Infectious virus was quantified by plaque assay using confluent MDCK (IAV) or Vero E6/TMPRSS2 (SCOV2) cells seeded in 12-well or 6-well plates, respectively, as previously described (26 (link), 66 (link)). Samples were incubated on cell monolayers for 1 h before removal of inoculum, and all plates were incubated at 37°C for 2 days prior to fixation with 70% ethanol and staining with crystal violet to observe plaques. Limit of detection for both viruses was 10 PFU.
For quantification of viral RNA, 140 μL of each sample (out of the total sample volume) was inactivated in 560 μL of AVL buffer (Qiagen) and stored at −80°C until extraction using the QIAamp 96 viral RNA mini-extraction kit and QIAcube HT automated high-throughput nucleic acid purification platform with a 100-μL elution volume (Qiagen). All samples were tested in duplicate by real-time RT-PCR using the CDC influenza virus/SARS-CoV-2 multiplex assay as previously described (26 (link)). Viral copy numbers in each specimen were quantified against a 10-fold serial dilution of IAV or SCOV2 viral RNA included on each plate. Mean viral RNA copy numbers were normalized and expressed as RNA copy number per milliliter (ferret specimens) or RNA copy number per liter of air (aerosol specimens); specimens with gene copy numbers of <1/μL of extracted RNA were declared negative.
For quantification of viral RNA, 140 μL of each sample (out of the total sample volume) was inactivated in 560 μL of AVL buffer (Qiagen) and stored at −80°C until extraction using the QIAamp 96 viral RNA mini-extraction kit and QIAcube HT automated high-throughput nucleic acid purification platform with a 100-μL elution volume (Qiagen). All samples were tested in duplicate by real-time RT-PCR using the CDC influenza virus/SARS-CoV-2 multiplex assay as previously described (26 (link)). Viral copy numbers in each specimen were quantified against a 10-fold serial dilution of IAV or SCOV2 viral RNA included on each plate. Mean viral RNA copy numbers were normalized and expressed as RNA copy number per milliliter (ferret specimens) or RNA copy number per liter of air (aerosol specimens); specimens with gene copy numbers of <1/μL of extracted RNA were declared negative.