Detection and Quantification of Infectious Severe Acute Respiratory Coronavirus-2 in Diverse Clinical and Environmental Samples from Infected Patients: Evidence to Support Respiratory Droplet, and Direct and Indirect Contact as Significant Modes of Transmission

Lin, Y.-C.; Malott, R. J.; Ward, L.; Kiplagat, L.; Pabbaraju, K.; Gill, K.; Berenger, B. M.; Hu, J.; Fonseca, K.; Noyce, R.; Louie, T.; Evans, D. H.; Conly, J. M.

Few studies have assessed for infectious SARS-CoV-2 in multiple types of clinical and environmental samples. In almost 500 samples from 75 hospitalized and community cases, we detected infectious virus with quantitative burdens varying from 5.0 plaque-forming units/mL (PFU/mL) up to 1.0x106 PFU/mL in clinical specimens and up to 1.3x106 PFU/mL on fomites including facial tissues, nasal prongs, call bells/cell phones, dentures, and sputum deposits with confirmation by plaque morphology, PCR, immunohistochemistry, and sequencing. Expectorated sputum samples had the highest percentage of positive samples and virus titers (71%, 2.9x102 to 5.2x105 PFU/mL), followed by saliva (58%, 10 to 4.6x104 PFU/mL), and cough samples without sputum (19%, 5 to 1.9x103 PFU/mL). We also detected infectious SARS-CoV-2 from patients' hands (28%, 60 to 2.3x102 PFU/mL) but no infectious virus was found in continuous speech samples despite finding high levels of infectious virus in the associated nasopharynx, throat, or saliva specimens. We demonstrated infectious virus stability in clinical samples, including those dried for prolonged periods of time. Infectious virus correlated with time since symptom onset with no detection after 7-8 days in immunocompetent hosts and with N-gene based Ct values [≤]25 significantly predictive of yielding plaques in culture. One PFU was associated with ~105 copies of N gene RNA across a diversity of samples and times from symptom onset. Clinical salivary isolates caused illness in a hamster model with a minimum infectious dose of [≤]14 PFU/mL. Our findings of high quantitative burdens of infectious virus, stability even with drying, and a very low minimal infectious dose suggest multiple modes of transmission are exploited by SARS-CoV-2, including direct contact, large respiratory droplet, and fomite transmission and in the context of a high binding avidity to human cellular receptors, offer an explanation of the high contagiousness of this virus.