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.