Oseltamivir carboxylate osv c

Madin-Darby canine kidney (MDCK) cells, human cervical cancer HeLa cells and human lung epithelial A549 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured at 37 °C/5% CO₂ in Minimum Essential Medium (MEM; Invitrogen, Carlsbad, CA, USA), Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen) or Roswell Park MEMorial Institute 1640 Medium (RPMI; Hyclone, Logan, UT, USA), respectively, supplemented with 10% FBS (Invitrogen). Influenza viruses A/Puerto Rico/8/34 (PR8), A/Hong Kong/8/68 (HK), and B/Lee/40 (Lee) were obtained from the ATCC. The two type A influenza strains were amplified in 10-day-old embryonated chicken eggs, while the type B strain was inoculated into MDCK cells in the presence of 2 μg/mL TPCK-treated trypsin at 37 °C for 3 days. The NA inhibitor, oseltamivir carboxylate (OSV-C; ≥98%), was purchased from United States Biological (Swampscott, MA, USA). The cell entry blocker (-)-epigallocatechin-3-gallate (EGCG, ≥95%) and the polymerase inhibitor ribavirin (RBV) were from Sigma-Aldrich (St. Louis, MO, USA). Stocks were prepared in water (for EGCG) or DMSO (for OSV-C and RBV); the final concentration of each was 50 mM.

Madin-Darby canine kidney (MDCK) cells and 293 T cells were purchased from ATCC (Rockville, MD) and maintained in minimum essential medium (MEM; Invitrogen, Carlsbad, CA) and in Dulbecco’s minimal essential medium (DMEM; Invitrogen), respectively, supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C in 5% CO₂. Influenza viruses PR8, HK and Lee were obtained from ATCC. Influenza A viral strains (PR8 and HK) were amplified by infection of 10-day-old chicken eggs at 37 °C for 3 days and influenza B virus (Lee) by infection of MDCK cells under serum-free conditions. Viruses were partially purified from harvested egg allantoic fluid or cell culture supernatant by centrifugation at 3000 rpm for 5 min. Viral titers were determined by HA assay or plaque assay, and stocks were stored at −70 °C before use. Antiviral agents, ribavirin (RBV) (Sigma-Aldrich) and oseltamivir carboxylate (OSV-C; US Biological, Swampscott, MA) were used as control compounds. The test extracts and purified compounds, with the exception of the water layer which was dissolved in water, were dissolved in DMSO to 40 mg/mL and stored at −20 °C.