Avicel microcrystalline cellulose

Plaque assays were performed as previously described (31 (link)). Briefly, whole lungs isolated from infected mice were isolated, weighed, and homogenized using a Tissue Lyser (Qiagen, Hilden, Germany). Serial dilutions of 10% homogenate were made in dilution media (1×MEM, 1 μg/mL TPCK-treated trypsin) and incubated for 1 hour atop confluent monolayers of Madin-Darby canine kidney cells (MDCKs) grown in 12 well plates for 1 hour at 37°C. Following infection, cell layers were washed with PBS and overlaid with MEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA), 0.04 M HEPES, 0.02 mM L-glutamine, 0.15% NaHCO₃ (w/v), and 1 μg/mL TPCK-treated trypsin. After 72 hours at 37°C, the overlay was removed, and the cells were washed with PBS, fixed by incubation with cold methanol/acetone (60:40%), and stained with crystal violet. Plaques were counted and plaque-forming units per mg of lung tissue determined.

Plaque reduction assays were performed in ST6Gal1-expressing MDCK cells as previously reported (Hu et al., 2017 (link)). Briefly, confluent cells were washed with PBS containing magnesium and calcium and infected with virus diluted in Dulbecco’s modified Eagle’s medium (DMEM) with 0.5% BSA for a final concentration of approximately 100 PFU per well. Viral infection was synchronized for 30 min at 4°C and then incubated for 1 h at 37°C in a 5% CO₂ atmosphere. Viruses were then aspirated, washed, and incubated in a DMEM media overlay containing 2μg/ml N-acetyl trypsin and 1.2% avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA) at 37°C in a 5% CO₂ atmosphere. Cells were stained 46 hpi with 0.2% crystal violet dye solution (0.2% crystal violet; 20% methanol). EC₅₀ values were determined by the average plaque area per well using ImageJ software.

Plaque reduction assays were performed in MDCK cells with the A/Soloman Island/3/2006 (H1N1) virus as previously described.^{36 (link),51 (link)–53 (link)} A/Soloman Island/3/2006 (H1N1) M2 contains the same sequence of M2 WT as A/Udorn/72. Briefly, confluent cells were washed with phosphate buffered saline (PBS) and infected with virus diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 0.5% BSA for a final concentration of approximately 100 plaque forming units (PFU) per well. Viral infection was synchronized for 30 min at 4 °C and then incubated for 1 h at 37 °C in a 5% CO₂ atmosphere. The inoculum was aspirated, and cells were washed and incubated in a DMEM overlay media containing different concentrations of compound, 2 μg/mL N-acetyl trypsin, and 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA) at 37 °C in a 5% CO₂ atmosphere. Cells were stained 2 days post infection with 0.2% crystal violet dye. EC₅₀ values were calculated by plotting the plaque area per well against the rimantadine concentration applied with a dose–response function in Prism 5.

The plaque reduction assay was performed as previously reported,^{21 (link)} except MDCK cells expressing ST6Gal I were used instead of regular MDCK cells.^{35 (link)} Briefly, confluent monolayer of ST6Gal MDCK cells were incubated with ~100 pfu virus samples in DMEM with 0.5% BSA for 1 hour at 4°C, then 37°C for 1 hour. The inoculums were removed, and the cells were washed with phosphate buffered saline (PBS). The cells were then overlaid with DMEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA) and NAT (2.0 μg/mL). To examine the effect of the compounds on plaque formation, the overlay media was supplemented compounds at testing concentration. At 2 days after infection, the monolayers were fixed and stained with crystal violet dye solution (0.2% crystal violet, 20% methanol). The viruses used for this assay were A/WSN/33 (H1N1), A/Switzerland/9715293/2013 (H1N1), and A/California/07/2009 (H1N1), all of which contain the M2-S31N mutant. Influenza A viruses, A/Switzerland/9715293/2013 X-247 (H3N2), FR-1366, and A/California/07/2009 (H1N1)pdm09, FR-201, were obtained through the Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA, USA.