressed STV replication and the release of mainly non-infectious DIPs. Interestingly, recent research indicates that DIPs may serve as an antiviral agent. We here report the discovery of a yet unknown type of influenza A virus-derived DIP (termed "OP7" virus) that contains numerous point mutations instead of large deletions in its genome. Furthermore, the underlying principles that render OP7 virions interfering and apparently defective seem to differ from conventional DIPs. In conclusion, we believe that OP7 virus might be a promising candidate for antiviral therapy. Moreover, it exerts strong effects, both on virus replication and on host cell response, and may have been overlooked in other IAV preparations.