UV-C irradiation is highly effective in inactivating SARS-CoV-2 replication

Abstract

The potential virucidal effects of UV-C irradiation on SARS-CoV-2 were experimentally evaluated for different illumination doses and virus concentrations (1000, 5, 0.05 MOI). At a virus density comparable to that observed in SARS-CoV-2 infection, an UV-C dose of just 3.7 mJ/cm2 was sufficient to achieve a more than 3-log inactivation without any sign of viral replication. Moreover, a complete inactivation at all viral concentrations was observed with 16.9 mJ/cm2. These results could explain the epidemiological trends of COVID-19 and are important for the development of novel sterilizing methods to contain SARS-CoV-2 infection.

Full text

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
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UV‑C irradiation is highly eective
in inactivating SARS‑CoV‑2
replication
Mara Biasin1, Andrea Bianco2, Giovanni Pareschi2, Adalberto Cavalleri3, Claudia Cavatorta3,
Claudio Fenizia1,6, Paola Galli2, Luigi Lessio4, Manuela Lualdi5, Enrico Tombetti1,
Alessandro Ambrosi6, Edoardo Maria Alberto Redaelli2, Irma Saulle1,7, Daria Trabattoni1,
Alessio Zanutta2 & Mario Clerici7,8*
The potential virucidal eects of UV‑C irradiation on SARS‑CoV‑2 were experimentally evaluated
for dierent illumination doses and virus concentrations (1000, 5, 0.05 MOI). At a virus density
comparable to that observed in SARS‑CoV‑2 infection, an UV‑C dose of just 3.7 mJ/cm2 was sucient
to achieve a more than 3‑log inactivation without any sign of viral replication. Moreover, a complete
inactivation at all viral concentrations was observed with 16.9 mJ/cm2. These results could explain
the epidemiological trends of COVID‑19 and are important for the development of novel sterilizing
methods to contain SARS‑CoV‑2 infection.
e COVID-19 pandemic caused by SARS-CoV-2 virus1 has had an enormous, as yet barely understood, impact
on health and economic outlook at the global level2. e identication of eective microbicide approaches is
of paramount importance in order to limit further viral spread, as the virus can be transmitted via aerosol3,4
and can survive for hours outside the body5–7. Non-contact disinfection technologies are highly desirable, and
UV radiation, in particular UV-C (200–280nm) has been suggested to be able to inactivate dierent viruses,
including SARS-CoV8–12. e interaction of UV-C radiations with viruses has been extensively studied13–15, and
direct absorption of the UV-C photon by the nucleic acid basis and/or capsid proteins leading to the genera-
tion of photoproducts that inactivate the virus was suggested to be one of the main UV-C-associated virucidal
mechanisms16,17. Some models have been proposed to correlate the nucleic acid structure with the required dose
to inactivate the virus, but a reliable model is still unavailable18. is is also due to the fact that UV-C measure-
ments were conducted using dierent viruses and diverse experimental conditions19–22. is led to an extremely
wide range of values for the same virus and, e.g., in the case of SARS-CoV-1 values reported in the literature
range from a few mJ/cm2 to hundreds mJ/cm219,22,23. Likewise, recent papers reported values for UVC inactivation
ranging from 3 to 1000mJ/cm224–27. A better understanding of the eects of UV-C on SARS-CoV-2, which take
into account all the key factors involved in the experimental setting (including culture medium, SARS-CoV-2
concentration, UV-C irradiance, time of exposure, and UV-C absorbance) will allow to replicate the results in
other laboratory with dierent devices. Moreover, as recent evidences suggest that UV light from sunlight are
ecient in inactivating the virus28, these measurements will be relevant for the setting up of further experiment
considering the role of UV-A and UV-B on SARS-CoV-2 replication.
Results and discussion
Herein, we report the eect of monochromatic UV-C (254nm) on SARS-CoV-2, showing that virus inactivation
can be easily achieved. Experiments were conducted using a custom-designed low-pressure mercury lamp system,
which has been spectral-calibrated providing an average intensity of 1.082 mW/cm2 over the illumination area
(see the details reported in the Method section). ree dierent illumination exposure times, corresponding to
3.7, 16.9 and 84.4mJ/cm2, were administered to SARS-CoV-2 either at a multiplicity of infection (MOI) of 0.05, 5,
1000. e rst concentration is equivalent to the low-level contamination observed in closed environments (e.g.
OPEN
Department of Biomedical and Clinical Sciences L. Sacco, University of Milan, Milan, Italy. Italian National
       Epidemiology and Prevention
Unit, IRCCS Foundation, Istituto Nazionale dei Tumori, Milan, Italy. Italian National Institute for Astrophysics
     Department of Imaging Diagnostic and Radioterapy,
IRCCS Foundation, Istituto Nazionale dei Tumori, Milan, Italy.       
Italy. Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy. Don C. Gnocchi
Foundation, IRCCS Foundation, Milan, Italy. *email: mario.clerici@unimi.it
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hospital rooms), the second one corresponds to the average concentration found in the sputum of COVID-19
infected patients, and the third one is a very large concentration, corresponding to that observed in terminally
diseased COVID-19 patients29. Aer UV-C exposure, viral replication was assessed by culture‐polymerase chain
reaction (C-RT‐PCR) targeting two regions (N1 and N2) of the SARS-CoV-2 nucleocapsid gene, as well as by
analyzing SARS-CoV-2-induced cytopathic eect. Analyses were performed in the culture supernatant of infected
cells at three dierent time points (24, 48 and 72h for SARS-CoV-2 at MOI 1000 and 5; 24, 48h and 6days for
SARS-CoV-2 at MOI 0.05), as well as on cell lysates at the end of cellular culture (72h: MOI 1000 and 5; 6days:
MOI 0.05). is approach allows to follow the kinetic of viral growth and to verify whether the used dose is
sucient to completely inactivate the virus over time. is is useful from a practical point of view, when UV-C
devices are used to disinfect surfaces and the environment.
e eect of the UV-C exposure on SARS-CoV-2 replication was extremely evident and independent from
the MOI employed; dose–response and time-dependent curves were observed. Figures1, 2 and 3 report for dif-
ferent MOI the number of SARS-CoV-2 copies for the three concentrations as a function of the UV-C dose and
time, quantied on a standard curve from a plasmid control. e corresponding normalised curves of the virus
copies are reported in the same gures.
Viral replication was not observed at the lowest viral concentration (0.05 MOI) in either untreated or in UV-
C-irradiated samples in the initial 48h (Fig.1). However, 6days aer infection, viral replication was distinctly
evident in the UV-C unexposed condition, but was completely absent following UV-C irradiation even at 3.7mJ/
cm2 both in cell culture supernatants (Fig.1A,B) and in cell lysate (Fig.1C). A two-way ANOVA analysing the
eect of UV-C dose and time of incubation failed to identify a signicant eect of the UV exposure on viral
replication. is is due to the fact that at very low MOI relevant increases in N1 and N2 copy numbers were
detectable only in a single condition—at six days in the absence of UV-C exposure—thus hampering the statisti-
cal power of the analysis.
At the intermediate viral concentration (5 MOI), a signicant reduction of copy number starting from the
3.7mJ/cm2 dose with a decrease of a factor of 2000 (> 3-log decrease) aer 24h was observed (Fig.2D). A
two-way ANOVA conrmed that this UV-C dose signicantly dampened viral replication (p = 0.000796, and
p = 0.000713 for N1 and N2 copies respectively). Even more important, the copy number did not increase over
time, suggesting an eective inactivation of the virus, which was further conrmed by cytopathic eect assess-
ment (Fig.3A–C).
Using a high viral input (MOI = 1000), the two-way ANOVA conrmed that all the tree UV doses ana-
lysed resulted in a signicant suppression of viral replication for both N1 (3.7mJ/cm2: p = 0.008455; 16.9 mJ/
cm2: p = 0.004216; and 84.4mJ/cm2: p = 0.000202) and N2 copies (3.7mJ/cm2: p = 6.43E−05; 16.9 mJ/cm2:
p = 1.68E−05; and 84.4mJ/cm2: p = 1.68E−05) (Fig. 4). Notably, a dierent course of infection was observed,
in which the inhibitory eect was not accompanied by viral suppression for the UV-C dose of 3.7mJ/cm2
Figure1. Viral replication of UV-irradiated SARS-CoV-2 (0.05 MOI) virus in invitro VeroE6 cells. Vero
E6 cells were infected with UV-C irradiated SARS-CoV-2 virus at a MOI of 0.05. Culture supernatants were
harvested at the indicated times (24, 48h and 6days) and virus titers were measured (A,B) by absolute copy
number quantication (Real-Time PCR). Viral replication was assessed even on cell lysate harvested at the end
of cell cultures (6days) (C). All cell culture conditions were seeded in duplicate. Panel (D) reports the plots
of the measured virus copies normalized at the untreated sample in the dierent conditions. For descriptive
purposes, mean values and whiskers representing the observed half-ranges are shown.
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(Fig.4A,B,D). Indeed, a relevant reduction in N1 e N2 copy numbers was observed in a UV-C dose-dependent
manner as early as 24h (by a factor of 103 at 3.7mJ/cm2 and 104 at 16.9mJ/cm2, Fig.4A,B,D), but longer culture
times resulted in an increase in N1 and N2 copy numbers for the UV-C dose of 3.7mJ/cm2. is indicates that
the residual viral input le by the 3.7mJ/cm2 was able to replicate and sucient to generate an eective infection.
is is not the case in cultures exposed to higher UV-C doses, as no replication could be detected in these condi-
tions. All the results were further conrmed by 2-ANOVA statistical analyses performed on viral replication at
intracellular level (3.7mJ/cm2 vs. untreated: N1, p = 0.008455; N2: p = 6.43E−05; 16.9mJ/cm2 vs. untreated: N1,
p = 0.004216; N2: p = 1.68E−05; 84.4mJ/cm2 vs. untreated: N1, p = 0.004216; N2: p = 1.68E−05) (Figs.1C, 2C, 4C).
We compared our results with data available in the literature and observed that our inactivating dose is much
smaller than that reported in Heilingloh etal.26 (1000mJ/cm2 for the complete inactivation). is discrepancy is
likely to be the consequence of the UV-C absorption by the medium used in Heilingloh etal., which has a fourfold
higher thickness compared to the one used in our experiments. is possibility is supported by the observation
that 200mJ/cm2 of UV-A, which is not absorbed by the medium, was sucient to reduce viral replication of 1-log.
As UV-A light is signicantly less ecient (order of magnitudes) than UV-C, the reported UV-C inactivating
dose (100mJ/cm2) seems to be questionable.
Two other papers measured the eect of UV-C light on SARS-CoV-2. In Ruetalo etal.25, the illumination of
254nm light was employed on a dried sample of SARS-CoV-2. Complete inactivation was obtained with 20mJ/
cm2, a value greater than ours, but in the same range. It has to be underlined that in the dried lm a shielding
Figure2. Viral replication of UV-irradiated SARS-CoV-2 (5 MOI) virus in invitro VeroE6 cells. Vero E6 cells
were infected with UV-C irradiated SARS-CoV-2 virus at a MOI of 5. Culture supernatants were harvested at
the indicated times (24, 48 and 72h) and virus titers were measured by absolute copy number quantication
(Real-Time PCR, A,B). Viral replication was assessed even on cell lysate harvested at the end of cell cultures
(72h) (C). All cell culture conditions were seeded in duplicate. Panel (D) reports the plots of the measured virus
copies normalized at the untreated sample in the dierent conditions. For descriptive purposes, mean values and
whiskers representing the observed half-ranges are shown.
Figure3. Analyses of virus induced cytopathic eect. (A) No cytopathic eect was observed in uninfected
cultured VeroE6 monolayers maintained in 50mJ/cm2 UV-treated complete medium for 72h. (B) Invitro
infection of SARS-CoV-2 (5 MOI) UV-C untreated VeroE6 cells resulted in an evident cytopathic eect. (C)
SARS-CoV-2 irradiation with 3.7mJ/cm2 UV-C rescued the cytopathic eect induced by UV-C untreated virus.
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eect by the organic component present in the liquid can occur, reducing the eciency of the UV-C light. is
was shown by Ratnesar-Shumate etal.28, who demonstrated that the dose required to obtain a similar degree
of viral inactivation was twice in dried samples from gMEM compared to the ones resuspended in simulated
saliva. Notably, the two mediums dier for their composition, mainly in terms of protein and solid percentage,
with higher values for the gMEM.
Inagaki etal. used a 285nm UV LED and showed that a dose of about 38mJ/cm2 was sucient to com-
pletely inactivate SARS-CoV-2. is dose is greater compared to the one we established; this discrepancy can be
explained by the observation that the 285nm is less ecient than the 254nm wavelength24. Finally, in an elegant
study Storm etal.27 compared the virucidal eect of UV-C in wet and dry systems. Results were based on the
use of a very small volume of viral stock in DMEM (5μl) and showed that a dose of 3.4mJ/cm2 inactivated wet
samples, whereas a dose that twice as high was needed in dried samples. ese results are comparable to the ones
herein, and the shielding eect in dried samples is almost evident. Such comparisons show how the experimental
conditions adopted signicantly impact on the denition of the dose of UV-C resulting in virus inactivation.
It is therefore crucial to accurately describe all the details of the experiments to perform a reliable comparison.
In conclusion, we report the results of a highly controlled experimental model that allowed us to identify the
UV-C radiation dose sucient to inactivate SARS-CoV-2. e response depends on both the UV-C dose and
the virus concentration. Indeed, for virus concentrations typical of low-level contaminated closed environment
and sputum of COVID-19 infected patients, a very small dose of less than 4mJ/cm2 was enough to achieve full
inactivation of the virus. Even at the highest viral input concentration (1000 MOI), viral replication was totally
inactivated with a dose ≥ 16.9mJ/cm2. ese results show how the SARS-CoV-2 is extremely sensitive to UV-C
light and they are important to allow the proper design and development of ecient UV based disinfection
methods to contain SARS-CoV-2 infection.
Methods
In vitro SARS‑CoV‑2 infection assay. 3 × 105 VeroE6 cells were cultured in DMEM (ECB7501L, Euro-
clone, Milan, Italy) with 2% FBS medium, with 100 U/ml penicillin and 100μg/ml streptomycin, in a 24-well
plate one day before viral infection assay. SARS-CoV-2 (Virus Human 2019-nCoV strain 2019-nCoV/Italy-
INMI1, Rome, Italy) at a multiplicity of infection (MOI) of 1000, 5 and 0.05 were treated with dierent doses of
UV-C radiation (see the dedicated section) before inoculum into VeroE6 cells. UV-C-untreated virus served as
positive controls. Cell cultures were incubated with the virus inoculum in duplicate for three hours at 37°C and
5% CO2. en, cells were rinsed three times with warm PBS, replenished with the appropriate growth medium
and observed daily for cytopathic eect. Viral replication in culture supernatants was assessed by an Integrated
Culture‐polymerase chain reaction (C-RT‐PCR) method30 at 24, 48, and 72h post-infection (hpi) while infected
cells were harvested for RNA collection at 72 hpi. Cell cultures from SARS-CoV-2 at 0.05 MOI were harvested
6days post infection. RNA was extracted from VeroE6 cell culture supernatant and cell lysate by the Maxwell
RSC Instrument with Maxwell RSC Viral Total Nucleic Acid Purication Kit (Promega, Fitchburg, WI, USA),
Figure4. Viral replication of UV-irradiated SARS-CoV-2 (1000 MOI) virus in invitro VeroE6 cells. Vero
E6 cells were infected with UV-C irradiated SARS-CoV-2 virus at a MOI of 1000. Culture supernatants were
harvested at the indicated times (24, 48 and 72h) and virus titers were measured (A,B) by absolute copy number
quantication (Real-Time PCR). Viral replication was assessed even on cell lysate harvested at the end of cell
cultures (72h) (C). All cell culture conditions were seeded in duplicate. Panel (D) reports the plots of the
measured virus copies normalized at the untreated sample in the dierent conditions. For descriptive purposes,
mean values and whiskers representing the observed half-ranges are shown.
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quantied by the Nanodrop 2000 Instrument (ermo Scientic) and puried from genomic DNA with RNase-
free DNase (RQ1 DNase; Promega). One microgram of RNA was reverse transcribed into rst-strand cDNA in
a 20-μl nal volume as previously described31,32.
Real-time PCR was performed on a CFX96 (Bio-Rad, CA, USA) using the 2019-nCoV CDC qPCR Probe
Assay emergency kit (IDT, Iowa, USA), which targets two regions (N1 and N2) of the nucleocapsid gene of
SARS-CoV-2. Reactions were performed according to the following thermal prole: initial denaturation (95°C,
10min) followed by 45 cycles of 15s at 95°C (denaturation) and 1min at 60°C (annealing-extension).
Viral copy quantication was assessed by creating a standard curve from the quantied 2019-nCoV_N posi-
tive Plasmid Control (IDT, Iowa, USA).
UV illumination test. e illumination of the virus solution was conducted using a low-pressure mercury
lamp mounted in a custom designed holder, which consist in a box with a circular aperture 50mm in diameter
placed at approximately 220mm from the source. e aperture works as a spatial lter to make the illumination
of the area behind more uniform. A mechanical shutter is also present to start the illumination process. e plate
is placed 30mm below the circular aperture and a single dwell (34.7mm in diameter), centered in respect to the
50mm aperture, has been irradiated from the top. e dwell was lled with 0.976ml of the virus suspended in
Dulbecco’s Modied Eagle’s Medium (DMEM) in order to have a 1mm thick liquid layer. Aer the irradiation,
the sample was treated as described in the previous section.
The intensity of the lamp and its spectral properties have been measured using an Ocean Optics
HR2000 + spectrometer (Ocean Optics Inc., Dunedin, USA). e HR2000 + spectrometer was calibrated against
a reference deuterium–halogen source (Ocean Optics Inc. Winter Park, Winter Park, Florida) and in compliance
with National Institute of Standards and Technology (NIST) practices recommended in NIST Handbook 150-2E,
Technical guide for Optical Radiation Measurements. e last calibration was performed in March 2019. e
detector of our spectrometer is a high-sensitivity 2048-element Charge-Coupled Device (CCD) array from Sony.
e spectral range is 200–1100nm with a 25μm wide entrance slit and an optical resolution of 1.4nm (FWHM).
e cosine-corrected irradiance probe, model CC-3-UV-T, is attached to the tip of a 1m long optical bre and
couples to the spectrometer. e intensity of the lamp has been measured by positioning the spectrometer in ve
positions: in the center and at the ends of a 20mm cross arm aer a warming up time of 30s. e spectra in the
ve positions are reported in Fig.5A together with a scheme of the dwell and illuminated area.
As expected, the emission is dominated by the UV-C line (Fig.5A) and its intensity was uniform in the area
with an average value of 1.082 mW/cm2. e stability of the lamp was evaluated in ± 11E−3mW/cm2 during a
130s measurement. According to this value, three exposure times were set: 5, 23 and 114s (with an accuracy of
0.2s), which correspond to following doses: 5.4, 25.0, 123.4mJ/cm2. is is the nominal UV doses provided to
the dwell, but we were interested in the eective doses (De) reaching the virus. It was necessary to calculate the
eective irradiance (Ie). is step was performed considering both the reection losses at the air/water interface
(Rw) and the Transmittance (Ts) of the DMEM solution at 254nm (from the spectrum in Fig.5B, considering the
cuvette losses, Ts = 0 0.70). It is important to notice that the spectrum was measured in a quartz cuvette (1mm
thick) by means of a Jasco V770 spectrophotometer and this thickness was the same of the solution in the dwell
during the UV irradiation step.
e reection loss was computed as follow:
where nw = 1.375 is the refractive index of water at 254nm. en, Ie was calculated:
(1)
R
w=(nw−1)
2
(
n
w
+1
)
2
,
(2)
I
e=
I
n
(1
−
R
w
)
×
T
s
.
Figure5. Mercury lamp spectrum measured in the ve positions (A). Inset: scheme of the illuminated dwell
and the measuring position. UV–vis transmission spectrum of the Dulbecco’s Modied Eagle’s Medium
(DMEM) in a 1mm quartz cuvette (B).
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e nal transmission of the DMEM solution was equal to 0.68 and the corresponding eective doses were
derived simply multiplying Ie by the exposure time.
According to this value, the eective doses provided to the viruses were: 3.7 ± 0.15, 16.9 ± 0.2 and 84.4 ± 0.9mJ/
cm2. We have to notice that we are neglecting here the absorption of the virus at this wavelength and the pos-
sible scattering. Such approximations are valid considering the relative low concentration of the virus and small
thickness of the layer (the solution appeared fully transparent).
Statistical analyses. To assess the eect of the dierent UV-C doses on N1 and N2 copy numbers, two-
way ANOVAs were performed. For the analysis of intracellular N1 and N2 doses in the supernatant, UV-C dose
and MOI represented the dependent variables, while for the analysis of N1 and N2 in the supernatant, dierent
analyses were performed for individual MOI, using UV-C dose and time as dependent variables.
Received: 3 July 2020; Accepted: 22 February 2021
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Acknowledgements
is research was partially supported by a grant from Falk Renewables and it has been carried out in the con-
text of the activities promoted by the Italian Government and in particular, by the Ministries of Health and of
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University and Research, against the COVID-19 pandemic. Authors are grateful to INAF’s President, Prof. N.
D’Amico, for the support and for a critical reading of the manuscript.
Author contributions
P.G., L.L. E.R. and A.Z. designed and produced the illumination system; A.C., C.C. and M.L. performed the lamp
calibration; A.B. performed the lamp setup and lamp dosimetry, wrote the main manuscript; M.B. performed
biological experiments, analyzed the data, wrote the main manuscript; C.F., I.S. designed and performed some
biological tests; E.T, A.A. performed the statistical analysis; D.T. discussed the results; G.P. and M.C. supervised
the study and review the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
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