Preparation of the SARS-CoV-2 and plaque reduction neutralization (PRNT) assay

SARS-Cov-2 USA-WA1/2020 isolate was passaged in Vero E6 cells from a seed stock (BEI Resources cat. no. NR-52281) at Bioqual. The challenge stock, which was assigned lot no. 061620-1000, was generated by the following method. The NR-52281 seed stock⁶⁹ was diluted 1:200 in DMEM/2% FBS and added to Vero E6 cell (ATCC^® CRL-1586) monolayers (90–100% confluency) in T150 flasks. The cells were incubated with 5 mL diluted virus for 1 h at 37 °C, 5% CO₂ with intermittent rocking. The virus was removed and replaced with DMEM, 2% FBS. The cells were incubated at 37 °C, 5% CO₂ for 5 days when CPE was observed for 80–90% of the cells. The medium was collected and centrifuged at 1500 rpm for 10 min at 4 °C. The supernatant was maintained at 4 °C while collecting while collecting cells. Five (5) mL of DMEM, 2% FBS was added to each flask, the cells scraped cells off and collected into a conical 50 mL tube. The cells were spun down at 1500 rpm for 10 min at 4 °C and the resulting pellet resuspended in 5 mL of DMEM, 2% FBS. The cells were freeze-thawed twice to release virus and the resulting cell lysate combined with the supernatant. This was mixed well, 0.5 mL aliquots were prepared in cryovials and stored at ≤−70 °C.

PRNT assay with hamster sera samples was performed at Bioqual. After heat inactivation (30 min at 56 °C), hamster sera samples were serially diluted, mixed with SARS-CoV-2 viral stock and placed on 80–100% confluent monolayer of Vero E6 cells. Viral stock was tested to generate 30 plaque forming units (pfu) per well in 24-well plate of Vero E6 cells. Plates were incubated for 1 h at 37 °C in 5% CO₂ incubator and media was discarded. Prewarmed 0.5% methylcellulose solution was added to each well and the plates were incubated at 37 °C in 5% CO₂ for 72 h. Media was discarded and plates were washed once with Phosphate Buffered Saline (PBS). The plates were fixed with 400 μL ice cold methanol per well at −20 °C for 30 min. After fixation, the methanol was discarded, and the monolayers stained with 250 μL per well of 0.2% crystal violet (20% MeOH, 80% dH₂O) for 30 min at ambient temperature. Plates were washed once with PBS and air dried for 15 min. The plaques in each well are recorded and the number of infectious units (pfu) calculated. The titers are reported as the serum dilutions that reduce the number of plaques by 50% (PRNT₅₀).