Challenge Virus in Nasal Secretions: Quantification (PCS1, PCS3, PMBC-influenza)
PCS1 & PCS3
Frozen nasal lavage samples that had been stored at -70°C were quickly thawed in a 36°C water bath. For each sample, serial 10-fold dilutions from 100 to 104 were prepared by adding 180 μl of 10% Eagle’s minimal essential medium (EMEM) to all wells of a 96-well plate, and an initial 20 μl of sample nasal lavage to 4 wells across the 100 position. Four wells were prepared as negative (cell) controls by adding 20 μl of 10% EMEM. The resulting mixtures were transferred to 96-well tissue culture plates that had been prepared with MRC-5 cells (media removed). Plates were then incubated for 7-10 days at 33°C in 5% CO2. Virus titer was calculated per milliliter.
The level of virus replication was determined by plaque assay. After two washings of MDCK cell monolayers with phosphate-buffered saline + bovine albumin, serial 10-fold dilutions of lavage specimen were inoculated in 0.1-ml amounts. After a 30 minute adsorption at 34°C, each culture received 5ml of agar overlay medium which consisted of EMEM + Earle’s balanced salt solution supplemented with glucose and vitamins; 2.2 mg/ml of NaHCO3; 100µg/ml of DEAE-dextran; 20µg/ml of crystalline trypsin; and 0.6% Inoagar No. 2. After incubating for 3 days at 34°C in 5% CO2, an equal amount of second agar overlay medium containing 0.003% neutral red was added and the plaques were counted 6 hours later. Virus titer was calculated in plaque-forming units (PFU) per milliliter.
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