Generation and Characterization of an Influenza D Reporter Virus.

Influenza D virus (IDV) can infect various livestock animals, such as cattle, swine, and small ruminants, and was shown to have zoonotic potential. Therefore, it is important to identify viral factors involved in the broad host tropism and identify potential antiviral compounds that can inhibit IDV infection. Recombinant reporter viruses provide powerful tools for studying viral infections and antiviral drug discovery. Here we present the generation of a fluorescent reporter IDV using our previously established reverse genetic system for IDV. The mNeonGreen (mNG) fluorescent reporter gene was incorporated into the IDV non-structural gene segment as a fusion protein with the viral NS1 or NS2 proteins, or as a separate protein flanked by two autoproteolytic cleavage sites. We demonstrate that only recombinant reporter viruses expressing mNG as an additional separate protein or as an N-terminal fusion protein with NS1 could be rescued, albeit attenuated, compared to the parental reverse genetic clone. Serial passaging experiments demonstrated that the mNG gene is stably integrated for up to three passages, after which internal deletions accumulate. We conducted a proof-of-principle antiviral screening with the established fluorescent reporter viruses and identified two compounds influencing IDV infection. These results demonstrate that the newly established recombinant IDV reporter virus can be applied for antiviral drug discovery and monitoring viral replication, adding a new molecular tool for investigating IDV.