Cell culture

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

Viruses

The 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 kindly provided by Dr. Saijo (National Institute of Infectious Diseases, Tokyo, Japan. These viruses were prepared using VeroE6/TMPRSS2 cells. All experiments using SARS-CoV-2 were performed at the Biosafety Level-3 (BSL-3) facility of the International Institute for Zoonosis Control ( approved number: 19(19), #21002-3), Hokkaido University and followed the standard operating procedures of BSL-3. In addition, all experimental designs using pathogens were approved by the Graduate school of dental medicine, Hokkaido University (approved number : R-2-4-1).

Reagents

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

Saliva from healthy volunteers

Saliva was provided from five healthy unvaccinated volunteers. All saliva samples were determined to be negative for SARS-CoV-2 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) prior to the experiments and mixed in one tube. This experiment was approved by the Institutional Ethics Committee of Hokkaido University to use human-derived materials. Informed consent was obtained from each volunteer before collecting saliva (approved number: 2021-2).

Cell survival assay

Cell viability of VeroE6/TMPRSS2 was measured by an MTS [3-(4,5-dimethylthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay using CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA) in the presence of different concentration of CPC (0–50 µg/mL) for 1 h at 37 °C. The absorbance was measured with GloMax Multiplus Plate Reader/ Luminometer (Promega). Three independent experiments were performed in triplicate.

Plaque assay

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

Virus entry assay

VeroE6/TMPRSS2 cells were seeded on 24-well plates at a density of 1.0 × 10⁵ cells/well. SARS-CoV-2 Wuhan strain was mixed with equal amount of CPC (Final concentration: 0–25 μg/mL). At each drug concentration, the wells were infected with 1.0 × 10³ PFU (MOI = 0.01) of virus. The mixtures were incubated for 30 min at room temperature. After incubation, the mixtures were inoculated into VeroE6/TMPRSS2 cells and incubated at 37 °C for 1 h with rotation. After 1 h of absorption, cells were washed twice with 1 × PBS to remove CPC and cultured in maintenance medium. At 24 h postinfection (hpi), total RNAs were extracted from inoculated cells using TRIzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and total RNA was extracted with RNeasy Mini Kit (QIAGEN, Hilden, Germany). The extracted RNAs were subjected to qRT-PCR analysis with the THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO, Osaka, Japan). The SARS-CoV-2 genome was quantified using primer probe sets for N2 (Takara, Shiga, Japan). Nonhuman primate β-actin was employed as endogenous control. The primer and probe sequences for nonhuman primate β-actin were described previously³³. Levels of N gene of SARS-CoV-2 were normalized with that of β-actin mRNA³⁴. Furthermore, viral RNA levels at 24 hpi were normalized with viral RNA levels at 0 hpi. All RT-PCR tests were carried out using the CFX96 Real-Time PCR System (BioRad, Hercules, CA, USA). Three independent experiments were performed in triplicate.

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

SARS-CoV-2 Wuhan strain was added in saliva collected from healthy volunteers and mixed with equal amount of CPC (Final concentration: 0–40 μg/mL). The saliva mixtures were diluted to 1/100 to reduce viscosity and filtered through 0.45 μm filters (Sartorius) to remove bacteria and fungi. Plaque assay was performed as previously described (Section “Plaque assay”). Three independent experiments were performed in triplicate.

Sucrose density-gradient analysis

The SARS-CoV-2 Wuhan strain was treated with CPC (50 μg/mL) or Triton X-100 (1%) at room temperature for 10 min. After incubation, the mixtures were loaded on top of 10%–50% sucrose density gradients. Following ultracentrifugation with Optima XE-90 (Beckman Coulter, Brea, CA, USA) for 6 h at 250,000×geach 100 μL of gradients were fractionated into 22 fractions and mixed with 100 μL of SDS-PAGE sample buffer and boiled at 95 °C for 5 min. After boiling, they were analyzed by 10% SDS-PAGE followed by immunoblot analysis using mouse monoclonal anti-SARS-CoV-2 S protein (GTX632604) or rabbit polyclonal N protein (GTX135357) antibody (GeneTex, Irvine, CA, USA). The membranes were cut at 100 kDa after the transfer and hybridized with S protein antibody and with N protein antibodies respectively and subjected to visualization. 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 min at room temperature. The mixtures were fixed with 2.5% glutaraldehyde at 4 °C for 24 h. Fixed sample (5 μL) was placed onto a sheet of Parafilm. Formvar Film (#10-1009, Okenshoji, Tokyo, Japan) was placed on each drop to adsorb virus for 5 min. Grids were washed with DDW and placed on a drop of filtered 2.0% uranyl acetate solution for an additional 1 min, air dried, and examined using a JEM-1400 TEM (JEOL, Tokyo, Japan) at 80 kV.

Statistics

Statistical analyses were performed using Graphpad Prism v9 (GraphPad Software Inc., San Diego, CA, USA). Data is presented as the mean values ​​± SD of biological triplicates. Statistical analysis was performed using one-way analysis of variance. For all data sets, a p value of less than 0.05 was considered significant.

Institutional review board

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

Informed consent

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