Table 6. Findings of included studies: viral culture.

Study ID	Threshold for viral culture	Timing of viral culture	Method used for viral culture	Cycle Threshold	Results of viral culture
Ben- Shmuel 2020	Not specified	Not specified	Applied 200 μL from 10-fold serial sample dilutions upon VERO E6 cell cultures in 24-well plates. After 1 h, wells were overlaid with 1 mL of MEM medium supplemented with 2% foetal calf serum (FCS), MEM non-essential amino acids, 2 mM L-glutamine, 100 units/mL penicillin, 0.1% streptomycin, 12.5 units/mL nystatin and 0.15% sodium bicarbonate. Cells were incubated for 5 days (37°C, 5% CO2), and CPEs were observed after fixation with crystal violet solution.	34 to 37.9	None of the samples was culturable. No viable virus was recovered from plastic or metal coupons after 4–14 days of incubation
Colaneri 2020a	All 26 samples were inoculated onto susceptible Vero E6 cells	Not specified	A 200-μL sample was inoculated onto a Vero E6 confluent 24-well microplate for virus isolation. After 1 hour of incubation at 33°C in 5% CO2 in air, the inoculum was discarded and 1 mL of medium for respiratory viruses was added (Eagle's modified minimum essential medium supplemented with 1% penicillin, streptomycin and glutamine, and 5 mg/mL trypsin) to each well. Cells were incubated at 33°C in 5% CO2 in air and observed by light microscopy every day for cytopathic effect. After a 7-day incubation, 200 μL of supernatant was used for molecular assays.	Not reported	None of the inoculated samples induced a cytopathic effect on day 7 of culture.
Döhla 2020	Not specified	Not specified	Seeded Vero E6 cells in 24 well plates or T25 flasks at a density of 70–80 %. Cells were incubated with 200µl (24 well) – 1000 µl (T25 flask) of the sample material supplemented with 1x penicillin/streptomycin/amphotericin B and incubated for 1 h at 37°C in 5 % CO2. For water samples, 10% (v/v) of inoculation volume was replaced by 10xPBS to obtain a final concentration of 1xPBS. After 1 h of incubation, the inoculum was removed, Dulbecco’s Modified Eagle’s medium (Gibco) with 3 % foetal bovine serum (Gibco) and 1x penicillin/streptomycin/ amphotericin B was added. Cells were incubated over several days at 37°C, 5% CO2 and observed for development of a cytopathic effect that typically occurs for growth of SARS-CoV-2 on Vero E6 cells.	Not reported	No infectious virus could be isolated under cell culture conditions from any sample
Feng 2020	Not specified	Not specified	Not reported	Not reported	Could not perform viral culture due to the low virus quantity in the positive samples.
Moore 2020	<34	Not specified	Vero E6 cells (Vero C1008; ATCC CRL-1586) in culture medium [MEM supplemented with GlutaMAX-I, 10% (v/v) fetal bovine serum (FBS), 1X (v/v) non-essential amino acids and 25 mM HEPES] were incubated at 37oC. Cells (1 x 106 cells/25 cm2 flask) were washed with 1X PBS and inoculated with ≤1 mL environmental sample and incubated at 37°C for 1 h. Cells were washed with 1X PBS and maintained in 5 mL culture medium (4% FBS) with added antibiotic– antimycotic (4X), incubated at 37°C for 7 days and monitored for cytopathic effects (CPE). Cell monolayers that did not display CPE were subcultured up to three times, providing continuous cultures of ~30 days.	28·8 to 39·1	No CPE or a decrease in Ct values across the course of three serial passages were observed suggesting the samples did not contain infectious virus
Ong 2020	Positive swabs from PCR	Not specified	Monolayers of Vero C1008 cells (ATCC-1586) in T25 flasks were inoculated with 1 mL inoculum (500 µL of the swab sample and 500 µL of Eagle’s MEM) and cultured at 37°C, 5% CO2 with blind passage every 7 days. Also, 140 µL cell culture was used for RNA extraction and real-time PCR twice per week to monitor changes in target SARS-CoV-2 genes as an indication of successful viral replication. In the absence of CPEs and real-time PCR indication of viral replication, blind passages continued for a total of 4 passages before any sample was determined to be negative of viable SARS-CoV-2 virus particles.	Not reported	All samples in common areas and staff pantry were negative on viral cell culture.
Peyrony 2020	Not specified	Not specified	Not specified	35.71 to 39.69	Because of weak amounts of viral RNA in positive samples, there was no attempt to isolate viruses in cell culture
Santarpia 2020	Subset of samples that were positive for viral RNA by RT-PCR	Days 5–9 of patient occupancy for one site and day 10 occupancy for the second site. No information is provided on the date of onset of patient symptoms	Vero E6 cells. Several indicators were utilized to determine viral replication including cytopathic effect (CPE), immunofluorescent staining, time course PCR of cell culture supernatant, and electron microscopy.	Not reported	Cultivation of virus on cell culture was not confirmed including the air sample.
Suzuki 2020	Some samples from which viral RNA was present	No details provided from time of symptom onset but ranged from 1–17 days after the cabin was vacated and at least 17 days after the quarantining to cabins was ordered and 8 days after the first cabin cleaning	Samples were mixed with Dulbecco’s modified Eagle medium supplemented with typical concentrations of penicillin G, streptomycin, gentamicin, amphotericin B and 5% fetal bovine serum. They were inoculated on confluent VeroE6/TMPRSS2 cells. Culture medium at 0- or 48-hours post-infection (hpi) were collected and diluted10-fold in water, then boiled for 5 minutes. CPE observation after 4 days.	26.21-38.99	No virus was cultured
Wang 2020a	Not specified	Not specified	Samples were obtained and inoculated on Vero-E6 cells for virus culture. The cytopathic effect (CPE) was observed after 96 h.	No positive samples	No positive samples
Zhou 2020	Ct value <30	Not specified	Vero E6 and Caco2 cells were used to culture virus. The cells were cultured in DMEM supplemented with heat inactivated fetal bovine serum (10%) and Penicillin/Streptomycin (10, 000 IU/mL &10, 000 µg/mL). For propagation, 200 µL of samples were added to 24 well plates. After 5–7 days, cell supernatants were collected, and RT-qPCR to detect SARS-CoV-2 performed. Samples with at least one log increase in copy numbers for the E gene (reduced Ct values relative to the original samples) after propagation in cells were considered positive.	>30.	No virus was cultured