Infectious bursal disease virus (IBDV) is responsible for the highly contagious infectious bursal disease in chickens. Previously, by blind passage, a vvIBDV Gx strain was attenuated to the Gt strain, and a strain CEF-9 with intermediate characters was obtained during attenuation. Since CEF-9 exhibited only two interesting amino acid mutations (Q253H and A284T) on loops P(DE) and P(FG) at the tip of VP2 spikes, we hypothesized that, either function separately or in combination, they define the cell tropism and virulence of vvIBDV. To test this hypothesis, Q253H and A284T were introduced individually or in combination into VP2 of the Gx or Gt strain to obtain six modified clones. Using reverse genetics, combined mutations of Q253H and A284T could adapt vvIBDV to non-permissive CEF cells (rGx-F9VP2) but any single mutation could not. In vivo, rGx-F9VP2 did not cause mortality while the Gx strain induced 66.7% mortality. Dual evidence from natural and rescued strains identified that the cell tropism of vvIBDV to CEF cells was determined by the combined VP2 mutations Q253H and A284T, but not by single mutation. The two residues were mainly responsible for the virulence of vvIBDV. These findings may be helpful in the design of new tailored IBDV vaccines.