Wen Siang Tan

Toll-like receptors (TLRs) form the primary host defense against avian influenza virus (AIV) in chickens, yet quantitative and mechanistic insights into their coordinated activation remain fragmented. This study provides an integrated meta-analytic and signaling architecture of TLR-driven antiviral immunity and its translational modulation by TLR agonists. Fifteen peer-reviewed studies (2010–2023) comprising 1,560 quantitative observations were systematically analysed. Fold-change data for TLR3, TLR7, TLR15, TLR21 cytokines were standardized to log₂ fold-change and pooled using random-effects models. Correlation matrices and network mapping defined MyD88/TRIF–IRF7–NFκB signaling architecture. A translational sub-analysis assessed viral-load reduction and cytokine induction following TLR agonist treatments. Pooled analysis showed significant upregulation of TLR3, TLR7 and TLR21 with minimal heterogeneity (I² < 50%). IRF7 and NFκB constituted the central amplification layer (mean LFC = 4.5), driving strong cytokines (IFN-β, IL-6) and ISG (Mx, OAS, PKR) induction. Co-expression analysis also identified conserved TLR3–IRF7–IFN-β–OAS and TLR7–IL-1β–Mx–PKR modules (ρ ≈ 0.7–0.8). Translational meta-aggregation revealed that CpG + poly I:C co-stimulation reduced viral titers by > 5 log₁₀ TCID₅₀/mL and decreased shedding by ~ 60%, representing a large therapeutic effect (Cohen’s d ≈ 1.1). This work establishes a unified quantitative framework of TLR-driven antiviral immunity in chickens, and demonstrates that TLR3, TLR7, and TLR21 act as a coordinated antiviral axis through IRF7/NFκB-dependent interferon signaling. The synergy of CpG + poly I:C co-stimulation provides a translational pathway for TLR-guided adjuvant design, bridging molecular immunology with applied poultry vaccine development for chicken health.

Avian influenza is an economically significant disease affecting poultry worldwide and is caused by influenza A viruses that can range from low to highly pathogenic strains. These viruses primarily target the respiratory, digestive, and nervous systems of birds, leading to severe outbreaks that threaten poultry production and pose zoonotic risks. The ectodomain of the avian influenza virus (AIV) matrix protein 2 (M2e), known for its high conservation across influenza strains, has emerged as a promising candidate for developing a universal influenza vaccine in a mouse model. However, the efficacy of such expression against poultry AIVs remains limited. The objective of this study was to evaluate the immunogenicity of nodavirus-like particles displaying the M2e proteins. In this study, three synthetic heterologous M2e genes originated from AIV strains H5N1, H9N2 and H5N2 were fused with the nodavirus capsid protein (NVC) of the giant freshwater prawn Macrobrachium rosenbergii (NVC-3xAvM2e) prior to immunogenicity characterisations in chickens. The expression vector pTRcHis-TARNA2 carrying the NVC-3xAvM2e gene cassette was introduced into E. coli TOP-10 cells. The recombinant proteins were purified, inoculated into one-week-old specific pathogen-free chickens subcutaneously and analysed. The recombinant protein NVC-3xAvM2e formed virus-like particles (VLPs) of approximately 25 nm in diameter when observed under a transmission electron microscope. Dynamic light scattering (DLS) analysis revealed that the VLPs have a polydispersity index (PDI) of 0.198. A direct ELISA upon animal experiments showed that M2e-specific antibodies were significantly increased in vaccinated chickens after the booster, with H5N1 M2e peptides having the highest mean absorbance value when compared with those of H9N2 and H5N2. A challenge study using low pathogenic AIV (LPAI) strain A/chicken/Malaysia/UPM994/2018 (H9N2) at 10^6.5 EID₅₀ showed significant viral load in the lung and cloaca, but not in the oropharyngeal of vaccinated animals when compared with the unvaccinated control group. Collectively, this study suggests that nodavirus-like particles displaying three heterologous M2e have the potential to provide protection against LPAI H9N2 in chickens, though the vaccine’s efficacy and cross-protection across different haemagglutinin (HA) subtypes should be further evaluated.

The displacement of an electroactive monitoring agent, i.e., polyamidoamine dendrimers encapsulated gold nanoparticles (PAMAM-Au) upon the presence of a target antibody via acoustic streaming has been studied. Acoustic streaming has been used to improve the mass transfer and reduce the sample incubation rate, thus this study investigated its ability in enhancing the PAMAM-Au displacement efficiency of our immunosensor. For this purpose, the bio-nanogate components of maltose-binding protein carrying the antigenic determinant (MBP-aD) of hepatitis B surface antigen (HBsAg) as a bioreceptor was functionalized, followed by the monitoring agent i.e. PAMAM-Au on the electrode prior to the incubation with targeted anti-hepatitis B surface antigen (anti-HBsAg) antibody. The modified electrode was then coupled with a piezotransducer and connected to the signal transducer to induce acoustic streaming upon sample incubation. Under optimal acoustic actuation, the sample incubation time has been reduced from 20 min to 8 min via the enhancement of PAMAM-Au displacement induced by acoustic streaming. The result also demonstrated that the specificity and selectivity of the sensing platform under acoustic actuation are comparable to the static incubation in detecting the targeted antibody.