Figure 3
(A) Inhibition of the infectivity of H5N1 pesudovirus by Cn-Jp-Hp (n = 12, 14, 16, 18, 20) peptides. The peptides in various concentrations were incubated with pseudo-typed particles for 30 min at 37 °C, before transferred to the MDCK cells. The mixture of virus, peptide and MDCK cells was incubated for another 48 h, and then the luciferase activity corresponding to the survival of viruses was measured in a microplate luminometer. (B) The VSV-G pseudovirus was constructed by employing VSV-glycoprotein encoded plasmid similar to that of H5N1 pseudovirus. C20-Jp-Hp was serially two-fold diluted from 50 to 0.78 μg/mL in culture medium and then incubated with VSVG pseudovirus at 37 °C for 30 min prior to transferring into the MDCK cells. With the same procedure as that of H5N1 pseudovirus, the inhibitory effect toward VSVG pseudovirus was determined. As a comparison, C20-Jp-Hp at various concentrations inhibiting the infectivity of H5N1 pseudovirus was used in the same experiment. Each data was expressed as the mean of three independent replicates. (C) Neuraminidase (NA) inhibition assay. The neuraminidase from influenza A/Puerto Rico/8/34 (H1N1) virus was used in this experiment. The reaction mixture consisting of the tested peptides and virus in MES buffer was incubated for 45 min, and then 4-MU-NANA was added into each reaction well. The cleavage reaction was conducted for an additional 1 h, then terminated with 100 μL 34 mM NaOH (83% ethanol). The resulting fluorescence of the mixture was recorded at the excitation wavelength of 340 nm and emission wavelength of 440 nm. Oseltamivir phosphate was employed as positive control.
