doi:10.1097/INF.0b013e318194596a. illness, meningitis, and encephalitis (9,C12). PeV-A3 has been the most common parechovirus recognized from cerebrospinal fluid (CSF) since its finding in 1999, emphasizing its importance as an growing agent of severe infections in young children (4, 6). To day, no specific therapies are available to treat PeV-A3-infected children. FDA-cleared antifungal medicines itraconazole (ITC) and posaconazole (POS) were previously identified as potent broad-spectrum inhibitors of enterovirus (13,C15), dengue disease (16), and PeV-A3 (17) at concentrations clinically attainable in pediatric individuals. This study explores the possible mechanism(s) of action for antiviral activity of POS against PeV-A3. RESULTS POS is an early-stage inhibitor of PeV-A3 replication. To determine at which stage in the PeV-A3 existence cycle POS and ITC exert their antiviral effects, a time program experiment was performed with compounds added at ?1, 0, +1, +2, +4, and +6?h relative to disease illness (Fig. 1). POS at 0.3?M and ITC at 1?M or 3?M were more effective inhibitors at earlier time points than the no-drug control. Open in a separate windowpane FIG 1 Antifungal azoles POS and ITC exert antiviral activity against PeV-A3 in the early stages of illness. The cytopathic effect of POS (0.3 and 1?M) and ITC (1 and 3?M) on PeV-A3 strain US-WI-09 was evaluated in a time program assay. Results symbolize means standard deviations (SD) from five self-employed experiments. To further Ac-DEVD-CHO characterize the mechanism of action of POS, we used a time-of-addition assay to evaluate the antiviral effect on disease replication of drug pretreatments, coaddition, and addition postinfection (Fig. 2A). The disease titer was decreased by 100-fold (2 log10) when cells were pretreated with POS prior to illness (Fig. 2B, pretreatment-1) and decreased by 31-fold (1.49 log10) when PeV-A3 and POS were added simultaneously (Fig. 2B, coaddition). When POS was added Ac-DEVD-CHO after disease infection, titers decreased by only 3-collapse (0.49 log10) (Fig. 2B, postinfection), which suggests Ac-DEVD-CHO POS is more effective earlier in the PeV-A3 illness cycle. To assess whether POS focuses on the PeV-A3 particle directly and not the sponsor cell, Vero-P cells were pretreated with POS and any unbound drug was washed aside prior to disease infection. A reduction of antiviral activity resulted when unbound POS was eliminated prior to addition of disease; only a moderate decrease of viral titer (0.44 log10; 2.75-fold) was observed (Fig. 2B, pretreatment-2). Open in a separate windowpane FIG 2 POS is more effective early in PeV-A3 existence cycle. (A) Schematics of the treatments for the time-of-drug-addition assays, utilizing Vero-P cell monolayers. Shaded areas show the presence of 0.5?M POS. One hundred CCID50 PeV-A3 (strain US-WI-09) or PeV-A1 (Harris strain) was utilized for all experiments. Titration results were acquired using the disease titration assay previously explained (45). (B) The pretreatment-1 experiment showed the effect of POS on Vero-P cells before PeV illness. Monolayers were incubated with 0.5?M POS, followed by the addition of PeV. Residual POS and unbound PeVs were eliminated by washing. The pretreatment-2 experiment showed the effect of POS on Vero-P cells with residual drug eliminated by washing before inoculation. PeV was added and incubated for 2 h, followed by a second wash to remove unbound disease. For the coaddition test, Vero-P monolayers were inoculated simultaneously with POS and PeV and incubated for 2 h, followed by washing to remove unbound POS and PeV. In the postinfection construction, Vero cells were infected with PeV, followed by removal of unbound disease by washing. POS was added and remained present EDNRB throughout incubation. Data demonstrated represent mean ideals from three self-employed experiments. POS blocks early methods in viral access by binding to disease particles and inhibiting viral attachment. The time program and time-of-drug-addition assays suggested that POS exerts its antiviral effect early in the PeV-A3 illness cycle, independent of connection with the Vero-P cells. To further demonstrate it directly targets the disease, we performed synchronized illness assays (18) to evaluate the antiviral effect of POS when combined with PeV-A3 prior to inoculation of cells (free disease particles), on PeV-A3 attachment, and on PeV-A3 access (Fig. 3A and ?andB).B). One hundred microliters of concentrated PeV-A3 (500 CCID50 [50% cell tradition infectious dose]) was incubated with 100 l (0.5?M) POS for 2?h at.