In untreated cells, CTGV formed smaller comet tails compared to VACV-WR (Fig. 4A). In the presence of increasing concentrations of ST-246, comet tails were reduced for both viruses, demonstrating the clear effect of ST-246 on extracellular virus production. Nevertheless, in CTGV-infected Fulvestrant in vitro cells, comet tails were barely detected at 0.015 μM ST-246 whereas VACV-WR still generated small comets and primary plaques at 0.05 μM. This result suggested

that the production of extracellular particles in CTGV-infected cells was more susceptible to the effect of ST-246 than in cells infected with VACV-WR. It is important to note that comet tails were visualized in CTGV-infected cells after 4 days of infection, whereas VACV-WR comets were better visualized after 3 days because of the difference in the sizes of virus plaques. Therefore, to measure the effect of ST-246 after similar period of treatment and infection, BSC-40 cells were infected in the presence of different concentrations of ST-246, and cell medium was harvested after 48 h. Medium samples were first depleted of contaminating IMV released from lysed cells by incubation with anti-A28 PI3K inhibitor neutralizing antibody and were subsequently

titrated on BSC-40 cells. As shown in Fig. 4B, ST-246 inhibited the production of infectious extracellular CTGV in a dose–response way. Extracellular yield was inhibited by nearly 64% at 0.01 μM ST-246, whereas a decrease of BCKDHA approximately 4% was detected for VACV-WR at this dose (p < 0.001). At 1 μM, the yield of extracellular CTGV dropped 3 logs when compared

with a 2-log reduction for VACV-WR (p < 0.001). We next investigated the antiviral effect of ST-246 on the replication of CTGV in vivo. To determine the best route of infection for producing measurable disease in mice, we tested intravenous injection into the tail vein, intranasal inoculation and scarification on the tail. Intravenous inoculations of BALB/c mice with up 5 × 104 PFU of CTGV by the tail vein did not generate lesions on the tail in contrast to inoculation with 5 × 103 PFU of VACV-WR, which produced visible lesions by day 13 post-infection (data not shown). Similarly, intranasal inoculation of mice with 105 or 5 × 107 PFU of CTGV did not produce clinical signs of disease in mice such as weight loss (weight was measured daily for 21 days), ruffled fur or lethargy (Reis, Moussatche and Damaso, unpublished data) whereas intranasal inoculation of mice with 105 PFU of VACV-WR produced measurable clinical disease with symptoms that have been used by others to describe disease severity ( Alcami and Smith, 1996 and Bray et al., 2000).