in-vitro-culture-for-h5n1-specific-duck-t-cells-detection-immune

<em>In Vitro</em> Culture for H5N1-Specific Duck T Cells and Detection of Immune Responses Using Intracellular Cytokine Staining Method

The in vitro culture of T cells is a critical method for studying immune responses, viral infections, and potential therapeutic strategies. However, no ...

South China Agricultural University

National Avian Influenza Para-Reference Laboratory

In recent years, the occurrence of fowl adenovirus 2 (FAdV-2) has been on the rise in China, posing a significant threat to the poultry industry. This study aimed to investigate the epidemiology, phylogenetic relationship, genomic characteristics, and pathogenicity of FAdV-2. The epidemiological analysis revealed the detection of multiple FAdV serotypes, including FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-8a, FAdV-8b, and FAdV-11 serotypes. Among them, FAdV-2 exhibited the highest proportion, accounting for 21.05% (8/38). The complete genomes of these 8 FAdV-2 strains were sequenced. Genetic evolution analysis indicated that these FAdV-2 strains formed a separate branch within the FAdV-D group, sharing 94.60 to 97.90% nucleotide similarity with the reference FAdV-2 and FAdV-11 strains. Notably, the recombination analysis revealed that 5 out of the 8 FAdV-2 strains, exhibited recombination events between FAdV-2 and FAdV-11. The recombination regions involved Hexon, Fiber, ORF19 genes and 3' end. Furthermore, pathogenicity experiments demonstrated that recombinant FAdV-2 XX strain is capable of inducing mortality rate of 66.70% and causing more severe hepatitis hydropericardium syndrome (HHS) in 6-wk-old specific-pathogen-free chickens. These findings contribute to our understanding of the prevalence, genomic characteristics, and the pathogenicity of FAdV-2, providing foundations for FAdV-2 vaccine development.

Prevalence, genomic characteristics, and pathogenicity of fowl adenovirus 2 in Southern China.

Emergence of a human co-infected with seasonal influenza A (H3N2) virus and avian influenza A (H10N5) virus, China, December 2023.

Although the use of inactivated vaccines has kept avian influenza (AI) outbreaks largely under control, they fail to prevent virus shedding. To enhance the efficacy of inactivated H9N2 AIV vaccines (InV), we constructed a multi-epitope recombinant baculovirus (BV-BNT) containing two B cell epitopes and nine T cell epitopes of H9N2 AIV for combined immunization with InV. The results showed that HI titer, IgG and IgM levels, and the percentage of B cells, CD4 T cells, CD8 T cells, and CD4CD8 T cells were significantly higher in the InV+BV-BNT immunization group than the InV immunization group. Besides, the expression levels of IL-1β, IFN-γ, IFN-α, IL-4, IL-13, and CXCLi1 were significantly higher in the InV+BV-BNT group than the InV group. Moreover, four conservative peptides (NP, NP, NS, and NP) significantly stimulated splenocytes to express IFN-γ in the InV+BV-BNT group instead of InV group. After heterologous virus challenging, the percentages of CD4 T and CD8 T cells were significantly upregulated in the InV+BV-BNT group compared to Inv group at 3 DPI. Viral loads in oropharyngeal of the InV+BV-BNT group was significantly lower than that in the InV group at 3 days post-infection (DPI). Furthermore, compared to the InV group, the virus positivity rate of oropharyngeal and cloacal swabs in the InV+BV-BNT group was lower at 5 DPI, with none positive at 7 DPI. Hence, this study indicated that the combined immunization of InV and BV-BNT could induce stronger humoral and cellular immune responses, shorten the detoxification period and reduce viral load compared to Inv alone, which suggests BV-BNT could act as a supplementary vaccine to potentially address the protection deficiency of the H9N2 inactivated vaccine.

Construction with recombinant epitope-expressing baculovirus enhances protective effects of inactivated H9N2 vaccine against heterologous virus.

The duck CD8 T-cell response effectively defends against H5N1 highly pathogenic avian influenza virus (HPAIV) infection, but the recognized peptide is rarely identified. Here, we found that the ratio of CD8 T cells and the expression of IFN-γ and cytotoxicity-associated genes, including granzyme A/K, perforin and IL2, at 7 days post-infection in peripheral blood mononuclear cells (PBMCs) from B1 haplotype ducks significantly increased in the context of defending against H5N1 AIV infection in vivo. Moreover, similar results were observed in cultured and sorted H5N1 AIV-stimulated duck CD8 T cells in vitro. Next, we selected 109 epitopes as candidate epitopes on the basis of the MHC-I restriction binding peptide prediction website database and further identified twelve CD8 T-cell epitopes that significantly increased IFN-γ gene expression after stimulating B1 haplotype duck memory PBMCs. In particular, NP, NP, M, PB1 and PA were highly conserved in H5N1, H5N6, H5N8, H7N9, and H9N2 AIVs. These findings provide directions for the development of universal T-cell epitope vaccines for AIV in ducks.

Revealing novel CD8 T-cell epitopes from the H5N1 avian influenza virus in HBW/B1 haplotype ducks.

Corrigendum to "Construction with recombinant epitope-expressing baculovirus enhances protective effects of inactivated H9N2 vaccine against heterologous virus" [Vet. Microbiol. 300 (2025) 110337].

Since 2020, the clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 viruses have caused unprecedented outbreaks in wild birds and domestic poultry globally, resulting in significant ecological damage and economic losses due to the disease and enforced stamp-out control. In addition to the avian hosts, the H5N1 viruses have expanded their host range to infect many mammalian species, potentially increasing the zoonotic risk. Here, we review the current knowns and unknowns of clade 2.3.4.4b HPAI H5N1 viruses, and we highlight common challenges in prevention. By integrating our knowledge of viral evolution and ecology, we aim to identify discrepancies and knowledge gaps for a more comprehensive understanding of the virus. Ultimately, this review will serve as a theoretical foundation for researchers involved in related avian influenza virus studies, aiding in improved control and prevention of H5N1 viruses.

Article	Year
Prevalence, genomic characteristics, and pathogenicity of fowl adenovirus 2 in Southern China. Poultry science\| PubMed ID: 37980763	2024
Emergence of a human co-infected with seasonal influenza A (H3N2) virus and avian influenza A (H10N5) virus, China, December 2023. Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi\| PubMed ID: 39048397	2024
Construction with recombinant epitope-expressing baculovirus enhances protective effects of inactivated H9N2 vaccine against heterologous virus. Veterinary microbiology\| PubMed ID: 39671758	2025
Revealing novel CD8 T-cell epitopes from the H5N1 avian influenza virus in HBW/B1 haplotype ducks. Veterinary research\| PubMed ID: 39695865	2024
Corrigendum to "Construction with recombinant epitope-expressing baculovirus enhances protective effects of inactivated H9N2 vaccine against heterologous virus" [Vet. Microbiol. 300 (2025) 1... Veterinary microbiology\| PubMed ID: 40059020	2025
Clade 2.3.4.4b highly pathogenic avian influenza H5N1 viruses: knowns, unknowns, and challenges. Journal of virology\| PubMed ID: 40340397	2025

Zimin Xie

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