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During the routine surveillance, we isolated nine H4N6 subtype avian influenza viruses (AIVs) in Jiangsu Province, China, in March 2023. Phylogenetic analysis revealed that nine H4N6 viruses belonged to the Eurasian lineage and underwent complex genetic recombination among Asian countries during their evolution. It is particularly noteworthy that the PB2 and PB1 genes of our representative virus were descended from clade 2.3.4.4b H5 high-pathogenic AIVs in Japan. Mutations of D3V and D622G in PB1, N66S in PB1-F2, N30D, I43M, and T215A in M1, and P42S and I106M in NS1 were observed in nine isolates, which may increase the pathogenicity of the viruses in mice. The receptor binding analysis showed that the tested H4N6 virus could bind to both avian-type and human-type receptors. Vitro infection kinetics revealed that the representative virus could efficiently replicate in mammalian cells, including MDCK and 293T cells. Pathogenicity tests in mice indicated that the representative virus could replicate in nasal turbinates and lungs without prior adaptation. Our data reveal the potential public health issues represented by H4N6 viruses from wild birds and highlight the need to strengthen routine surveillance of wild birds. [ABSTRACT FROM AUTHOR]

Background: The COVID-19 pandemic is a persistent global threat to public health. As for the emerging variants of SARS-CoV-2, it is necessary to develop vaccines that can induce broader immune responses, particularly vaccines with weak cellular immunity. Methods: In this study, we generated a double-layered N-S1 protein nanoparticle (N-S1 PNp) that was formed by desolvating N protein into a protein nanoparticle as the core and crosslinking S1 protein onto the core surface against SARS-CoV-2. Results: Vaccination with N-S1 PNp elicited robust humoral and vigorous cellular immune responses specific to SARS-CoV-2 in mice. Compared to soluble protein groups, the N-S1 PNp induced a higher level of humoral response, as evidenced by the ability of S1-specific antibodies to block hACE2 receptor binding and neutralize pseudovirus. Critically, N-S1 PNp induced Th1-biased, long-lasting, and cross-neutralizing antibodies, which neutralized the variants of SARS-CoV-2 with minimal loss of activity. N-S1 PNp induced strong responses of CD4+ and CD8+ T cells, mDCs, Tfh cells, and GCs B cells in spleens. Conclusions: These results demonstrate that N-S1 PNp vaccination is a practical approach for promoting protection, which has the potential to counteract the waning immune responses against SARS-CoV-2 variants and confer broad efficacy against future new variants. This study provides a new idea for the design of next-generation SARS-CoV-2 vaccines based on the B and T cells response coordination. [ABSTRACT FROM AUTHOR]