Ethanol vapor inhalation treatment inhibits lethal respiratory viral infection with Influenza A

Abstract

Ethanol (EtOH) effectively inactivates enveloped viruses in vitro, including influenza and SARS-CoV-2. Inhaled EtOH vapor may inhibit viral infection in mammalian respiratory tracts, but this has not yet been demonstrated. Here we report that unexpectedly low EtOH concentrations in solution, approximately 20% (v/v), rapidly inactivate influenza A virus (IAV) at mammalian body temperature (37°C) and are not toxic to lung epithelial cells upon apical exposure. Furthermore, brief exposure to 20% (v/v) EtOH decreases production of infectious progeny viruses in IAV-infected cells. Using an EtOH vapor exposure system that is expected to expose murine respiratory tracts to 20% (v/v) EtOH solution by gas-liquid equilibrium at 37°C, we demonstrate that brief EtOH vapor inhalation twice a day protects mice from lethal IAV respiratory infection by reducing viruses in the lungs without harmful side effects. Our data suggest that EtOH vapor inhalation may provide a versatile therapy against various respiratory viral infectious diseases.

Full text

Effect of Ethanol Vapor Inhalation Treatment on Lethal
Respiratory Viral Infection With Inuenza A
Miho Tamai,
1
Seita Taba,
2
Takeshi Mise,
2
Masao Yamashita,
2
Hiroki Ishikawa,
1,a
and Tsumoru Shintake
2,a
1
Immune Signal Unit, Okinawa Institute of Science and Technology, Tancha 1919-1, Onna-son, Okinawa 904-0495, Japan; and
2
Quantum Wave Microscopy Unit, Okinawa Institute of Science and
Technology, Tancha 1919-1, Onna-son, Okinawa 904-0495, Japan
Ethanol (EtOH) effectively inactivates enveloped viruses in vitro, including inuenza and severe acute respiratory syndrome
coronavirus 2. Inhaled EtOH vapor may inhibit viral infection in mammalian respiratory tracts, but this has not yet been
demonstrated. Here we report that unexpectedly low EtOH concentrations in solution, approximately 20% (vol/vol), rapidly
inactivate inuenza A virus (IAV) at mammalian body temperature and are not toxic to lung epithelial cells on apical exposure.
Furthermore, brief exposure to 20% (vol/vol) EtOH decreases progeny virus production in IAV-infected cells. Using an EtOH
vapor exposure system that is expected to expose murine respiratory tracts to 20% (vol/vol) EtOH solution by gas-liquid
equilibrium, we demonstrate that brief EtOH vapor inhalation twice a day protects mice from lethal IAV respiratory infection
by reducing viruses in the lungs without harmful side effects. Our data suggest that EtOH vapor inhalation may provide a
versatile therapy against various respiratory viral infectious diseases.
Keywords. ethanol vapor; inuenza; inhalation treatment; respiratory infection; enveloped viruses.
Respiratory viral infections are global health and economic risks
that are difficult to control [1, 2], as illustrated by the coronavirus
disease 2019 (COVID-19) pandemic [3, 4]. Effective vaccines can
reduce the risk of specific respiratory infections in many people
[5, 6], but additional therapeutic strategies should be developed
to protect those who cannot access such vaccines. Furthermore,
to address the risks of emerging mutant viruses that are resistant
to vaccine-induced immune responses and new pandemic virus-
es in the future [7–10], therapeutic strategies applicable to a wide
range of respiratory infectious diseases are needed. Inhalation of
compounds that are cheap and that effectively target general viral
properties or functions in the respiratory tract might be useful to
control respiratory infections [11].
Ethanol (EtOH) is widely used for disinfection of environ-
mental and body surfaces in daily life because it effectively inac-
tivates bacteria and enveloped viruses [12]. For example,
inuenza A virus (IAV) and severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) are inactivated by exposure to
about 30% EtOH in approximately 1 minute [13–15].
Theoretically, inhalation of EtOH vapor can expose the respira-
tory epithelium to enough EtOH to inactivate enveloped viruses
[16]. Indeed, a molecular imaging study detected substantial
amounts of EtOH in the lungs of rats following inhalation of
EtOH vapor [17]. These observations support the therapeutic
potential of EtOH vapor inhalation against respiratory infectious
diseases, but there is no direct evidence that inhaled EtOH inhib-
its viral respiratory infections without damaging epithelial cells.
METHODS
Cells and Mice
Madin-Darby canine kidney (MDCK) cells (NBL-2; JCRB9029)
were maintained in minimum essential medium (MEM;
11095080; Thermo Fisher Scientific) containing 10% fetal bovine
serum (173012; Sigma-Aldrich). The human lung cancer cell line
A549 (RCB0098) was provided by the RIKEN BioResource
Research Center through the National Bio-Resource Project of
the Ministry of Education, Culture, Sports, Science and
Technology/Japan Agency for Medical Research and
Development, Japan, and was maintained in Dulbecco’s MEM
(DMEM; D6429; Sigma-Aldrich) containing 10% fetal bovine se-
rum. Female 6–8-week-old, specific pathogen-free C57BL/6J mice
were obtained from Japan SLC. Protocols of all mouse experiments
were approved by the Animal Care and Use Committee at the
Okinawa Institute of Science and Technology Graduate University.
Influenza Virus
Inuenza A/PR/8/34 virus (IAV; American Type Culture
Collection, VR-95) was propagated in MDCK cells cultured in
Received 02 December 2022; editorial decision 27 March 2023; accepted 03 April 2023; pub-
lished online 27 April 2023
a
H. I. and T. S. contributed equally to this work.
Correspondence: Hiroki Ishikawa, PhD, Immune Signal Unit, Okinawa Institute of Science
and Technology, Tancha 1919-1, Onna-son, Okinawa 904-0495, Japan (hiroki.ishikawa@oist.
jp); Tsumoru Shintake, PhD, Quantum Wave Microscopy Unit, Okinawa Institute of Science
and Technology, Tancha 1919-1, Onna-son, Okinawa 904-0495, Japan (tsumoru.shintake@
oist.jp).
The Journal of Infectious Diseases
®
© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases
Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-
NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial reproduction and distribution of the work, in any medium, pro-
vided the original work is not altered or transformed in any way, and that the work properly
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https://doi.org/10.1093/infdis/jiad089
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MAJOR ARTICLE
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DMEM containing 0.35% bovine serum albumin (BSA; A7979;
Sigma-Aldrich) and 0.12% sodium bicarbonate (199-05985;
Wako). Medium was centrifuged at 2000g for 30 minutes at 4°C, fol-
lowed by ultracentrifugation at 18 000g for 30 minutes at 4°C to re-
move cell debris. Supernatant was then layered on top of 5 mL of a
chilled 5% sucrose cushion and ultracentrifuged at 112 000g for 90
minutes at 4°C to purify the virus. The viral pellet was resuspended
in phosphate-buffered saline (PBS) and stored at −80°C. The titer of
the viral stock was determined as described below.
IAV Titration
Titers of viral stock were measured by indirect immunouores-
cence [18], while those of infectious viruses in other test samples
were determined by median tissue culture infective dose
(TCID
50
) assay [19]. Briey, conuent monolayers of MDCK cells
cultured in 96-well tissue culture plates were infected with viral
samples serially diluted 10-fold in infection medium (DMEM sup-
plemented with 0.35% BSA and 0.12% sodium bicarbonate) for
1 hour at 37°C. The inoculum was then replaced with infection me-
dium supplemented with 0.75 μg/mL tosyl phenylalanyl chloro-
methyl ketone–trypsin. In the indirect immunouorescence
assay, 8 hours after infection, cells were fixed, permeabilized,
and blocked with 2.5% BSA (wt/vol) in PBS. Cells were reacted
with 0.7 μg/mL anti-nucleoprotein (H16-L10-4R5) antibody
(GTX14213; GeneTex) and then with Alexa Fluor Plus 555–conju-
gated anti-mouse immunoglobulin G secondary antibody
(A32727, Thermo Fisher Scientific), and analyzed using uores-
cence microscopy (BZ-X710; Keyence). In the TCID
50
assay,
on day 4 after infection, cells were fixed with 4% paraformaldehyde
in PBS and stained with crystal violet dye (C3886; Sigma-Aldrich).
The TCID
50
was determined with the Reed–Muench method.
Cell Viability Assay
Cell viability was assessed with a Cell Counting Kit-8 assay
(CCK-8; Dojindo laboratories), according to the manufactur-
er’s instructions. Briey, cells were incubated with CCK-8 sol-
ution for 1 hour at 37°C, and absorbance was measured at
450 nm using a microplate reader (iMark; Bio-Rad).
Transepithelial Electrical Resistance Assay
To evaluate the integrity of the epithelial monolayer culture, we
measured transepithelial electrical resistance was measured us-
ing a Millicell ERS-2 (Millipore). During the measurement,
cells on a cell culture insert in a tissue culture plate were incu-
bated with PBS (300 μL in the apical compartment and 700 μL
in the basolateral compartment) at 37°C. Relative transepithe-
lial electrical resistance values were calculated based on the val-
ues before EtOH treatment.
EtOH Vapor Exposure System
A modified anesthesia induction chamber (MK-ICS; Muramachi)
connected to an ultrasonic humidifier (TF003; Repti Zoo; ow
rate, 26 L/min) was used as an exposure chamber, which allowed
simultaneous exposure of up to 5 mice. In all animal experiments,
50% (vol/vol) EtOH solution preheated to 50°C was vaporized in
high-power mode (atomizing amount when water was vaporized,
3.1 mL/min). As a control, distilled water preheated to 33°C was
vaporized in medium-power mode (atomizing amount, 1.8 mL/
min). In these conditions, about 30 mL of solvents were vaporized
in 10 minutes, and the temperature in the exposure chamber was
about 30°C.
Measurement of EtOH Concentrations
Concentrations of EtOH solutions were measured using a re-
fractometer calibrated for EtOH (Yieryi; THE01507), accord-
ing to the manufacturer’s instructions. Briey, 5–40-μL
samples were analyzed, and standard curves were generated
with known concentrations of EtOH diluted in distilled water
or PBS. EtOH gas concentration was measured using an optical
interferometer (FI-8000; Riken Instruments).
Henry’s Law: Estimation of EtOH Concentration in Solution Exposed to
EtOH gas
The amount of EtOH gas dissolved in a liquid is proportional to
its partial pressure above the liquid, as follows:
Ca=KH·p,
where C
a
is a concentration of EtOH in solution, K
H
is
Henry’s coefficient of EtOH for water, and p is the partial pres-
sure of EtOH gas. At 25°C (absolute temperature [T] = 298 K),
the average EtOH measured value is 194 mol/L/atmospheric
pressure (atm; 1 atm = 101.3 kPa) [20]. The temperature de-
pendence of the coefficient is calculated as follows:
d[ln(KH)]/d(1/T)=6274.
At 37°C (T = 310 K), we have 87.2 mol/L/atm. EtOH 4% (vol/
vol) gas has a partial pressure of 4 kPa (0.04 atm), and the solu-
ble EtOH in water becomes 87.2 × 0.04 = 3.5 mol/L, which is
equal to 20.4% EtOH (vol/vol) in solution, where the molecular
weight of EtOH is 46.1 g/mol, and the density is 0.79 g/mL.
EtOH Vapor Inhalation Treatment of Mice
A maximum of 5 mice were placed in the exposure chamber
and exposed to EtOH vapor for 10 minutes twice a day. Mice
were housed in cages between treatments and weighed daily.
Mouse Blood EtOH Measurement
Mouse blood EtOH concentration was assessed with an
Alcohol Assay Kit (Colorimetric; STA-620; Cell Biolabs).
Briey, blood samples collected from tails were incubated at
room temperature for 30 minutes, followed by centrifugation
at 2500g for 20 minutes. Serum was collected and diluted in
1× assay buffer (1:50). Absorbance was measured at 570 nm
using a microplate reader (iMark; Bio-Rad).
2 • JID • Tamai et al
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IAV Infection of Mice
Mice were anesthetized with isourane and intranasally inject-
ed with 1 × 10
3
uorescence-forming units of IAV in 30 μL
PBS. For viral titration, mice were euthanized on day 3 after in-
fection, and their lungs and nasal mucosae were isolated and
homogenized using a PowerMasher II homogenizer (891300;
Nippi) at room temperature, stored at −80°C, and subjected
to TCID
50
titration assays.
Histopathological Analysis
Lung samples were fixed in 4% paraformaldehyde phosphate
buffer. Sectioning and hematoxylin-eosin staining were per-
formed with GenoStaff. Hematoxylin-eosin–stained sections
of the lung were analyzed with an all-in-one uorescence mi-
croscope (BZ-X710; Keyence).
Statistical Analysis
Statistical analysis was performed using GraphPad Prism soft-
ware (version 9.4.0), and statistical details are provided in the
figure legends.
RESULTS
Inactivation of IAV by Ethanol Solution at Room Temperature
We reasoned that inhalation of EtOH vapor protects the host
from respiratory viral infection if the EtOH concentration in
the respiratory epithelial lining uid is sufficient to inactivate
the viruses without being toxic to epithelial cells. Because in
vivo virucidal activity of EtOH remains to be determined, we
first sought to address this by exposing IAV to various concen-
trations of aqueous EtOH solution at body temperature (37°C)
or room temperature (24°C) for 1 minute. Consistent with pre-
vious reports [14], the lowest concentration of EtOH solution
that completely inactivated IAV at 24°C was 36% (vol/vol)
(Figure 1A). In contrast, surprisingly, 22.5% (vol/vol) EtOH sol-
ution effectively inactivated IAV to undetectable levels at 37°C
(Figure 1B). Furthermore, even 18% (vol/vol) EtOH solution re-
duced infectious IAV >10-fold at 37°C (Figure 1B). In the ab-
sence of EtOH, the infectivity of IAV decreased in a
temperature-dependent manner above 55°C, reaching undetect-
able levels at 61°C (Figure 1C). Minimal EtOH concentrations
required to inactivate IAV to undetectable levels were inversely
proportional to the reaction temperature (Figure 1D), suggesting
that thermal and chemical energies contribute cooperatively to
viral inactivation. Taken together, these data suggest that an ap-
proximately 20% (vol/vol) EtOH solution might be sufficient to
inactivate IAV in the respiratory tract.
Effect of Apical Exposure of Lung Epithelial Cells to EtOH on Cell Viability
and Progeny Virus Production
Next, we investigated susceptibility of respiratory epithelial
cells to EtOH. When compounds are inhaled, respiratory epi-
thelial cells are exposed from the apical side, which is covered
by the epithelial lining uid in the lumen of respiratory tracts
[21]. To assess cytotoxic effects of apical EtOH exposure, we ex-
posed A549 cells to 22.5% (vol/vol) EtOH from the apical side
(Figure 2A). The concentration of EtOH solution in the apical
uid was slightly decreased in 10 minutes, while that in the ba-
solateral compartment was undetectable (Figure 2B). Notably,
apical exposure to 22.5% (vol/vol) EtOH solution for 10 min-
utes did not affect A549 cell viability (Figure 2C). However,
there was a significant reduction in cell viability on exposure
to 22.5% EtOH from both the apical and basolateral sides
(Figure 2C) or apical exposure to concentrations of EtOH
>27.0% (vol/vol) (Supplementary Figure 1A).
To investigate whether EtOH exposure inhibits progeny vi-
rus production in IAV-infected cells, we treated IAV-infected
A549 cells with apical exposure to 22.5% (vol/vol) EtOH for
1 minute at 6 hours after infection. Interestingly, EtOH signifi-
cantly reduced viral production over the course of 18 hours af-
ter treatment (Figure 2D) without affecting cell viability
(Supplementary Figure 1B). Antiviral innate immunity inhibits
intracellular IAV infection process [22], but we observed that
EtOH treatment did not affect infection-induced expression
of genes critical for innate immunity, such as the gene for inter-
feron regulatory factor 7 (IRF7) [23–25] (Supplementary
Figure 1C). These results suggest that respiratory epithelial
cells are tolerant to brief apical exposure to EtOH solution
up to about 20% (vol/vol) and that EtOH inhibits intracellular
IAV infection process.
Generation of EtOH Vapor as an In Vivo Virucide
Based on our in vitro results, we hypothesized that inhaled
EtOH vapor would inactivate enveloped viruses in respiratory
epithelial lining uid without cytotoxicity by increasing EtOH
concentration in the uid to about 20% (vol/vol). To verify our
hypothesis, we developed an EtOH exposure system that can si-
multaneously expose up to 5 mice to EtOH vapor generated
from an ultrasonic humidifier (Figure 3A). From Henry’s
law, it was estimated that exposure of solvent to 4% (vol/vol)
EtOH gas would result in generation of about 20% (vol/vol)
EtOH solution at 37°C by gas-liquid equilibrium. We first test-
ed whether the EtOH exposure system can generate EtOH gas
concentrations of 4% (vol/vol) or higher. Because EtOH solu-
tions at concentrations of ≥67% (vol/vol) are considered haz-
ardous under the Japanese Fire Service Law, we decided to
use 50% (vol/vol) EtOH solutions as a vapor source. We ob-
served that vaporization of 50% (vol/vol) EtOH solution pre-
heated to 50°C was sufficient to generate 4% (vol/vol) EtOH
gas (Figure 3B). Furthermore, exposure to the EtOH gas gener-
ated in this condition increased EtOH concentrations in the
apical uid of A549 cell culture to >20% (vol/vol) within 6 min-
utes (Figure 3C). These results suggest that an optimized EtOH
vaporizing protocol can generate enough EtOH gas to increase
the EtOH concentration in respiratory epithelial lining uid to
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about 20% (vol/vol), a level sufficient to inactivate IAV at body
temperature.
Safety of EtOH Vapor Inhalation
To evaluate the safety of EtOH vapor inhalation treatment, we
exposed mice to EtOH vapor generated by vaporizing 50% (vol/
vol) EtOH solution preheated to 50°C for 10 minutes twice a
day. We observed that mice walked unsteadily after a brief
(10-minute) exposure to EtOH vapor but recovered to normal
within 20 minutes (data not shown). Blood alcohol concentra-
tions (BACs) were about 0.052% (0.019%) (mean [standard de-
viation (SD)]) (wt/vol) at 15 minutes after EtOH vapor
inhalation treatment but decreased to undetectable levels in
5 hours (Figure 4A and Supplementary Figure 2A). We also de-
tected a mean (SD) of 0.024 (0.016) mg of EtOH in the bron-
choalveolar lavage uid (BALF) immediately after a brief
EtOH vapor exposure (Supplementary Figure 2B). Assuming
that the volume of the epithelial lining uids in murine lungs
is 80 μL (thickness of the uid, 0.1 μm; surface area of the lungs,
800 cm
2
), the mean (SD) concentrations of EtOH in the lung
epithelial lining uid was estimated to be 0.030% (0.020%)
(wt/vol). Brief EtOH vapor exposure twice a day did not alter
body weight or serum levels of aspartate aminotransferase
and alanine aminotransferase until 3 weeks of treatment
(Figure 4B and 4C). Moreover, EtOH vapor treatment also
did not cause detectable tissue damage or inammation in
the nasal cavity and lungs, nor did it result in hepatic steatosis
(Figure 4D and Supplementary Figure 2C and 2D). These re-
sults indicate that short-term use of daily brief inhalation of
EtOH vapor does not have adverse effects on mice.
Protection of Mice from Lethal IAV Infection by EtOH Vapor Inhalation
Finally, we assessed the in vivo virucidal activity of EtOH vapor
inhalation treatment using a mouse respiratory infection model
of IAV. Mice were treated with EtOH vapor twice a day from
the day before intranasal administration of IAV (1 × 10
3
uorescence-forming units per mouse). Mice treated with
EtOH vapor exhibited less weight loss than control mice
A
C
B
2
3
4
5
6
04.59.0
13.5
18.0
22.5
27.0
31.5
36.0
40.5
***
24°C
IAV, Log TCID50/mL
IAV, Log TCID50/mL
IAV, Log TCID50/mL
EtOH, % (vol/vol)
EtOH, % (vol/vol) EtOH, % (vol/vol)
2
3
4
5
6
04.59.0
13.5
18.0
22.5
27.0
31.5
36.0
40.5
***
37°C
0 20 40 60 80
0
10
20
30
40 R² = 0.9916
y = –0.9380x + 59.88
24 45 47 49 51 53 55 57 59 61 63 65
2
3
4
5
6
7
Temperature, °C Temperature, °C
***
**
D
Figure 1. Virucidal efficacy of ethanol (EtOH) depends on temperature. A–C, Influenza A virus (IAV) suspended in 50 μL of phosphate-buffered saline (PBS) was mixed with
450 μL of PBS containing 0%–45% (vol/vol) EtOH preheated to reaction temperatures and incubated for 1 minute at 24°C (A), 37°C (B), or various temperatures (C). Final
concentrations of EtOH and reaction temperatures are indicated in each panel. The reaction was terminated by adding 9 volumes of Dulbecco’s minimum essential medium
supplemented with 0.35% bovine serum albumin, 0.12% sodium bicarbonate, 1% antibiotic-antimycotic, and 0.75 μg/mL tosyl phenylalanyl chloromethyl ketone–trypsin–
trypsin, and viral titers were determined. Data are expressed as means with standard deviations (n = 3). **P < .01; ***P < .001 (calculated using 1-way analysis of variance
with Dunnett multiple comparison tests). Abbreviation: TCID
50
, median tissue culture infective dose. D, Scatterplot showing an inverse linear relationship between EtOH
concentrations required for inactivation of IAV and reaction temperature. Data are representative of ≥2 independent experiments.
4 • JID • Tamai et al
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(Figure 5A). By day 10 after infection, 63% (17 of 27) of the un-
treated mice reached the humane end point (20% loss of orig-
inal body weight), compared with only 11% (3 of 27) of the
mice treated with EtOH vapor (Figure 5B). In our EtOH expo-
sure system, mouse bodies are exposed to EtOH vapor, which
may lead to oral ingestion of EtOH adhering to the fur. To
rule out the possibility that ingested EtOH inhibits lethal IAV
infection, we applied a 22.5% EtOH solution to the body sur-
face of mice. Application of EtOH to the body surface did
not increase BAC as high as EtOH vapor inhalation treatment,
nor did it inhibit lethal IAV infection (Supplementary
Figure 3A and 3B).
Notably, EtOH vapor treatment decreased viral titers in the
lungs, but not in the nasal cavity, on day 3 after infection
(Figure 5C). EtOH vapor treatment also reduced leukocyte in-
filtration and damage in the lungs (Figure 5D and
Supplementary Figure 4A). Consistent with this, EtOH vapor
treatment decreased numbers of monocytes and macrophages
in BALF (Supplementary Figure 4B–D), although it tended to
increase the viability of these cells (Supplementary
PBS
or
EtOH
ALI
culture ALI
culture
A
Heat
block
: 37°C
C
B
EtOH
0 10 0 10
0
5
10
15
20
25
EtOH, % (vol/vol)
BasolateralApical
Time, min
CCK-8 Absorbance
(450 nm)
Basolateral
22.5% vol/vol
EtOH
Apical –
IAV, Log TCID50/mL
–
+
–
+
+
– + – +
NS
***
0
0.2
0.4
0.6
Apical exposure
to 22.5% vol/vol EtOH
D
18 h After
treatment
0
2
4
6
8
NS *
Before
***
NS
22.5% vol/vol EtOH
Figure 2. Resistance of lung epithelial cells to apical exposure to ethanol (EtOH). A, Schematic representation of experimental settings. A549 cells (3 × 10
4
) seeded on a
cell culture insert in a 24-well tissue culture plate were grown under air-liquid interface (ALI) culture conditions in which basolateral surfaces of the cells were in contact with
culture medium. Culture medium was changed every 2–3 days, and cells were cultured for ≥25 days until EtOH exposure. Cell numbers and integrity of epithelial monolayers
were assessed with a Cell Counting Kit-8 (CCK-8) and transepithelial electrical resistance (TEER) assays, respectively, and cells with CCK-8 values >0.5 and TEER values >20
were used for EtOH exposure assays. After washing cells on the cell culture insert with phosphate-buffered saline (PBS), the insert was placed on a tube (352059; Falcon)
containing 12.25 mL of PBS with (for exposure from both the apical and basolateral sides) or without various concentrations of EtOH (for exposure from the apical side only),
and 500 μL of PBS containing EtOH was added to the apical compartment of the insert. B, EtOH concentrations in apical and basolateral compartments were measured after
10 minutes of exposure of cells to 22.5% EtOH from the apical side only (n = 3). C, Analysis of the viability of cells unexposed (indicated by minus symbols) or exposed to
22.5% EtOH from the apical side only or from both the apical and basolateral sides (indicated by plus symbols). After 10-minute exposure, cells were washed with PBS and
cultured under ALI conditions for 24 hours. Cell viability was assessed with a CCK-8 assay (n = 3). D, Analysis of progeny virus production in cells infected with influenza A
virus (IAV) and then unexposed (indicated by minus symbols) or exposed (indicated by plus symbols) to EtOH. A549 cells were infected with IAV at a multiplicity of infection of
1. At 6 hours after infection, cells were apically exposed to 22.5% (vol/vol) EtOH for 1 minute. After washing with PBS, cells were cultured for 18 hours, and viral titers in
culture medium were measured (n = 4). Abbreviation: TCID
50
, median tissue culture infective dose. B–D, Data are expressed as means with standard deviations and are
representative of ≥2 independent experiments. *P < .05; ***P < .001; NS, not significant (calculated using 1-way analysis of variance with Dunnett multiple comparison
tests).
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Figure 4E). Furthermore, EtOH vapor treatment significantly
decreased expression of IAV-induced genes associated with in-
nate immunity, such as Irf7, in lung tissues (Supplementary
Figure 4F). These results indicate that EtOH vapor inhalation
reduces viral load in the lungs and protects mice from lethal
IAV infection.
DISCUSSION
The current study demonstrates that short-term use of EtOH
vapor inhalation treatment, initiated before infection, is safe
and effectively protects mice from lethal respiratory infection
with IAV. Brief EtOH vapor inhalation treatment twice a day
significantly reduced viral titers in the lungs and ameliorated
lung damage caused by IAV infection. On the other hand,
when healthy mice were treated with brief EtOH vapor inhala-
tion twice a day for 3 weeks, there were no detectable adverse
effects. These results confirm the safety of short-term use of
EtOH vapor inhalation treatment and provide evidence of
EtOH’s virucidal activity in vivo.
Our results suggest that the virucidal efficacy of low concen-
trations of EtOH solution for the respiratory tract is higher than
that for environmental surfaces, owing to temperature effects.
Inactivation of IAV at room temperature requires EtOH solu-
tions more concentrated than 30% (vol/vol) [13–15]. However,
at body temperature, IAV is inactivated within 1 minute after
exposure to 22.5% (vol/vol) EtOH solution. Interestingly, api-
cal exposure of IAV-infected cells to 22.5% (vol/vol) EtOH
for 1 minute significantly reduced progeny virus yield over
the course of 18 hours after exposure. Given these observations,
we speculated that, if EtOH vapor inhalation increases concen-
trations of EtOH in the respiratory epithelial lining uids to
about 20%, EtOH not only directly inactivates extracellular
IAV but also suppresses progeny virus production in the respi-
ratory tract. Inactivation of extracellular IAV by EtOH is likely
due to its ability to disrupt viral envelope [26, 27], but how
Exposure chamber
(25 × 10 × 10 cm)
EtOH
vapor Air-flow fan
EtOH (50%)
Ultrasonic transducer
AB
0246810
0
1
2
3
4
5
Time, min
EtOH Gas, % (vol/vol)
25°C
37°C
50°C
C
EtOH vapor exposure system
In the air
In the apical fluid
0 2 4 6 8 10
0
10
20
30
Time, min
EtOH, % (vol/vol)
Cell
culture
insert
Heat block
: 37°C
PBS
Apical fluid
(40 μL of PBS)
Drying agent
(CaCl")
Optical
interferometer
EtOH vapor
A549
cells
Heated to 37°C
by a water bath
EtOH gas
Pump
Figure 3. Ethanol (EtOH) vapor-mediated gas-liquid equilibrium. A, Schematic diagram of the EtOH vapor exposure system. EtOH solutions are vaporized in an ultrasonic
humidifier, and vaporized EtOH flows into the exposure chamber for mice and is released from the air outlet. Dimensions of the chamber represent width × depth × height. B,
Analysis of concentrations of EtOH gas generated by the EtOH vapor exposure system; 50% EtOH solutions preheated to 25°C, 37°C, or 50°C were vaporized in the EtOH
exposure system, as shown in A. The concentration of EtOH gas released from the exposure chamber was analyzed by an optical interferometer connected via a tube con-
taining calcium chloride (CaCl
2
) as a desiccant. Schematic diagram on left shows the experimental design; graph right, the change in EtOH gas concentrations (vol/vol in the
air) for 10 minutes after EtOH vaporization. C, Analysis of EtOH concentrations in the apical fluid of A549 cells exposed to EtOH gas at 37°C. EtOH gas produced by vaporizing
a 50% EtOH solution preheated to 50°C in the EtOH vapor exposure system was released on the apical fluid (40 μL of phosphate-buffered saline [PBS]) of A549 cells cultured
on a cell culture insert placed in PBS at 37°C, via the pump of the optical interferometer shown in B. Schematic diagram on left shows the experimental design; graph on right,
changes in EtOH concentration (vol/vol) in the apical fluid. B, C, Data are expressed as means with standard deviations (n = 3) and are representative of 2 independent
experiments.
6 • JID • Tamai et al
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EtOH regulates progeny virus production is enigmatic.
Although we observed that IAV-induced expression of type I
interferon-related genes was not affected by EtOH, further
studies are needed to determine whether EtOH enhances anti-
viral immunity, because EtOH modulates several
immune-related pathways [28–33]. Given previously reported
EtOH activity [34–36], it is also important to evaluate the effect
of EtOH on activity of viral proteins or cellular metabolism in
virus-infected epithelial cells.
We used an EtOH exposure system that can generate about
4% (vol/vol) EtOH gas for EtOH inhalation treatment of mice.
Although the lower explosion limit (LEL) of EtOH gas is 3.5%
(vol/vol) in dry conditions, in our preliminary experiments, 4%
(vol/vol) EtOH gas generated by vaporization of 50% (vol/vol)
EtOH solution did not explode on ignition, probably owing to
abundant water vapor. Consistent with estimation from
Henry’s law, we observed that on exposure to 4% (vol/vol)
EtOH gas, about 20% (vol/vol) EtOH solution was generated
in the apical compartment of lung epithelial cell culture at
37°C. This supports the hypothesis that EtOH vapor inhalation
protects mice from lethal IAV infection by raising the concen-
trations of EtOH in the respiratory epithelial lining uids to
20% (vol/vol). However, it is challenging to demonstrate this
in vivo since it is impossible to directly measure EtOH concen-
trations in the respiratory tract with current technology [37].
Based on our data, the mean (SD) concentration of EtOH in
the lung epithelial lining uid was estimated to be 0.030%
(0.020%) (wt/vol), the same range as for BAC, in mice treated
with EtOH vapor. As observed with other compounds [37],
EtOH may be rapidly absorbed from the lungs into the systemic
circulation during a few minutes of BALF collection. EtOH
pharmacokinetics in the lung should be further studied using
computational uid dynamics simulation.
Our data support the safety of brief EtOH vapor inhalation.
Although cytotoxic effects of EtOH were previously observed
in various cells [38], we found that respiratory epithelial cells
are tolerant to exposure of their apical sides to 20% (vol/vol)
EtOH solution, as long as their basolateral sides are not exposed
to such concentrations. Consistent with this, brief EtOH vapor
inhalation twice a day did not cause body weight changes, dam-
age in the nasal cavity and lungs, hepatic steatosis, or elevation
of serum alanine aminotransferase and aspartate aminotrans-
ferase levels until 3 weeks of treatment. In mice treated with
EtOH vapor, BAC increased up to about 0.05% (wt/vol) and de-
creased rapidly in a few hours. Assuming a similar effect in hu-
mans, EtOH vapor inhalation may transiently raise BAC to the
Japanese legal limit for motor vehicle operation (0.03% [wt/
vol]), but not to the United States legal limit (0.08% [wt/
vol]). These results suggest that short-term use of brief EtOH
vapor inhalation treatment does not likely cause adverse side
UT
1
2
3
3
2
1
DW EtOH
7
8
9
4
5
6
9
8
7
6
5
4
1 mm
200 μm
A
D
70
80
90
100
110
120
Time After Treatment, d
Body Weight, %
–1 Day 0 0.5 2.5 5.0
0
0.02
0.04
0.06
Time, h
Blood EtOH (BAC),
% (wt/vol)
B
0 7 14 21
0 3 0 3 0 3
0
20
40
60
80
100
ALT, U/L
NS NS NS
DW
Weeks
UT EtOH
C
: Untreated
: DW vapor
: EtOH vapor
0
50
100
150
200
AST, U/L
NS NS NS
0 3 0 3 0 3
DW
Weeks
UT EtOH
NS NS
***
**
*
***
*
Figure 4. Ethanol (EtOH) vapor inhalation treatment does not damage bronchial
tissues. Female C57Bl/6 mice were placed in the exposure chamber and exposed to
EtOH or distilled water (DW) vapor for 10 minutes every 12 hours for 3 weeks. A,
Blood EtOH concentration (blood alcohol concentration [BAC]) was measured after
EtOH vapor inhalation. *P < .05; **P < .01; ***P < .001 (calculated using 1-way
analysis of variance [ANOVA] with Tukey’s multiple comparison tests). B, Body
weight changes in mice treated with EtOH or DW vapor inhalation or untreated
(UT) (n = 5). *P < .05; NS, not significant (calculated using 2-way ANOVA with
Tukey’s multiple comparison tests). C, Serum alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) were measured (n = 5); P values were calculated
using unpaired t tests followed by correction using Benjamini-Hochberg false dis-
covery rate for multiple comparisons. D, Histopathological analysis of lungs isolat-
ed from mice treated without or with DW or EtOH vapor inhalation on day 21.
Enlarged images of areas shown in open boxes in the top panels are shown in pan-
els 1–9. All data in A–D are representative of ≥2 independent experiments.
Ethanol Inhibits Airway Inuenza Infection • JID • 7
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effects, including alcohol-related chronic diseases, although the
systemic effects of long-term usage need to be evaluated.
Our data suggest promising therapeutic potential of EtOH
vapor inhalation against respiratory viral infection, but the vi-
rucidal efficacy of the current treatment protocol is still limited.
EtOH vapor treatment reduced viral load 10-fold in the lung
but not in the nasal cavities of IAV-infected mice. Because
the surface-to-volume ratio of the epithelial lining uids in the
nasal cavity is much lower than that in the lung, where the
thin surfactant covers a vast respiratory surface [21], brief expo-
sure to 4% (vol/vol) EtOH gas might not be enough to saturate
the nasal cavity uids with EtOH. In addition, since tempera-
tures in the nasal cavity are lower than in the lungs [39], mini-
mum concentrations of EtOH required for IAV inactivation
A
Time after Infection, d
Body Weight, %
**
**
DW EtOH
Nasal
mucosa
DW EtOH
Lung
IAV, Log TCID50/mL
IAV, Log TCID50/mL
C
NS
2
4
6
8
**
2
4
6
8
0 2 4 6 8 10 12 14
80
90
100
110
:
DW
:
EtOH
B
***
Time after Infection, d
Survival, %
0 2 4 6 8 10 12 14
0
25
50
75
100
:
DW
:
EtOH
Time after Infection, d
0 2 4 6 8 10 12 14
Time after Infection, d
0 2 4 6 8 10 12 14
:
DW :
EtOH
80
90
100
110
80
90
100
110
D
DW/IAV
EtOH/IAV
*
*
*
6
*
*
5
**
4
*
**
3
*
*
*
1
*
*
2
*
*
2
3
1
4
6
5
1 mm 200 μm
Figure 5. Ethanol (EtOH) vapor inhalation treatment suppresses pulmonary infection with influenza A virus (IAV). From 1 day before the IAV infection (day −1), mice were
subjected to distilled water (DW) or EtOH vapor inhalation treatment for 10 minutes twice a day throughout the experiment, as in Figure 4. On day 0, mice were intranasally
injected with 1 × 10
3
fluorescence-forming units of IAV. On the same day, EtOH vapor inhalation treatment was performed 4–6 hours before and after infection. A, B, Body
weight changes (A) and survival rates (B) in mice treated with DW or EtOH vapor inhalation. Mice were weighed daily, and those showing 20% weight loss were considered
to be dying and were humanely euthanized. Data are pooled from 4 independent experiments (n = 27). A, Left graph shows means with standard deviations (SDs) of weights
in each group; middle and right graphs, weights of individual mice treated with DW vapor or EtOH vapor, respectively. (** P < .01 (calculated with multiple Mann-Whitney
tests). B, Kaplan-Meier survival curves were calculated. **P < .01; ***P < .001 (calculated with log rank test). C, Viral titers in the lungs and nasal cavity on day 3. Data are
pooled from 2 independent experiments and expressed as means with SDs (n = 8). **P < .01; NS, not significant (calculated using unpaired t tests). Abbreviation: TCID
50
,
median tissue culture infective dose. D, Histopathological analysis of lungs isolated from mice treated without or with EtOH vapor inhalation on day 3. IAV-induced lung
inflammation is characterized by leukocytic infiltration (asterisks), alveolar collapse (arrows), and focal inflammation in the lung (arrowheads). Zoomed-in images of areas
indicated in open boxes in the left panels are shown in panels 1–6. Representative results are shown for 1 mouse of 5 in each group (others shown in Supplementary
Figure 4A).
8 • JID • Tamai et al
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are likely higher in the nasal cavity than in the lungs. It is worth
evaluating whether the virucidal activity of EtOH vapor treat-
ment is enhanced by increasing the treatment time or generating
higher concentrations of EtOH gas.
In conclusion, EtOH vapor inhalation optimized to expose the
respiratory tract to about 4% (vol/vol) EtOH gas can limit lethal
IAV respiratory infection without damaging epithelial cells.
Because various enveloped viruses, including SARS-CoV-2, are
more susceptible to EtOH than inuenza viruses [14], EtOH va-
por inhalation treatment may be effective against respiratory in-
fection of these viruses, which enter through and bud from the
apical surface of the respiratory epithelium [40, 41]. Future stud-
ies should evaluate the therapeutic effects of EtOH vapor inhala-
tion against various respiratory infectious diseases, including
SARS-CoV-2 and avian IAV.
Supplementary Data
Supplementary materials are available at The Journal of
Infectious Diseases online. Consisting of data provided by the au-
thors to benefit the reader, the posted materials are not copyed-
ited and are the sole responsibility of the authors, so questions or
comments should be addressed to the corresponding author.
Notes
Acknowledgments. We thank Mary K. Collins, Tadashi
Yamamoto, Amy Shen, Jun-ichiro Inoue, and Kiyoshi
Kurokawa for discussion and encouragement. We also thank
Steven D. Aird for editing the manuscript. We are also grateful
to the Okinawa Institute of Science and Technology Graduate
University for its generous funding of the Quantum Wave
Microscopy Unit and the Immune Signal Unit.
Author contributions. M. T. performed most of the experi-
ments using mouse and cell line models. S. T. and
M. Y. conducted the in vitro inuenza A virus inactivation
assay. T. M. assisted with experiments of ethanol vapor inhala-
tion treatment of mice. H. I. and T. S. conceived, designed, and
supervised the study. M. T., M. Y., H. I., and T. S. wrote the
manuscript.
Financial support. This study was supported by subsidy
funding from the Cabinet Office, Government of Japan to the
Okinawa Institute of Science and Technology.
Potential conicts of interest. All authors: No reported con-
icts. All authors have submitted the ICMJE Form for
Disclosure of Potential Conicts of Interest. Conicts that the
editors consider relevant to the content of the manuscript
have been disclosed.
References
1. Molinari NA, Ortega-Sanchez IR, Messonnier ML, et al.
The annual impact of seasonal inuenza in the US: measur-
ing disease burden and costs. Vaccine 2007; 25:5086–96.
2. Uyeki TM, Bernstein HH, Bradley JS, et al. Clinical practice
guidelines by the Infectious Diseases Society of America:
2018 update on diagnosis, treatment, chemoprophylaxis,
and institutional outbreak management of seasonal inu-
enza. Clin Infect Dis 2019; 68:e1–e47.
3. Bartsch SM, Ferguson MC, McKinnell JA, et al. The poten-
tial health care costs and resource use associated with
COVID-19 in the United States. Health Aff (Millwood)
2020; 39:927–35.
4. Di Fusco M, Shea KM, Lin J, et al. Health outcomes and
economic burden of hospitalized COVID-19 patients in
the United States. J Med Econ 2021; 24:308–17.
5. Tregoning JS, Flight KE, Higham SL, Wang Z, Pierce BF.
Progress of the COVID-19 vaccine effort: viruses, vaccines
and variants versus efficacy, effectiveness and escape. Nat
Rev Immunol 2021; 21:626–36.
6. Fiolet T, Kherabi Y, MacDonald CJ, Ghosn J,
Peiffer-Smadja N. Comparing COVID-19 vaccines for
their characteristics, efficacy and effectiveness against
SARS-CoV-2 and variants of concern: a narrative review.
Clin Microbiol Infect 2022; 28:202–21.
7. Petrova VN, Russell CA. The evolution of seasonal inuen-
za viruses. Nat Rev Microbiol 2018; 16:47–60.
8. Harvey WT, Carabelli AM, Jackson B, et al. SARS-CoV-2
variants, spike mutations and immune escape. Nat Rev
Microbiol 2021; 19:409–24.
9. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T.
Bats: important reservoir hosts of emerging viruses. Clin
Microbiol Rev 2006; 19:531–45.
10. Morse SS, Mazet JA, Woolhouse M, et al. Prediction and
prevention of the next pandemic zoonosis. Lancet 2012;
380:1956–65.
11. Zhou QT, Leung SS, Tang P, Parumasivam T, Loh ZH,
Chan HK. Inhaled formulations and pulmonary drug de-
livery systems for respiratory infections. Adv Drug Deliv
Rev 2015; 85:83–99.
12. Kampf G. Efficacy of ethanol against viruses in hand disin-
fection. J Hosp Infect 2018; 98:331–8.
13. Kratzel A, Todt D, V’Kovski P, et al. Inactivation of severe
acute respiratory syndrome coronavirus 2 by
WHO-recommended hand rub formulations and alcohols.
Emerg Infect Dis 2020; 26:1592–5.
14. Nomura T, Nazmul T, Yoshimoto R, Higashiura A, Oda K,
Sakaguchi T. Ethanol susceptibility of SARS-CoV-2 and
other enveloped viruses. Biocontrol Sci 2021; 26:177–80.
15. Meister TL, Fortmann J, Todt D, et al. Comparable envi-
ronmental stability and disinfection profiles of the current-
ly circulating SARS-CoV-2 variants of concern B.1.1.7 and
B.1.351. J Infect Dis 2021; 224:420–4.
16. Shintake T. Possibility of disinfection of SARS-CoV-2
(COVID-19) in human respiratory tract by controlled eth-
anol vapor inhalation. ArXiv 12444 [Preprint: not peer
Ethanol Inhibits Airway Inuenza Infection • JID • 9
Downloaded from https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiad089/7103191 by guest on 05 May 2023

reviewed]. 15 March 2020. Available from: https://doi.org/
10.48550/arXiv.2003.12444.
17. Castro-Balado A, Mondelo-Garcia C, Barbosa-Pereira L,
et al. Development and characterization of inhaled ethanol
as a novel pharmacological strategy currently evaluated in a
phase II clinical trial for early-stage SARS-CoV-2 infection.
Pharmaceutics 2021; 13:342.
18. Rodriguez L, Nogales A, Martinez-Sobrido L. Inuenza A
virus studies in a mouse model of infection. J Vis Exp
2017; 127:55898.
19. Lei C, Yang J, Hu J, Sun X. On the calculation of TCID50 for
quantitation of virus infectivity. Virol Sin 2021; 36:141–4.
20. Henry’s law data. Available from: https://webbook.nist.gov/
cgi/cbook.cgi?ID=C64175&Mask=10#Solubility. Data com-
piled by Rolf Sander. Accessed 13 April 2023.
21. Knight DA, Holgate ST. The airway epithelium: structural
and functional properties in health and disease.
Respirology 2003; 8:432–46.
22. Wu W, Metcalf JP. The role of type I IFNs in inuenza: an-
tiviral superheroes or immunopathogenic villains? J Innate
Immun 2020; 12:437–47.
23. Honda K, Ohba Y, Yanai H, et al. Spatiotemporal regula-
tion of MyD88-IRF-7 signalling for robust type-I interfer-
on induction. Nature 2005; 434:1035–40.
24. Honda K, Yanai H, Negishi H, et al. IRF-7 is the master reg-
ulator of type-I interferon-dependent immune responses.
Nature 2005; 434:772–7.
25. Takeuchi O, Akira S. Pattern recognition receptors and in-
ammation. Cell 2010; 140:805–20.
26. Watts S, Ramstedt M, Salentinig S. Ethanol inactivation of
enveloped viruses: structural and surface chemistry in-
sights into phi6. J Phys Chem Lett 2021; 12:9557–63.
27. Patra M, Salonen E, Terama E, et al. Under the inuence of
alcohol: the effect of ethanol and methanol on lipid bilay-
ers. Biophys J 2006; 90:1121–35.
28. Azizov V, Dietel K, Steffen F, et al. Ethanol consumption
inhibits T
FH
cell responses and the development of autoim-
mune arthritis. Nat Commun 2020; 11:1998.
29. Sibley D, Jerrells TR. Alcohol consumption by C57BL/6
mice is associated with depletion of lymphoid cells from
the gut-associated lymphoid tissues and altered resistance
to oral infections with Salmonella typhimurium. J Infect
Dis 2000; 182:482–9.
30. Mason CM, Dobard E, Zhang P, Nelson S. Alcohol exacer-
bates murine pulmonary tuberculosis. Infect Immun 2004;
72:2556–63.
31. Karavitis J, Murdoch EL, Gomez CR, Ramirez L, Kovacs EJ.
Acute ethanol exposure attenuates pattern recognition re-
ceptor activated macrophage functions. J Interferon
Cytokine Res 2008; 28:413–22.
32. Nurmi K, Virkanen J, Rajamaki K, Niemi K, Kovanen PT,
Eklund KK. Ethanol inhibits activation of NLRP3 and
AIM2 inammasomes in human macrophages–a novel
anti-inammatory action of alcohol. PLoS One 2013; 8:
e78537.
33. Goral J, Choudhry MA, Kovacs EJ. Acute ethanol exposure
inhibits macrophage IL-6 production: role of p38 and
ERK1/2 MAPK. J Leukoc Biol 2004; 75:553–9.
34. Yoshizawa S, Arakawa T, Shiraki K. Dependence of ethanol
effects on protein charges. Int J Biol Macromol 2014; 68:
169–72.
35. Hoek JB, Cahill A, Pastorino JG. Alcohol and mitochon-
dria: a dysfunctional relationship. Gastroenterology 2002;
122:2049–63.
36. Steiner JL, Lang CH. Etiology of alcoholic cardiomyopathy:
mitochondria, oxidative stress and apoptosis. Int J
Biochem Cell Biol 2017; 89:125–35.
37. Borghardt JM, Kloft C, Sharma A. Inhaled therapy in respi-
ratory disease: the complex interplay of pulmonary kinetic
processes. Can Respir J 2018; 2018:2732017.
38. Tapani E, Taavitsainen M, Lindros K, Vehmas T, Lehtonen
E. Toxicity of ethanol in low concentrations. Experimental
evaluation in cell culture. Acta Radiol 1996; 37:923–6.
39. Foxman EF, Storer JA, Fitzgerald ME, et al.
Temperature-dependent innate defense against the com-
mon cold virus limits viral replication at warm temperature
in mouse airway cells. Proc Natl Acad Sci U S A 2015; 112:
827–32.
40. Dou D, Revol R, Ostbye H, Wang H, Daniels R. Inuenza A
virus cell entry, replication, virion assembly and move-
ment. Front Immunol 2018; 9:1581.
41. Pinto AL, Rai RK, Brown JC, et al. Ultrastructural insight
into SARS-CoV-2 entry and budding in human airway ep-
ithelium. Nat Commun 2022; 13:1609.
10 • JID • Tamai et al
Downloaded from https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiad089/7103191 by guest on 05 May 2023