P igs have long been considered a crucial genetic mixing vessel for influenza A viruses (IAVs) of different hosts (1) because of the dual expression of human (SAα-2,6Gal) and avian (SAα-2,3Gal) viral receptors on their respiratory epithelium. Swine IAVs such as H1N1 and H3N2 subtypes sporadically infect humans and are prone to cause bidirectional interspecies transmission at the swine-human interface (2-5). So far, Eurasian avian-like (EA) H1N1 has dominated prevalence in pig herds in China and caused >10 human infections (6-9). In particular, the dominant genotype 4 (G4) EA H1N1 containing 2009 pandemic influenza A(H1N1) polymerase basic (PB) 1 and 2, polymerase acid (PA), nucleoprotein (NP), and matrix (M) genes, plus the triple-reassortant (TR) nonstructural (NS) gene, is thought to be a candidate virus of potential pandemic (10,11). Indeed, a case of human infection with G4 EA H1N1 was reported in Yunan Province, China, in 2021 (8). It is imperative to conduct surveillance on swine IAVs and evaluate their risk to public health.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81 EHPSA 85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. Key points • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81 EHPSA 85 on NP protein