ArticlePDF Available

Chloroquine is a potent inhibitor of SARS coronavirus infection and spread


Abstract and Figures

Severe acute respiratory syndrome (SARS) is caused by a newly discovered coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available. We report, however, that chloroquine has strong antiviral effects on SARS-CoV infection of primate cells. These inhibitory effects are observed when the cells are treated with the drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of infection and spread of SARS CoV at clinically admissible concentrations. Chloroquine is effective in preventing the spread of SARS CoV in cell culture. Favorable inhibition of virus spread was observed when the cells were either treated with chloroquine prior to or after SARS CoV infection. In addition, the indirect immunofluorescence assay described herein represents a simple and rapid method for screening SARS-CoV antiviral compounds.
Content may be subject to copyright.
BioMed Central
Page 1 of 10
(page number not for citation purposes)
Virology Journal
Open Access
Chloroquine is a potent inhibitor of SARS coronavirus infection and
Martin J Vincent1, Eric Bergeron2, Suzanne Benjannet2, Bobbie R Erickson1,
Pierre E Rollin1, Thomas G Ksiazek1, Nabil G Seidah2 and Stuart T Nichol*1
Address: 1Special Pathogens Brach, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road,
Atlanta, Georgia, 30333, USA and 2Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Ave West,
Montreal, QCH2W1R7, Canada
Email: Martin J Vincent -; Eric Bergeron -; Suzanne Benjannet -;
Bobbie R Erickson -; Pierre E Rollin -; Thomas G Ksiazek -;
Nabil G Seidah -; Stuart T Nichol* -
* Corresponding author
severe acute respiratory syndrome coronaviruschloroquineinhibitiontherapy
Background: Severe acute respiratory syndrome (SARS) is caused by a newly discovered
coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available.
Results: We report, however, that chloroquine has strong antiviral effects on SARS-CoV infection
of primate cells. These inhibitory effects are observed when the cells are treated with the drug
either before or after exposure to the virus, suggesting both prophylactic and therapeutic
advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal
pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensin-
converting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the
infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of
infection and spread of SARS CoV at clinically admissible concentrations.
Conclusion: Chloroquine is effective in preventing the spread of SARS CoV in cell culture.
Favorable inhibition of virus spread was observed when the cells were either treated with
chloroquine prior to or after SARS CoV infection. In addition, the indirect immunofluorescence
assay described herein represents a simple and rapid method for screening SARS-CoV antiviral
Severe acute respiratory syndrome (SARS) is an emerging
disease that was first reported in Guangdong Province,
China, in late 2002. The disease rapidly spread to at least
30 countries within months of its first appearance, and
concerted worldwide efforts led to the identification of
the etiological agent as SARS coronavirus (SARS-CoV), a
novel member of the family Coronaviridae [1]. Complete
genome sequencing of SARS-CoV [2,3] confirmed that
this pathogen is not closely related to any of the
Published: 22 August 2005
Virology Journal 2005, 2:69 doi:10.1186/1743-422X-2-69
Received: 12 July 2005
Accepted: 22 August 2005
This article is available from:
© 2005 Vincent et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Virology Journal 2005, 2:69
Page 2 of 10
(page number not for citation purposes)
previously established coronavirus groups. Budding of the
SARS-CoV occurs in the Golgi apparatus [4] and results in
the incorporation of the envelope spike glycoprotein into
the virion. The spike glycoprotein is a type I membrane
protein that facilitates viral attachment to the cellular
receptor and initiation of infection, and angiotensin-con-
verting enzyme-2 (ACE2) has been identified as a func-
tional cellular receptor of SARS-CoV [5]. We have recently
shown that the processing of the spike protein was
effected by furin-like convertases and that inhibition of
this cleavage by a specific inhibitor abrogated cytopathic-
ity and significantly reduced the virus titer of SARS-CoV
Due to the severity of SARS-CoV infection, the potential
for rapid spread of the disease, and the absence of proven
effective and safe in vivo inhibitors of the virus, it is impor-
tant to identify drugs that can effectively be used to treat
or prevent potential SARS-CoV infections. Many novel
therapeutic approaches have been evaluated in laboratory
studies of SARS-CoV: notable among these approaches are
those using siRNA [7], passive antibody transfer [8], DNA
vaccination [9], vaccinia or parainfluenza virus expressing
the spike protein [10,11], interferons [12,13], and mono-
clonal antibody to the S1-subunit of the spike glycopro-
tein that blocks receptor binding [14]. In this report, we
describe the identification of chloroquine as an effective
pre- and post-infection antiviral agent for SARS-CoV.
Chloroquine, a 9-aminoquinoline that was identified in
1934, is a weak base that increases the pH of acidic vesi-
cles. When added extracellularly, the non-protonated por-
tion of chloroquine enters the cell, where it becomes
protonated and concentrated in acidic, low-pH
organelles, such as endosomes, Golgi vesicles, and lyso-
somes. Chloroquine can affect virus infection in many
ways, and the antiviral effect depends in part on the extent
to which the virus utilizes endosomes for entry. Chloro-
quine has been widely used to treat human diseases, such
as malaria, amoebiosis, HIV, and autoimmune diseases,
without significant detrimental side effects [15]. Together
with data presented here, showing virus inhibition in cell
culture by chloroquine doses compatible with patient
treatment, these features suggest that further evaluation of
chloroquine in animal models of SARS-CoV infection
would be warranted as we progress toward finding effec-
tive antivirals for prevention or treatment of the disease.
Preinfection chloroquine treatment renders Vero E6 cells
refractory to SARS-CoV infection
In order to investigate if chloroquine might prevent SARS-
CoV infection, permissive Vero E6 cells [1] were pre-
treated with various concentrations of chloroquine (0.1–
10 µM) for 20–24 h prior to virus infection. Cells were
then infected with SARS-CoV, and virus antigens were vis-
ualized by indirect immunofluorescence as described in
Materials and Methods. Microscopic examination (Fig.
1A) of the control cells (untreated, infected) revealed
extensive SARS-CoV-specific immunostaining of the mon-
olayer. A dose-dependant decrease in virus antigen-posi-
tive cells was observed starting at 0.1 µM chloroquine, and
concentrations of 10 µM completely abolished SARS-CoV
infection. For quantitative purposes, we counted the
number of cells stained positive from three random loca-
tions on a slide. The average number of positively stained
control cells was scored as 100% and was compared with
the number of positive cells observed under various chlo-
roquine concentrations (Fig. 1B). Pretreatment with 0.1,
1, and 10 µM chloroquine reduced infectivity by 28%,
53%, and 100%, respectively. Reproducible results were
obtained from three independent experiments. These data
demonstrated that pretreatment of Vero E6 cells with
chloroquine rendered these cells refractory to SARS-CoV
Postinfection chloroquine treatment is effective in
preventing the spread of SARS-CoV infection
In order to investigate the antiviral properties of chloro-
quine on SARS-CoV after the initiation of infection, Vero
E6 cells were infected with the virus and fresh medium
supplemented with various concentrations of chloro-
quine was added immediately after virus adsorption.
Infected cells were incubated for an additional 16–18 h,
after which the presence of virus antigens was analyzed by
indirect immunofluorescence analysis. When chloro-
quine was added after the initiation of infection, there was
a dramatic dose-dependant decrease in the number of
virus antigen-positive cells (Fig. 2A). As little as 0.1–1 µM
chloroquine reduced the infection by 50% and up to 90–
94% inhibition was observed with 33–100 µM concentra-
tions (Fig. 2B). At concentrations of chloroquine in excess
of 1 µM, only a small number of individual cells were ini-
tially infected, and the spread of the infection to adjacent
cells was all but eliminated. A half-maximal inhibitory
effect was estimated to occur at 4.4 ± 1.0 µM chloroquine
(Fig. 2C). These data clearly show that addition of chloro-
quine can effectively reduce the establishment of infection
and spread of SARS-CoV if the drug is added immediately
following virus adsorption.
Electron microscopic analysis indicated the appearance of
significant amounts of extracellular virus particles 5–6 h
after infection [16]. Since we observed antiviral effects by
chloroquine immediately after virus adsorption, we fur-
ther extended the analysis by adding chloroquine 3 and 5
h after virus adsorption and examined for the presence of
virus antigens after 20 h. We found that chloroquine was
still significantly effective even when added 5 h after infec-
tion (Fig. 3); however, to obtain equivalent antiviral
Virology Journal 2005, 2:69
Page 3 of 10
(page number not for citation purposes)
effect, a higher concentration of chloroquine was required
if the drug was added 3 or 5 h after adsorption.
Ammonium chloride inhibits SARS-CoV infection of Vero
E6 cells
Since chloroquine inhibited SARS-CoV infection when
added before or after infection, we hypothesized that
another common lysosomotropic agent, NH4Cl, might
also function in a similar manner. Ammonium chloride
has been widely used in studies addressing endosome-
mediated virus entry. Coincidently, NH4Cl was recently
shown to reduce the transduction of pseudotype viruses
decorated with SARS-CoV spike protein [17,18]. In an
attempt to examine if NH4Cl functions similarly to chlo-
roquine, we performed infection analyses in Vero E6 cells
before (Fig. 4A) and after (Fig. 4B) they were treated with
various concentrations of NH4Cl. In both cases, we
observed a 93–99% inhibition with NH4Cl at 5 mM.
These data indicated that NH4Cl ( 5 mM) and chloro-
quine ( 10 µM) are very effective in reducing SARS-CoV
infection. These results suggest that effects of chloroquine
and NH4Cl in controlling SARS CoV infection and spread
might be mediated by similar mechanism(s).
Effect of chloroquine and NH4Cl on cell surface expression
of ACE2
We performed additional experiments to elucidate the
mechanism of SARS-CoV inhibition by chloroquine and
NH4Cl. Since intra-vesicular acidic pH regulates cellular
functions, including N-glycosylation trimming, cellular
trafficking, and various enzymatic activities, it was of
interest to characterize the effect of both drugs on the
processing, glycosylation, and cellular sorting of SARS-
CoV spike glycoprotein and its receptor, ACE2. Flow
cytometry analysis was performed on Vero E6 cells that
were either untreated or treated with highly effective anti-
SARS-CoV concentrations of chloroquine or NH4Cl. The
results revealed that neither drug caused a significant
change in the levels of cell-surface ACE2, indicating that
the observed inhibitory effects on SARS-CoV infection are
not due to the lack of available cell-surface ACE2 (Fig.
5A). We next analyzed the molecular forms of endog-
Prophylactic effect of chloroquineFigure 1
Prophylactic effect of chloroquine. Vero E6 cells pre-treated with chloroquine for 20 hrs. Chloroquine-containing media
were removed and the cells were washed with phosphate buffered saline before they were infected with SARS-CoV (0.5 mul-
tiplicity of infection) for 1 h. in the absence of chloroquine. Virus was then removed and the cells were maintained in Opti-
MEM (Invitrogen) for 16–18 h in the absence of chloroquine. SARS-CoV antigens were stained with virus-specific HMAF, fol-
lowed by FITC-conjugated secondary antibodies. (A) The concentration of chloroquine used is indicated on the top of each
panel. (B) SARS-CoV antigen-positive cells at three random locations were captured by using a digital camera, the number of
antigen-positive cells was determined, and the average inhibition was calculated. Percent inhibition was obtained by considering
the untreated control as 0% inhibition. The vertical bars represent the range of SEM.
Virology Journal 2005, 2:69
Page 4 of 10
(page number not for citation purposes)
enous ACE2 in untreated Vero E6 cells and in cells that
were pre-incubated for 1 h with various concentrations of
either NH4Cl (2.5–10 mM) or chloroquine (1 and 10 µM)
and labeled with 35S-(Met) for 3 h in the presence or
absence of the drugs (Fig. 5B and 5C). Under normal con-
ditions, we observed two immunoreactive ACE2 forms,
migrating at ~105 and ~113 kDa, respectively (Fig. 5B,
lane 1). The ~105-kDa protein is endoglycosidase H sen-
sitive, suggesting that it represents the endoplasmic retic-
ulum (ER) localized form, whereas the ~113-kDa protein
is endoglycosidase H resistant and represents the Golgi-
modified form of ACE2 [19]. The specificity of the anti-
body was confirmed by displacing the immunoreactive
protein bands with excess cold-soluble human recom-
binant ACE2 (+ rhACE2; Fig. 5B, lane 2). When we ana-
lyzed ACE2 forms in the presence of NH4Cl, a clear
stepwise increase in the migration of the ~113-kDa pro-
tein was observed with increasing concentrations of
NH4Cl, with a maximal effect observed at 10 mM NH4Cl,
resulting in only the ER form of ACE2 being visible on the
gel (Fig. 5B, compare lanes 3–5). This suggested that the
trimming and/or terminal modifications of the N-glyco-
sylated chains of ACE2 were affected by NH4Cl treatment.
In addition, at 10 mM NH4Cl, the ER form of ACE2
migrated with slightly faster mobility, indicating that
NH4Cl at that concentration might also affect core glyco-
Post-infection chloroquine treatment reduces SARS-CoV infection and spreadFigure 2
Post-infection chloroquine treatment reduces SARS-CoV infection and spread. Vero E6 cells were seeded and
infected as described for Fig. 1 except that chloroquine was added only after virus adsorption. Cells were maintained in Opti-
MEM (Invitrogen) containing chloroquine for 16–18 h, after which they were processed for immunofluorescence. (A) The con-
centration of chloroquine is indicated on the top. (B) Percent inhibition and SEM were calculated as in Fig. 1B. (C) The effec-
tive dose (ED50) was calculated using commercially available software (Grafit, version 4, Erithacus Software).
Virology Journal 2005, 2:69
Page 5 of 10
(page number not for citation purposes)
sylation. We also examined the terminal glycosylation sta-
tus of ACE2 when the cells were treated with chloroquine
(Fig. 5C). Similar to NH4Cl, a stepwise increase in the
electrophoretic mobility of ACE2 was observed with
increasing concentrations of chloroquine. At 25 µM chlo-
roquine, the faster electrophoretic mobility of the Golgi-
modified form of ACE2 was clearly evident. On the basis
of the flow cytometry and immunoprecipitation analyses,
Timed post-infection treatment with chloroquineFigure 3
Timed post-infection treatment with chloroquine. This experiment is similar to that depicted in Fig. 2 except that cells
were infected at 1 multiplicity of infection, and chloroquine (10, 25, and 50 µM) was added 3 or 5 h after infection.
NH4Cl inhibits SARS-CoV during pre or post infection treatmentFigure 4
NH4Cl inhibits SARS-CoV during pre or post infection treatment. NH4Cl was added to the cells either before (A) or
after (B) infection, similar to what was done for chloroquine in Figs 1 and 2. Antigen-positive cells were counted, and the
results were presented as in Fig. 1B.
Virology Journal 2005, 2:69
Page 6 of 10
(page number not for citation purposes)
it can be inferred that NH4Cl and chloroquine both
impaired the terminal glycosylation of ACE2, while
NH4Cl resulted in a more dramatic effect. Although ACE2
is expressed in similar quantities at the cell surface, the
variations in its glycosylation status might render the
ACE2-SARS-CoV interaction less efficient and inhibit
virus entry when the cells are treated with NH4Cl and
To confirm that ACE2 undergoes terminal sugar modifica-
tions and that the terminal glycosylation is affected by
NH4Cl or chloroquine treatment, we performed immuno-
preipitation of 35S-labeled ACE2 and subjected the immu-
Effect of lysomotropic agents on the cell-surface expression and biosynthesis of ACE2Figure 5
Effect of lysomotropic agents on the cell-surface expression and biosynthesis of ACE2. (A) Vero E6 cells were cul-
tured for 20 h in the absence (control) or presence of chloroquine (10 µM) or NH4Cl (20 mM). Cells were labeled with anti-
ACE2 (grey histogram) or with a secondary antibody alone (white histogram). (B) Biosynthesis of ACE2 in untreated cells or
in cells treated with NH4Cl. Vero E6 cells were pulse-labeled for 3 h with 35S-Met, and the cell lysates were immunoprecipi-
tated with an ACE2 antibody (lane 1). Preincunbation of the antibody with recombinant human ACE2 (rhACE2) completely
abolished the signal (lane 2). The positions of the endoglycosidase H-sensitive ER form and the endoglycosidase H-resistant
Golgi form of ACE2 are emphasized. Note that the increasing concentration of NH4Cl resulting in the decrease of the Golgi
form of ACE2. (C) A similar experiment was performed in the presence of the indicated concentrations of chloroquine. Note
the loss of terminal glycans with increasing concentrations of chloroquine. (D) The terminal glycosidic modification of ACE2
was evaluated by neuraminidase treatment of immunoprecipitated ACE2. Here cells were treated with 1–25 µM concentra-
tions of chloroquine during starvation, pulse, and 3-h chase.
Virology Journal 2005, 2:69
Page 7 of 10
(page number not for citation purposes)
noprecipitates to neuraminidase digestion. Proteins were
resolved using SDS-PAGE (Fig 5D). It is evident from the
slightly faster mobility of the Golgi form of ACE2 after
neuraminidase treatment (Fig 5D, compare lanes 1 and
2), that ACE2 undergoes terminal glycosylation; however,
the ER form of ACE2 was not affected by neuraminidase.
Cells treated with 10 µM chloroquine did not result in a
significant shift; whereas 25 µM chloroquine caused the
Golgi form of ACE2 to resolve similar to the neuramini-
dase-treated ACE2 (Fig 5D, compare lanes 5 and 6). These
data provide evidence that ACE2 undergoes terminal gly-
cosylation and that chloroquine at anti-SARS-CoV con-
centrations abrogates the process.
Effect of chloroquine and NH4Cl on the biosynthesis and
processing of SARS-CoV spike protein
We next addressed whether the lysosomotropic drugs
(NH4Cl and chloroquine) affect the biosynthesis, glyco-
sylation, and/or trafficking of the SARS-CoV spike glyco-
protein. For this purpose, Vero E6 cells were infected with
SARS-CoV for 18 h. Chloroquine or ammonium chloride
was added to these cells during while they were being
starved (1 h), labeled (30 min) or chased (3 h). The cell
lysates were analyzed by immunoprecipitation with the
SARS-specific polyclonal antibody (HMAF). The 30-min
pulse results indicated that pro-spike (proS) was synthe-
sized as a ~190-kDa precursor (proS-ER) and processed
into ~125-, ~105-, and ~80-kDa proteins (Fig. 6A, lane 2),
a result identical to that in our previous analysis [6].
Except for the 100 µM chloroquine (Fig. 6A, lane 3), there
was no significant difference in the biosynthesis or
processing of the virus spike protein in untreated or chlo-
roquine-treated cells (Fig. 6A, lanes 4–6). It should be
noted that chloroquine at 100 µM resulted in an overall
decrease in biosynthesis and in the levels of processed
virus glycoprotein. In view of the lack of reduction in the
Effects of NH4Cl and chloroquine (CQ) on the biosynthesis, processing, and glycosylation of SARS-CoV spike proteinFigure 6
Effects of NH4Cl and chloroquine (CQ) on the biosynthesis, processing, and glycosylation of SARS-CoV spike
protein. Vero E6 cells were infected with SARS-CoV as described in Fig. 2. CQ or NH4Cl was added during the periods of
starvation (1 h) and labeling (30 min) with 35S-Cys and followed by chase for 3 h in the presence of unlabeled medium. Cells
were lysed in RIPA buffer and immunoprecipitated with HMAF. Virus proteins were resolved using 3–8% NuPAGE gel (Invitro-
gen). The cells presented were labeled for 30 min (A) and chased for 3 h (B). The migration positions of the various spike
molecular forms are indicated at the right side, and those of the molecular standards are shown to the left side. proS-ER and
proS-Golgi are the pro-spike of SARS-Co in the ER and Golgi compartments, respectively and proS-ungly is the unglycosylated
pro-spike ER.
Virology Journal 2005, 2:69
Page 8 of 10
(page number not for citation purposes)
biosynthesis and processing of the spike glycoprotein in
the presence of chloroquine concentrations (10 and 50
µM) that caused large reductions in SARS-CoV replication
and spread, we conclude that the antiviral effect is proba-
bly not due to alteration of virus glycoprotein biosynthe-
sis and processing. Similar analyses were performed with
NH4Cl, and the data suggested that the biosynthesis and
processing of the spike protein were also not negatively
affected by NH4Cl (Fig. 6A, lanes 7–12). Consistent with
our previous analysis [6], we observed the presence of a
larger protein, which is referred to here as oligomers.
Recently, Song et al. [20] provided evidence that these are
homotrimers of the SARS-CoV spike protein and were
incorporated into the virions. Interestingly, the levels of
the homotrimers in cells treated with 100 µM chloroquine
and 40 and 20 mM NH4Cl (Fig. 6A, lanes 3, 9, and 10)
were slightly lower than in control cells or cells treated
with lower drug concentrations.
The data obtained from a 30-min pulse followed by a 3-h
chase (Fig. 6B, lanes 2 and 8) confirmed our earlier obser-
vation that the SARS-CoV spike protein precursor (proS-
ER) acquires Golgi-specific modifications (proS-Golgi)
resulting in a ~210-kDa protein [6]. Chloroquine at 10,
25, and 50 µM had no substantial negative impact on the
appearance of the Golgi form (Fig. 6B, compare lane 2 to
lanes 4–6). Only at 100 µM chloroquine was a reduction
in the level of the Golgi-modified pro-spike observed
(lane 3). On the other hand, NH4Cl abrogated the appear-
ance of Golgi-modified forms at 10 mM (compare lane
8 with 9–11) and had a milder effect at 1 mM (lane 12).
These data clearly demonstrate that the biosynthesis and
proteolytic processing of SARS-CoV spike protein are not
affected at chloroquine (25 and 50 µM) and NH4Cl (1
mM) doses that cause virus inhibitory effects. In addition,
with 40, 20, and 10 mM NH4Cl, there was an increased
accumulation of proS-ER with a concomitant decrease in
the amount of oligomers (Fig. 6B, lanes 9–11). When we
examined the homotrimers, we found that chloroquine at
100 µM and NH4Cl at 40 and 20 mM resulted in slightly
faster mobility of the trimers (Fig. 6B, lanes 3, 9, and 10),
but lower drug doses, which did exhibit significant antivi-
ral effects, did not result in appreciable differences. These
data suggest that the newly synthesized intracellular spike
protein may not be a major target for chloroquine and
NH4Cl antiviral action. The faster mobility of the trimer at
certain higher concentration of the drugs might be due the
effect of these drugs on the terminal glycosylation of the
We have identified chloroquine as an effective antiviral
agent for SARS-CoV in cell culture conditions, as evi-
denced by its inhibitory effect when the drug was added
prior to infection or after the initiation and establishment
of infection. The fact that chloroquine exerts an antiviral
effect during pre- and post-infection conditions suggest
that it is likely to have both prophylactic and therapeutic
advantages. Recently, Keyaerts et al. [21] reported the anti-
viral properties of chloroquine and identified that the
drug affects SARS-CoV replication in cell culture, as evi-
denced by quantitative RT-PCR. Taken together with the
findings of Keyaerts et al. [21], our analysis provides fur-
ther evidence that chloroquine is effective against SARS-
CoV Frankfurt and Urbani strains. We have provided
evidence that chloroquine is effective in preventing SARS-
CoV infection in cell culture if the drug is added to the
cells 24 h prior to infection. In addition, chloroquine was
significantly effective even when the drug was added 3–5
h after infection, suggesting an antiviral effect even after
the establishment of infection. Since similar results were
obtained by NH4Cl treatment of Vero E6 cells, the under-
lying mechanism(s) of action of these drugs might be
Apart from the probable role of chloroquine on SARS-
CoV replication, the mechanisms of action of chloroquine
on SARS-CoV are not fully understood. Previous studies
have suggested the elevation of pH as a mechanism by
which chloroquine reduces the transduction of SARS-CoV
pseudotype viruses [17,18]. We examined the effect of
chloroquine and NH4Cl on the SARS-CoV spike proteins
and on its receptor, ACE2. Immunoprecipitation results of
ACE2 clearly demonstrated that effective anti-SARS-CoV
concentrations of chloroquine and NH4Cl also impaired
the terminal glycosylation of ACE2. However, the flow
cytometry data demonstrated that there are no significant
differences in the cell surface expression of ACE2 in cells
treated with chloroquine or NH4Cl. On the basis of these
results, it is reasonable to suggest that the pre-treatment
with NH4Cl or chloroquine has possibly resulted in the
surface expression of the under-glycosylated ACE2. In the
case of chloroquine treatment prior to infection, the
impairment of terminal glycosylation of ACE2 may result
in reduced binding affinities between ACE2 and SARS-
CoV spike protein and negatively influence the initiation
of SARS-CoV infection. Since the biosynthesis, processing,
Golgi modification, and oligomerization of the newly
synthesized spike protein were not appreciably affected by
anti-SARS-CoV concentrations of either chloroquine or
NH4Cl, we conclude that these events occur in the cell
independent of the presence of the drugs. The potential
contribution of these drugs in the elevation of endosomal
pH and its impact on subsequent virus entry or exit could
not be ruled out. A decrease in SARS-CoV pseudotype
transduction in the presence of NH4Cl was observed and
was attributed to the effect on intracellular pH [17,18].
When chloroquine or NH4Cl are added after infection,
these agents can rapidly raise the pH and subvert on-going
Virology Journal 2005, 2:69
Page 9 of 10
(page number not for citation purposes)
fusion events between virus and endosomes, thus inhibit-
ing the infection.
In addition, the mechanism of action of NH4Cl and chlo-
roquine might depend on when they were added to the
cells. When added after the initiation of infection, these
drugs might affect the endosome-mediated fusion, subse-
quent virus replication, or assembly and release. Previous
studies of chloroquine have demonstrated that it has mul-
tiple effects on mammalian cells in addition to the eleva-
tion of endosomal pH, including the prevention of
terminal glycosyaltion of immunoglobulins [22]. When
added to virus-infected cells, chloroquine inhibited later
stages in vesicular stomatitis virus maturation by inhibit-
ing the glycoprotein expression at the cell surface [23],
and it inhibited the production of infectious HIV-1 parti-
cles by interfering with terminal glycosylation of the glyc-
oprotein [24,25]. On the basis of these properties, we
suggest that the cell surface expression of under-glyco-
sylated ACE2 and its poor affinity to SARS-CoV spike pro-
tein may be the primary mechanism by which infection is
prevented by drug pretreatment of cells prior to infection.
On the other hand, rapid elevation of endosomal pH and
abrogation of virus-endosome fusion may be the primary
mechanism by which virus infection is prevented under
post-treatment conditions. More detailed SARS CoV
spike-ACE2 binding assays in the presence or absence of
chloroquine will be performed to confirm our findings.
Our studies indicate that the impact of NH4Cl and chloro-
quine on the ACE2 and spike protein profiles are signifi-
cantly different. NH4Cl exhibits a more pronounced effect
than does chloroquine on terminal glycosylation, high-
lighting the novel intricate differences between chloro-
quine and ammonium chloride in affecting the protein
transport or glycosylation of SARS-CoV spike protein and
its receptor, ACE2, despite their well-established similar
effects of endosomal pH elevation.
The infectivity of coronaviruses other than SARS-CoV are
also affected by chloroquine, as exemplified by the
human CoV-229E [15]. The inhibitory effects observed on
SARS-CoV infectivity and cell spread occurred in the pres-
ence of 1–10 µM chloroquine, which are plasma concen-
trations achievable during the prophylaxis and treatment
of malaria (varying from 1.6–12.5 µM) [26] and hence are
well tolerated by patients. It recently was speculated that
chloroquine might be effective against SARS and the
authors suggested that this compound might block the
production of TNFα, IL6, or IFNγ [15]. Our data provide
evidence for the possibility of using the well-established
drug chloroquine in the clinical management of SARS.
Chloroquine, a relatively safe, effective and cheap drug
used for treating many human diseases including malaria,
amoebiosis and human immunodeficiency virus is effec-
tive in inhibiting the infection and spread of SARS CoV in
cell culture. The fact that the drug has significant inhibi-
tory antiviral effect when the susceptible cells were treated
either prior to or after infection suggests a possible pro-
phylactic and therapeutic use.
SARS-CoV infection, immunofluorescence, and
immunoprecipitation analyses
Vero E6 cells (an African green monkey kidney cell line)
were infected with SARS-CoV (Urbani strain) at a multi-
plicity of infection of 0.5 for 1 h. The cells were washed
with PBS and then incubated in OPTI-MEM (Invitrogen)
medium with or without various concentrations of either
chloroquine or NH4Cl (both from Sigma). Immunofluo-
rescence staining was performed with SARS-CoV-specific
hyperimmune mouse ascitic fluid (HMAF) [8] followed
by anti-mouse fluorescein-coupled antibody.
Eighteen hours after infection, the virus-containing super-
natants were removed, and the cells were pulsed with 35S-
(Cys) for 30 min and chased for 3 h before lysis in RIPA
buffer. Clarified cell lysates and media were incubated
with HMAF, and immunoprecipitated proteins were sepa-
rated by 3–8% NuPAGE gel (Invitrogen); proteins were
visualized by autoradiography. In some experiments, cells
were chased for 3 h with isotope-free medium. Clarified
cell supernatants were also immunoprecipitated with
SARS-CoV-specific HMAF.
ACE2 flow cytometry analysis and biosynthesis
Vero E6 cells were seeded in Dulbecco's modified Eagle
medium (Invitrogen) supplemented with 10% fetal
bovine serum. The next day, the cells were incubated in
Opti-MEM (Invitrogen) in the presence or absence of 10
µM chloroquine or 20 mM NH4Cl. To analyze the levels
of ACE2 at the cell surface, cells were incubated on ice
with 10 µg/mL affinity-purified goat anti-ACE2 antibody
(R&D Systems) and then incubated with FITC-labeled
swine anti-goat IgG antibody (Caltag Laboratories).
Labeled cells were analyzed by flow cytometry with a FAC-
SCalibur flow cytometer (BD Biosciences). For ACE2 bio-
synthesis studies, Vero E6 cells were pulsed with 250 µCi
35S-(Met) (Perkin Elmer) for 3 h with the indicated con-
centrations of chloroquine or NH4Cl and then lysed in
RIPA buffer. Clarified lysates were immunoprecipitated
with an affinity-purified goat anti-ACE2 antibody (R&D
systems), and the immunoprecipitated proteins were sep-
arated by SDS-polyacrylamide gel electrophoresis.
Competing interests
The author(s) declare that they have no competing
Virology Journal 2005, 2:69
Page 10 of 10
(page number not for citation purposes)
Authors' contributions
MV did all the experiments pertaining to SARS CoV infec-
tion and coordinated the drafting of the manuscript. EB
and SB performed experiments on ACE2 biosynthesis and
FACS analysis. BE performed data acquisition from the
immunofluorescence experiments. PR and TK provided
critical reagents and revised the manuscript critically. NS
and SN along with MV and EB participated in the plan-
ning of the experiments, review and interpretation of data
and critical review of the manuscript. All authors read and
approved the content of the manuscript.
We thank Claudia Chesley and Jonathan Towner for critical reading of the
manuscript. This work was supported by a Canadian PENCE grant (T3),
CIHR group grant #MGC 64518, and CIHR grant #MGP-44363 (to NGS).
1. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S,
Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE,
Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota PB, Fields B,
DeRisi J, Yang JY, Cox N, Hughes J, LeDuc JW, Bellini WJ, Anderson
LJ, SARS Working Group: A novel coronavirus associated with
severe acute respiratory syndrome. N Engl J Med 2003,
2. Marra MA, Jones SJ, Astell CR, Holt RA, Brooks-Wilson A, Butterfield
YS, Khattra J, Asano JK, Barber SA, Chan SY, Cloutier A, Coughlin SM,
Freeman D, Girn N, Griffith OL, Leach SR, Mayo , McDonald H,
Montgomery SB, Pandoh PK, Petrescu AS, Robertson AG, Schein JE,
Siddiqui A, Smailus DE, Stott JM, Yang GS, Plummer F, Andonov A,
Artsob H, Bastien N, Bernard K, Booth TF, Bowness D, Czub M,
Drebot M, Fernando L, Flick R, Garbutt M, Gray M, Grolla A, Jones S,
Fe ldm ann H, M eye rs A, K aba ni A , Li Y, N orm and S, Str ohe r U, Tip ple s
GA, Tyler S, Vogrig R, Ward D, Watson B, Brunham RC, Krajden M,
Petric M, Skowronski DM, Upton C, Roper RL: The Genome
sequence of the SARS-associated coronavirus. Science 2003,
3. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle
JP, Penaranda S, Bankamp B, Maher K, Chen MH, Tong S, Tamin A,
Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TC,
Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B,
Limor J, McCaustland K, Olsen Rasmussen M, Fouchier R, Gunther S,
Osterhaus AS, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ:
Characterization of a novel coronavirus associated with
severe acute respiratory syndrome. Science 2003,
4. Ng ML, Tan SH, See EE, Ooi EE, Ling AE: Proliferative growth of
SARS coronavirus in Vero E6 cells. J Gen Virol 2003,
5. Li M, Moore WJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasunda-
ran M, Sullivan JL, Luzuriaga K, Greenough TC, Choe H, Farzan M:
Angiotensin-converting enzyme 2 is a functional receptor for
the SARS coronavirus. Nature 2003, 426:450-454.
6. Bergeron E, Vincent MJ, Wickham L, Hamelin J, Basak A, Nichol ST,
Chrétien M, NG Seidah: Implication of proprotein convertases
in the processing and spread of severe acute respiratory syn-
drome coronavirus. Biochem Biophys Res Comm 2005,
7. Zhang Y, Li T, Fu L, Yu C, Li Y, X u X , W ang Y, Ning H, Zh ang S, Ch en
W, Babiuk LA, Chang Z: Silencing SARS-CoV spike protein
expression in cultured cells by RNA interference. FEBS Lett
2004, 560:141-146.
8. Subbarao K, McAuliffe J, Vogel L, Fahle G, Fischer S, Tatti K, Packard
M, Shieh WJ, Zaki S, Murphy B: Prior infection and passive trans-
fer of neutralizing antibody prevent replication of severe
acute respiratory syndrome coronavirus in the respiratory
tract of mice. J Virol 2004, 78:3572-3577.
9. Yang ZY, Kong WP, Huang Y, Roberts A, Murphy BR, Subbarao K,
Nabel GJ: A DNA vaccine induces SARS coronavirus neutral-
ization and protective immunity in mice. Nature 2004,
10. Bisht H, Roberts A, Vogel L, Bukreyev A, Collins PL, Murphy BR, Sub-
barao K, Moss B: Severe acute respiratory syndrome corona-
virus spike protein expressed by attenuated vaccinia virus
protectively immunizes mice. Proc Natl Acad Sci USA 2004,
11. Bukreyev A, Lamirande EW, Buchholz UJ, Vogel LN, Elkins WR, St.
Claire M, Murphy BR, Subbarao K, Collins PL: Mucosal immuniza-
tion of African green monkeys (Cercopithecus aethiops)
with an attenuated parainfluenza virus expressing the SARS
coronavirus spike protein for the prevention of SARS. Lancet
2004, 363:2122-2127.
12. Sainz B Jr, Mossel EC, Peters CJ, Garry RF: Interferon-beta and
interferon-gamma synergistically inhibit the replication of
severe acute respiratory syndrome-associated coronavirus
(SARS-CoV). Virology 2004, 329:11-17.
13. Stroher U, DiCaro A, Li Y, Strong JE, Aoki F, Plummer F, Jones SM,
Feldmann H: Severe acute respiratory syndrome-related coro-
navirus is inhibited by interferon- alpha. J Infect Dis 2004,
14. Sui J, Li W, Murakami A, Tamin A, Matthews LJ, Wong SK, Moore MJ,
Tallarico AS, Olurinde M, Choe H, Anderson LJ, Bellini WJ, Farzan M,
Marasco WA: Potent neutralization of severe acute respira-
tory syndrome (SARS) coronavirus by a human mAb to S1
protein that blocks receptor association. Proc Natl Acad Sci USA
2004, 101:2536-2541.
15. Savarino A, Boelaert JR, Cassone A, Majori G, Cauda R: Effects of
chloroquine on viral infections: an old drug against today's
diseases? Lancet Infect Dis 2003, 3:722-727.
16. Ng ML, Tan SH, See EE, Ooi EE, Ling AE: Early events of SARS
coronavirus infection in vero cells. J Med Virol 2003, 71:323-331.
17. Simmons G, Reeves JD, Rennekamp AJ, Amberg SM, Piefer AJ, Bates
P: Characterization of severe acute respiratory syndrome-
associated coronavirus (SARS-CoV) spike glycoprotein-
mediated viral entry. Proc Natl Acad Sci USA 2004, 101:4240-4245.
18. Yang ZY, Huang Y, Ganesh L, Leung K, Kong WP, Schwartz O, Sub-
barao K, Nabel GJ: pH-dependent entry of severe acute respi-
ratory syndrome coronavirus is mediated by the spike
glycoprotein and enhanced by dendritic cell transfer through
DC-SIGN. J Virol 2004, 78:5642-5650.
19. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ: A
human homolog of angiotensin-converting enzyme. Cloning
and functional expression as a captopril-insensitive
carboxypeptidase. J Biol Chem 2000, 275:33238-33243.
20. Song HC, Seo MY, Stadler K, Yoo BJ, Choo QL, Coates SR, Uematsu
Y, Harada T, Greer CE, Polo JM, Pileri P, Eickmann M, Rappuoli R,
Abrignani S, Houghton M, Han JH: Synthesis and characterization
of a native, oligomeric form of recombinant severe acute
respiratory syndrome coronavirus spike glycoprotein. J Virol
2004, 78:10328-10335.
21. Keyaerts E, Vijgen L, Maes P, Neyts J, Ranst MV: In vitro inhibition
of severe acute respiratory syndrome coronavirus by
chloroquine. Biochem Biophys Res Commun 2004, 323:264-268.
22. Thorens B, Vassalli P: Chloroquine and ammonium chloride
prevent terminal glycosylation of immunoglobulins in
plasma cells without affecting secretion. Nature 1986,
23. Dille BJ, Johnson TC: Inhibition of vesicular stomatitis virus
glycoprotein expression by chloroquine. J Gen Virol 1982,
24. Tsai WP, Nara PL, Kung HF, Oroszlan S: Inhibition of human
immunodeficiency virus infectivity by chloroquine. AIDS Res
Hum Retroviruses 1990, 6:481-489.
25. Savarino A, Lucia MB, Rastrelli E, Rutella S, Golotta C, Morra E, Tam-
burrini E, Perno CF, Boelaert JR, Sperber K, Cauda RC: Anti-HIV
effects of chloroquine: inhibition of viral particle glycosyla-
tion and synergism with protease inhibitors. J Acquir Immune
Defic Syndr 2004, 35:223-232.
26. Ducharme J, Farinotti R: Clinical pharmacokinetics and metab-
olism of chloroquine. Focus on recent advancements. Clin
Pharmacokinet 1996, 31:257-274.
... At the time this study was designed, a potential candidate which met the criteria for medication repurposing towards treating or preventing COVID-19 was hydroxychloroquine (HCQ). HCQ alters the cellular receptor for SARS-CoV-2, can inhibit the entry of SARS-CoV-2 into the cell, and can inhibit both endocytosis and replication within cells [16][17][18][19][20]. Additionally, HCQ has previously been used effectively for malaria prophylaxis with a favourable side effect profile at low doses, concentrates in the lung tissues and has a pharmacokinetic profile that lends itself to weekly, rather than daily, dosing [21,22]. ...
Full-text available
Background Coronavirus disease 2019 (COVID-19) has exposed the disproportionate effects of pandemics on frontline workers and the ethical imperative to provide effective prophylaxis. We present a model for a pragmatic randomised controlled trial (RCT) that utilises Bayesian methods to rapidly determine the efficacy or futility of a prophylactic agent. Methods We initially planned to undertake a multicentre, phase III, parallel-group, open-label RCT, to determine if hydroxychloroquine (HCQ) taken once a week was effective in preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in healthcare workers (HCW) aged ≥ 18 years in New Zealand (NZ) and Ireland. Participants were to be randomised 2:1 to either HCQ (800 mg stat then 400 mg weekly) or no prophylaxis. The primary endpoint was time to Nucleic Acid Amplification Test-proven SARS-CoV-2 infection. Secondary outcome variables included mortality, hospitalisation, intensive care unit admissions and length of mechanical ventilation. The trial had no fixed sample size or duration of intervention. Bayesian adaptive analyses were planned to occur fortnightly, commencing with a weakly informative prior for the no prophylaxis group hazard rate and a moderately informative prior on the intervention log hazard ratio centred on ‘no effect’. Stopping for expected success would be executed if the intervention had a greater than 0.975 posterior probability of reducing the risk of SARS-CoV-2 infection by more than 10%. Final success would be declared if, after completion of 8 weeks of follow-up (reflecting the long half-life of HCQ), the prophylaxis had at least a 0.95 posterior probability of reducing the risk of SARS-CoV-2 infection by more than 10%. Futility would be declared if HCQ was shown to have less than a 0.10 posterior probability of reducing acquisition of SARS-CoV-2 infection by more than 20%. Discussion This study did not begin recruitment due to the marked reduction in COVID-19 cases in NZ and concerns regarding the efficacy and risks of HCQ treatment in COVID-19. Nonetheless, the model presented can be easily adapted for other potential prophylactic agents and pathogens, and pre-established collaborative models like this should be shared and incorporated into future pandemic preparedness planning. Trial registration The decision not to proceed with the study was made before trial registration occurred.
... Hydroxychloroquine and chloroquine have been labeled as potential -game-changers‖ in the popular press for COVID-19. [4] The reason behind the direct use of these two drugs was inhibitory effects on other coronaviruses [5,6] , such as SARS-CoV-1 i. e. Severe Acute Respiratory Syndrome coronavirus first emerged in 2002 in China and was subsequently first identified in 2003. [7] Chloroquine and Hydroxychloroquine have been used in the treatment of various diseases like lupus erythematosus, rheumatoid arthritis, malaria, amebiasis that is occurring outside the intestines, rheumatoid arthritis, and lupus erythematosus for more than 70 years. ...
Full-text available
Chloroquine and Hydroxychloroquine have been used in the treatment of various diseases like lupus erythematosus, rheumatoid arthritis, malaria, amebiasis that is occurring outside the intestines, rheumatoid arthritis, and lupus erythematosus for more than 70 years. On 11 of march in the afternoon World Health Organization (WHO) declared that covid-19 was a pandemic disease and they also declared that already the number of cases outside China was increased 13-fold, and the number of affected countries has tripled. But at that time no such medical treatments were available for covid-19. So, in this emergency, the only option available the use of existing medications. This systematic review aims to summarize the chemical structure, mechanism of action, pharmacokinetics, and available evidence regarding the role of chloroquine and hydroxychloroquine in treating coronavirus infection. In this review, we discuss the available evidence for the use of chloroquine (CQ) and hydroxychloroquine (HCQ) against COVID-19. Chloroquine and hydroxychloroquine both have unusual pharmacokinetic properties with enormous apparent volumes of distribution (chloroquine > hydroxychloroquine) and very slow elimination from the body. There is theoretical, experimental, preclinical, and clinical evidence of the effectiveness of chloroquine in patients affected with COVID-19. There is adequate evidence of drug safety from the long-time clinical use of chloroquine and hydroxychloroquine in covid-19.
... It has been reported that modulation of autophagy inhibits coronavirus infection, making it a promising target for anticoronavirus treatment. For example, autophagy inhibitor chloroquine blocked autophagic flux and prevented the formation of autophagosome, thereby inhibiting the replication of SARS-CoV and SARS-CoV-2 (Vincent et al., 2005;Mauthe et al., 2018;Yao et al., 2020). In addition, autophagy induced by rapamycin can suppress the infection of MERS-CoV and TGEV (Kindrachuk et al., 2015;Guo et al., 2016). ...
Full-text available
Porcine epidemic diarrhea virus (PEDV) could cause lethal diarrhea and dehydration in suckling piglets, which can adversely affect the development of the global swine industry. The lack of effective therapeutical and prophylactic treatment especially for PEDV variant strains underlines the importance of effective antiviral strategies, such as identification of novel antiviral agents. In the present study, the antiviral activity of cinchonine against PEDV was investigated in Vero CCL81 and LLC-PK1 cells at a non-cytotoxic concentration determined by Cell Counting Kit-8 assay in vitro. We found that cinchonine exhibited a significant suppression effect against PEDV infection and its inhibitory action was primarily focused on the early stage of PEDV replication. Moreover, we also observed that cinchonine could significantly induce autophagy by detecting the conversion of LC3-I to LC3-II by using western blot analysis. Cinchonine treatment could inhibit PEDV replication in a dose-dependent manner in Vero CCL81 cells, while this phenomenon disappeared when autophagy was attenuated by pre-treatment with autophagy inhibitor 3MA. Consequently, this study indicated that cinchonine can inhibit PEDV replication via inducing cellular autophagy and thus from the basis for successful antiviral strategies which potentially suggest the possibility of exploiting cinchonine as a novel antiviral agent.
... It should be noted that most of them exert anti-viral activity by regulating autophagy as well as other mechanisms. Chloroquine (CQ) and its less toxic derivative, hydroxychloroquine (HCQ), two FDA-approved anti-malarial drugs, exhibit autophagy inhibition, immunosuppressive functions, anti-inflammatory properties as well as anti-viral activity, which has been proposed against SARS-CoV, 287 HIV, 288 and Zika. 289 The previous study has shown that they can inhibit autophagic flux by disturbing autophagosomelysosome fusion 290 and endosomal/lysosomal acidification, 291 resulting in accumulative autophagosomes-induced cell death and cessation of virus replication. ...
Full-text available
The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has dramatically influenced various aspects of the world. It is urgent to thoroughly study pathology and underlying mechanisms for developing effective strategies to prevent and treat this threatening disease. It is universally acknowledged that cell death and cell autophagy are essential and crucial to maintaining host homeostasis and participating in disease pathogenesis. At present, more than twenty different types of cell death have been discovered, some parts of which have been fully understood, whereas some of which need more investigation. Increasing studies have indicated that cell death and cell autophagy caused by coronavirus might play an important role in virus infection and pathogenicity. However, the knowledge of the interactions and related mechanisms of SARS-CoV-2 between cell death and cell autophagy lacks systematic elucidation. Therefore, in this review, we comprehensively delineate how SARS-CoV-2 manipulates diverse cell death (including apoptosis, necroptosis, pyroptosis, ferroptosis, and NETosis) and cell autophagy for itself benefits, which is simultaneously involved in the occurrence and progression of COVID-19, aiming to provide a reasonable basis for the existing interventions and further development of novel therapies.
Background: The Royal College of Ophthalmologists has produced guidelines for screening for hydroxychloroquine retinopathy. New imaging modalities had suggested an increased prevalence of retinopathy compared with previous reports. The aim of this study is to identify the real-life prevalence of hydroxychloroquine retinopathy in patients attending Manchester Royal Eye Hospital screening service over a 2-year period using The RCOphth diagnostic criteria. Methods: Data were collected prospectively from all patients attending Manchester Royal Eye Hospital hydroxychloroquine screening service over the audit period. Results of Humphrey visual field tests, spectral-domain optical coherence tomography and fundus autofluorescence were collected as well as data on dose, indication, duration of treatment, and additional risk factors. Cases were identified as having definite, possible, or no retinopathy based on the 2018 RCOphth criteria. The data are not publicly available due to information that could compromise research participant privacy and confidentiality but are available upon request from the corresponding author. Results: 910 patients attended for screening. 566 were identified as being at risk of retinopathy (543 had been on treatment >5 years, 10 had renal impairment, 12 were on doses of >5 mg/kg/day, and one was concurrently on tamoxifen). The prevalence of HCQ retinopathy was 10/910 (1.09%) of all those screened, and 1.76% of those at risk (10/566). Six patients of those deemed at risk were identified as having definite hydroxychloroquine retinopathy, while four had possible retinopathy. Conclusions: Our results show a prevalence of retinopathy largely consistent with reports from regional audits yet reveal a far lower estimate compared to previously reported figures.
Giriş ve Amaç: Bu çalışmada Eğitim Aile Sağlığı Merkezlerine (EASM) kayıtlı ve COVID-19 nedeniyle evde hidroksiklorokin (HCQ) tedavisi alan hastaların ilaç uyumlarını ve geri bildirimlerinin değerlendirilmesi amaçlandı. Gereç ve Yöntem: Hastanemize bağlı Eğitim Aile Sağlığı Merkezlerinde kayıtlı COVID-19 nedeniyle evde tedavi gören ve HCQ tedavisi gören hastalar dahil edildi. Evde takip edilen bu hastalara ev takiplerinin 6. ve 14. günleri arasında bir kez ulaşıldı. Hastalara HCQ tedavisine uyumları, ilaç yan etkileri, geri bildirimler, ateş ve öksürük gibi semptomların süreleri, hastaneye yeniden sevklerle ilgili bilgileri içeren 31 maddeden oluşan anket soruları yöneltildi. Anket yoluyla toplanan veriler istatistiksel analiz ile değerlendirildi. İstatistiksel analizler SPSS 17.0 programı ile yapıldı. Bulgular: Çalışmaya 37'si erkek 30'u kadın toplam 67 hasta katıldı. Ortalama yaş 45.34 ± 19.01 yıldır. Orta derecede semptomları olan 7 hasta ve hafif semptomları olan 60 hasta vardı. Bir hasta ilaca bağlı cilt döküntüsü nedeniyle tedaviyi bıraktı. 63 hastanın semptomlarında azalma oldu. Hastaneye yatan 23 hastanın ortalama yatış süresi 12.96 ± 5.92 gündü. Tekrar hastaneye sevk edilen 5 hasta vardı. Azitromisin kullanımı olanlarda tekrar hastaneye sevk oranı daha düşük bulundu. Sonuç: HCQ kullanan evdeki takip edilen COVİD-19 hastalarının çoğunluğunun semptomlarında hafifleme olmuştur. HCQ ile kombine azitromisin tedavisi alanlarda tekrar hastaneye yatma oranları daha düşük gözlenmiştir.
The COVID-19 pandemic has brought fear and havoc into the world in ways we have never imagined. More than 10 million people have been infected and more than 500,000 have died worldwide. Until now no definite cure has been discovered and no vaccine is yet available for its prevention. Efforts and resources have been poured in to address these challenges. However, the future is still not secured, and lives and economies all over the world are still at risk of continued destruction. In this book chapter, we present the various mono and combination therapies currently being used by health professionals to combat this disease. To date, no single therapy has been shown to completely eradicate the infection, but interim data on the use of combination treatments have shown better outcome.
Greener and sustainable synthetic strategies have been evolving as the demanding domain of organic transformations during the last decade. The division of an environmentally benign methodology to construct bioactive heterocyclic scaffolds has always been a perpetual subject of interests for medicinal and synthetic chemists. Thus, the newer and novel synthetic methodologies of quinoline have drawn the attention of synthetic organic or medicinal chemists as evident from the growing numbers of publications. The current review focuses on some of the notable synthetic methodologies carried out in the last decade with the inherent objective to attain sustainability towards the synthesis. The key aspects of the review would include to highlight sustainable and environmentally benign approaches like the solvent‐free reactions, use of alternate reaction media (e. g., water, fluorous alcohols, polyethylene glycols, and ionic liquids), and alternate modes of synthesis such as microwave‐assisted synthesis and flow reactions, nano‐catalysts, etc. Coverage of the green synthetic reports for the synthesis of quinolines have been discussed along with their classification of sustainable alternatives, merits and demerits of the reported synthetic protocols, and special emphasis on water, microwave, ionic liquids, photocatalysts, and nano‐catalysts assisted organic transformations.
Coronavirus Disease 2019 (COVID-19) pandemic has been on the agenda of humanity for more than 2 years. In the meantime, the pandemic has caused economic shutdowns, halt of daily lives and global mobility, overcrowding of the healthcare systems, panic, and worse, more than 6 million deaths. Today, there is still no specific therapy for COVID-19. Research focuses on repurposing of antiviral drugs that are licensed or currently in the research phase, with a known systemic safety profile. However, local safety profile should also be evaluated depending on the new indication, administration route and dosage form. Additionally, various vaccines have been developed. But the causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has undergone multiple variations, too. The premise that vaccines may suffice to eradicate new and all variants is unreliable, as they are based on earlier versions of the virus. Therefore, a specific medication therapy for COVID-19 is crucial and needed in order to prevent severe complications of the disease. Even though there is no specific drug that inhibits the replication of the disease-causing virus, among the current treatment options, systemic antivirals are the most medically appropriate. As SARS-CoV-2 directly targets the lungs and initiates lung damage, treating COVID-19 with inhalants can offer many advantages over the enteral/parenteral administration. Inhaled drug delivery provides higher drug concentration, specifically in the pulmonary system. This enables the reduction of systemic side effects and produces a rapid clinical response. In this article, the most frequently (systemically) used antiviral compounds are reviewed including Remdesivir, Favipiravir, Molnupiravir, Lopinavir-Ritonavir, Umifenovir, Chloroquine, Hydroxychloroquine and Heparin. A comprehensive literature search was conducted to provide insight into the potential inhaled use of these antiviral drugs and the current studies on inhalation therapy for COVID-19 was presented. A brief evaluation was also made on the use of inhaler devices in the treatment of COVID-19. Inhaled antivirals paired with suitable inhaler devices should be considered for COVID-19 treatment options.
Full-text available
In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
Full-text available
A novel human zinc metalloprotease that has considerable homology to human angiotensin-converting enzyme (ACE) (40% identity and 61% similarity) has been identified. This metalloprotease (angiotensin-converting enzyme homolog (ACEH)) contains a single HEXXH zinc-binding domain and conserves other critical residues typical of the ACE family. The predicted protein sequence consists of 805 amino acids, including a potential 17-amino acid N-terminal signal peptide sequence and a putative C-terminal membrane anchor. Expression in Chinese hamster ovary cells of a soluble, truncated form of ACEH, lacking the transmembrane and cytosolic domains, produces a glycoprotein of 120 kDa, which is able to cleave angiotensin I and angiotensin II but not bradykinin or Hip-His-Leu. In the hydrolysis of the angiotensins, ACEH functions exclusively as a carboxypeptidase. ACEH activity is inhibited by EDTA but not by classical ACE inhibitors such as captopril, lisinopril, or enalaprilat. Identification of the genomic sequence of ACEH has shown that the ACEH gene contains 18 exons, of which several have considerable size similarity with the first 17 exons of human ACE. The gene maps to chromosomal location Xp22. Northern blotting analysis has shown that the ACEH mRNA transcript is approximately 3. 4 kilobase pairs and is most highly expressed in testis, kidney, and heart. This is the first report of a mammalian homolog of ACE and has implications for our understanding of cardiovascular and renal function.
Full-text available
A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak. We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens. None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population. A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.
Full-text available
We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.
An isolate of SARS coronavirus (strain 2003VA2774) was obtained from a patient and used to infect Vero E6 cells. The replication cycle of the virus was followed from 1 to 30 h post-infection (p.i.). It was surprising to observe the swift growth of this human virus in Vero cells. Within the first hour of infection, the most obvious ultrastructural change was the proliferation of the Golgi complexes and related vesicles accompanied by swelling of some of the trans-Golgi sacs. Extracellular virus particles were present by 5 h p.i. in about 5 % of the cells and this increased dramatically to about 30 % of the cell population within an hour (6 h p.i.). Swollen Golgi sacs contained virus nucleocapsids at different stages of maturation. These virus precursors were also in large vacuoles and in close association with membrane whorls. The membrane whorls could be the replication complexes, since they appeared rather early in the replication cycle. As infection progressed from 12 to 21 h p.i., the cytoplasm of the infected cells was filled with numerous large, smooth-membraned vacuoles containing a mixture of mature virus and spherical cores. Several of these vacuoles were close to the cell periphery, ready to export out the mature progeny virus particles via exocytosis. By 24 to 30 h p.i., crystalline arrays of the extracellular virus particles were seen commonly at the cell surface.
Cellular entry of enveloped viruses is often dependent on attachment proteins expressed on the host cell surface. Viral envelope proteins bind these receptors, and, in an incompletely understood process, facilitate fusion of the cellular and viral membranes so as to introduce the viral core into the cytoplasm. Only a small fraction of viral receptors have been identified so far. Recently, a novel coronavirus was identified as the etiological agent of severe acute respiratory syndrome (SARS). The fusion protein gene of SARS coronavirus (SARS-CoV) was cloned and characterized, and shortly thereafter, angiotensin-converting enzyme 2 (ACE2) was shown to be its functional receptor. Identification of ACE2 as a receptor for SARS-CoV will likely contribute to the development of antivirals and vaccines. It may also contribute to the development of additional animal models for studying SARS pathogenesis, and could help identify the animal reservoir of SARS-CoV.
The effect of chloroquine, a drug known to affect intracellular exocytic pathways, was studied in two retroviral systems: human immunodeficiency virus (HIV-1) and avian reticuloendotheliosis virus (REV-A). With chloroquine treatment of virus-infected cells, significant size reduction of the cell- and virus-associated surface glycoproteins, gp90 of REV-A and gp120 of HIV-1, was observed. In the case of HIV-1, extracellular virions derived from treated cells contained very little gp120. Infectivity and reverse transcriptase assays of HIV-1 demonstrated that by chloroquine treatment the majority of the virions released was noninfectious and the total virus yield was also reduced. The data suggest that chloroquine inhibition of infectious virus production is most likely due to interference with terminal glycosylation in the trans-Golgi network.
The generation of an acidic pH in intracellular organelles is required for several membrane and protein recycling processes. For instance, the internalization of ligands by receptor-mediated endocytosis is followed by the development of an acidic pH inside endosomes; this allows dissociation of the ligand, which is then transported to the lysosomes, from the receptor, which is recycled to the cell surface. There is evidence that part of this recycling process involves the distal region of the Golgi complex, where terminal glycosylation occurs: when the plasma membrane transferrin receptor is desialylated by neuraminidase treatment, it acquires new sialic acid molecules after endocytosis and before cell-surface re-expression. Golgi membranes have been shown to contain a proton pump and the distal Golgi cisternae appear to have an acidic content. Here, we have studied the effects of chloroquine and ammonium chloride, which raise the pH of acidic intracellular compartments, on the processing and secretion of immunoglobulins by plasma cells. Sialic acid transfer to terminal galactose residues, a reaction known to occur in the distal Golgi shortly before secretion, is completely and rapidly inhibited in the presence of these drugs, without significant modification of the secretion rate. This effect is accompanied by a dilatation of the Golgi cisternae and is not rapidly reversible.
Addition of 50 micrograms/ml chloroquine to neuroblastoma cells 1 h before infection with temperature-sensitive mutant ts G31 (III) of vesicular stomatitis virus (VSV) prevented virus-induced cell fusion from occurring. Interestingly, addition of chloroquine after infection still inhibited cell fusion. Based on the number of fusion events required to produce the polykaryocytes observed, cell fusion was inhibited 92% when chloroquine was added 1 h post-infection and 77% when chloroquine was added 2 h post-infection. The inhibition of virus-induced cell fusion could not be accounted for by an inhibition of virus protein synthesis because the virus protein synthesis measured 6 h post-infection was 90% of that in untreated, infected cells with chloroquine added 1 h post-infection, and the same as untreated, infected cells when chloroquine was added 2 h post-infection. No virus proteins were made, however, when chloroquine was added before infection, which is consistent with a chloroquine-mediated inhibition of virus uncoating. The release of infectious virions was completely inhibited when chloroquine was added before infection or 1 or 2 h post-infection, which indicated an inhibition of virus maturation in the later stages of virus assembly. By indirect immunofluorescence the virus glycoprotein (G protein) could not be detected on the surface of chloroquine-treated, infected cells, but the G protein was present inside the treated cells. With 125I-labelled anti-G protein IgG, 16% of the G protein found on the surface of untreated, infected cells was on the cell surface when chloroquine was added 2 h post-infection. When chloroquine was removed from infected cells, the G protein accumulated at the cell surface, and this accumulation could not be prevented by tunicamycin, an inhibitor of glycosylation. Furthermore, galactose was incorporated into the G protein in the presence of chloroquine. Therefore, the VSV G protein was being synthesized and glycosylated in the presence of chloroquine but the drug prevented the expression of the glycoprotein at the cell surface during the final stages of G protein assembly.U
This paper presents the current state of knowledge on chloroquine disposition, with special emphasis on stereoselectivity and microsomal metabolism. In addition, the impact of the patient’s physiopathological status and ethnic origin on chloroquine pharmacokinetics is discussed. In humans, chloroquine concentrations decline multiexponentially. The drug is extensively distributed, with a volume of distribution of 200 to 800 L/kg when calculated from plasma concentrations and 200 L/kg when estimated from whole blood data (concentrations being 5 to 10 times higher). Chloroquine is 60% bound to plasma proteins and equally cleared by the kidney and liver. Following administration chloroquine is rapidly dealkylated via cytochrome P450 enzymes (CYP) into the pharmacologically active desethylchloroquine and bisdesethylchloroquine. Desethylchloroquine and bisdesethylchloroquine concentrations reach 40 and 10% of chloroquine concentrations, respectively; both chloroquine and desethylchloroquine concentrations decline slowly, with elimination half-lives of 20 to 60 days. Both parent drug and metabolite can be detected in urine months after a single dose. In vitro and in vivo, chloroquine and desethylchloroquine competitively inhibit CYP2D1/6-mediated reactions. Limited in vitro studies and preliminary data from clinical experiments and observations point to CYP3A and CYP2D6 as the 2 major isoforms affected by or involved in chloroquine metabolism. In vitro efficacy studies did not detect any difference in potency between chloroquine enantiomers but, in vivo in rats, S(+)-chloroquine had a lower dose that elicited 50% of the maximal effect (ED950) than that of R(−)-chloroquine. Stereoselectivity in chloroquine body disposition could be responsible for this discrepancy. Chloroquine binding to plasma proteins is stereoselective, favouring S(+)-chloroquine (67% vs 35% for the R-enantiomer). Hence, unbound plasma concentrations are higher for R(−)-chloroquine. Following separate administration of the individual enantiomers, R(−)-chloroquine reached higher and more sustained blood concentrations. The shorter half-life of S(+)-chloroquine appears secondary to its faster clearance. Blood concentrations of the S(+)-forms of desethylchloroquine always exceeded those of the R(−)-forms, pointing to a preferential metabolism of S(+)-chloroquine.