Fig 8. The contribution of NS2B/NS3 substitutions on replication advantage of GI virus in amplifying hosts.

(A) The schematic diagram displayed the location of GI virus-specific NS2B/NS3 substitutions. (B-E) The single, double, or triple substitutions were introduced into NS2B/NS3 proteins of rGIII viruses. Single NS2B or NS3 substitutions with green or blue color. Double or triple substitutions with cyan or dark blue color. The rGIII, rGI, and rGIII/ GI NS1-3 viruses with white, red, and yellow color. The mutant rGIII viruses infected C6/36 (B), VERO (C), PK-15 (D), and DF-1 (E) cells at 0.5 of MOI and replicated at 28°C (C6/36) or 41°C (VERO, PK-15 and DF-1). The viral titer in the supernatant was detected at 48 HPI by the micro-antigen focus assay. Mean with SEM of the triplicates was displayed. (F) 10⁴ ffu of the rJEVs were subcutaneously inoculated into one-day old chickens (n = 6 per group). The viral titer in plasma was detected at 48 HPI by the micro-antigen focus assay. A dot and horizontal line represent an individual animal and mean of a group. Error bars indicate SEM. A dotted line indicates the detection limit. The statistical analysis was calculated with one-way ANOVA followed by Dunnett’s Multiple Comparison Test utilizing the control of rGIII virus. A significant difference was indicated as an asterisk (B-E) or shown as a red dot (F) (P< 0.05).