Influenza virus plaque assay

Lungs were homogenized using sterilized sand and a mortar and pistil, 1% FBS in PBS was added to obtain a 10% weight/volume suspension. Samples were spun down at 600 G for 15 min at 4 °C, and the supernatant was transferred to a new tube and kept on ice until use.

Madin-Darby Canine Kidney epithelial (MDCK) cells were used for influenza plaque assay and grown in complete medium. 4.5 × 10⁴ MDCK cells in 100 μl medium were grown in 96-well plates overnight. For the plaque assay, 10-fold dilutions of the lung suspensions were prepared using an influenza growth medium containing DMEM 1965 medium with 2 mM L-glutamine, 200 IU/ml penicillin, 50 μg/ml streptomycin, 0.2% BSA, 1% sodium-pyruvate and 5 units/ml TPCK Trypsin. MDCK-cells were first washed twice with PBS and then incubated with 50 μl of virus dilution for 2 hour at 37 °C, 5% CO2. Samples were then removed, and an overlay medium containing 2x minimum essential medium (MEM) eagle supplemented with 0.4% BSA, 10% NaHCO3, 2% Streptomycin, 2% penicillin and 5 units/ml TPCK trypsin mixed 1:1 with 1.8% methylcellulose was added to the cells. Cells were incubated for 48 hour at 37 °C, 5% CO2. Then overlay was removed and wells were washed 2x with PBS. Cells were fixated with 4% formaldehyde in PBS for 30 min, RT. After fixation cells were washed twice with PBS and permeabilized with warm 0.5% Triton-X in Hanks balanced salt solution medium for 10 min, RT. Cells were subsequently washed twice with PBS. Next, cells were incubated with primary α-influenza nucleocapsid A mAb (Nordic Biosite) diluted 1:1500 in 10% FBS in PBS for 1 hour at 37 °C, 5% CO2. Antibody was removed and cells washed 5x. This was followed by incubation with a secondary goat α-mouse HRP conjugated mAb (Dako) diluted 1:500 in 10% FBS in PBS for 1 hour at 37 °C, 5% CO2. After secondary antibody incubation cells were washed 5x with PBS. 200 μl substrate solution containing 3 mg/ml 3-amino-9-ethylcarbazole and 0.07% H₂O₂ and 5 mM citrate phosphate buffer pH5 was added to wells for 30 minutes, RT. Substrate was removed and cells were washed once with PBS before counting. All samples were run in duplicates. Plaque forming units per g lung tissue were calculated accordingly:

Detection level was calculated to be 1000 PFU/mg, corresponding to 3.0 log PFU/mg