TABLE 2.

Interference with DEN replication by recombinant dsSIN viruses with single and multiple tandem antisense RNA inserts^ab

dsSIN insertc	Insert size (nt)	% Inhibition in C6/36 cells infected with:				% Inhibition in A. aegypti infected with:
		DEN-1	DEN-2	DEN-3	DEN-4	DEN-1	DEN-2	DEN-3	DEN-4
Single inserts (−RNA)
D1/prM	249	>99				100
D1/NS5/GDD	240	>99				100
D1/NS5/5′	180	0				NDd
D2/prM	290		>99				100
D3/prM	286			>99				100
D3/NS3/5′	216			85				ND
D4/C	240				>99				100
Double inserts (5′-3′)
D1/prM/D3/prM	540	97		97		99		97
D1/GDD/D3/GDD	490	98		90		91		92
D4/C/D1/GDD	480	>99			80	ND			ND
Triple inserts (5′-3′)
D1/GDD/D4/C/D2/prM	770	>99	0		70	ND	ND		ND
Variable-size inserts
D1/GDD	240	>99		50		100
D1/GDDFsh	160	>99		50		100
D1/GDDRsh	183	98		50		89
D1/GDDFshRsh	105	85		0		70

^aAdapted from Adelman et al., unpublished. 

^bAssays of interference with DEN replication in C6/36 mosquito cell cultures were performed as described in Table 1. Challenge with DEN was at a MOI of 0.1. The proportion of cells escaping interference was determined, 7 days after DEN challenge, by the indirect-immunofluorescence assay using a primary monoclonal antibody specific for DEN E protein. For interference assays in A. aegypti, adult female mosquitoes were inoculated intrathoracically simultaneously with 10⁵ TCID₅₀ recombinant dsSIN plus 3 × 10³ PFU of DEN of the indicated serotype. The mosquitoes were held for 14 days at 28°C, and then the heads were removed and examined by the indirect-immunofluorescence assay for the presence of the DEN E protein, indicating escape from interference. 

^cInserts expressed from the subgenomic dsSIN promoter are designated by the DEN virus serotype and genome region from which the antisense RNA was derived. 

^dND, not determined.