Characterization of bovine-derived H5N1 viruses expressing fluorescent and luminescent reporter proteins

Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses present a broad host range, with recent spillover and sustained transmission in dairy cattle reported in the USA. Replication-competent reporter viruses are critical tools that enable real-time monitoring of virus replication, facilitating high-throughput screens. In this study, we engineered three recombinant H5N1 clade 2.3.4.4b reporter viruses expressing nanoluciferase (NLuc) and two fluorescent reporter proteins, miniGFP2 and UnaG within the open reading frame of the nonstructural gene of the bovine A/Cattle/Texas/063224-24-1/2024 (TX2/24) virus. All reporter viruses replicated efficiently in vitro , presenting replication kinetics comparable to the parental rTX2/24 virus, but exhibited smaller plaque sizes, suggesting reduced cell-to-cell spread. In vivo infection studies in mice showed comparable pathogenicity among all four viruses, although rTX2/24-miniGFP2 and rTX2/24-UnaG exhibited decreased virus shedding relative to rTX2/24 and rTX2/24-NLuc. Virus titrations and in situ localization of virus replication sites demonstrated robust replication in respiratory tissues, with slightly attenuated systemic dissemination of all three reporter viruses. Fluorescent virus neutralization assays using miniGFP2 and UnaG reporter viruses accurately quantified neutralizing antibody titres in sera from naturally infected dairy cattle, consistent with wild-type virus assays. Additionally, the utility of the NLuc reporter virus for antiviral screening was validated against oseltamivir in vitro . Collectively, these results establish the H5N1 TX2/24-based reporter viruses as versatile and biologically relevant tools for investigating H5N1 pathogenesis and for use in serological and antiviral drug screens.