Nasopharyngeal swabs or nasal wash from individuals who were influenza A positive during the 2019–20 Northern Hemisphere influenza season were used for virus isolation on primary cells. The apical side of hNEC wells were washed twice with 300ul of phosphate buffered saline (PBS) and 100ul of sample was added to the cells and incubated for two hours. The sample was then aspirated and cells were washed twice with 300ul of PBS. At three, five, and seven days post-infection 300ul of hNEC infection media (DMEM supplemented with .3% BSA (Sigma), 100 units/ml pen/strep (Life Technologies), 2mM Glutamax) was added to the well and incubated for ten minutes. TCID50 was performed on collected media and stocks were made from the collected media when virus was detected at concentrations greater than 10^4 TCID50/mL.
To generate viral stocks, T75 flasks (Corning) were seeded with MDCK-SIAT cells and grown to confluence in complete media. Viral isolates from the previous step were diluted to a multiplicity of infection (MOI) of 0.001 in infection media (hNEC infection media plus 5μg/ml N-acetyl trypsin). Cells were washed twice with PBS plus 100mg/L each of anhydrous calcium chloride and magnesium chloride hexahydrate (PBS +/+) and inoculum was added to the flasks and incubated at 33°C, rocking every 15 minutes. Inoculum was then aspirated, and 13mL of infection media was added to the flask. After flasks showed 75% cell death (~3 days post infection), media was collected and centrifuged at 500g for 10 minutes to remove cell debris. Stocks were aliquoted and stored at −80 °C.
Viral RNA was extracted using the QIAamp viral RNA mini extraction kit, and Illumina RNA Prep with Enrichment(L) Tagmentation was used for library preparation. Quality was checked using the Qubit and Agilent Bioanalyzer, and samples were sequenced with using a MiSeq Illumina sequencer. Consensus sequences were generated using the DRAGEN RNA Pathogen Detection pipeline.
To generate viral stocks, T75 flasks (Corning) were seeded with MDCK-SIAT cells and grown to confluence in complete media. Viral isolates from the previous step were diluted to a multiplicity of infection (MOI) of 0.001 in infection media (hNEC infection media plus 5μg/ml N-acetyl trypsin). Cells were washed twice with PBS plus 100mg/L each of anhydrous calcium chloride and magnesium chloride hexahydrate (PBS +/+) and inoculum was added to the flasks and incubated at 33°C, rocking every 15 minutes. Inoculum was then aspirated, and 13mL of infection media was added to the flask. After flasks showed 75% cell death (~3 days post infection), media was collected and centrifuged at 500g for 10 minutes to remove cell debris. Stocks were aliquoted and stored at −80 °C.
Viral RNA was extracted using the QIAamp viral RNA mini extraction kit, and Illumina RNA Prep with Enrichment(L) Tagmentation was used for library preparation. Quality was checked using the Qubit and Agilent Bioanalyzer, and samples were sequenced with using a MiSeq Illumina sequencer. Consensus sequences were generated using the DRAGEN RNA Pathogen Detection pipeline.
