LAUSR

The balance of the segmented genome derived from naturally occurring influenza A viruses (IAVs) is delicate and vulnerable to foreign insertions, thus most reporter IAVs up to date are generated using the backbone of the laboratory‐adapted strains. In this study, we constructed a reporter influenza A/H3N2 virus (A/NY‐HiBiT) which was derived from a clinical isolate, by placing a minimized HiBiT tag to the N‐terminus of the viral nuclear‐export protein (NEP). Here, we show that this 11‐amino acid HiBiT tag did not adversely impact the viral genome balance, and the recombinant A/NY‐HiBiT virus maintains its relative stability. Moreover, the replication profile of the HiBiT‐tagged virus can be measured by a simple Nano‐Glo assay, providing a robust high‐throughput screening (THS) platform. We used this platform to evaluate a collection of the pre‐purified fractions which were derived from rare Chinese medicinal materials, and we identified three fractions, including wild Trametes robiniophila (50% methanol fraction), Ganoderma (water fraction), and wild Phellinus igniarius (ethyl acetate fraction), as potent anti‐IAV actives. Our results demonstrate that this IAV reporter can be used as a powerful HTS platform for antiviral development.