Streptomycin mixture

Cell lines were originally obtained from the American Type Culture Collection (ATCC). Vero cell lines were propagated in Eagle’s Minimum Essential Medium (EMEM, Lonza, Belgium), supplemented with 6% fetal bovine serum (FBS) (Euroclone), 100 U/mL penicillin and 100 μg/mL streptomycin mixture (Lonza, Belgium). HEp-2 cells (human larynx epidermoid carcinoma cell lines) and PKR-deficient HEp-2 cells (PKR^−/−), kindly provided by Professor Zhou G. (Shenzhen International Institute for Biomedical Research, Shenzhen, Guangdong, China), were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza, Belgium) and supplemented with 10% of FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin mixture. All cell lines were grown at 37 °C in a 5% CO₂ incubator.

Hep-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza or Gibco) supplemented with 10 % fetal bovine serum (FBS) (Greiner or Atlanta Biologicals), 100 IU/ml penicillin 100 µg/ml streptomycin mixture (Lonza), 200 mM L-glutamine (Lonza), 1.5 mg/ml sodium bicarbonate (Lonza), 10 mM HEPES (Lonza) and 0.25 mg/ml fungizone (Invitrogen). The cells were maintained at 37 °C and 5 % CO₂.

RSV-B-positive samples were titrated on Hep-2 cells for the quantification of infectious virus, as defined by the culturability of RSV. Briefly, Hep-2 cells were grown to confluency in 96 well plates overnight. Subsequently, cells were spin-inoculated (15 min, 2000 rpm) with 100 µl of 10-fold serial dilutions of RSV-B positive samples and incubated at 37 °C, 5 % CO₂. One hour after inoculation, cells were washed once and cultured in serum-reduced (2 %) Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 100 IU/ml penicillin 100 µg/ml streptomycin mixture (Lonza), 200 mM L-glutamine (Lonza), 1.5 mg/ml sodium bicarbonate (Lonza), 10 mM HEPES (Lonza) and 0.25 mg/ml fungizone (Invitrogen). After 7 days of incubation, positive samples were identified by immunofluorescence assays using a FITC labelled polyclonal antibody directed against RSV (Fisher Scientific). Infectious virus titers were calculated from four replicates as tissue culture infective dose (TCID₅₀) by the Spearman-Karber method.