No virus-specific siRNAs could be selleck compound detected in mosquitoes mock-injected with cell culture medium or injected with TE/3’2J/B2, indicating that B2 protein could inhibit targeted degradation of the SINV genome in the context of infected mosquitoes (Figure 3B). Effects of B2 protein expression on SINV replication The inhibition of siRNA accumulation showed that B2 protein check details could inhibit RNAi in mosquito cells. To determine the effects that RNAi inhibition may have on SINV replication, we first examined the ability of SINV RNA to accumulate in infected cells. Using the same total RNA samples used for siRNA detection, we examined the accumulation of viral genomic and subgenomic RNA species in Aag2

cells and mosquitoes by Northern blot analysis (Figure 4A and 4B). Starting at 24

hours post-infection, three viral RNA species were detected in cells infected with TE/3’2J, TE/3’2J/GFP, and TE/3’2J/B2 viruses. These bands represent the genomic, first subgenomic, and second subgenomic RNAs produced during virus infection. The second subgenomic RNA, expressed from the most 3′ virus promoter, is the PLX-4720 mw most highly transcribed RNA species for all three viruses, consistent with previous reports [22]. The observed inhibition of siRNA accumulation in TE/3’2J/B2-infected cells corresponded with a distinct increase in viral RNA accumulation. Considerably more viral RNA was detected in cells and mosquitoes infected with TE/3’2J/B2 virus beginning at 24 hours post-infection and continuing throughout all time points tested. Much less viral RNA accumulated in TE/3’2J/GFP-infected cells and mosquitoes, an expected outcome

considering the increase in genome size and accompanying decrease in Ribose-5-phosphate isomerase replication efficiency [23]. No bands were observed in RNA from mock-infected cells. Figure 4 Detection of viral RNAs in Aag2 cells (A) and Ae. aegypti mosquitoes (B). Monolayers of Aag2 cells were mock-infected or infected with TE/3’2J, TE/3’2J/GFP, or TE/3’2J/B2 virus at MOI = 0.01. Mosquitoes were intrathoracically-inoculated with cell culture medium, TE/3’2J, TE/3’2J/GFP, or TE/3’2J/B2 virus. At indicated times post infection, total RNA was isolated and an E1-specific riboprobe was used to detect virus genomic and subgenomic RNA. Ethidium bromide-stained ribosomal RNA below each blot serves as a loading control. Time post infection for each virus in (A) is 0, 24, 48, and 72 hrs, and in (B) 0, 48, and 96 hrs. G = genomic; S1 = first subgenomic; S2 = second subgenomic. Because siRNA accumulation was inhibited and viral RNA amounts increased in TE/3’2J/B2 virus-infected cells, we tested if suppression of RNAi by B2 would cause more infectious virus to be produced during infection. We performed two-step growth curve analysis in Aag2 and Vero cells to determine the effects of B2 protein expression on infectious virus production (Figure 5A).