FIG 6.

Screening strategy for compensatory mutations of NS4B NS58QN62Q. (A) Schematic representation of procedures used to screen compensatory mutations of NS4B N58QNS62Q mutant virus from mammalian BHK-21 cells (left panel) and mosquito C6/36 cells (right panel). BHK-21 cells were electroporated with WT or N58QN62Q mutant FL RNA (10 μg). At 5 dpe, one-fifth of the electroporated cells from P1 were subcultured to a new culture dish and named P2. The culture fluid was transferred to naive BHK-21 cells at 3-day intervals until P1I3 and P2I5, thus enriching any rescued virus. C6/36 cells were electroporated with WT or N58QN62Q mutant FL RNA (10 μg), and the culture fluids were harvested at 14, 21, and 28 dpe. (B and C) Analysis of rescued virus from BHK-21 cells (B) and C6/36 cells (C). For panel B, culture fluids of 5 dpe, P1I3, and P2I5 for NS4B N58QN62Q FL RNA were used for plaque assays. The plaque assay of the culture fluid of P2I5 for WT FL RNA was used for comparison. Two isolated plaques from P1I3 contained an NS4B Y66A second-site mutation, and independent plaques from P2I5 contained NS4B S59F or A137T second-site mutations. For panel C, culture fluids of 14, 21, and 28 dpe for NS4B N58QN62Q FL RNA were used for the plaque assay. For comparison, a culture fluid of 14 dpe for WT FL RNA was used for the plaque assay. NS4B S59Y and A137 second-site mutations were identified in isolated plaques from 28 dpe, with two clones for each mutation.