Study on the Antiviral Activities and Hemagglutinin-Based Molecular Mechanism of Novel Chlorogenin 3-O-β-Chacotrioside Derivatives against H5N1 Subtype Viruses

Figures

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  Figure 1. Chemical structures of chlorogenin 3-O-β-chacotrioside derivatives.

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  Figure 2. Dose–response inhibition curves of the derivatives against H5N1 avian influenza virus. Three curves represent the dose-response curve of XC-27-2, XC-27-1, and UA-Nu-ph-5 against H5N1 virus from left to right in turn. The error bars represent mean ± standard deviation (three replicates). The horizontal axis represents the log concentration of the derivatives, and the vertical axis represents the inhibition percentage.

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  Figure 3. Dose–response inhibition curves of the derivatives against H5N1 and VSVG pseudoviruses. (a,b) represent the dose-response curves of XC-27-1, XC-27-2 against pseudoviruses, respectively. The error bars represent mean ± standard deviation (three replicates). The horizontal axis represents the log concentration of the derivative, and the vertical axis represents the inhibition percentage.

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  Figure 4. Dose–response curves of the derivatives inhibiting the neuraminidase activity of H5N1 pseudovirus. (a,b) represent the dose-response inhibition curves of XC-27-1, XC-27-2 to NA, respectively. The error bars represent mean ± standard deviation (three replicates). The horizontal axis represents the log concentration of the derivative, and the vertical axis represents the inhibition percentage.

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  Figure 5. The inhibition rate of derivatives for the H5N1 viral plaques forming amounts. (a–c) respectively represent the average inhibition rate of XC-27-1, XC-27-2, and UA-Nu-ph-5 for H5N1 viral plaque-forming units (PFUs). Each group was investigated in triplicate for each experiment. Data pooled from three independent experiments (n = 3, mean ± S.E.M.).

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  Figure 6. Assay of hemagglutinin titer for the H5N1 virus antigen. For the antigen group, the H5 antigen was added to the wells from the 1st to the 11th column. Among them, the 1st well had the original solution added, and the wells from the 2nd to the 11th column were two-fold serial diluted in turn, the final well had PBS added.

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  Figure 7. The hemagglutination inhibition effects of derivatives. (a,b) respectively represent the activity of XC-27-1 and XC-27-2 in inhibition of H5N1 influenza virus-induced aggregation of chicken erythrocytes. In the plates, the H5-antigen group in the the 2nd column had the H5 antigen added. The positive control group in the 3rd column had the H5 serum added. From the 4th to the 10th column, wells were treated from left to right with XC-27-1 and XC-27-2 at 1.57, 3.125, 6.25, 12.5, 25, 50, 100 μM, respectively. The results of the hemagglutination inhibition assay are shown in the photograph of Figure 7. The experiments were repeated independently at least three times.

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  Figure 8. Computational structural prediction for potential binding sites between the derivative XC-27 and HA protein. Using the hemagglutinin crystal structure (2IBX) of the virus VN1194 (H5N1) as a reference structure, all of the available HA sequences and highly homologous HA crystal structure were retrieved from NCBI and PDB databases respectively, based on this, accurate 3D space structures of HA were constructed by using Modeller 9.x software, the molecular docking between XC-27 and HA was performed using AutoDock software.

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  Figure 9. The infectivity of mutant H5N1 pseudovirus strains to MDCK cells. The infection ability of HA mutant pseudoviruses to MDCK cells after co-transfected for 48 h was measured using the luciferase assay method. The RLU of HA mutant pseudovirus at 10-fold diluted are means of three independent experiments (n = 3, mean ± S.E.M.). Difference was considered statistically significant when ** p < 0.01 vs. vehicle control group.

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  Figure 10. Inhibition rate curves of compounds against H5N1 pseudoviruses. (a,b) represent the dose–response curves of XC-27-1, XC-27-2 against wildtype and mutant H5N1 pseudovirus strains, respectively. The error bars represent mean ± standard deviation (three replicates). The horizontal axis represents the log concentration of the derivatives, and the vertical axis represents the inhibition percentage.