16 showing a 1.2 log10 reduction at this concentration (Fig. 4A). In previous studies 226/8.1 showed a higher neutralizing activity than 133/3.16 (Takada et al., 2003), clearly corresponding to our results. For testing of the DsiRNA, 293 cells were pretransfected with a DsiRNA directed against L (targeting the same region as the siRNA EK1, that has been successfully used to protect both guinea pigs and NHPs against lethal EBOV challenge (Geisbert et al., 2006 and Geisbert et al., 2010)) check details or control DsiRNAs, and then infected with 100
TCID50 (equivalent to an MOI of 0.005) of rgEBOV-luc2. After two days, reporter activity was measured, and as expected we observed a clear drop in reporter activity of about 2 log10 at the highest amount of DsiRNA used, and smaller reductions of reporter activity at lower amounts of DsiRNA (Fig. 4B). No effects of the control DsiRNAs were observed, indicating that the reduction in reporter activity was due to a sequence specific effect of the DsiRNA on virus replication. Antiviral screening of EBOV poses unique challenges. While reporter-expressing recombinant EBOVs have enabled rapid detection of infection, the need for a BSL4 laboratory when working with live virus remains, making fully automated high-throughput screenings for
these viruses challenging. However, screening of libraries www.selleckchem.com/products/ldn193189.html containing several thousand compounds in a 96-well format is feasible, as was recently demonstrated
(Panchal et al., 2012), and rgEBOV-luc2 is highly amenable to be used in such a screen. rgEBOV-luc2 also has several advantages over eGFP-expressing EBOVs, including its ease of use (no requirement for removal of samples from BSL4, very little labor intensive), low equipment costs and the ability to use either a much lower infectious dose, or alternatively the much faster readout times when using higher infectious doses. These faster readout times, in addition to obvious practical advantages, also mean that compounds with a low stability in culture medium can be more reliably screened. However, too short readout times also have to be avoided, since otherwise the virus does not have time to complete a full life cycle, which CYTH4 would results in inhibitors of late stages of the virus life cycle (e.g. budding inhibitors) not being recognized in the screen. In contrast, high-content screening, which so far is the most extensively used screening approach that has been performed with EBOV-GFP, requires extensive and costly automated imaging equipment. Until now this kind of screening has relied on a multistep approach in which cells are first infected in a BSL4 laboratory for several days, and then fixed for several days in formalin before they are analyzed under BSL2 conditions (of course we cannot exclude the possibility that despite the very complex technology high-content imaging will in future become available under BSL4 conditions).