TE/3’2J/B2 replicated to a maximum titer of 8.8 log10 PFU/ml at 48 hours post-infection Salubrinal concentration in Aag2 (Figure 5, top panel). This was more than 10-fold higher than TE/3’2J (7.4 log10 PFU/ml) and 100-fold higher than TE/3’2J/GFP (6.6 log10 PFU/ml). TE/3’2J/GFP

replicated less efficiently than TE/3’2J, suggesting that virus encoding an insert may be less able to replicate in Aag2 cells. A marked decrease in titer was observed at later time points during TE/3’2J/B2 virus infection of Aag2, coinciding with the presence of cytopathic effects not observed in TE/3’2J- or TE/3’2J/GFP-infected cells (Figure 5B). Notwithstanding, the titer of TE/3’2J/B2 virus was greater than the titers of TE/3’2J and TE/3’2J/GFP at all time points tested in this cell line. Figure 5 Growth of TE/3’2J, TE/3’2J/GFP, and TE/3’2J/B2 viruses in invertebrate and vertebrate cells. A) Triplicate flasks containing cell monolayers of Aag2 cells (A, top panel) and Vero cells (A, bottom panel) were infected at MOI = 0.01. Titers were determined by Forskolin plaque formation on Vero cells. Black circles = TE/3’2J, Black squares = TE/3’2J/GFP, Black triangles = TE/3’2J/B2. B) Cytopathic effect of TE/3’2J, TE/3’2J/GFP, and TE/3’2J/B2 on Aag2 cells at 72 Enzalutamide supplier hrs post infection (MOI = 0.01). Growth curve analysis was also performed in Vero cells to determine the effects of B2 protein expression on SINV replication in vertebrate cells (Figure 5A, bottom panel). Surprisingly,

replication of all three viruses was similar in this cell line. Peak titers of 7.1, 7.0, and 6.7 log10 PFU/ml were reached at 48 hours post-infection for TE/3’2J, TE/3’2J/GFP, and TE/3’2J/B2 viruses, respectively. The similar replication kinetics observed for all three viruses suggests that RNAi may not be as important for antiviral immunity in vertebrate cells compared to mosquito cells. Based Progesterone on our data showing increased replication of TE/3’2J/B2 in Aag2 cells, we tested whether TE/3’2J/B2 would increase virus

replication in mosquitoes following an infectious oral bloodmeal. At four and seven days post infection (dpi), midguts were dissected from 48 mosquitoes per group and, along with remaining mosquito carcasses, were titrated on Vero cells. Titers of infectious virus represent the extent to which virus replicated in individual mosquitoes while the total number of infected midguts and carcasses represent the infection and dissemination rates, respectively (Figure 6). Because electroporation-derived recombinant SINVs and invertebrate cell-derived viruses produced from TE/3’2J inefficiently infect mosquito midguts following oral infection, virus was passed once in Vero cells prior to use in blood feeds [24, 25]. TE/3’2J/B2 virus exhibited the highest rates of infection and dissemination and the highest average titers at both time points. Of 48 mosquitoes tested, 12 (25%) had detectable TE/3’2J/B2 virus in the midgut at four dpi, significantly more compared to TE/3’2J and TE/3’2J/GFP (P = 0.