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Indomethacin Has a Potent Antiviral Activity against Sars Coronavirus

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Severe acute respiratory syndrome (SARS) is a newly emerging, highly transmissible and fatal disease caused by a previously unknown coronavirus (SARS-CoV). Existing in non-identified animal reservoirs, SARS-CoV continues to represent a threat to humans because there is no effective specific antiviral therapy for coronavirus infections. Starting from the observation that cyclopentenone cyclooxygenase (COX) metabolites are active against several RNA viruses, we investigated the effect of the COX inhibitor indomethacin on coronavirus replication. Work involving infectious SARS-CoV was performed in biosafety level 3 facilities. SARS-CoV was grown in monkey VERO cells and human lung epithelial A549 cells, while canine coronavirus (CCoV) was grown in A72 canine cells. Antiviral activity was analysed by determining infective virus titres by TCID50, viral RNA synthesis by Northern blot analysis and real-time RT-PCR, and viral protein synthesis by SDS-PAGE analysis after 35S-methionine-labelling. Antiviral efficacy in vivo was determined by evaluating virus titres in CCoV-infected dogs treated orally with 1 mg/kg body weight indomethacin (INDO). Unexpectedly, we found that INDO has a potent direct antiviral activity against the coronaviruses SARS-CoV and CCoV. INDO does not affect coronavirus binding or entry into host cells, but acts by blocking viral RNA synthesis at cytoprotective doses. This effect is independent of cyclooxygenase inhibition. INDO's potent antiviral activity (>1,000-fold reduction in virus yield) was confirmed in vivo in CCoV-infected dogs. The results identify INDO as a potent inhibitor of coronavirus replication and suggest that, having both anti-inflammatory and antiviral activity, INDO could be beneficial in SARS therapy.
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Severe acute respiratory syndrome (SARS) is a newly
emerging, highly transmissible and fatal disease caused
by a previously unknown coronavirus (SARS-CoV).
Existing in non-identified animal reservoirs, SARS-CoV
continues to represent a threat to humans because there
is no effective specific antiviral therapy for coronavirus
infections.
Objectives: Starting from the observation that cyclopen-
tenone cyclooxygenase (COX) metabolites are active
against several RNA viruses, we investigated the effect of
the COX inhibitor indomethacin on coronavirus replication.
Methods: Work involving infectious SARS-CoV was
performed in biosafety level 3 facilities. SARS-CoV was
grown in monkey VERO cells and human lung epithelial
A549 cells, while canine coronavirus (CCoV) was grown in
A72 canine cells. Antiviral activity was analysed by deter-
mining infective virus titres by TCID
50
, viral RNA synthesis
by Northern blot analysis and real-time RT-PCR, and viral
protein synthesis by SDS-PAGE analysis after
35
S-methio-
nine-labelling. Antiviral efficacy in vivo was determined
by evaluating virus titres in CCoV-infected dogs treated
orally with 1 mg/kg body weight indomethacin (INDO).
Results: Unexpectedly, we found that INDO has a potent
direct antiviral activity against the coronaviruses SARS-
CoV and CCoV. INDO does not affect coronavirus binding
or entry into host cells, but acts by blocking viral RNA
synthesis at cytoprotective doses. This effect is indepen-
dent of cyclooxygenase inhibition. INDO’s potent
antiviral activity (>1,000-fold reduction in virus yield)
was confirmed in vivo in CCoV-infected dogs.
Conclusions: The results identify INDO as a potent
inhibitor of coronavirus replication and suggest that,
having both anti-inflammatory and antiviral activity,
INDO could be beneficial in SARS therapy.
Indomethacin has a potent antiviral activity against
SARS coronavirus
Carla Amici
1
, Antonino Di Caro
2
, Alessandra Ciucci
1
, Lucia Chiappa
1
, Concetta Castilletti
2
, Vito Martella
3
,
Nicola Decaro
3
, Canio Buonavoglia
3
, Maria R Capobianchi
2
and M Gabriella Santoro
1
*
1
Department of Biology, University of Rome Tor Vergata, Rome, Italy
2
Laboratory of Virology, National Institute for Infectious Diseases ‘L. Spallanzani’, Rome, Italy
3
Faculty of Veterinary Medicine, University of Bari, Bari, Italy
*Corresponding author: Tel: +39 06 7259 4822; Fax: +39 06 7259 4821; E-mail: santoro@bio.uniroma2.it
Antiviral Therapy 11:1021–1030
Severe acute respiratory syndrome (SARS) is a new
disease with a high fatality rate that emerged in
Southern China in late 2002. Within a remarkably
short period of time following the worldwide epidemic
that caused over 780 fatalities in 2002–2003, the SARS
causative agent was identified as a novel highly infec-
tious coronavirus named SARS-CoV [1,2].
Coronaviruses are enveloped, positive-strand RNA
viruses highly diffused among humans and mammals
and commonly associated with respiratory or enteric
infections [3]. SARS-CoV infection causes an unusually
severe febrile illness, with myalgia, headaches and
respiratory symptoms, followed by progression to
acute respiratory distress and respiratory failure [4].
Although the SARS epidemic has been controlled by
conventional measures, the animal reservoir of SARS-
CoV has not been identified and sporadic cases continue
to arise in Southern China, possibly because of human
contact with the animal host [4,5]. The possibility that
the virus could be reintroduced into the human popula-
tion is therefore likely, and effective antiviral drugs
against coronaviruses are urgently needed.
We have shown that cyclopentenone prostaglandins
have a potent antiviral activity against several RNA
viruses by interfering with stress-sensitive cellular signal
transduction pathways and transcription factors,
including the heat shock factor type-1 and nuclear
factor-κB [6–9]. During experiments aimed at identi-
fying cyclooxygenase (COX) metabolites active against
coronaviruses in a model of canine coronavirus (CCoV)
infection, we utilized the COX inhibitors indomethacin
(INDO) and aspirin as negative controls.
In the present report we describe how, unexpectedly,
INDO was found to possess a potent antiviral activity
against CCoV, being able to dramatically inhibit virus
replication and protect the host cell from virus-induced
damage. A remarkable antiviral activity was also found
in vivo in the natural host. We then found that the
Introduction
© 2006 International Medical Press 1359-6535
1021
8_0044_santoro.qxp 24/11/06 09:45 Page 1021
antiviral activity is not limited to CCoV and that
INDO is also very effective against human SARS-CoV.
Materials and methods
Cell culture and treatment
Human A549 alveolar type II-like epithelial cells and
African green monkey kidney VERO E6 cells were
obtained from LGCPromochem-ATCC (Milan, Italy).
Canine adenocarcinoma (A72) cells were obtained
from Dr M Ferrari (Centro Substrati Cellulari, Istituto
Zooprofilattico Sperimentale della Lombardia e
Emilia-Romagna, Brescia, Italy). VERO, A72 and
A549 cells were grown at 37°C in a 5% CO
2
humidi-
fied atmosphere in Modified Eagle Medium (MEM)
(VERO cells), DMEM medium (A72 cells) or F-12K
medium (A549) (GIBCO) supplemented with 10%
fetal calf serum (FCS), 2 mM glutamine and antibiotics
(complete medium). Indomethacin (Sigma Aldrich,
Milan, Italy) was dissolved in absolute ethanol at a
concentration of 40 mM and diluted in culture medium
immediately before use. Unless otherwise specified,
INDO was added immediately after the 1 h adsorption
period and maintained in the medium for the duration
of the experiment. Controls received equal amounts of
ethanol diluent, which did not affect cell viability or
virus replication (data not shown). Aspirin and
ribavirin (RBV) (Sigma), as well as interferon α (IFN-
α) (Hu-rIFNα, Intron A; Schering-Plough, Milan, Italy)
were added immediately after the 1 h adsorption period
and maintained in the medium for the duration of the
experiment. Controls received equal amounts of appro-
priate diluent. Cell viability was determined by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) to MTT formazan conversion assay
(Sigma), as described [10].
Coronavirus infection and titration
Canine CoV (CCoV, strain S-378) and SARS-CoV
(TOR-2 isolate, kindly provided by Heinz Feldmann,
National Microbiology Laboratory,Winnipeg, Canada)
were prepared by infecting A72 and VERO cell
cultures, respectively. The maintenance medium
consisted of MEM without FCS. Virus yields were
determined by plaque forming units (PFU) and 50%
tissue culture infective dose (TCID
50
) titrations in
confluent cells as previously described [11,12], and
virus stocks were stored at –80°C. For in vitro infec-
tion, confluent cell monolayers were infected with
CCoV or SARS-CoV for 1 h at 37°C at a multiplicity
of infecion (MOI) of 10 PFU/cell, unless otherwise
stated. After the adsorption period, the viral inoculum
was removed and cell monolayers were washed three
times with PBS and incubated at 37°C with medium
without FCS for CCoV or with 2% FCS for SARS-CoV.
Virus yields were determined by TCID
50
assay. All work
involving infectious SARS-CoV was performed in a
biosafety level 3 facility in accordance with World
Health Organization recommendations.
Animal maintenance and infection
A group of four mixed-breed, medium-sized, 8-month-
old dogs that tested negative for the presence of CCoV
RNA in faeces and for CCoV antibodies in serum
samples, were selected and randomly assigned to two
groups of two dogs each. Dogs were housed individu-
ally in separate boxes, fed twice daily with a commer-
cial dry dog food and provided water ad libitum. After
an acclimatization period of 5 days, each dog was
administered oronasally with 4 ml of a viral suspension
of a CCoV field strain, with a titre of 10
5.50
TCID
50
and
6.00×10
6
RNA copies/ml. To evaluate CCoV shedding,
faecal samples were collected daily for 12 days from
treated and non-treated CCoV-infected dogs and
subjected to real-time RT-PCR analysis, as described
below. For all experiments, humane standards were
adhered to.
DNA and RNA synthesis
Cells were labelled for 24 h with
3
H-thymidine or
3
H-
uridine (10 µCi/10
5
cells) and the radioactivity incor-
porated into acid-soluble and -insoluble material was
determined as previously described [13]. Briefly, cells
were washed three times with PBS and 0.4 ml 5%
trichloroacetic acid (TCA) was added to each culture.
The radioactivity in acid-soluble material was deter-
mined after 1 h at 4°C. Acid-insoluble radioactivity
was measured after washing the TCA precipitates three
times with ethanol, drying under an infrared lamp and
dissolving the samples in 0.4 ml of a solution
containing 0.1 M NaOH and 0.5% SDS.
Protein synthesis and PAGE analysis
CCoV- and mock-infected A72 cell monolayers were
labelled with
35
S-methionine (10 µCi/10
5
cells, 2 h pulse)
at 22 h post-infection (p.i.). At 24 h p.i., cells were lysed
in L buffer (20 mM Tris-Cl pH 7.4, 0.1 M NaCl, 5 mM
MgCl
2
, 1% NP40, 0.5% SDS), and the radioactivity
incorporated was determined as described [11]. Samples
containing the same amount of radioactivity were sepa-
rated by SDS/PAGE (3% stacking gel, 10% resolving
gel) and processed for autoradiography [11].
RNA extraction, Northern blot and real-time RT-PCR
analysis
Total RNA from uninfected and virus-infected cells was
isolated by the guanidinium isothiocianate method [13]
and stored at –20°C. For detection of CCoV mRNA by
Northern blot analysis, total RNA was fractionated
(5 µg) on 1% agarose/formaldehyde gels and transferred
C Amici et al.
© 2006 International Medical Press
1022
8_0044_santoro.qxp 24/11/06 09:45 Page 1022
onto Hybond-N nylon membranes (Amersham
Biosciences, Piscataway, NJ, USA). For detection of
CCoV mRNA, filters were hybridized with
32
P-labelled
pCR21-M-CCoV probe [14]. After being stripped,
filters were rehybridized with a plasmid specific for the
β-actin gene 5 end-labelled by T4 kinase with γ-AT
32
P
(Amersham), as a loading control [11]. Measurement
of SARS-CoV genomic RNA was performed by quan-
titative real time RT-PCR using the commercial kit
RealArt
TM
HPA-Coronavirus LC RT-PCR (Artus,
Hamburg, Germany) on a LightCycler Instrument
(Roche Diagnostic, Basel, Switzerland) [15]. For in
vivo experiments, total RNA was extracted from each
faecal sample with QIAamp
®
RNeasy Mini Kit (Qiagen
GmbH, Hilden, Germany) in accordance with the
manufacturer’s protocol. The starting material
consisted of 10 mg of faeces for each sample. Template
RNAs were eluted in 50 µl of RNase-free water and
stored at –70°C. Real-time RT-PCR analysis was
performed as previously described [14].
Statistical analysis
Statistical analysis was performed using Student’s t-test
for unpaired data. Data were expressed as the mean ±
SD and P-values of <0.05 were considered significant.
Results
INDO is a potent inhibitor of CCoV replication in vitro
Canine A72 cell monolayers were infected with CCoV
(10 TCID
50
/cell) and treated with different concentra-
tions (10, 25, 50, 100, 200 and 400 µM) of INDO after
the 1 h adsorption period. The effect of INDO on cell
viability was determined by MTT assay in confluent
cells. CCoV infection was highly cytopathic, causing
cell shrinkage and loss of adhesion at 24 h p.i. (Figure
1A). Surprisingly, INDO was found to possess a
remarkable antiviral activity, reducing viral particle
production dose-dependently with an IC
50
of 5 µM, an
LD
50
of 550 µM and a selectivity index of 110 (Figure
1B). At 400 µM, INDO caused a dramatic (more than
3 log) reduction in viral yield that lasted for at least 48 h
p.i. (Figure 1C). At this concentration, INDO also had
a remarkable cytoprotective effect in CCoV-infected
cells, which did not show any sign of infection up to 36
h p.i. (Figure 1A). Treatment with RBV up to a concen-
tration of 1 mM did not affect CCoV replication under
the same conditions (data not shown). In addition, no
effect on coronavirus replication was found in cells
treated with the non-steroidal anti-inflammatory drug
(NSAID) aspirin up to a concentration of 2 mM (Figure
2A), suggesting that INDO antiviral activity is indepen-
dent of the block of cyclooxygenase function.
To investigate whether treatment of cells with INDO
before virus adsorption was able to protect the cells
Antiviral Therapy 11:8
1023
Indomethacin against coronavirus infection
Figure 1. INDO is a potent inhibitor of CCoV replication
(A) Cytoprotective effect of INDO (400 µM) in CCoV-infected A72 cells 36 h
p.i., bar, 50 µm. (B) CCoV yield (G) in the supernatant of infected cells
treated with different concentrations (10, 25, 50, 100, 200 and 400 µM) of
INDO, as determined by infectivity assay at 24 h p.i.. Data expressed in
TCID
50
/ml represent the mean ± SD of duplicate samples from a representa-
tive experiment of three with similar results. *P=0.021; **P<0.001. Cell
viability ( ) determined by MTT assay in uninfected INDO-treated cells is
expressed as percentage of MTT conversion in untreated control. (C) CCoV
yield in the supernatant of infected cells treated with 400 µM INDO (G) or
control diluent ( ) was determined by infectivity assay at different times
p.i.. Data expressed in TCID
50
/ml represent the mean ± SD of three indepen-
dent experiments. *P<0.001. CCoV, canine coronavirus; INDO, indomethacin;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; p.i.,
post-infection; TCID, tissue culture infective dose.
Uninfected
CCoV-infected
– INDO + INDO
10
7
10
5
**
**
**
**
10
6
10
4
10
3
0
25
50
75
100
10
2
10
1
0
INDO, µM
CCoV yield, TCID
50
Cell viability, %
10
2
10
3
10
7
10
5
*
*
*
*
10
6
10
4
10
3
10
2
3216 480
Time p.i., h
CCoV yield, TCID
50
*
*
A
C
B
8_0044_santoro.qxp 24/11/06 09:45 Page 1023
C Amici et al.
© 2006 International Medical Press
1024
Figure 2. Inhibition of CCoV RNA and protein expression by indomethacin
(A) A72 cells were infected with CCoV and treated with different concentrations of the non-steroidal anti-inflammatory drugs (NSAIDs) indomethacin (G) (10, 100
and 400 µM) or aspirin ( ) (10, 100, 400, 800 and 2,000 µM) after the 1 h adsorption period. CCoV yield was determined by infectivity assay in the supernatant of
infected cells at 24 h p.i.. Data expressed in TCID
50
/ml represent the mean ± SD of duplicate samples from a representative experiment of two with similar results.
(B) A72 cells were treated with 400 µM INDO (filled bars) at the indicated times before infection (Pre), immediately after the adsorption period (Post) or only during
the adsorption period (Ads, grey bar). The empty bar represents the untreated infected control (Control). CCoV yield was determined at 24 h p.i.. Data represent the
mean ± SD of triplicate samples. *P<0.001. (C) Uninfected (U, empty bars) or CCoV-infected (CCoV, filled bars) A72 cells treated with the indicated amounts of INDO
were labelled with
35
S-methionine (2 h pulse) at 22 h p.i..
35
S-Methionine incorporation into proteins is expressed as means ± SD cpm/10
6
cells (lower panel).
*P<0.001 as compared with uninfected control. Samples containing an equal amount of radioactivity were processed for SDS-PAGE analysis and autoradiography
(upper panel). The major viral proteins E2/S, N and E1/M are indicated. (D) RNA extracted from uninfected (U) or CCoV-infected (CCoV) A72 cells, treated with the
indicated amounts of INDO for 18 h, was analysed by Northern blot using a
32
P-labelled pCR21-M-CCoV probe (upper panel). Levels of β-actin mRNA in the same
samples are shown as control (lower panel).
10
7
10
5
10
6
10
4
10
3
10
2
10
1
0
NSAID, µM
CCoV yield, TCID
50
10
2
10
3
10
4
10
7
10
5
10
6
10
4
10
3
10
2
Pre, h
CCoV yield
Post
Ads
Control
18 14 9 6 3
*
60
40
20
0
cpm x 10
-3
E1/M
N
E2/S
0
100 400 0
*
*
100 400 µM INDO
0100
U
CCoV
400 0 100 400 µM INDO
5.6 kb
β
-Actin
6.6 kb
9.4 kb
U
CCoV
100
0
0
400 µM INDO
A
C
D
B
8_0044_santoro.qxp 24/11/06 09:45 Page 1024
from viral infection, A72 cells were treated with
400 µM INDO for 18, 14, 9, 6 and 3 h. At the end of
the treatment period, the drug was removed by
washing the cell monolayers three times with culture
medium, and cells were then infected with CCoV
(10 TCID
50
/cell). One set of cultures was treated with
400 µM INDO immediately after the virus adsorption
period, as a positive control. As shown in Figure 2B,
treatment of A72 cells with 400 µM INDO prior to
viral infection had no effect on CCoV replication, indi-
cating that, differently from interferon, INDO is not
priming the host cell to raise a defence response against
the invading virus. Moreover, treatment of the viral
suspension with high concentrations (1 mM for 1 h) of
INDO (data not shown) or the addition of 400 µM
INDO only during the 1 h adsorption period had no
effect on virus replication (Figure 2B), indicating that
the drug is not affecting virus infectivity, binding or
entry into target cells.
In the same experiment, INDO caused a dramatic
(approximately four logs) reduction of virus yield when
added soon after the adsorption period and kept for the
next 24 h (Figure 2B). To investigate how early after
infection INDO treatment needs to be started to be
effective, CCoV-infected cells were treated with 400
µM INDO immediately after the adsorption period
(time 0) or at 3, 6 and 12 h p.i., and virus yield were
determined after 24 h. Treatment with INDO started at
0 or 3 h p.i. was extremely effective in inhibiting virus
replication (control = 3.14 ±0.58×10
6
TCID
50
/cell;
INDO at time 0 = <10
2
TCID
50
/cell; INDO at 3 h p.i. =
2.04 ±0.01×10
2
TCID
50
/cell), further indicating that
INDO is not affecting virus entry, and suggesting that
the drug has no effect on virus uncoating. Treatment
started at 6 h p.i. was less effective, but still able to
inhibit virus replication (INDO at 6 h p.i. = 3.25
±0.30×10
3
TCID
50
/cell), whereas the drug lost most of
its effect when administered at 12 h p.i. (INDO at 12 h
p.i. = 2.15 ±0.80×10
5
TCID
50
/cell). In a different exper-
iment, to investigate whether the antiviral effect was
retained upon removal of the drug, CCoV-infected cells
were treated with 400 µM INDO soon after the adsorp-
tion period. At 3, 6 or 9 h p.i., the drug was removed by
washing as described above, and culture medium devoid
of the drug was added. Virus titres were determined at
24 h p.i.. Removal of the drug up to 9 h p.i. resulted in
a complete reversal of the antiviral effect; treatments
longer than 9 h were necessary in order to block virus
replication (data not shown).
In order to determine whether INDO was affecting
CCoV protein synthesis, confluent monolayers of A72
cells were mock-infected or infected with CCoV
(10 TCID
50
/cell) and treated with 100 or 400 µM
INDO after the adsorption period. Uninfected and
CCoV-infected cells were labelled with
35
S-methionine
(2 h pulse) at 22 h p.i., and labelled proteins were
analysed by SDS-PAGE and autoradiography. At this
time, INDO did not inhibit protein synthesis in unin-
fected cells up to a concentration of 400 µM (Figure
2C, lower panel) and did not cause any detectable
change in the pattern of cell protein synthesis (Figure
2C, upper panel). As expected, CCoV infection caused
a dramatic inhibition of host cell protein synthesis
(Figure 2C). In infected cells, INDO caused a dose-
dependent inhibition of viral protein synthesis (Figure
2C, upper panel). CCoV proteins were barely
detectable at concentration of 400 µM. At this concen-
tration, INDO was also able to protect the host cell
from the virus-induced shut-off of host protein
synthesis (Figure 2C, lower panel).
CCoV RNA levels were then analysed in parallel
samples by Northern blot analysis. Data shown in Figure
2D demonstrate that INDO profoundly affects viral
RNA synthesis. No viral RNA is detected at the cyto-
protective concentration of 400 µM, indicating that the
drug-induced inhibition of virus protein synthesis is due
to a dramatic decrease in the viral RNA levels.
Antiviral activity of INDO in vivo
In order to evaluate whether INDO could also be effec-
tive against coronaviruses in vivo, a group of six
mixed-breed, medium-sized, 8-month-old dogs were
tested for the presence of CCoV RNA in faeces by a
real-time RT-PCR assay [14] and for CCoV antibodies
in serum samples by an ELISA test [16]. Of these, four
dogs that had tested negative for CCoV RNA and anti-
bodies were selected and randomly divided into two
groups (A and B). All animals were infected with CCoV
(10
5.50
TCID
50
/dog) and dogs in group A were treated
orally with INDO (1 mg/kg body weight) daily for 4
days, starting on day 4 p.i., whereas dogs in group B
served as infected non-treated controls. The antiviral
efficacy of INDO was determined by evaluating the
CCoV shedding in the faeces of the infected dogs for a
period of 12 days. In the first 2–3 days p.i., all infected
dogs experienced mild diarrhoea, according to previous
observations [14,17]. As shown in Figure 3, virus shed-
ding in faeces was already detectable 1 day p.i., and the
pattern was similar in all animals before starting treat-
ment, with viral loads ranging from 10
4
to 10
5
RNA
copies/µl, according to previous results [14].
Subsequently, the titre of shed virus in control non-
treated dogs increased, reaching a peak at day 7 p.i.
(mean titre = 2.11×10
5
RNA copies/µl). In contrast, in
INDO-treated dogs, viral RNA titres in the faeces
decreased rapidly after starting treatment, reaching
minimal levels at day 7 p.i. (mean titre = 1.16×10
2
RNA
copies/µl), in concomitance with the peak observed in
non-treated dogs (Figure 3, closed circles). INDO
antiviral effect was reversed upon suspension of treat-
Antiviral Therapy 11:8
1025
Indomethacin against coronavirus infection
8_0044_santoro.qxp 24/11/06 09:45 Page 1025
ment. In fact, at the end of the 4-day treatment, CCoV
RNA titres increased progressively, reaching the same
values as control dogs on day 10 p.i. (data not shown).
During and after the INDO treatment, no systemic or
local adverse reactions were observed in treated dogs.
These results indicated that INDO treatment is effective
against coronavirus infection in vivo as well as in vitro.
INDO has a potent antiviral activity against SARS-CoV
On the basis of these observations, we investigated
whether INDO could also inhibit SARS-CoV replica-
tion. VERO cell monolayers were infected with
2 TCID
50
/cell SARS-CoV (TOR-2 isolate) for 30 min at
37°C, and then incubated in complete medium,
containing different concentrations (100, 200 and
400 µM) of INDO or control diluent. In parallel, the
effect of INDO (50, 100, 200 and 400 µM) on VERO
cell viability was determined by MTT assay on mock-
infected VERO cell monolayers. As shown by infec-
tious virus titration, INDO inhibited SARS-CoV
replication dose-dependently with an IC
50
of 50 µM
(Figure 4A, closed circles). A greater than 99%
decrease in virus yield was detected at concentrations
that were non-toxic for uninfected cells as shown by
MTT assay (Figure 4A, open circles). INDO treatment
also partially protected cells by the cytopathic effect
caused by SARS-CoV infection for a period of 24 h
(Figure 4B). To determine whether treatment with
INDO could inhibit cellular DNA or protein synthesis,
in a parallel experiment VERO cells were treated with
different concentrations (50, 100, 200 and 400 µM) of
INDO and then labelled with
3
H-thymidine (10
µCi/10
5
cells) or
35
S-methionine (10 µCi/10
5
cells) for
the next 24 h. At this time, the radioactivity incorpo-
rated into acid-insoluble material was determined. As
shown in Figure 4C, INDO did not significantly alter
DNA or protein synthesis in uninfected cells up to a
concentration of 400 µM.
Since we have recently set up a model of SARS-CoV
infection in human lung epithelial cells (A549 cell line),
we investigated whether INDO was also able to inhibit
SARS-CoV replication in human cells. The results indi-
cated that INDO is able to inhibit SARS-CoV replica-
tion at the same concentrations as in VERO cells
(control cells = 4.39 ±1.2×10
3
; 100 µM INDO treated
cells = <10
2
TCID
50
/ml), demonstrating that the
antiviral activity of INDO is not dependent on the type
of cells.
Next, we investigated the effect of IFN and RBV,
which have been used in the clinic to treat SARS-CoV
infection [18], in the VERO cell model. VERO cell
monolayers were infected with 2 TCID
50
/cell SARS-
CoV for 30 min at 37°C, and then incubated in
complete medium containing different concentrations of
IFN-α (500 and 5,000 IU/ml), RBV (40 and 400 µM) or
INDO (100 and 400 µM). The results shown in Figure
5A indicate that treatment with IFN-α or RBV after the
adsorption period had no major effect on SARS-CoV
replication up to a concentration of 5,000 IU/ml for
IFN-α and 400 µM for RBV, respectively. The effect of
the NSAID aspirin was then investigated in a parallel
experiment. As shown above for the canine coronavirus
(Figure 2A), treatment with aspirin had no effect on
SARS-CoV replication up to a concentration of 2 mM
(Figure 5B).
Treatment of SARS-CoV suspension for 1 h with
1 mM INDO (data not shown), or the addition of
300 µM INDO prior to viral infection and/or during
the 1 h virus adsorption period had no effect on virus
yield, indicating that, as shown above for CCoV, INDO
is not affecting SARS-CoV binding or entry into target
cells (Figure 5C).
As for other coronaviruses, the expression of the
SARS-CoV genome is mediated by translation of the
genomic RNA and a ‘nested’ set of subgenomic
messenger RNAs, produced by a unique mechanism
involving discontinuous transcription during negative-
C Amici et al.
© 2006 International Medical Press
1026
Figure 3. Antiviral activity of INDO in vivo
Shedding of CCoV RNA in the faeces of dogs infected with CCoV and treated
(G, group A) or non-treated ( , group B) with INDO (1 mg/kg body weight,
oral administration) for 4 days. Arrow indicates the first day of treatment. Viral
titres are expressed as RNA copy numbers per µl of template. Each sample was
tested in duplicate. INDO, indomethacin.
10
6
10
1
0
RT-PCR (copy number)
10
5
10
4
10
3
10
2
864
Time p.i., days
2
8_0044_santoro.qxp 24/11/06 09:45 Page 1026
strand RNA synthesis [3]. To determine whether, as in
the case of CCoV, INDO was acting by blocking viral
RNA synthesis, VERO cells were infected with SARS-
CoV and treated with different concentrations (100,
200 and 400 µM) of INDO soon after the virus adsorp-
tion period. Total RNA was extracted 24 h p.i. and
analysed by RT-PCR. In order to investigate whether
INDO was affecting cellular RNA synthesis, mock-
infected cells were treated identically in parallel and
labelled with
3
H-uridine (10 µCi/10
5
cell) for the next
24 h. As shown above for CCoV, INDO treatment
caused a decrease of intracellular SARS-CoV RNA levels
dose-dependently, reaching an inhibition of more than
95% of control at concentrations of INDO that did not
affect RNA synthesis in uninfected cells (Figure 5D).
Discussion
Coronaviruses cause a wide spectrum of diseases in
humans and animals, primarily infecting the respira-
Antiviral Therapy 11:8
1027
Indomethacin against coronavirus infection
Figure 4. Antiviral activity of INDO during SARS-CoV infection
VERO cells infected with SARS-CoV (2 TCID
50
/cell) were incubated in complete MEM medium containing the indicated amounts of INDO or control diluent for 48 h.
(A) SARS-CoV titres (G) in TCID
50
/ml represent the mean ± SD of duplicate samples from a representative experiment of three with similar results. *P<0.003;
**P<0.0001. Cell viability ( ) determined by MTT assay in uninfected cells is expressed as in Figure 1. (B) Cytoprotective effect of INDO (400 µM) in SARS-CoV-
infected VERO cells at 24 h p.i., bar, 100 µm. (C) Effect of INDO on DNA and protein synthesis in uninfected cells as determined by
3
H-thymidine ( ) and
35
S-methionine ( ) incorporation (24 h pulse), and expressed as mean ± SD cpm/10
6
cells. INDO, indomethacin.
*
**
**
1 0
4
0
25
50
75
1 00
1 0
3
1 00 200 300 0
IND O , µM
CCoV yield, T CID
50
Cell viabilit y , %
400
1 0
5
1 0
6
1 0
7
1 0
0
1 00 200 300 0
IND O , µM
cpm x 1 0
–3
/ 1 0
6
cells
400
20
30
40
A
C
B
8_0044_santoro.qxp 24/11/06 09:45 Page 1027
tory and gastrointestinal mucosa; however, it was not
until the SARS epidemic that a large scientific interest
focused on this family of viruses. The human coron-
aviruses usually cause mild self-resolving upper respi-
ratory tract infections, most of which result in the
common cold, and have occasionally been associated
with pneumonia or with neurological symptoms and
myocarditis [3]. Before the SARS epidemic, no effec-
tive specific antiviral therapy for coronavirus infec-
tions was known.
The results described in the present report demon-
strate that the NSAID INDO has a potent antiviral
activity against different coronaviruses, being effective
against the canine (CCoV) and the human (SARS-CoV)
coronaviruses. A dramatic antiviral effect was also
found in a model of in vitro feline coronavirus infec-
C Amici et al.
© 2006 International Medical Press
1028
Figure 5. INDO inhibits SARS-CoV RNA expression
(A,B) SARS-CoV-infected VERO cells were treated with different concentrations of IFN-α, RBV, INDO (A) or aspirin (B) soon after the adsorption period. SARS-CoV
yield was determined at 24 h p.i.. Data expressed as TCID
50
/ml represent the mean ± SD of duplicate samples from a representative experiment. The empty bar repre-
sents the untreated infected control. (C) VERO cells were treated with 300 µM INDO (filled bars) 9 h before virus infection (Pre), only during the adsorption period
(Ads) or soon after virus adsorption (Post). The empty bar represents the untreated infected control (–). SARS-CoV yield was determined at 36 h p.i.. Data are
expressed as percent of virus yield in untreated cells. *P =0.0002. Untreated infected control = 1.74×10
6
TCID
50
/ml. (D) Intracellular SARS-CoV replication as
measured by real time RT-PCR targeted to the viral polymerase gene and expressed as percentage of genomic RNA copy number in untreated infected cells (G).
*P<0.025; **P=0.017. Untreated infected control = 1.696×10
3
copies/cell. RNA synthesis in uninfected cells was assayed by
3
H-uridine labelling (24 h pulse). Data are
expressed as
3
H-uridine incorporation/total cellular uptake ratio ( ). IFN-α, interferon α; INDO, indomethacin; RBV, ribavirin.
10
4
10
3
RBV,
µM
INDO,
µM
SARS-CoV yield, TCID
50
10
5
10
6
10
7
10
8
400
100
400
40
5,000
500
Control
10
5
10
4
Aspirin, µM
SARS-CoV yield, TCID
50
10
6
10
7
2,000
1,000
200
0
25
50
0
+
Pre
+
Ads
+ INDO
Post
*
SARS-CoV yield, %
75
100
25
50
0
0 100 200 300 400
**
*
*
INDO, ×M
75
40
50
30
20
10
0
100
RT-PCR (
RNA synthesis (
(
(
IFN-α,
IU/ml
A
C
D
B
8_0044_santoro.qxp 24/11/06 09:45 Page 1028
tion, FCoV type II in feline kidney CRFK cells (C Amici
and MG Santoro, unpublished observations). In
addition, oral administration of INDO at the concen-
tration of 1 mg/kg body weight, which is in the range
of the therapeutic dose in humans [19] and dogs [20],
to CCoV-infected dogs was found to cause a dramatic
decrease in virus titres in the faeces of the treated
animals during a 4-day treatment period, also demon-
strating a potent antiviral activity of the drug in vivo.
No local or systemic adverse reactions were observed
during or after treatment with INDO. It should be
pointed out that, since coronavirus infection in dogs
causes a mild disease characterized by the occurrence of
moderate diarrhoea followed by a rapid recovery of the
infected animals, this model does not allow us to define
the effectiveness of INDO on the outcome of the
disease, but only the effect of the drug on virus replica-
tion. In addition, due to the difficulty in finding pups
negative for CCoV RNA and anti-CCoV antibodies (C
Buonavoglia, personal communication), only a small
number of animals could be tested.
The mechanism of antiviral action appears to be
novel. We have shown that treatment of target cells
with INDO prior to viral infection has no effect on
coronavirus replication, indicating that, as opposed to
IFN, INDO is not priming the host cell to raise a
defence response against the invading virus. Moreover,
INDO does not affect virus infectivity, binding or entry
into target cells, but acts early on the coronavirus repli-
cation cycle, selectively blocking viral RNA synthesis.
INDO has been used for a long time as a potent
anti-inflammatory drug, acting by blocking COX-1
and COX-2 activity and inhibiting pro-inflammatory
prostaglandin synthesis [21]. The antiviral effect,
however, appears to be cyclooxygenase-independent,
since it occurs at concentrations higher than those
needed for COX inhibition (10
–8
,10
–9
M) [22]; in addi-
tion, the antiviral activity cannot be mimicked by the
potent COX inhibitor aspirin, which has no effect on
either CCoV or SARS-CoV replication up to
millimolar concentrations.
Although the mechanisms underlying the antiviral
activity of INDO against coronavirus infection require
further investigation, the results shown herein may
have important implications. In fact, SARS resurgence
is still a threat, since no definitive animal reservoir has
been identified, and sporadic cases have been reported
since the epidemic period [4,5]. The recently reported
evasion of antibody neutralization by SARS-CoV
raises concerns about the efficacy of SARS-CoV
vaccines [23]. Moreover, although different clinical
approaches to SARS therapy have been tried using
steroidal anti-inflammatory agents and broad-spec-
trum antivirals such as RBV and IFN, no effective
treatment protocol has been established [18]. As
reported above, surprisingly, INDO was found to be
a more effective antiviral agent than IFN-α up to a
concentration of 5,000 IU/ml [23], and RBV up to
concentrations of 400 µM. In addition INDO was
also found to be effective during SARS-CoV infection
of human pulmonary epithelial cells.
Altogether, the results described suggest that
indomethacin, possessing both anti-inflammatory
properties and a direct antiviral activity against SARS-
CoV, could be effective in the treatment of SARS.
Acknowledgements
This work was supported by the Italian Ministry of
Public Health (ISS project ‘Lotta alla SARS’), Ricerca
Finalizzata and Ricerca Corrente to I.R.C.C.S., the
Italian Ministry of University (MIUR PRIN and FIRB
projects), and the EC EPISARS project (NSP22-CT-
2004-511063).
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... Already at the beginning of 2020, some of us were expressing an opinion on the subject by saying that "while waiting for definite EBM-based facts for the treatment of COVID-19, it would be absolutely unethical to leave patients at home without treatment, accepting the risk that the disease could worsen, instead we should be looking to try other, even off-label, pharmacological treatments with available wellproven drugs for other conditions, which might prove effective against COVID-19 as well, based on the pathophysiological mechanisms of the disease, which were gradually becoming known and on the well-known actions of these drugs" [1]. Even back then, Indomethacin was already indicated as one of the drugs that could bring benefits in the treatment of COVID-19, due to both its powerful anti-inflammatory action and, above all, its known antiviral action [2]. ...
... Italian physicians who performed home-based therapies during the pandemic have already produced two retrospective studies. The first [7] chose indomethacin for the early home treatment of 153 patients with mild-to-moderate COVD-19 since the beginning of the pandemic, both for its potent anti-inflammatory action and for its documented anti-viral action against various viruses and, in particular, also in cells in vitro and in vivo (in dogs) against coronavirus [2] and more recently also against SARS-CoV-2 [32,33]. Our multitherapeutic treatment is based on a therapeutic rationale combining various molecules with anti-inflammatory, antiviral and antioxidant properties, such as indomethacin, hesperidin, quercetin, aspirin in an antiplatelet dosage, and gastric protection with omeprazole [34]. ...
... Furthermore, "doctors should be given the possibility of proposing on-and offlabel drugs to patients, as well as useful indications for the prevention and treatment of diseases that can complicate the course of COVID-19, also by way of general evidencebased nutritional education and specific advice on nutrition (also to ensure a proper intake of vitamins and flavonoids, without ruling out the use of supplements write where necessary)". Among the drugs that may prove useful, according to the doctor's judgement in individual cases for the prompt home management of patients with COVID-19, are NSAIDS and, in particular, indomethacin, that in addition to the anti-inflammatory and antibradykinin actions, also demonstrated a clear antiviral action in vitro and in vivo against SARS-CoV-2 [2,7,31,32,34,65]. While a recent meta-analysis, performed by analyzing 40 studies, showed that the use of NSAIDs did not reduce mortality outcomes among people with COVID-19, it has also shown that NSAIDs can be used safely among patients positive to SARS-CoV-2 [66]. ...
... A stock solution of Indomethacin (Sigma Aldrich, Israel; cat #17378) was prepared by dissolving in absolute ethanol at a concentration of 40 mM. Further, it was diluted to the desired concentrations (0.1 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 250 μM, 500 μM, 750 μM and 1000 μM) in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, USA) culture medium [8]. ...
... [16], SARS-CoV [8], HSV-1 [51], and vesicular stomatitis virus [9]. Apart from its anti-inflammatory properties, Indomethacin had been shown in-vitro and in-vivo to decrease viral replication in SARS-CoV [8] and SARS-CoV-2 [94]. ...
... [16], SARS-CoV [8], HSV-1 [51], and vesicular stomatitis virus [9]. Apart from its anti-inflammatory properties, Indomethacin had been shown in-vitro and in-vivo to decrease viral replication in SARS-CoV [8] and SARS-CoV-2 [94]. Notably, in this study, we have observed Indomethacin influences 85 genes (Supplementary file 4) associated with the COVID signature, indicating a significant role it may play in host responses. ...
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the worldwide spread of coronavirus disease 19 (COVID-19), and till now, it has caused death to more than 6.2 million people. Although various vaccines and drug candidates are being tested globally with limited to moderate success, a comprehensive therapeutic cure is yet to be achieved. In this study, we applied computational drug repurposing methods complemented with the analyses of the already existing gene expression data to find better therapeutics in treatment and recovery. Primarily, we identified the most crucial proteins of SARS-CoV-2 and host human cells responsible for viral infection and host response. An in-silico screening of the existing drugs was performed against the crucial proteins for SARS-CoV-2 infection, and a few existing drugs were shortlisted. Further, we analyzed the gene expression data of SARS-CoV-2 in human lung epithelial cells and investigated the molecules that can reverse the cellular mRNA expression profiles in the diseased state. LINCS L1000 and Comparative Toxicogenomics Database (CTD) were utilized to obtain two sets of compounds that can be used to counter SARS-CoV-2 infection from the gene expression perspective. Indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), and Vitamin-A were found in two sets of compounds, and in the in-silico screening of existing drugs to treat SARS-CoV-2. Our in-silico findings on Indomethacin were further successfully validated by in-vitro testing in Vero CCL81 cells with an IC50 of 12 μM. Along with these findings, we briefly discuss the possible roles of Indomethacin and Vitamin-A to counter the SARS-CoV-2 infection in humans.
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... It is suggested that naproxen combines a broad-spectrum antiviral activity with its well-known anti-inflammatory properties to improve severe respiratory mortality associated with COVID-19 [96]. There is also a similar report regarding the antiviral properties of indomethacin [97]. These reports could be the beginning of future research in COVID treatment. ...
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