Methods
Informed consent to monitor, treat and report the subject was obtained from the family and approved by the West Palm Beach VA Research & Education Committee. Nasopharyngeal swab specimens were obtained from the patient. Samples were shipped to Orlando for performance of the Gene Xpert Cepheid Innovation XPRSARS-COv2-10 RT PCR assay measuring viral N2 sequence abundance. The limit of detection was 0.01 plaque forming units/mL (CFU/mL) or 250 viral RNA copies/mL with first cycle number above background (Cq) of 39. Additional sample was shipped on dry ice to University of California San Diego for viral culture in a Biosafety Level-3 facility. For measurement of infectious virus, Vero E6 were obtained from American Type Culture Collection, Rockville, MD and grown in Dulbecco’s Minimal Essential Medium (DMEM, Corning) with 10% fetal calf serum and Penicillin-Streptomycin (Gibco). The clinical sample was thawed, and 200uL per well was added to row A (column 2-12) of a 96-well plate. Then, 100ul of serum-free DMEM was added to rows B-H and clinical samples were 2-fold serially diluted by transferring 100ul down the rows of the plate (B-H). Then, the entire volume of each well was transferred to a 96 well plate containing 20,000 Vero E6 cells per well in 100ul of DMEM, 10% fetal bovine serum, 2× Penicillin-Streptomycin, 2× Antibiotic-Antimycotic, and 2× Amphotericin B (Gibco) and gently mixed. Inoculated cultures were grown in a humidified 37°C incubator with 5% CO2 and observed for cytopathic effects (CPEs) for a total of 6 days. No CPE was observed after 6 days, and thus the samples were blind passaged onto a fresh 96 well plate containing 20,000 Vero E6 cells per well and cultured at 37°C with 5% CO2 and observed for CPE for an additional 6 days. Infectious SARS-CoV-2 produce CPE on Vero E6 cells. All work with potentially infectious SARS-CoV-2 clinical material was conducted in Biosafety Level-3 conditions at the University of California San Diego following guidelines approved by the Institutional Biosafety Committee.