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Titre: In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant
Auteur: Ilenia Delogu
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Antiviral Research 90 (2011) 99–107
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/antiviral
In vitro antiviral activity of arbidol against Chikungunya virus and characteristics
of a selected resistant mutant
Ilenia Delogu 1, Boris Pastorino ⇑,1, Cécile Baronti, Antoine Nougairède, Emilie Bonnet, Xavier de Lamballerie
Unité des Virus Emergents, UMR190 ‘‘Emergence des pathologies virales’’ Université de la Méditerranée, Institut de Recherche pour le Développement,
EHSP French School of Public Health, Faculté de Médecine, Marseille, France
a r t i c l e
i n f o
Received 10 February 2011
Revised 14 March 2011
Accepted 17 March 2011
Available online 1 April 2011
a b s t r a c t
Arbidol (ARB) is an antiviral drug originally licensed in Russia for use against inﬂuenza and other respiratory viral infections. Although a broad-spectrum antiviral activity has been reported for this drug, there
is until now no data regarding its effects against alphavirus infection. Here, the in vitro antiviral effect of
ARB on Chikungunya virus (CHIKV) replication was investigated and this compound was found to present
potent inhibitory activity against the virus propagated onto immortalized Vero cells or primary human
ﬁbroblasts (MRC-5 lung cells) (IC50 < 10 lg/ml). A CHIKV resistant mutant was then selected and adapted
to growth in the presence of 30 lg/ml ARB in MRC5 cells; its complete sequence analysis revealed a single
amino acid substitution (G407R) localized in the E2 envelope protein. To conﬁrm the G407R role in the
molecular mechanism of ARB resistance, a CHIKV infectious clone harboring the same substitution was
engineered, tested, and was found to display a similar level of resistance. Finally, our results demonstrated the effective in vitro antiviral activity of ARB against CHIKV and gave some tracks to understand
the molecular basis of ARB activity.
Ó 2011 Elsevier B.V. All rights reserved.
Chikungunya virus (CHIKV) is an arthropod-borne viral disease
ﬁrst described in Tanzania in 1952 (Robinson, 1955) which has
reemerged since 2005 in Eastern Africa, the Indian Ocean, India
and South-East Asia and even reached Europe in 2007 (Arankalle
et al., 2007; Rezza et al., 2007). From 2005, this new variant has been
responsible for millions of cases of CHIKV disease. The adaptation to
its new vector, Aedes albopictus (Santhosh et al., 2008; Schuffenecker
et al., 2006) rendered possible the spread of the virus in new territories in which Aedes aegypti was absent (e.g., Reunion Island, Mauritius, and the south of Europe). CHIKV infection is commonly an
acute disease marked by febrile arthralgia and a frequent rash, but
persisting arthralgia has been reported in a signiﬁcant number of
cases (Borgherini et al., 2008). Lethal infections are rare but severe
cases have been described including neurological presentations
and neonatal contaminations which were documented during the
outbreak in Reunion Island (Economopoulou et al., 2009; Lemant
et al., 2008). Current treatments of Chikungunya fever are for
symptoms with no effective licensed vaccine nor speciﬁc antiviral
drug available. The utilization of the antimalarial chloroquine
⇑ Corresponding author. Address: UMR190 Unité des Virus Emergents, Faculté de
Médecine, de Marseille 27, Bd Jean Moulin, 13005 Marseille cedex 05, France. Tel.:
+33 4 91 32 44 20; fax: +33 4 91 32 44 21.
E-mail address: firstname.lastname@example.org (B. Pastorino).
These authors contributed equally to this work.
0166-3542/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
proved to be poorly active in vivo despite it’s in cellulo antiviral effect
on CHIKV infection (de Lamballerie et al., 2008; Michault and Staikowsky, 2009; Thiboutot et al., 2010). Similarly, it has been shown
that the combination of interferon-alpha and ribavirin is effective
on CHIKV replication in vitro but these compounds have not been
tested in animal models and/or clinical trials (Briolant et al., 2004;
de Lamballerie et al., 2009).
The antiviral drug arbidol (ARB) (1-methyl-2-phenyl-thiomethyl-3-carbotoxy-4-dimetylaminomethyl-5-hydroxy-6-bromoindolehydrochloride monohydrate) (Fig. 1) was originally
developed at the Russian Research Chemical and Pharmaceutical
Institute about 20 years ago (Panisheva et al., 1988) and since
1990 this drug has been used in Russia for prophylaxis and treatment of acute respiratory infections including inﬂuenza. Until
now, it has been shown that ARB exhibits a wide range of activity
against a number of RNA, DNA, enveloped and non-enveloped
viruses (Boriskin et al., 2008). This suggests that ARB targets
common critical step(s) in virus–cell interaction. Recent data
showed that ARB incorporates into cellular membranes leading
to perturbed membrane structures and inhibition of virusmediated fusion (Villalain, 2010). In case of inﬂuenza viruses or
hepatitis C virus (HCV), ARB blocks virus entry into target cells
but exploits different modalities proving its effective broadspectrum antiviral activity (Leneva et al., 2009; Pecheur et al.,
In this study, we investigated the in cellulo antiviral ARB activity
against CHIKV. Several cell lines were assayed (MRC-5 and Vero), in
I. Delogu et al. / Antiviral Research 90 (2011) 99–107
Fig. 1. Chemical structures of arbidol ARB (A), HZ1 (B) and HZ3 (C).
various conditions (pre and post-infection treatments) and using
different ARB metabolites to demonstrated the in vitro inhibitory
effects of ARB on CHIKV replication (IC50 < 10 lg/ml). To further
characterize the mechanism of ARB action, we also selected ARBresistant mutant of CHIKV, identiﬁed a single drug-resistant mutation in the E2 envelope viral protein (G407R) and conﬁrmed its role
in the virus resistance using infectious clones in in vitro assays.
2. Materials and methods
2.1. Cells and viruses
The MRC-5 cells (ATCC number CCL-171) were grown in Basal
Medium Eagle (BME) supplemented with 10% fetal calf serum
(FCS), 2 mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin sulfate, under 5% CO2. The Vero cell line (ATCC number
CCL-81) was grown in minimal essential medium (MEM) supplemented with 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin,
100 lg/ml streptomycin sulfate, under 5% CO2. HEK-293 cells
(ATCC number CRL-1573) were cultured in Dulbecco’s modiﬁed Eagle medium (DMEM) containing 4.5 g/l of D-glucose, 1 mM of sodium pyruvate and 2 mM of L-glutamine, supplemented with 10%
decomplemented fetal calf serum (FCS) and antibiotics.
The CHIKV strain used in this study for antiviral assays or the
construction of infectious clone was LR2006 OPY1 (GenBank accession number DQ443544), isolated from a patient during the outbreak on Reunion Island in 2006 (Parola et al., 2006).
Puriﬁed arbidol (HZ2 = ARB) and two derived metabolites, HZ1
(6-bromo-4-(dimethylaminomethyl)-5-hydroxy-1-methyl-2-(phenylsulphonylmethyl)-1H-indole-3-carboxylate) and HZ3 (6-bromo4-(dimethylaminomethyl)-5-hydroxy-1-methyl-2-(methylphenylsulphoxyde)-1H-indole-3-carboxylate) (Fig. 1) were provided by
Stragen Pharma SA (Geneva, Switzerland). HZ2, HZ1 and HZ3 powders were dissolved to completion in dimethyl sulfoxide (DMSO)
at a ﬁnal concentration of 10 mg/ml followed by dilution in sterile
distilled water to prepare stocks at 1 mg/ml. After storage at
20 °C, these samples were used for preparation of required drug
solutions in all experiments. The ﬁnal 0.005% maximum DMSO concentration was also added to all mock control samples.
2.3. Cell viability assay
The ARB cytotoxicity in MRC5 and Vero cells was evaluated
using neutral red (NR) dye uptake assays and microscopic observations (Repetto et al., 2008). Brieﬂy, for NR dye uptake assays, 96well tissue culture plates were seeded with cells then exposed at
90% conﬂuence to varying ARB concentrations (0–100 lg/ml).
Plates were then incubated at 37 °C, 5% CO2 for 18 or 48 h (respectively, for Vero or MRC5 cells), at which times medium containing
neutral red (40 lg/ml) was added to each well. After 3 h of incubation, the dye was extracted with acidiﬁed ethanol solution and
optical densities (OD) were read using a microplate spectrophotometer at 540 nm (TECAN Sunrise). Results were expressed as a
percentage of OD value of treated cell cultures with respect to
untreated ones and the 50% cytotoxic (CC50) concentrations of
ARB for MRC5 and Vero cells were determined by regression
2.4. Selection of an ARB-resistant mutant
MRC-5 cells grown in 12-well plates were infected with different dilutions of CHIKV (LR2006 OPY1 strain) at a multiplicity of
infection (MOI) of 1–0.001 TCID50/cell. Virus growth in the presence or absence of ARB was examined at each passage by direct
microscopic observations of cytopathic effect (CPE) on MRC-5 cells.
The clariﬁed supernatant from the highest dilution providing CPE
was used for subsequent passage. Initially, CHIKV did not grow
in the presence of ARB at a concentration >10 lg/ml; its concentration was increased gradually from 4 to 30 lg/ml during successive
virus passages using 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22,
24, 26, 28 and 30 lg/ml ARB. By the 17th passage, the virus
appeared to have adapted to 30 lg/ml ARB in MRC-5 cell. This speciﬁc clariﬁed supernatant was stored at 80 °C for further analysis.
2.5. Sequence analysis
Viral RNA was extracted from samples using the EZ1 virus mini
kit on an EZ1 Biorobot workstation (Qiagen). Viral genomes were
reverse-transcribed and ampliﬁed by use a one-step RT-PCR kit
(Access RT-PCR core reagent kit, Promega) according to the
manufacturer’s instructions. PCR products were puriﬁed using
the Qiagen PCR extraction kit and sequenced using the ABI PRISM
I. Delogu et al. / Antiviral Research 90 (2011) 99–107
BigDye terminator cycle sequencing kit on an ABI Prism 31310X
Genetic Analyser sequencer. The sequencing primers were those
already used for the complete sequencing of the LR2006 OPY1
CHIK strain (Parola et al., 2006).
was determined. The IC50 value (i.e. the concentration of compound required to inhibit cell infection by 50%) was determined
by plotting the percentage inhibition of cell infection as a function
of ARB concentration after 4 independent experiments.
2.6. Construction of CHIKV infectious clones
2.9.2. Virucidal activity
To evaluate a putative virucidal action of ARB on CHIKV, the
same experiments were performed using CHIKV pre incubated
during 0.5 or 1 h with ARB concentrations ranging from 1 to
30 lg/ml before MRC5 infection. Each assay was realized in duplicate, and 2 independent experiments were performed. The IC50 values were determined as described previously.
The preparation of an infectious clone from the LR2006 CHIKV
strain was reported elsewhere (Tsetsarkin et al., 2006). The original
construct was modiﬁed by adding a CMV promoter and the
Hepatitis D virus ribozyme at the 50 and 30 ends of the genome,
respectively, in order to be able to transfect DNA and generate in
cellulo infectious viral RNAs. The resulting construct (set-up in a
pBR322 plasmid possessing an ampicilline resistance gene) was
named ‘‘Tonile’’. The infectious CHIKV clone containing the mutation G407R called ‘‘Tonile-ARB-R’’ was produced by standard
molecular biology techniques. The PCR fragments containing the
mutation were cloned and inserted between the unique restriction
sites AgeI and XhoI into the Tonile plasmid backbone (Vanlandingham et al., 2005) (Fig. 5). The Tonile and Tonile-ARB-R infectious
clones were sequenced completely as described previously to
validate the constructions.
2.9.3. Kinetics of ARB cell treatment
To evaluate the inﬂuence of pre- and post-infection ARB treatment on virus replication, the same experiments were performed
using MRC5 cells with the addition of ARB 1, 3, 5, 8, 24 h before
and post infection. Each assay was realized in duplicate and 2 independent experiments were performed. The IC50 values were determined as described previously. The Mann–Whitney U statistical
test was then used to analyze IC50 values obtained from different
times in comparison with the chosen reference one (IC50 value corresponding to the time 1 h post infection).
2.7. Transfection and production of CHIKV infectious clones
HEK-293 cells were seeded on 75 cm2 ﬂask in complete DMEM
without antibiotics. The day after, cells were transfected with the
CHIKV infectious clones (Tonile or Tonile-ARB-R) using Lipofectamine 2000 (Invitrogen) with a ratio of 1 lg of DNA per 1.5 ll of
Lipofectamine. Four hours post-transfection, transfected cells were
washed three times in Hanks balanced salt solution (HBSS) then
incubated for 16 h in complete DMEM supplemented with antibiotics and 30 lg/ml ARB for cells transfected with Tonile-ARB-R.
For each viral production, stocks were stored at 80 °C and a sample was used to perform complete genome sequencing and virus
2.8. Virus titration
Virus titers were determined by the Tissue Culture Infectious
Dose50 (TCID50) method in Vero cell cultures (Reed and Muench,
1938). Brieﬂy, the TCID50 assay was performed on Vero cells seeded
in 96-well plates. When the cells reached 80% conﬂuence, six replicates were infected with 150 ll of ten-fold serial dilutions of the
virus sample, and then incubated 7 days before microscopic observations and positive CPE well counting. For each supernatant sample, the infectivity titer was expressed as TCID50/ml using the
2.9. In vitro antiviral activity of ARB
2.9.1. IC50 determination using Indirect Immunoﬂuorescence Assays
MRC-5 cells were grown in an 8-microchamber Lab-Tek II slide
(Nalge Nunc international) to reach 80% conﬂuence. Cells were
then infected with different dilutions of CHIKV at MOIs from 1 to
0.0001 TCID50/cell. One hour after infection, the viral inoculum
was removed and cells were washed one time with PBS. Complete
medium (250 ll) supplemented with 0.9% ﬁnal concentration of
Methocult (H4100, StemCell Technologies INC), containing different concentration of ARB (0.9; 1.8; 3.75; 7.5; 15 and 30 lg/ml)
were added to each well and cells were grown for 48 h. After PBS
washing, cells were ﬁxed with acetone for 20 min at room temperature and viral antigens were detected by IFA using CHIKV-speciﬁc
immune human serum (1:20) and ﬂuorescein-conjugated anti-human IgG (1:400). The percentage of ﬂuorescent cells in each well
2.9.4. IC50 determination using comparative quantitative RT-PCR
Antiviral assays were carried out in Vero cells in 48-well plates
in duplicate and two independent experiments were performed.
Brieﬂy, 1 day after seeding, cells were infected with 100 ll of the
viral inoculum (at MOIs of either 0.1 or 0.01 TCID50/cell) for
90 min. at 37 °C, 5% CO2. Following incubation, the viral inoculum
was removed and cultures were washed once with HBSS after
which 500 lL of fresh complete MEM medium supplemented with
0, 10, 20, 30, 40 or 50 lg/ml ARB (HZ2) were added to the wells.
After 18 h p.i., supernatants were harvested and viral RNA was extracted from 100 ll of cell culture clariﬁed supernatant using the
NucleoSpin 96 virus kit according to the manufacturer’s protocol
(Macherey–Nagel, Duren, Germany) and an epMotion 5075 workstation (Eppendorf France SARL). One-step qRT-PCR was performed
on the Applied Biosystems 7900HT Fast Real-Time PCR System
using primers and probes already described (Pastorino et al.,
2005). For comparative quantiﬁcation, data were expressed as
the percentage of untreated virus control, and log reduction values
were calculated. The IC50 value (i.e. the concentration of compound
required to inhibit viral RNA load by 50%) was determined by plotting the percentage inhibition of cell infection as a function of ARB
2.9.5. IC50 determination using virus titration assays
Supernatant used in antiviral assays and stored at 80 °C were
titrated using the method described above. For each sample, the
viral titer was represented as percentage of positive control (viral
titer from infected cell supernatant sample without antiviral compound). The IC50 value (i.e. the concentration of compound required to inhibit infectious virus titer by 50%) was determined by
plotting the percentage inhibition of cell infection as a function
of ARB concentration.
2.10. Hemagglutination assay
Hemagglutination titration of the CHIKV strain LR2006 OPY1
was ﬁrst performed using standard methods (Clarke and Casals,
1958): twofold serial dilution of virus samples (cell supernatant)
on U-bottom microplates were carried out with 0.4% bovine albumin/borate saline pH 9.0 solution (ﬁnal volume: 35 ll/well).
Thirty-ﬁve microliters of pre-diluted goose red blood cells (1/150
I. Delogu et al. / Antiviral Research 90 (2011) 99–107
using the ﬁnal pH 6.0 adjusting diluents) were added, the mixture
was homogenized, incubated 45 min at room temperature and
then read using four scoring symbols: ++ for complete hemagglutination, + for partial hemagglutination, +/ for trace hemagglutination and for negative hemagglutination. The titer was the
reciprocal of the last dilution in which + was observed. To assess
the effect of ARB on hemagglutination, we then performed a hemagglutination assay in the same condition with 3 amounts of virus
(8, 4 and 2 UHA/well). Each concentration of virus was used in triplicate in presence of various ARB concentrations (0, 15, 30 and
60 lg/ml). Negative controls contained no virus and allowed us
to observe the effect of ARB on goose red blood cells sedimentation.
3.1. Effect of arbidol and its metabolites HZ1 and HZ3 on CHIKV strain
The cytotoxic effect of ARB was evaluated using NR cytotoxicity
assay and microscopic observations. Fig. 2C shows the 50% cytotoxic concentration values (CC50) obtained for conﬂuent Vero and
MRC5 cells after, respectively, 18 or 48 h of ARB treatment.
Then, the effect of ARB and two sulfone and sulfoxide metabolites (HZ1 and HZ3) (Fig. 1) on CHIKV replication was determined
using speciﬁc indirect immunoﬂuorescent assays (IFA). As shown
in Fig. 2A, ARB was found to inhibit CHIKV infection with IC50 values at 6.49 ± 1.17 lg/ml. This was further conﬁrmed using infected
Vero cells and qRT-PCR assays which also provided estimated IC50
values 610 lg/ml (Fig. 2B). ARB selectivity indices (CC50/IC50) calculated using Vero and MRC5 cell line, provided values about 28
and 36, respectively (Fig. 2C). Moreover, subconﬂuent monolayers
of Vero and MRC5 cells treated for 18 or 48 h with ARB at concentrations of 0–30 lg/ml did not show any microscopically visible
changes in cell morphology or cell density.
In the case of arbidol HZ1 and HZ3 compounds, a weak antiviral
activity was observed, with IC50 values reaching 30 lg/ml. Furthermore, pre-incubation of ARB for 12 h at 37 °C did not improve its
antiviral effect on MRC5 cells (HZ2a, IC50 value 6.21 ± 0.73 lg/ml)
(see Fig. 2A). These results suggested that the arbidol antiviral
activity against CHIKV was due to the HZ2 molecule and was not
extended to its metabolites or degradation products.
Finally, to investigate the direct inactivating effect of ARB,
CHIKV was pre treated for 0.5 or 1 h with concentrations ranging
from 1 to 30 lg/ml. The IC50 obtained (respectively, 12.3 ±
2.67 lg/ml and 16.85 ± 3.87 lg/ml) indicated that the antiviral
activity of ARB on CHIKV infection was not due to a virucidal
activity (Fig. 2A).
3.2. Time-of-drug-addition studies
To examine the mechanism of viral inhibition by ARB, a time-ofdrug-addition experiment was carried out. Various concentrations
of ARB HZ2 were added to CHIKV-MRC5 infected cells at several
time points before or post infection. As shown in Fig. 3, a decrease
in the IC50 values was observed from times 0 to 24 before infection
and the IC50 value at 24 h before infection reached a statistically
signiﬁcant difference from IC50 observed 1 h post infection
(P < 0.05). Moreover, antiviral activity was progressively reduced
when ARB was added at post infection stages and the increased
IC50 values at 3, 5 and 8 h post infection were statistically different
Fig. 2. Antiviral activity of ARB against CHIKV strain LR2006 OPY1. (A) The IC50 values were determined using indirect Immunoﬂuorescence Assay (IFA) and MRC5 cells.
HZ2 + virus (30 or 60 min.) represented experiments where CHIKV was pre incubated during 30 or 60 min. with various ARB concentrations before cell infection. HZ2a
represented experiments where ARB was pre-incubated for 12 h at 37 °C before cell addition. aMean ± SD values are determined from four independent experiments.
(B) Effect of ARB on CHIKV replication using Vero cells and comparative qRT-PCR assays. Data were expressed as the percentage of untreated virus control and each point
represents the mean of two replicate in two independent experiments). (C) Cytotoxicity of ARB in our experimental conditions. aThe 50% cytotoxic (CC50) concentrations of
ARB for MRC5 and Vero cells were determined using neutral red (NR) dye uptake assays after, respectively, 18 or 24 h ARB treatment. bSelectivity index (SI) is expressed as the
I. Delogu et al. / Antiviral Research 90 (2011) 99–107
Fig. 3. Time-of-addition study. Experiments were performed using MRC5 cells with the addition of ARB 1, 3, 5, 8, 24 h before and post infection. The IC50 values were
determined using IFA as described previously. Each point represents the mean of two replicate in two independent experiments. ⁄P < 0.05 vs. reference value at 1 h post
infection; ⁄⁄P < 0.01 vs. reference value at 1 h post infection.
from IC50 observed 1 h post infection (P < 0.01). These results suggested that ARB interferes with the earliest stages of the viral replication cycle (i.e., virus attachment/entry).
3.3. Effect of ARB on CHIKV hemagglutination
The ability of viruses to agglutinate erythrocyte is a potentially
simple model for the study of virus attachment to cellular receptors. Therefore, to make precise the mechanism of ARB action,
hemagglutination assays were performed and analyzed. As shown
in Fig. 4, ARB concentration equal or higher than 15 lg/ml inhibited the hemagglutination of CHIKV onto goose red blood cells
depending on the amount of virus. With 2 UHA/well and 30 lg/
ml ARB, the hemagglutination-inhibition reached around 50%.
When 60 lg/ml ARB was added in the assay, no CHIKV hemagglutination was detected. Clearly, these results showed that ARB prevents the interaction of CHIKV with goose red blood which could
suggest by analogy that the drug blocks the CHIKV replication cycle
at the cell adsorption step.
Fig. 5. Schematic representation of the infectious clone Tonile-ARB-R containing
the G407R mutation. Tonile-ARB-R derives from the Tonile Infectious clone which
contains the whole genome of the LR2006 CHIKV strain. The promoter CMV (pCMV)
and the Hepatitis Delta virus ribozyme (HDR) allow to generate in cellulo infectious
viral RNAs. All of them are inserted into a pBR322 plasmid possessing an ampicilline
resistance gene. The unique restriction sites AgeI and XhoI were used to add the
G407R mutation. Positions indicated in this ﬁgure are based on the complete
3.4. Selection and characteristics of an ARB-resistant mutant
CHIKV LR2006 OPY1 strain was used to select an ARB-resistant
variant following passage on MRC5 cells with increasing
concentrations of the drug (from 4 to 30 lg/ml). Efﬁcient virus
propagation was ultimately obtained at the 17th passage after
4 months of culture. The resistant mutant (called CHIKV-R) was
puriﬁed by end-point dilution in the presence of 30 lg/ml ARB
and a virus stock was prepared under the same concentration of
Fig. 4. Hemagglutination assay. Three amounts of virus (8, 4 and 2 UHA/well) were used in triplicate and in presence of various ARB concentrations (0, 15, 30 and 60 lg/ml) to
observe modiﬁcation on goose red blood cells hemagglutination. Negative controls represented wells without virus.
I. Delogu et al. / Antiviral Research 90 (2011) 99–107
drug. Complete nucleotide sequence of CHIKV-R revealed a single
amino acid substitution G407R localized in the E2 viral envelope
protein (Fig. 5). CHIKV-R was further characterized for ARB resistance and replication ﬁtness. For both studies, CHIKV replication
was determined on Vero cells using comparative qRT-PCR. As described in Fig. 6, CHIKV-R was highly resistant to ARB, with no signiﬁcant difference in virus replication after 18 h post infection
when exposed to 10 from 50 lg/ml ARB. The ﬁtness of CHIK
OPY1 and CHIKV-R was compared in infection assays using the
same MOIs (0.1 and 0.01 TCID50/cell) for both viruses. Viral RNA
load was quantiﬁed every eight hours for four days in the absence
of any antiviral drug using speciﬁc one-step qRT-PCR detection
(Pastorino et al., 2005) (Fig. 7). Despite slight initial difference in
the numbers of viral RNA copies, the growth curves exhibited the
same pattern with a similar exponential growth phase until reaching a plateau at the end of the second day and CPE apparition at
day 3. The rate of viral growth was identical between hours 8
and 56 for both viruses with no signiﬁcant difference in viral
RNA copies (Fig. 7).
However, in the absence of ARB selection pressure, CHIKV-R
seemed to be unstable as the reversion G407R G407 was rapidly
detected: when CHIKV-R was propagated on Vero cells without
ARB in the medium culture, sequence analysis of viral production
revealed the emergence of the G407 revertant as soon as 3 days
post infection. This suggests that the wild-type virus is much better adapted to replication than the G407R variant in our experimental conditions.
3.5. Effect of arbidol on Tonile-ARB-R, an infectious clone of CHIKV
containing the mutation G407R
Tonile and Tonile-ARB-R CHIKV infectious clones were used to
infect Vero cells at the same MOIs (0.1 and 0.01 TCID50/cell) in
the presence of various ARB concentrations (0–50 lg/ml). Eighteen
Fig. 6. Effect of ARB on CHIKV-R mutant replication. The antiviral activity of ARB on CHIKV-R mutant replication was evaluated using Vero cells and comparative qRT-PCR
assays as described previously. Data were expressed as the percentage of untreated virus control and each point represents the mean of two replicate in two independent
Fig. 7. Replication kinetics of CHIKV OPY1 and CHIKV-R mutant. Vero cells were infected with CHIKV OPY1 or CHIKV-R strains at the same MOIs (0.1 and 0.01 TCID50/cell).
The viral growth in the absence of antiviral compound was followed by measuring viral RNA copies every 8 h for 4 days using speciﬁc real-time RT-PCR assay.
I. Delogu et al. / Antiviral Research 90 (2011) 99–107
Fig. 8. Effect of ARB on CHIKV infectious clones replication. (A) The effect of ARB on Tonile and Tonile-ARB-R replication was assayed using Vero cells and comparative qRTPCR assays as already described. Data were expressed as the percentage of untreated virus control and each point represents the mean of two replicate in two independent
experiments. (B) The effect of ARB on Tonile and Tonile-ARB-R infectivity were determined using Vero cells and titration of cell culture supernatant. Data were expressed as
the percentage of untreated virus control and each point represents the mean of two replicates in two independent experiments.
hours post infection, infected cell supernatants were harvested and
compared using viral RNA quantiﬁcation and virus titration assays.
As expected (see Fig. 8A), a strong viral load decrease was observed
for Tonile CHIKV when arbidol was added to the culture medium. A
90% reduction of viral RNA was observed after addition of 10 lg/ml
ARB in the culture medium. The same experiment using TonileARB-R CHIKV infectious clone resulted in no signiﬁcant difference
for viral load when 0 to 30 lg/ml of arbidol was added to the culture medium. A 70% reduction of viral RNA was only observed
when the medium was supplemented with 40 or 50 lg/ml of the
drug. Virus titration assays conﬁrmed the results obtained for viral
RNA quantiﬁcation (Fig. 8B) with a complete suppression of Tonile
CHIKV production using 10 lg/ml ARB whereas the infectivity of
Tonile-ARB-R was only signiﬁcantly reduced with a 40 lg/ml ARB
Arbidol is a small indole-derivatives molecule that was ﬁrst
marketed in Russia in 1993 and in China in 2006 for prophylaxis
and treatment of infections by inﬂuenza A and B viruses (Boriskin
et al., 2008; Brooks et al., 2004). This drug proved to be efﬁcient to
reduce the duration of illness and to prevent the development of
post-inﬂuenza complications (Leneva et al., 2009). Moreover, despite a clinical use for more than 15 years, no ARB-resistant viruses
have been isolated so far and clinical trials revealed that the drug
was well tolerated with minor side effects (Liu et al., 2009). More
than 17 tonnes of ARB are used yearly in Russia for the treatment
of acute respiratory infectious syndromes. In addition to possible
immune-modulatory effects, ARB demonstrated a broad-spectrum
antiviral activity against a number of enveloped and non-enveloped viruses which could in part be due to its membranotropism
(Shi et al., 2007). Indeed, it has been shown that ARB was an entry
inhibitor of inﬂuenza virus infection by stabilizing the inﬂuenza HA
and preventing the endosomal membrane fusion (Leneva et al.,
2009). Similarly, ARB proved to be active in vitro against HCV infection and the antiviral mechanism was related to inhibition of the
HCV glycoprotein conformational changes needed for the membrane fusion process (Boriskin et al., 2008; Pecheur et al., 2007;
Teissier et al., 2011).
Since 2004, CHIKV has been identiﬁed as a reemerging arbovirus with a high epidemic potential in relation with its possible dissemination by rapid long-distance travels and its transmissibility
by urban mosquito vectors (Chevillon et al., 2008). Originally conﬁned to the developing nations, the virus began to encroach into
the boundaries of the developing world but there is currently no
vaccine available or effective treatment for CHIKV infection. In this
context, the development of prophylactic and therapeutic
strategies for CHIKV infection is important (Couderc et al., 2009;
Thiboutot et al., 2010).
Until now, our understanding of the interactions of CHIKV with
human cells remains limited. However, CHIKV is capable of replication in vitro in a variety of mammalian cells with the production of
characteristic CPE. Among these cells, continuous Vero and MRC5
ones are highly permissive to CHIKV infection. Primary human
ﬁbroblasts as MRC5 are cells of speciﬁc interest since human
ﬁbroblasts are known targets of CHIKV infection (Couderc et al.,
2008; Sourisseau et al., 2007).
In the current study, we used these two cell lines to demonstrate that ARB was able to inhibit CHIKV replication when added
before infection. The IC50 values obtained 610 lg/ml indicated that
I. Delogu et al. / Antiviral Research 90 (2011) 99–107
ARB was active at concentrations which were signiﬁcantly lower
than its cytotoxic concentrations (CC50 P 200 lg/ml), and similar
to those observed for the inﬂuenza A virus. The high CC50 values
measured could be explained by the short time of drug exposition
(18 or 48 h) in our experiments. This choice was justiﬁed on the
one hand by the high rate of CHIKV growth and on the other hand
by the low half-life of ARB in cultured cells (about 18 h). Moreover,
in relation with its ester prodrug nature, it is known that ARB antiviral activity may be produced by one or several hydrolyzed
metabolites. Among them oxidized sulfone and sulfoxide forms
of ARB are strongly represented (Anisimova et al., 1995; Boriskin
et al., 2008). However, our results did not conﬁrm this hypothesis
in the case of CHIKV, with no effect of two derived synthetic HZ2
products (HZ1, HZ3) on CHIKV replication. In addition, no virucidal
effect of ARB HZ2 for CHIKV was observed, in opposition with previous studies on inﬂuenza A virus, respiratory syncitial virus, human rhinovirus type 14 and coxsackie virus B3 (Shi et al., 2007)
but in agreement with data reported for Hantaan virus (Deng
et al., 2009). This result combined with those obtained in our
time-of-drug-addition experiment indicated that ARB blocks the
earliest stages of the CHIKV replication cycle (i.e., virus attachment
and/or virus entry) as previously demonstrated for other enveloped viruses (Boriskin et al., 2008). To further characterize the
mechanism of ARB action, we selected a CHIKV mutant and identiﬁed a single crucial amino acid substitution G407R localized in the
E2 viral envelope protein. Sequence alignments revealed that this
residue G407 was not conserved throughout the alphavirus genus.
Based on recent structural analysis of the CHIKV glycoprotein, this
mutation was shown to be localized in the domain A of E2 and
more precisely in the ‘‘wings’’ insertion which could be involved
in alphavirus interactions with cell receptors (Voss et al., 2010).
Accordingly, the mechanism or arbidol antiviral activity may be related to cell adsorption. This hypothesis was reinforced by the observed inhibition of CHIKV hemagglutination by ARB. However,
structural studies have also demonstrated that CHIKV E2 protein
was tightly associated with E1, a viral protein mostly implicated
in membrane fusion (Kielian, 2010; Li et al., 2010). Although the
E2 domain B has been identiﬁed as the covered E1 fusion loop, it
cannot be excluded that residue 407 may be indirectly involved
in the fusion process. Altogether, while a precise mechanism
remains to be fully elucidated, our results strongly suggested that
arbidol interferes with the early stages of Chikungunya virus
infection (virus attachment or entry) by targeting the cellular
membrane. In conclusion, arbidol, a molecule which has been
extensively used previously for the treatment of viral respiratory
infections in humans, was found to be a potent inhibitor against
in vitro CHIKV infection. In addition to the elucidation of its cellular
mode of action, this drug should be further evaluated for the
prevention of CHIKV infection and for the management of severe
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