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Monoclonal antibodies (mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein (NP) of influenza A virus (IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10E9 and 3F3, were generated with recombinant truncated NP proteins (NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10E9 and 3F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10E9 and 3F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10E9 and 3F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10E9 and 3F3 can be used in immunoprecipitation assay, and 10E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10E9-recognized 84SAGKDP89 epitope and 3F3-recognized 320ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Some H5 viruses isolated in poultry or wild birds between 2020 and 2021 were found to be antigenically different from the vaccine strains (H5-Re11 and H5-Re12) used in China. In this study, we generated three new recombinant vaccine seed viruses by using reverse genetics and used them for vaccine production. The vaccine strain H5-Re13 contains the hemagglutinin (HA) and neuraminidase (NA) genes of an H5N6 virus that bears the clade 2.3.4.4h HA gene, H5-Re14 contains the HA and NA genes of an H5N8 virus that bears the clade 2.3.4.4b HA gene, and H7-Re4 contains the HA and NA genes of H7N9 virus detected in 2021. We evaluated the protective efficacy of the novel H5/H7 trivalent inactivated vaccine in chickens, ducks, and geese. The inactivated vaccine was immunogenic and induced substantial antibody responses in the birds tested. Three weeks after vaccination, chickens were challenged with five different viruses detected in 2020 and 2021: three viruses (an H5N1 virus, an H5N6 virus, and an H5N8 virus) bearing the clade 2.3.4.4b HA gene, an H5N6 virus bearing the clade 2.3.4.4h HA gene, and an H7N9 virus. All of the control birds shed high titers of virus and died within 4 days post-challenge, whereas the vaccinated chickens were completely protected from these viruses. Similar protective efficacy against H5 viruses bearing the clade 2.3.4.4h or 2.3.4.4b HA gene was observed in ducks and geese. Our study indicates that the newly updated H5/H7 vaccine can provide solid protection against the H5 and H7N9 viruses that are currently circulating in nature.

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5N6) (DK/GZ/S4184/17) (a clade 2.3.4.4d virus), A/chicken/Liaoning/SD007/2017(H5N1) (CK/LN/SD007/17) (a clade 2.3.2.1d virus), and A/chicken/Guangxi/SD098/2017(H7N9) (CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.

H5N1 influenza represents one of the great challenges to public health. Some H5N1 viruses (i.e., A/goose/Hubei/65/05, GS/65) are weakly pathogenic, while the others (i.e., A/duck/Hubei/49/05, DK/49) are highly pathogenic to their natural hosts. Here, we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus. We demonstrated that, compared to the GS/65 virus, the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways (i.e., RIG-I and MDA5) in ducks. We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes, while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus. We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis (WGCNA). Moreover, we first demonstrated that the duck BCL2L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently. These analyses, in combination with our comprehensive transcriptomic data, provided global view of the molecular architecture for the interaction between host and H5N1 viruses.

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.