N-Desmethylclozapine, Fluoxetine, and Salmeterol Inhibit Postentry Stages of the Dengue Virus Life Cycle

Medigeshi, Guruprasad R.; Kumar, Rinki; Dhamija, Ekta; Agrawal, Tanvi; Kar, Meenakshi

For RSV stock preparation, HEp-2 cells (ATCC) were infected with an MOI 35 of 0.1 pfu/cell for one hour in MEM containing 2% FBS. Cells were washed once 36 with PBS, cultured in MEM-2% FBS and observed everyday for cytopathic effect 37 and syncytial formation (3-4 days). Cells were scraped in 3-4 ml of culture 38 medium and centrifuged at 1000 x g for 10 min at 4°C. Cell pellet was 39 resuspended in 1 ml of MEM-2%FBS and passed through a 26G syringe 40 attached to a one ml needle 10 times and centrifuged at 1000 x g for 10 min at 41 4°C. Multiple aliquots of the supernatant were prepared and stored at -80°C as 42 stock virus. RSV titers were estimated by plaque assays on HEp-2 cells by 43 following the same procedure as for DENV. Cells were washed with PBS twice and permeabilized using IMF buffer (2) 53 overnight at 4° C. Cells were stained with mouse anti-RSV antibody (AbD 54 Serotec) diluted in IMF buffer for 1 hour at 4° C followed by donkey-anti-mouse 55 antibody conjugated with alexaflour-488 (Invitrogen) for 1 hour at 4° C. 56