Antiviral effect of cetylpyridinium chloride in mouthwash on SARS-CoV-2

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Antiviral effect of cetylpyridinium chloride in mouthwash on SARS-CoV-2

武田, 遼

北海道大学. 博士(歯学) 甲第15504号

2023-03-23

10.14943/doctoral.k15504

http://hdl.handle.net/2115/89929

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theses (doctoral)

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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

博 士 論 文

Antiviral effect of cetylpyridinium chloride
in mouthwash on SARS-CoV-2
（洗口液中成分セチルピリジニウム塩化物水
和物の SARS-CoV-2 に対する抗ウイルス効果）

令和５年３月申請

北海道大学
大学院歯学院口腔医学専攻

武田

遼

www.nature.com/scientificreports

OPEN

Antiviral effect of cetylpyridinium
chloride in mouthwash
on SARS‑CoV‑2
Ryo Takeda1,2, Hirofumi Sawa3,4,5, Michihito Sasaki3, Yasuko Orba3,4, Nako Maishi1,
Takuya Tsumita1, Natsumi Ushijima6, Yasuhiro Hida7, Hidehiko Sano8,
Yoshimasa Kitagawa2 & Kyoko Hida1*
Cetylpyridinium chloride (CPC), a quaternary ammonium compound, which is present in mouthwash,
is effective against bacteria, fungi, and enveloped viruses. This study was conducted to explore the
antiviral effect of CPC on SARS-CoV-2. There are few reports on the effect of CPC against wild-type
SARS-CoV-2 at low concentrations such as 0.001%–0.005% (10–50 µg/mL). Interestingly, we found
that low concentrations of CPC suppressed the infectivity of human isolated SARS-CoV-2 strains
(Wuhan, Alpha, Beta, and Gamma) even in saliva. Furthermore, we demonstrated that CPC shows
anti-SARS-CoV-2 effects without disrupting the virus envelope, using sucrose density analysis and
electron microscopic examination. In conclusion, this study provided experimental evidence that CPC
may inhibit SARS-CoV-2 infection even at lower concentrations.
According to the recent information from the coronavirus resource center, Johns Hopkins University of
­Medicine1, COVID-19 is responsible for more than 420 million cases and around 6 million deaths worldwide.
SARS-CoV-2 was originally reported in Wuhan, ­China2 and some variants of interest and variants of concern
(VOCs) have also been ­reported3. In addition, it is concerned that some variants like Delta and Omicron might
have the ability to evade vaccine-induced i­ mmunity4–6. Therefore, scientists concern that SARS-CoV-2 pandemic
may continue even after the increase in vaccination coverage.
It has been reported that SARS-CoV-2 infects epithelial cells of oral mucosa and salivary glands, which
express viral entry factors, angiotensin-converting enzyme 2 (ACE2), and the trans- membrane protease serine (TMPRSS) m
­ embers7. Thus, in this fashion oral cavity plays a crucial role in infection and transmission of
SARS-CoV-2. Although the symptom of COVID-19 related to oral cavity is dysgeusia and ­stomatitis8,9, many
SARS-CoV-2-infected people could be asymptomatic, resulting in its transmission to other people.
SARS-CoV-2 can replicate in oral cavity and release into ­saliva7. In addition, SARS-CoV-2 can replicate
in respiratory ­epithelium10 and may be transmitted to oral cavity by coughing. Transmission of SARS-CoV-2
through droplets and/or aerosol causes its infection and replication in lung alveolar epithelial cells, resulting
in alveolar d
­ amage11. Furthermore, it is reported that SARS-CoV-2 transmission occurs through droplets from
expiratory activities, such as talking, coughing, and s­ neezing12,13. Interestingly, the SARS-CoV-2 infected people
may become a source of transmission even during the asymptomatic incubation period of the v­ irus14. Thus, we
need to investigate the prophylaxis strategy against COVID-19. Furthermore, the relationship between aspiration
of droplets from saliva containing SARS-CoV-2 and COVID-19 aggravation has been ­reported15. Therefore, oral
care is important for prevention of transmission of SARS-CoV-2.
Mouthwash has been focused on preventing microbiome ­infection16. In addition, several components of
mouthwash have recently been reported to reduce SARS-CoV-2 virions in the oral c­ avity17,18. Cetylpyridinium
chloride (CPC) is widely used as one of the bactericidal components of mouthwash, tablets, sprays, and drops.
CPC can disrupt the lipid membrane through physicochemical interactions. CPC has already been reported to
1

Vascular Biology and Molecular Pathology, Faculty of Dental Medicine and Graduate School of Dental
Medicine, Hokkaido University, Sapporo, Japan. 2Oral Diagnosis and Medicine, Faculty of Dental Medicine and
Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan. 3Division of Molecular Pathobiology,
International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. 4International Collaboration
Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. 5One Health Research
Center, Hokkaido University, Sapporo, Japan. 6Support Section for Education and Research, Graduate School
of Dental Medicine, Hokkaido University, Sapporo, Japan. 7Community Service and Welfare Network, Hokkaido
University Hospital, Sapporo, Japan. 8Restorative Dentistry, Faculty of Dental Medicine and Graduate School of
Dental Medicine, Hokkaido University, Sapporo, Japan. *email: khida@den.hokudai.ac.jp
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Figure 1.  Antiviral efficacy of CPC against SARS-CoV-2 by plaque assay using Vero E6 cells expressing the
TMPRSS2 gene (VeroE6/TMPRSS2). The virus titers were counted and the virus titer of SARS-CoV-2 Wuhan
(a), Alpha (b), Beta (c) and Gamma (d) strains treated by CPC (0–40 μg/mL) at room temperature for 30 min
were quantified and represented as PFU/mL. Plaque assay was also performed in the presence of PBS, CPC
(50 μg/mL) or Triton X-100 (1%) for 10 min. Thereafter, samples were filtered by PD-10 columns to eliminate
reagents (e). Statistical analysis was performed using one-way analysis of variance. (*p < 0.05).
have bactericidal effects as well as antiviral effects against influenza ­virus19 and c­ oronaviruses20–22. Compared to
other ingredients in mouthwashes, including povidone iodine and chlorhexidine (CHX); CPC is tasteless, odourless, and thus suitable for applications in oral care products. To date, there are few reports depicting virucidal
activity of CPC against SARS-CoV-2. Seneviratne et al. reported that CPC reduced viral load of SARS-CoV-2 in
the saliva of four patients with COVID-1923 compared to control water, but the viral infectivity in saliva was not
described. Recent report showed the effect of CPC at much lower concentration than that of CPC in commercially
available mouthwashes against p
­ seudovirus24. But there is no report on the effect of CPC at low concentrations
such as 0.001%–0.005% (10–50 µg/mL) against wild-type SARS-CoV-2 in saliva. In Japan, the concentration of
CPC in commercially available mouthwashes is almost 30–50 µg/mL, which is much lower than in the mouthwashes used in the previous r­ eports24,25. Therefore, we examined the antiviral effects of CPC on SARS-CoV-2 at
low concentrations. In addition, we also examined the mechanism of CPC’s anti-SARS-CoV-2 activity by sucrose
density analysis and electron microscopical observation.

Results

CPC suppressed SARS‑CoV‑2 infectivity.  We have examined the SARS-CoV-2 strains, including
Wuhan, Alpha, Beta, and Gamma, which belong to VOC. The plaque assay demonstrated that CPC significantly
suppressed the infectivity of all examined SARS-CoV-2 directly in a dose-dependent manner (Figs. 1a–d, S1).
CPC (50 μg/mL) treatment completely inactivated SARS-CoV-2 Wuhan strain similarly as Triton X-100 (1%)
(Fig.  1e). A commercial mouthwash (SP-T medical gargle: SP-T) containing the same concentration of CPC
showed a better antiviral effect than CPC solution with no interfering ingredients. The virus titer of SARS-CoV-2
treated with SP-T was below the limit of detection of 2.0 × ­103 PFU/mL (Fig. S2). These results indicated that
the lower concentrations of CPC (10–40 μg/mL) than those of commercially available mouthwash (50 μg/mL)
exhibited anti-SARS-CoV-2 effects in many strains, including VOC.
Next, we assessed effect of CPC on cell entry of SARS-CoV-2. The VeroE6/TMPRSS2 cells were infected with
CPC-treated SARS-CoV-2 Wuhan strain at multiplicity of infection (MOI) of 0.01. Viral RNA expression level
in the cells was significantly reduced by CPC via dose-dependent manner at 24 h postinfection (Fig. 2). The viral
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Figure 2.  Antiviral efficacy of CPC against SARS-CoV-2 by qRT-PCR. VeroE6/TMPRSS2 cells were inoculated
with SARS-CoV-2 Wuhan strain at a multiplicity of infection (MOI) of 0.01 after mixing equal amount CPC.
At 24 h postinfection, the relative levels of viral N protein RNA were evaluated quantitatively by qRT-PCR.
(*p < 0.05).

Figure 3.  Antiviral efficacy of CPC against SARS-CoV-2 with saliva by plaque assay using Vero E6 cells
expressing the TMPRSS2 gene (VeroE6/TMPRSS2). SARS-CoV-2 Wuhan strain was added in saliva and mixed
with equal amount CPC. (*p < 0.05).
RNA copy number was reduced to around one-thirtieth by CPC at the concentration of 15 μg/mL compared to
control. These data indicated that the amounts of infectious virions were decreased by CPC before cell entry. All
experiments have been performed using CPC at the concentration which did not cause cytotoxicity (Fig. S3).

CPC has antiviral activity against SARS‑CoV‑2 even in saliva.  To address whether CPC is effective

on SARS-CoV-2 in saliva that contains many proteins and is highly viscous, we measured infectivity of SARSCoV-2 Wuhan strain by plaque assay after incubation with CPC in saliva collected from healthy volunteers.
Plaque assay demonstrated the inhibitory effect of CPC (25–40 μg/mL) against SARS-CoV-2 in saliva significantly in a dose-dependent manner (Fig. 3). ...

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