Background/Objectives: Eurasian avian-like (EA) H1N1 swine influenza viruses, with their persistent evolution and zoonotic potential, seriously threaten both swine and human health. The objective was to develop an effective vaccine against these viruses. Methods: A cold-adapted, temperature-sensitive live-attenuated influenza vaccine (LAIV) candidate, GX18ca, was developed. It was derived from the wild-type EA H1N1 strain A/swine/Guangxi/18/2011 (GX18) through serial passaging in embryonated eggs at temperatures decreasing from 33 °C to 25 °C. Its characteristics were studied in mice, including attenuation, immune responses (mucosal IgA, serum IgG, IFN-γ+ CD4⁺/CD8⁺ T-cell responses), and protective efficacy against homologous (GX18), heterologous EA H1N1 (LN972), and human 2009/H1N1 (SC1) viruses. Results: GX18ca showed cold-adapted and temperature-sensitive phenotypes. In mice, it was attenuated, with viral titers in the nasal turbinates and lungs reduced 1000–10,000-fold compared to the wild-type strain, and it cleared by day 5 post infection. Intranasal immunization elicited strong cross-reactive immune responses. Mucosal IgA had broad reactivity, and serum IgG titers reached high levels. IFN-γ+ CD4^+/CD8⁺ T-cell responses were detected against all the tested viruses. A single dose of GX18ca fully protected against GX18 and LN972 challenges, and two doses significantly reduced SC1 lung viral loads, preventing mortality and weight loss. Conclusions: GX18ca is a promising LAIV candidate. It can induce broad immunity, addressing the cross-protection gaps against evolving EA H1N1 SIVs and zoonotic H1N1 variants, which is crucial for swine influenza control and pandemic preparedness.

The H1N1 swine influenza viruses CQ91 and CQ445, isolated from pigs in China, exhibited distinct virulence in mice despite sharing similar genomic constellations. CQ91 demonstrated higher pathogenicity (MLD₅₀: 5.4 log₁₀ EID₅₀) and replication efficiency in mice compared to CQ445 (MLD₅₀: 6.6 log₁₀ EID₅₀). Through reverse genetics, we found that the attenuation of CQ445 was due to a single substitution of glutamic acid (E) with lysine (K) at position 343 in the PB2 protein. Introducing the CQ445-PB2 (343K) into CQ91 significantly reduced viral replication and pathogenicity in mice, while replacing CQ445-PB2 with CQ91-PB2 (343E) restored virulence. In vitro studies showed that the K343E mutation impaired viral replication in MDCK and A549 cells and reduced polymerase activity in minigenome assays. Mechanistically, the amino acid at position 343 in the PB2 affects the transcription stage of the viral replication process. Structural modeling indicated that the charge reversal caused by E343K altered local electrostatic interactions without major conformational changes. Phylogenetic analysis revealed that PB2-343E is highly conserved (>99.9%) in human and swine H1/H3 influenza viruses, suggesting that PB2-343E confers an adaptive advantage. This study identifies PB2-343E as a critical determinant of influenza virus pathogenicity in mammals, highlighting its role in host adaptation.

Gyrovirus homsa1 (GyH1) causes immunosuppression and multi-organ damage in young poultry. The VP1 capsid protein (VP1) is a key target for subunit vaccine development; however, recombinant VP1 alone elicits limited immunogenicity and requires an efficient delivery system. Here, we developed a liposomal formulation associated with the GyH1 VP1 protein (LNP-VP1) and evaluated its immunogenicity and preliminary safety in specific-pathogen-free chickens. The prepared LNP-VP1 had a mean particle size of 230.11 ± 5.95 nm, a polydispersity index of 0.204 ± 0.023, a zeta potential of −36.55 ± 0.99 mV, and an apparent encapsulation efficiency of 84.03% ± 2.10%. In addition, immunization of chicks with LNP-VP1 induced robust antigen-specific IgG responses and significantly enhanced the infiltration of CD4⁺ and CD8⁺ T cells in the spleen. Furthermore, cytokine analysis revealed upregulation of IFN-γ, IL-4, and IL-17 levels following vaccination. Importantly, no vaccine-associated histopathological changes were observed in major immune or metabolic organs after immunization. Overall, LNP-VP1 effectively elicited both humoral and cellular immune responses with a favorable safety profile, indicating its potential as a candidate vaccine against GyH1 and providing experimental evidence supporting lipid nanoparticles as a delivery platform for avian subunit vaccines.

The H9N2 virus has severely harmed the livestock and bird farming industry. Currently, it is mainly prevented through vaccination immunization. However, conventional vaccines often fail to induce durable immune responses and long-lasting immunoprotection. In this research, we used Simvastatin (Sim) and CpG as adjuvants for the H9N2 inactivated vaccine to evaluate the vaccine’s immunogenicity in chickens. We evaluate vaccine immunogenicity through antibody testing, T lymphocyte phenotyping, and RNA-sequencing analysis. The results indicated that the Sim + CpG/H9N2 formulation significantly enhanced specific IgY and hemagglutination inhibition (HI) antibody titers. It also increased the proportions of CD4⁺ T cells and CD8⁺ T cells, promoted immune organ development, and stimulated the formation of germinal centers. RNA-sequencing analysis revealed that Sim + CpG/H9N2 vaccination significantly upregulated immune-related genes, which were enriched in pathways associated with stress response activation, immune cell recruitment, and inflammatory signaling. Overall, these findings demonstrate that Sim + CpG/H9N2 markedly enhances the immunogenicity of the inactivated H9N2 vaccine and provides new insights into the application of vaccine adjuvants for improved immune protection.