Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China, and low pathogenic H7N9 viruses have disappeared nationwide. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) based on the sequence of the HA gene. Deduced amino acid sequences of the HA gene revealed that both strains had a four-amino-acid (KRTA) insertion at position 339-342 and an I335V mutation in the cleavage site to make the motif PEVPKRKRTAR↓GLF. Remarkably, both strains gained the F102V and N157D mutations (H3 numbering) in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attentions should be paid to the importance of continual surveillance of the H7N9 AIVs.

H7N9 avian influenza vaccines induce high levels of non-neutralizing (nonNeu) antibodies against the haemagglutinin (HA). However, the antigenic epitopes underlying this particular antibody response are still undefined. In this study, a panel of 15 monoclonal antibodies (mAbs) against the HA protein of H7N9 virus was generated and 14 of them had no hemagglutination inhibition and virus neutralizing activities. Four antigenic epitopes, including one linear and three conformational epitopes, in HA were identified using peptide-based enzyme-linked immunosorbent assay and biopanning of phage display random peptide library. More importantly, two mAbs (4B7 4D5 and 1B10 1D1) strongly inhibited HA-binding of chicken nonNeu antisera against viral-vectored H7N9 vaccine, whereas lower inhibition was observed for chicken neutralizing antisera. In contrast, there was weak competition between the mAb and murine nonNeu antisera against inactivated H7N9 antigen. The epitopes targeted by these two mAbs were defined as the immunodominant epitopes underpinning the elicitation of nonNeu antibodies by viral-vectored H7N9 vaccine. Additionally, the identified stalk epitopes were conserved among the H1-H17 subtypes and the stalk-reactive mAbs exhibited cross-reactivity with different subtypes. In conclusion, four novel nonNeu epitopes in H7N9 HA were identified, and two dominant epitopes underlying the induction of nonNeu antibodies by viral-vectored H7N9 vaccine were identified. Our results add new knowledge to the molecular basis for antibody immunity against H7N9 vaccines and provide useful implications for vaccine design and modification.