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Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 μM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 μM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4. [ABSTRACT FROM AUTHOR]

Due to the rapid mutation of porcine epidemic diarrhea virus (PEDV), existing vaccines cannot provide sufficient immune protection for pigs. Therefore, it is urgent to design the affinity peptides for the prevention and control of this disease. In this study, we made use of a molecular docking technology for virtual screening of affinity peptides that specifically recognized the PEDV S1 C-terminal domain (CTD) protein for the first time. Experimentally, the affinity, cross-reactivity and sensitivity of the peptides were identified by an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR) test, separately. Subsequently, Cell Counting Kit-8 (CCK-8), quantitative real-time PCR (qRT-PCR), Western blot and indirect immunofluorescence were used to further study the antiviral effect of different concentrations of peptide 110766 in PEDV. Our results showed that the P/N value of peptide 110766 at 450 nm reached 167, with a KD value of 216 nM. The cytotoxic test indicated that peptide 110766 was not toxic to vero cells. Results of the absolute quantitative PCR revealed that different concentrations (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of peptide 110766 could significantly reduce the viral load of PEDV compared with the virus group (p < 0.0001). Similarly, results of Western blot and indirect immunofluorescence also suggested that the antiviral effect of peptide 110766 at 3.125 is still significant. Based on the above research, high-affinity peptide 110766 binding to the PEDV S1-CTD protein was attained by a molecular docking technology. Therefore, designing, screening, and identifying affinity peptides can provide a new method for the development of antiviral drugs for PEDV. [ABSTRACT FROM AUTHOR]

Gram-positive enhancer matrix (GEM) nanoparticles are often used in mucosal immunity, preparation of subunit vaccines or as an immune adjuvant due to its good immunological activities in recent years. Here, we designed and screened out a high affinity peptide ligand PL23, which could specifically target the non-epitope region of Classic Swine Fever Virus (CSFV) E2 protein, by virtual screening technology, enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) test. The OD value of PL23 at 450 nm was reached 1.982, and the K D value of it was 90.12 nM. Its binding capacity to protein was verified by SDS-PAGE as well. PL23 was subsequently conjugated to GEM nanoparticles by dehydration synthesis generating GEM-PL23 particles, and the GEM-PL-E2 particles were assembled after incubated with CSFV E2 protein. The cytotoxic test indicated that PL23, CSFV E2 protein, GEM nanoparticles, GEM-PL23 particles and GEM-PL-E2 particles were not toxic to cells and GEM nanoparticles could significantly promote the growth of APCs at high concentration for 1 h, p<0.001. In addition, GEM nanoparticles could promote the uptake of antigen by APCs. The cytokines tests suggested that GEM-PL-E2 particles could promote innate immune responses, regulate adaptive immune responses generated by T cells and APCs, and promote the differentiation and maturation of dendritic cells without producing inflammasomes. The results of immunological activity identification showed GEM-PL-E2 particles induced higher levels of both neutralizing antibodies and anti-CSFV antibodies than CSFV E2 protein in mice. This strategy provided a new, simpler, faster and cheaper method for assembling GEM nanoparticles, using an affinity peptide ligand replaced the protein anchor (PA), and provided a better application prospect for the application of GEM particles. [ABSTRACT FROM AUTHOR]