The diseases caused by genotype VII Newcastle disease virus (NDV), H9N2 avian influenza virus (AIV), and fowl adenovirus serotype 4 (FAdV-4) continue to threaten the global poultry industry. However, no broad-spectrum vaccines provide simultaneous protection against these three pathogens. This study employed bioinformatics and immunoinformatics approaches to design a multi-epitope vaccine, named NFAF, which consists of B-cell, cytotoxic T lymphocyte (CTL) epitopes, and helper T lymphocyte (HTL) epitopes derived from hemagglutinin-neuraminidase (HN) and fusion (F) proteins of genotype VII NDV, hemagglutinin (HA) protein of H9N2, and Fiber2 protein of FAdV-4. The vaccine candidate was predicted to have non-allergenic properties, non-toxicity, high antigenicity, and favorable solubility. Each of its constituent antigenic epitopes has a high degree of conservation. Molecular docking demonstrated stable binding between NFAF and chicken Toll-like receptor (TLRs) and major histocompatibility complex (MHC) molecules. NFAF was expressed in soluble form in Escherichia coli and purified. Polyclonal antibodies against all three target viruses showed specific binding to NFAF. In vitro experiments revealed that NFAF effectively stimulated chicken peripheral blood mononuclear cells (PBMCs) and induced Th1, Th2, and pro-inflammatory cytokine production, confirming its immunogenicity, and increased the mRNA expression of the key signaling molecules MyD88 and NF-κB. These results suggested that NFAF could therefore be an efficacious multi-epitope vaccine against genotype VII NDV, H9N2, and FAdV-4 infections.

Genotype VII Newcastle disease virus (NDV) has been confirmed as the predominant epidemic strain in China. Traditional vaccine strains fail to provide complete immune protection when challenged with an epidemic strain. NDV vaccines with phylogenetic relationships closer to those of the endemic viruses demonstrate improved protective efficacy in reducing viral shedding and transmission. This research seeks to develop attenuated vaccine strains that are specifically aligned with NDV genotype VII. A reverse genetics system for the genotype VII NDV HB strain was developed, successfully rescuing the attenuated recombinant virus aHB by substituting the fusion protein (F) cleavage site motif “¹¹²R-R-Q-K-R↓F¹¹⁷” with “¹¹²G-R-Q-G-R↓L¹¹⁷.” Recombinant aHB virus attenuation was verified by assessing the mean death time (MDT) and intracerebral pathogenicity index (ICPI). The attenuated aHB strain demonstrated greater proliferation titers than did the virulent HB and rHB strains both in vivo and in vitro. Furthermore, the genome exhibited significant genetic stability even after 10 passages in chicken embryos. When challenged with the HB strain of NDV genotype VII, the aHB-inactivated vaccine provided 100% protection to chickens and effectively prevented viral shedding. These findings indicate that recombinant aHB may serve as an effective vaccine candidate.

H9N2 avian influenza virus (AIV) continues to mutate, leading to immunosuppression and secondary infections in poultry. Traditional inactivated vaccines mainly induce humoral immunity and have limited cross-protection efficacy against various subtypes of virus strains. In this study, we targeted the HA2 and M1 proteins of H9N2 as antigens and used immunoinformatics methods to design a broad-spectrum multi-epitope vaccine (MEV) that can simultaneously activate humoral and cellular immunity. Firstly, through systematic evolutionary analysis and sequence comparison, highly conserved amino acid sequence regions were selected from HA2 and M1 proteins. B-cell epitopes were predicted in the HA2 conserved sequence, and cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes were predicted in the M1 conserved sequence. Three candidate vaccines containing different epitope combinations were constructed. After secondary structure and physicochemical property comparisons, HM1 was determined as the optimal scheme. HM1 contains three B cell epitopes, two CTL epitopes, and three HTL epitopes, and was connected to chicken β-defensin at the N-terminus as a molecular adjuvant; a dendritic cell-targeting peptide was added at the C-terminus. The HM1 tertiary structure optimized by GalaxyRefine met the standards of a reliable model. The molecular docking results indicated that HM1 can form stable binding with chicken TLR2, TLR4, MHC I, and MHC II molecules, with binding free energies of −7.1 kcal/mol and −6.1 kcal/mol, respectively, and can form multiple hydrogen bonds and salt bridges. Normal mode analyses revealed that the HM1–TLR complex exhibits favorable dynamic properties at the computational level. The immune simulation prediction results showed that after vaccination with HM1, specific antibodies can be induced, B cells, helper T cells, and cytotoxic T cells can be activated, and IFN-γ and IL-2 can be secreted. In summary, the HM1 designed based on the conserved regions of HA2 and M1 proteins has good physicochemical stability and immunogenicity, providing a theoretical basis for the development of broad-spectrum and highly effective H9N2 vaccines.

Pigeon paramyxovirus type 1 (PPMV-1) poses a significant threat to pigeon farming in China, and understanding its biological characteristics and pathogenicity is critical for vaccine development and disease control. In this study, we characterized a PPMV-1 QY strain, performed full-length genome sequencing, and constructed a phylogenetic tree based on the F gene. Then, the biological properties and the pathogenicity of the QY strain were assessed and evaluated in vitro and in vivo. The results showed that phylogenetic analysis classified the QY strain within subgenotype VI.2.1.1.2.2, the predominant circulating strain in China. The QY strain exhibited a 50% egg infectious dose (EID₅₀) of 10^−6.8/0.1 mL, mean death time (MDT) in chicken embryos of 68.7 ± 2.1 h, and intracerebral pathogenicity index (ICPI) in one-day-old chicks of 1.12, which indicate it is a moderately virulent strain. Animal experiments showed that the QY strain resulted in a mortality rate of 66.7% in healthy pigeons. Necropsy findings included cerebral congestion and swelling, hemorrhagic glandular stomach papillae, tracheal ring hemorrhages, and duodenal congestion and swelling. Histopathological analysis revealed extensive inflammatory infiltration in the lungs and liver, widespread intestinal erosion, and severe necrosis of splenic red pulp cells. In conclusion, the QY strain belongs to subgenotype VI.2.1.1.2.2 and exhibits moderate virulence, causing high mortality and severe pathological lesions in infected pigeons. These findings provide valuable insights into the pathogenicity of PPMV-1 and the specific mutations in the F protein can serve as potential attenuation targets in vaccine development against the emerging subgenotype VI.2.1.1.2.2.

Hepatitis-hydropericardium syndrome, caused by fowl adenovirus serotype 4(FAdV-4), and inclusion body hepatitis, caused by fowl adenovirus 8b and 11 (FAdV-