Fig. 3
Antiviral activity of TLR7 ligand, ssRNA against H4N6 LPAIV replication is attributable to IL-1β production. Avian macrophages, MQ-NCSU cells, were cultured in 12-well plates for 24 h and stimulated with either ssRNA (10 µg/ml) or growth medium (control) including 6 replicates per treatment. The resultant MQ-NCSU cell culture supernatants were collected at 24 h post-treatment and transferred (250 µl) to DF-1 cell monolayers. Before transferring MQ-NCSU cell culture supernatants, 3 wells from ssRNA and control groups in DF-1 cells were incubated with 1.0 µg/ml of IL-1Ra for 30 min. Twenty-four hours later, the DF-1 cells were infected with H4N6 LPAIV (0.1 MOI). Twenty-four hours post-infection, the infected DF-1 cell culture supernatants were collected from each well and titrated in MDCK cell monolayers in tenfold serial dilution. The plates were stained with 1% crystal violet after 48 h and resulting plaques were counted. The experiment was repeated with the same number of replicates with similar results and the data were pooled. The representative picture of stained well in each group at 10−1 dilution are shown. The one-way ANOVA test followed by Bonferroni’s posttest for selected comparison was performed to identify group differences and the differences were considered significant at P < 0.05. The bars represent mean ± SEM