The H9N2 subtype of avian influenza virus (AIV) causes enormous economic losses and poses a significant threat to public health; the development of vaccines against avian influenza is ongoing. To study the immunogenicity of hemagglutinin (HA) protein, we constructed a recombinant pET-32a-HA plasmid, induced HA protein expression with isopropyl β-D-1-thiogalactopyranoside (IPTG), verified it by SDS–PAGE and Western blotting, and determined the sensitivity of the recombinant protein to acid and heat. Subsequently, mice were immunized with the purified HA protein, and the immunization effect was evaluated according to the hemagglutination inhibition (HI) titer, serum IgG antibody titer, and cytokine secretion level of the mice. The results showed that the molecular weight of the HA protein was approximately 84 kDa, and the protein existed in both soluble and insoluble forms; in addition, the HA protein exhibited good acid and thermal stability, the HI antibody titer reached 6 log2–8 log2, and the IgG-binding antibody titer was 1:1,000,000. Moreover, the levels of IL-2, IL-4, and IL-5 in the immunized mouse spleen cells were significantly increased compared with those in the control group. However, the levels of IL-1β, IL-6, IL-13, IFN-γ, IL-18, TNF-α, and GM-CSF were decreased in the immunized group. The recombinant HA protein utilized in this study exhibited good stability and exerted beneficial immune effects, providing a theoretical basis for further research on influenza vaccines.

H9N2 avian influenza virus (AIV) is widely prevalent in poultry in China. To understand the genetic characteristics and evolution of H9N2 AIVs in Guangxi, southern China, the complete genomes of H9N2 AIVs from 1999–2023 were systematically analysed. Maximum likelihood (ML) trees indicated that H9N2 AIV gene sublineage diversity contributed to genotype diversity, yielding 17 genotypes (G1–G17). Since 2010, genotype G14 (also known as genotype S or G57) has become predominant in poultry in Guangxi. Phylogenetic analysis in the HA has resulted in the distancing of recent Guangxi isolates from the vaccine strains. This study also revealed that the genotypes of H9N2 AIVs infecting swine, equines and canines in Guangxi were consistent with those found in avian species at the same time, highlighting the capacity of H9N2 AIVs to be transmitted across species. The antigenic residues in the HA head region and NA protein of the Guangxi isolates from 2020–2023 changed significantly compared to the vaccine strains, suggesting possible antigenic drift in these viruses. Amino acid analysis of the HA protein revealed that 84.9% (73/86) of H9N2 AIV isolates from Guangxi, including those from live poultry markets, preferentially bound to α-2,6 sialic acid receptors. Considerable attention should be given to cross-species transmission of H9N2 AIV in the region. On the basis of these findings, strengthening the monitoring of H9N2 AIV in poultry in Guangxi is essential.

Gyrovirus galga 1 (GyG1) can infect a variety of animals and humans, but prevention and control strategies are limited, which endangers the healthy development of the poultry breeding industry and has a potential impact on public health safety. The live poultry market (LPM) connects the production and consumption ends, and the pathogen may spread across regions through transportation and personnel flow. To understand the prevalence of GyG1 in Guangxi, 3482 samples from LPMs, namely, 2693 chicken throat and cloacal swabs and 789 environmental samples collected in Guangxi from December 2019 to December 2024, were assayed by PCR. The results revealed that GyG1 was present in chicken and environmental samples from LPMs in Guangxi, China, with positivity rates of 17.08% and 13.31%, respectively. Eight GyG1-positive samples were randomly selected, including 5 chicken swab samples and 3 environmental samples for whole-genome amplification. The amino acids encoded by the three ORFs were analysed, and some mutation sites unique to these 8 variants were found. The homology between the 8 GyG1 genomes and 36 reference sequences was 96.8–99.8%. The homology of the VP1 gene sequence was 96.5–99.9%, and the homology of the amino acid sequence was 99.4–100%. A phylogenetic tree was constructed on the basis of the 8 GyG1 genomes and 36 GyG1 reference genome sequences from 14 different species (8 from zoos) in this study. The 44 sequences were divided into three branches constituting groups A, B and C, with the 8 novel strains classified into group A2. Recombination analysis predicted that two recombination events in the GyG1 sequence were associated with the emergence of Guangxi strain GX-AGV2-202109-5. This study clarified the prevalence and molecular characteristics of GyG1 in LPMs in Guangxi, China, were clarified for the first time, providing important data supporting the prevention and control of GyG1 infection and providing a reference for further understanding the epidemiology and genetic diversity of GyG1.

Fowl adenovirus serotype 4 (FAdV-4) has been identified as the primary pathogen responsible for hydropericardium-hepatitis syndrome (HHS), resulting in significant economic losses in major poultry-producing countries since 2015. Timely and accurate diagnosis of FAdV-4 infection is essential for the effective prevention and control of HHS. In this study, the two nonstructural genes of FAdV-4, 100K and 22K, were inserted into the expression vector pET-32a (+) respectively. The expressed recombinant proteins were used as coating antigens to develop two indirect ELISA methods, designated as 100K-ELISA and 22K-ELISA. Both ELISAs demonstrated high specificity, showing no cross-reactivity with serum samples positive for other avian diseases. Both ELISAs yielded positive results when applied to 50 serum samples from SPF chickens experimentally infected with FAdV-4 and negative results when applied to 50 serum samples from SPF chickens immunized with an inactivated FAdV-4 vaccine. Similarly, the field sample testing results demonstrated a significant ability to distinguish between vaccinated and infected samples. The 100K-ELISA and 22K-ELISA, which are based on nonstructural proteins, may be effective tools for differentiating between FAdV-4 infection and vaccination, offering a promising approach for differentiating infected from vaccinated animals (DIVA) strategies in poultry.

The global emergence of gyrovirus galga1 (GyVg1) across diverse regions and species un-derscores an urgent demand for rapid diagnostics. This study aimed to engineer a field-deployable diagnostic platform for rapid pathogen detection. We established two visual detection methods by integrating recombinase-aided amplification (RAA) and CRISPR/Cas12a technologies: RAA‒CRISPR/Cas12a combined with fluorescence and RAA‒CRISPR/Cas12a combined with lateral flow strips. By systematically optimizing the reaction conditions, designed primers and crRNA enabled target recognition within 1 hour, and demonstrated no cross-reactivity with other relevant avian pathogens. RAA‒CRISPR/Cas12a combined with fluorescence achieved a detection limit of 2 copies/µL (10 copies/µL visually under UV), and RAA-CRISPR/Cas12a-lateral flow strips demonstrated a detection limit of 5×10² copies/µL. Clinical validation using 192 samples revealed ~10% positivity rates across both novel methods and fluorescence quantitative PCR, with high concordance in positive identifications. The results suggest that the two RAA‒CRISPR/Cas12a visual detection methods established in this study are highly efficient, specific and sensitive, and can be used for the rapid field detection of GyVg1, providing a cost-effective and powerful diagnostic tool for grassroot workers.

Fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium-hepatitis syndrome (HHS) and has resulted in severe economic losses to the poultry industry in China since 2015. In this study, we isolated a naturally occurring strain with reduced virulence designated GXYL-03, which caused no gross or histological HHS lesions in 4-week-old specific pathogen-free (SPF) chickens infected at 10⁶ TCID₅₀, whereas clinical signs and partial mortality were observed at higher doses of 10⁷–10⁸ TCID₅₀. After a single immunization with 5 × 10⁴ TCID₅₀ of GXYL-03, all ten immunized SPF chickens survived throughout the 14-day observation following lethal GX2017-005 challenge, whereas all ten challenge-control chickens died. Integrated transcriptome and translatome analyses were performed on the livers of chickens infected with FAdV-4 strains of differing virulence by RNA sequencing (RNA-seq) and polysome profiling, with three biological replicates per group. The polysome/non-polysome (P/NP) ratio did not differ significantly among groups (p > 0.05), indicating stable global translation flux. The GXYL-03 group had 2,131 transcriptomic differentially expressed genes (DEGs) but only 71 translatomic DEGs; the GX2017-005 group had 7767 and 148, respectively, indicating that only 3.3% and 1.9% of transcriptional perturbations were transmitted to translation, revealing a pronounced translational buffering effect. Translation efficiency (TE) changes were predominantly suppressive, and downregulated genes accounted for 58.6% and 61.8% of TE changes in the two strains. The functional enrichment of genes whose expression was synchronously upregulated at both levels (TE+RNA+) revealed that GXYL-03 was enriched mainly in protein-coding genes involved in cellular stress, DNA damage repair, vesicle transport, and protein homeostasis (HSPA2, HSPB9, FANCD2, EHD2, and Vault), whereas GX2017-005 was enriched in liver injury-related genes (CD74, ATG2B, FKBP5, ND1, and COX1) that were not differentially expressed in GXYL-03. This study provides insights into the development of FAdV-4 strains with reduced virulence and virus–host interactions.