Antiviral effects of cetylpyridinium chloride in mouthwash against SARS-CoV-2

cell culture

Vero E6 (ATCC, Manassas, VA, USA) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (v/v) at 37 °C, 5% CO2. was incubated with Vero E6 stably expressing human TMPRSS2 (VeroE6/TMPRSS2) cells³² was also used in this study.

virus

SARS-CoV-2 Wuhan (WK-521; EPI_ISL_408667), alpha (QK002; EPI_ISL_768526), ​​beta (TY8-612; EPI_ISL_1123289), and gamma (TY7-501; EPI_ISL_833366) strains were obtained from Dr. Saijo (National Institute of Infectious Diseases). ,Tokyo Japan). These viruses were prepared using VeroE6/TMPRSS2 cells. All experiments with SARS-CoV-2 were performed in accordance with his Biosafety Level-3 (BSL-3) at the Institute for International Zoonosis Control, Hokkaido University (approval number: 19 (19), #21002-3). Performed in a facility and adheres to standards. BSL-3 operating instructions. All plans using pathogens have been approved by the Hokkaido University Graduate School of Dentistry (approval number: R-2-4-1).

reagent

CPC (TCI, Tokyo, Japan) was dissolved in deionized distilled water (DDW) and sterilized with a filter (diameter 0.45 µm) (Sartorius, Göttingen, Germany). In addition, a detergent, Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control. PBS was used as a negative control.

saliva of healthy volunteers

Saliva was provided by 5 healthy, unimmunized volunteers. All saliva samples were tested negative for SARS-CoV-2 by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) prior to experimentation and mixed in one tube. This experiment was approved by the ethics committee of Hokkaido University and used human-derived materials. Informed consent was obtained from each volunteer before collecting saliva (approval number: 2021-2).

Cell viability assay

VeroE6/TMPRSS2 cell viability was measured by MTS. [3-(4,5-dimethylthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA) is used to assay in the presence of different concentrations of CPC (0-50 μg/mL) at 37 °C for 1 hour. Absorbance was measured with a GloMax Multiplus Plate Reader/Luminometer (Promega). Three independent experiments were performed in triplicate.

plaque assay

SARS-CoV-2 strain was mixed with an equal volume of CPC solution (final concentration: 0–50 μg/mL in DMEM containing 2% FBS) or SP-T medical mouthwash (Lion Co., Ltd., Tokyo, Japan). , similar to the diluted CPC, to a concentration of 50 µg/mL in PBS. The mixture was incubated at room temperature for 30 minutes and diluted 1/10 with 2% FBS DMEM to reduce CPC in the mixture. The diluted mixture was inoculated into VeroE6/TMPRSS2 cells and incubated for 1 h at 37 °C with rotation. After incubation, cells were washed twice with 1 × PBS to remove CPCs and overlaid with 2% FBS DMEM containing 1.2% Bacto Agar (Becton Dickinson, Franklin Lakes, NJ, USA). After 48 hours of incubation at 37°C, cells were fixed overnight with 3.7% buffered formaldehyde. Fixed cells were stained with 1% crystal violet. Cells infected with SARS-CoV-2 exhibit cytopathic effects and infected cell clusters can be seen as unstained areas such as plaques.

Viral entry assay

VeroE6/TMPRSS2 cells were seeded in 24-well plates at a density of 1.0 × 10^Five cell/well. SARS-CoV-2 Wuhan strain was mixed with an equal volume of CPC (final concentration: 0-25 μg/mL). At each drug concentration, wells were 1.0 x 10³ Viral PFU (MOI = 0.01). The mixture was incubated at room temperature for 30 minutes. After incubation, the mixture was inoculated onto VeroE6/TMPRSS2 cells and incubated for 1 h at 37 °C with rotation. After 1 h of absorption, cells were washed twice with 1 × PBS to remove CPCs and cultured in maintenance medium. Twenty-four hours post-infection (hpi), total RNA was extracted from inoculated cells using TRIzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and RNeasy Mini Kit (QIAGEN, Hilden, Germany). total RNA was extracted using Extracted RNA was subjected to qRT-PCR analysis using the THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO, Osaka, Japan). The SARS-CoV-2 genome was quantified using the N2 primer probe set (Takara, Shiga, Japan). Non-human primate β-actin was used as an endogenous control.Primer and probe sequences for non-human primate β-actin were previously described³³Levels of the SARS-CoV-2 N gene were normalized with levels of β-actin mRNA³⁴Additionally, viral RNA levels at 24 hpi were normalized with viral RNA levels at 0 hpi. All RT-PCR tests were performed using the CFX96 real-time PCR system (BioRad, Hercules, CA, USA). Three independent experiments were performed in triplicate.

Analysis of virucidal activity of CPC against SARS-CoV-2 in saliva

The SARS-CoV-2 Wuhan strain was spiked into saliva collected from healthy volunteers and mixed with an equal volume of CPC (final concentration: 0-40 μg/mL). The saliva mixture was diluted 1/100 to reduce viscosity and filtered through a 0.45 μm filter (Sartorius) to remove bacteria and fungi. Plaque assays were performed as previously described (section “plaque assay”). Three independent experiments were performed in triplicate.

Sucrose density gradient analysis

SARS-CoV-2 Wuhan strain was treated with CPC (50 μg/mL) or Triton X-100 (1%) for 10 minutes at room temperature. After incubation, the mixture was loaded onto a 10% to 50% sucrose density gradient. After 250,000 × 6 hours ultracentrifugation using Optima XE-90 (Beckman Coulter, Brea, CA, USA)g, each 100 μL gradient was fractionated into 22 fractions, mixed with 100 μL SDS-PAGE sample buffer and boiled at 95 °C for 5 min. After boiling, they were analyzed by 10% SDS-PAGE, followed by immunoblotting using mouse monoclonal anti-SARS-CoV-2 S protein (GTX632604) or rabbit polyclonal N protein (GTX135357) antibodies (GeneTex, Irvine, CA). I did the analysis. Membranes were cleaved at 100 kDa after transcription, hybridized with S and N protein antibodies, respectively, and visualized. An ImageQuant LAS 4000 mini (Fujifilm Corporation, Tokyo, Japan) was used for imaging (Fig. S4).

transmission electron microscopy

SARS-CoV-2 Wuhan strain was treated with 1 × PBS, CPC (10, 50, and 250 μg/mL) and Triton X-100 (1%) for 10 minutes at room temperature. The mixture was fixed with 2.5% glutaraldehyde for 24 h at 4 °C. A fixed sample (5 µL) was placed on a sheet of Parafilm. A foambar film (#10-1009, Ouken Shoji, Tokyo, Japan) was placed on each drop and allowed to adsorb virus for 5 minutes. The grids were washed with DDW, placed on a drop of filtered 2.0% uranyl acetate solution for another minute, air-dried, and examined at 80 kV using a JEM-1400 TEM (JEOL, Tokyo, Japan).

statistics

Statistical analysis was performed using Graphpad Prism v9 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± SD of biological triplicates. Statistical analysis was performed using one-way ANOVA. For all datasets, p Values ​​less than 0.05 were considered significant.

Institutional Review Board

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Hokkaido University (approval number: 2021–2).

informed consent

Informed consent was obtained from all subjects involved in the study.