Cells, instruments, reagents, and viruses
VERO-E6 cells bought from ATCC (Manassas, VA, USA; Cat. (ATCC® CRL-1586™) were stored in the laboratory of Zhejiang University. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), and phosphate-buffered saline were bought from GIBCO (Grand Island, NY, USA). MB is a product of Jichuan Pharmaceutical Group Co., Ltd. (Taixin, China). Human plasma was donated by volunteers, and written informed consents were obtained. All experimental operations involving live viruses were carried out in Zhejiang University’s Biosafety Level 3 (BSL-3) Laboratory. The research protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine.
Methylene blue-light inactivated virus
To 180 mL of healthy human plasma, 20 mL of live SARS-CoV-2 spike virus was added. Diluted MB was added to 200 mL of the plasma-virus mixture and mixed to a final MB concentration of 1, 2, or 4 μM. The “BX-1 AIDS treatment instrument” was used at room temperature, and illumination was adjusted to 55,000 ± 0.5 million Lux. The plasma samples were irradiated for 0, 2, 5, 10, 20, or 40 mins under light at a single wavelength of 630 nm. About 1 mL of plasma was taken at each time point, diluted 10-fold with DMEM medium containing 2% FBS, and filtered through a 0.45-μm filter (Millipore, Billerica, MA, USA) for virus titer detection. At the same time, a virus control was set (only virus in the plasma, left at room temperature for 40 mins as an untreated control), a control with pure MB was set (virus and 4 μM MB added to the plasma, with no light treatment, and allowed to stand for 40 mins as a control for the effect of MB on the virus), and finally a light-only control was set (only the virus was added to the plasma, and light was used for 40 mins as a control of the effect of light on the virus). This experiment was repeated three times.
Virus titer measurement
After trypsinization of VERO-E6 cells, 1 × 104 cells/well were inoculated into 96-well cell culture plates in 100 μL of culture medium per well. After the cells grew into a single layer in the 96-well plate, the culture medium was discarded and the treated plasma was seeded into the wells. The treated plasma was log-diluted with 2% FBS in DMEM medium from 102–107times. This process was repeated for 4 wells per dilution, 200 μL/well. Normal cell control wells were established (with cells, virus-free). The 96-well plate was placed in an incubator with 5% CO2 at 37 °C. After 3 h of incubation, the supernatant was washed off, and 200 μL /well of 2% FBS DMEM medium was added. Cell lesions were observed every 24 h until 6 d. The TCID50 (median tissue culture infectious dose) was calculated according to the Reed-Muench method. The cell culture supernatant at 6 d of the culture was pipetted, and the viral nucleic acid load was measured. This experiment was repeated three times.
Three generations of blind tests
One milliliter of the test plasma with virus and 1,2, and 4 μM MB was irradiated for 40 min. It was then diluted 10 times with 2% FBS DMEM medium, filtered through a 0.45-μm filter, and added to VERO-E6 cells. After 48 h, 1 mL of the supernatant of the culture was diluted 10 times with 2% FBS DMEM medium, and added to VERO-E6 cells for three passages. These cells were observed for cytopathic effects. For the duration of the 3rd generation of cell blind transmission, the sample was deemed positive (+) if cytopathic lesions appeared at any time, indicating that the virus was not completely inactivated; samples were deemed negative (−) if no cytopathic lesions appeared, indicating that the virus had been completely inactivated. This experiment was repeated three times.
Viral load of culture supernatant measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Nucleic acid extraction: Using 200 μL of virus culture supernatant, the MVR01 magnetic bead method nucleic acid extraction kit was used (article number: ZM-0044, Shanghai Zhijiang Biotechnology Co., Ltd., Shanghai, China) in an EX2400 automatic nucleic acid extraction instrument (Shanghai Zhijiang Biotechnology Co., Ltd). Virus RNA was extracted, and the elution volume was about 50 μL.
qRT-PCR: A new coronavirus nucleic acid assay kit (Cat. No. Z-RR-0479-02-50, Shanghai Zhijiang Biotechnology Co., Ltd.) was used for qRT-PCR to detect the viral load, following the manufacturer’s instructions. Briefly, the qRT-PCR amplification reaction tube was placed on a LightCycler® 480II (Roche, Basel, Switzerland) qPCR instrument, and FAM and VIC (or TEXAS RED) fluorescence channels were selected for detection. The recommended cycle parameter settings were: 45 °C for 10 min and 95 °C for 15 min; then 45 cycles of 95 °C for 15 s and 60 °C for 60 s; with single-point fluorescence detection at 60 °C. A cycle threshold (CT) value below 35 was considered effective amplification, and a CT value above 35 was considered undetected.
Statistical analysis
The statistical analyzes were performed using SPSS 20.0 (IBM Corp., Armonk, NY USA). Student’s t-test was employed if the measurement data exhibited a normal distribution, and non-parametric test if it did not exhibit a normal distribution. P < 0.05 was considered to indicate statistical significance.
