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Liu et al. Cell Discovery (2020) 6:16 Cell Discovery
https://doi.org/10.1038/s41421-020-0156-0 www.nature.com/celldisc
CORRESPONDENCE Open Access
Hydroxychloroquine, a less toxic derivative
of chloroquine, is effective in inhibiting
SARS-CoV-2 infection in vitro
Jia Liu
1
,RuiyuanCao
2
, Mingyue Xu
1,3
,XiWang
1
, Huanyu Zhang
1,3
,HengruiHu
1,3
,YufengLi
1,3
, Zhihong Hu
1
,
Wu Zhong
2
and Manli Wang
1
Dear Editor,
The outbreak of coronavirus disease 2019 (COVID-19)
caused by the severe acute respiratory syndrome cor-
onavirus 2 (SARS-CoV-2/2019-nCoV) poses a serious
threat to global public health and local economies. As of
March 3, 2020, over 80,000 cases have been confirmed in
China, including 2946 deaths as well as over 10,566
confirmed cases in 72 other countries. Such huge num-
bers of infected and dead people call for an urgent
demand of effective, available, and affordable drugs to
control and diminish the epidemic.
We have recently reported that two drugs, remdesivir
(GS-5734) and chloroquine (CQ) phosphate, efficiently
inhibited SARS-CoV-2 infection in vitro
1
. Remdesivir is a
nucleoside analog prodrug developed by Gilead Sciences
(USA). A recent case report showed that treatment with
remdesivir improved the clinical condition of the first
patient infected by SARS-CoV-2 in the United States
2
,
and a phase III clinical trial of remdesivir against SARS-
CoV-2 was launched in Wuhan on February 4, 2020.
However, as an experimental drug, remdesivir is not
expected to be largely available for treating a very large
number of patients in a timely manner. Therefore, of the
two potential drugs, CQ appears to be the drug of choice
for large-scale use due to its availability, proven safety
record, and a relatively low cost. In light of the pre-
liminary clinical data, CQ has been added to the list of
trial drugs in the Guidelines for the Diagnosis and
Treatment of COVID-19 (sixth edition) published by
National Health Commission of the People’s Republic
of China.
CQ (N4-(7-Chloro-4-quinolinyl)-N1,N1-diethyl-1,4-
pentanediamine) has long been used to treat malaria and
amebiasis. However, Plasmodium falciparum developed
widespread resistance to it, and with the development of
new antimalarials, it has become a choice for the pro-
phylaxis of malaria. In addition, an overdose of CQ can
cause acute poisoning and death
3
. In the past years, due to
infrequent utilization of CQ in clinical practice, its pro-
duction and market supply was greatly reduced, at least in
China. Hydroxychloroquine (HCQ) sulfate, a derivative of
CQ, was first synthesized in 1946 by introducing a
hydroxyl group into CQ and was demonstrated to be
much less (~40%) toxic than CQ in animals
4
. More
importantly, HCQ is still widely available to treat auto-
immune diseases, such as systemic lupus erythematosus
and rheumatoid arthritis. Since CQ and HCQ share
similar chemical structures and mechanisms of acting as a
weak base and immunomodulator, it is easy to conjure up
the idea that HCQ may be a potent candidate to treat
infection by SARS-CoV-2. Actually, as of February 23,
2020, seven clinical trial registries were found in Chinese
Clinical Trial Registry (http://www.chictr.org.cn) for using
HCQ to treat COVID-19. Whether HCQ is as efficacious
as CQ in treating SARS-CoV-2 infection still lacks the
experimental evidence.
To this end, we evaluated the antiviral effect of HCQ
against SARS-CoV-2 infection in comparison to CQ
in vitro. First, the cytotoxicity of HCQ and CQ in African
green monkey kidney VeroE6 cells (ATCC-1586) was
measured by standard CCK8 assay, and the result showed
© The Author(s) 2020
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Correspondence: Zhihong Hu (huzh@wh.iov.cn) or Wu Zhong
(zhongwu@bmi.ac.cn) or Manli Wang (wangml@wh.iov.cn)
1
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety
Mega-Science, Chinese Academy of Sciences, 430071 Wuhan, China
2
National Engineering Research Center for the Emergency Drug, Beijing
Institute of Pharmacology and Toxicology, 100850 Beijing, China
Full list of author information is available at the end of the article.
These authors contributed equally: Jia Liu, Ruiyuan Cao, Mingyue Xu
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Fig. 1 (See legend on next page.)
Liu et al. Cell Discovery (2020) 6:16 Page 2 of 4
that the 50% cytotoxic concentration (CC
50
) values of CQ
and HCQ were 273.20 and 249.50 μM, respectively, which
are not significantly different from each other (Fig. 1a). To
better compare the antiviral activity of CQ versus HCQ,
the dose–response curves of the two compounds against
SARS-CoV-2 were determined at four different multi-
plicities of infection (MOIs) by quantification of viral
RNA copy numbers in the cell supernatant at 48 h post
infection (p.i.). The data summarized in Fig. 1a and
Supplementary Table S1 show that, at all MOIs (0.01,
0.02, 0.2, and 0.8), the 50% maximal effective concentra-
tion (EC
50
) for CQ (2.71, 3.81, 7.14, and 7.36 μM) was
lower than that of HCQ (4.51, 4.06, 17.31, and 12.96 μM).
The differences in EC
50
values were statistically significant
at an MOI of 0.01 (P< 0.05) and MOI of 0.2 (P< 0.001)
(Supplementary Table S1). It is worth noting that the
EC
50
values of CQ seemed to be a little higher than that in
our previous report (1.13 μM at an MOI of 0.05)
1
, which
is likely due to the adaptation of the virus in cell culture
that significantly increased viral infectivity upon con-
tinuous passaging. Consequently, the selectivity index
(SI =CC
50
/EC
50
) of CQ (100.81, 71.71, 38.26, and 37.12)
was higher than that of HCQ (55.32, 61.45, 14.41, 19.25)
at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These
results were corroborated by immunofluorescence
microscopy as evidenced by different expression levels of
virus nucleoprotein (NP) at the indicated drug con-
centrations at 48 h p.i. (Supplementary Fig. S1). Taken
together, the data suggest that the anti-SARS-CoV-2
activity of HCQ seems to be less potent compared to CQ,
at least at certain MOIs.
Both CQ and HCQ are weak bases that are known to
elevate the pH of acidic intracellular organelles, such as
endosomes/lysosomes, essential for membrane fusion
5
.In
addition, CQ could inhibit SARS-CoV entry through
changing the glycosylation of ACE2 receptor and spike
protein
6
. Time-of-addition experiment confirmed that
HCQ effectively inhibited the entry step, as well as the
post-entry stages of SARS-CoV-2, which was also found
upon CQ treatment (Supplementary Fig. S2). To further
explore the detailed mechanism of action of CQ and HCQ
in inhibiting virus entry, co-localization of virions with
early endosomes (EEs) or endolysosomes (ELs) was ana-
lyzed by immunofluorescence analysis (IFA) and confocal
microscopy. Quantification analysis showed that, at
90 min p.i. in untreated cells, 16.2% of internalized virions
(anti-NP, red) were observed in early endosome antigen 1
(EEA1)-positive EEs (green), while more virions (34.3%)
were transported into the late endosomal–lysosomal
protein LAMP1
+
ELs (green) (n> 30 cells for each group).
By contrast, in the presence of CQ or HCQ, significantly
more virions (35.3% for CQ and 29.2% for HCQ; P<
0.001) were detected in the EEs, while only very few vir-
ions (2.4% for CQ and 0.03% for HCQ; P< 0.001) were
found to be co-localized with LAMP1
+
ELs (n> 30 cells)
(Fig. 1b, c). This suggested that both CQ and HCQ
blocked the transport of SARS-CoV-2 from EEs to ELs,
which appears to be a requirement to release the viral
genome as in the case of SARS-CoV
7
.
Interestingly, we found that CQ and HCQ treatment
caused noticeable changes in the number and size/mor-
phology of EEs and ELs (Fig. 1c). In the untreated cells,
most EEs were much smaller than ELs (Fig. 1c). In CQ-
and HCQ-treated cells, abnormally enlarged EE vesicles
were observed (Fig. 1c, arrows in the upper panels), many
of which are even larger than ELs in the untreated cells.
This is in agreement with previous report that treatment
with CQ induced the formation of expanded cytoplasmic
vesicles
8
. Within the EE vesicles, virions (red) were loca-
lized around the membrane (green) of the vesicle. CQ
treatment did not cause obvious changes in the number
and size of ELs; however, the regular vesicle structure
seemed to be disrupted, at least partially. By contrast, in
HCQ-treated cells, the size and number of ELs increased
significantly (Fig. 1c, arrows in the lower panels).
Since acidification is crucial for endosome maturation
and function, we surmise that endosome maturation
might be blocked at intermediate stages of endocytosis,
resulting in failure of further transport of virions to the
ultimate releasing site. CQ was reported to elevate the pH
(see figure on previous page)
Fig. 1 Comparative antiviral efficacy and mechanism of action of CQ and HCQ against SARS-CoV-2 infection in vitro. a Cytotoxicity and
antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays. Vero E6 cells were treated with
different doses of either compound or with PBS in the controls for 1 h and then infected with SARS-CoV-2 at MOIs of 0.01, 0.02, 0.2, and 0.8. The virus
yield in the cell supernatant was quantified by qRT-PCR at 48 h p.i. Y-axis represents the mean of percent inhibition normalized to the PBS group. The
experiments were repeated twice. b,cMechanism of CQ and HCQ in inhibiting virus entry. Vero E6 cells were treated with CQ or HCQ (50 μM) for 1 h,
followed by virus binding (MOI =10) at 4 °C for 1 h. Then the unbound virions were removed, and the cells were further supplemented with fresh
drug-containing medium at 37 °C for 90 min before being fixed and stained with IFA using anti-NP antibody for virions (red) and antibodies against
EEA1 for EEs (green) or LAMP1 for ELs (green). The nuclei (blue) were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs
in each group (n> 30 cells) was quantified and is shown in b. Representative confocal microscopic images of viral particles (red), EEA1
+
EEs (green),
or LAMP1
+
ELs (green) in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows
indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance (ANOVA) with
GraphPad Prism (F=102.8, df =5,182, ***P< 0.001).
Liu et al. Cell Discovery (2020) 6:16 Page 3 of 4
of lysosome from about 4.5 to 6.5 at 100 μM
9
. To our
knowledge, there is a lack of studies on the impact of
HCQ on the morphology and pH values of endosomes/
lysosomes. Our observations suggested that the mode of
actions of CQ and HCQ appear to be distinct in certain
aspects.
It has been reported that oral absorption of CQ and
HCQ in humans is very efficient. In animals, both drugs
share similar tissue distribution patterns, with high con-
centrations in the liver, spleen, kidney, and lung reaching
levels of 200–700 times higher than those in the plasma
10
.
It was reported that safe dosage (6–6.5 mg/kg per day) of
HCQ sulfate could generate serum levels of 1.4–1.5 μMin
humans
11
. Therefore, with a safe dosage, HCQ con-
centration in the above tissues is likely to be achieved to
inhibit SARS-CoV-2 infection.
Clinical investigation found that high concentration of
cytokines were detected in the plasma of critically ill
patients infected with SARS-CoV-2, suggesting that
cytokine storm was associated with disease severity
12
.
Other than its direct antiviral activity, HCQ is a safe and
successful anti-inflammatory agent that has been used
extensively in autoimmune diseases and can significantly
decrease the production of cytokines and, in particular,
pro-inflammatory factors. Therefore, in COVID-19
patients, HCQ may also contribute to attenuating the
inflammatory response. In conclusion, our results show
that HCQ can efficiently inhibit SARS-CoV-2 infection
in vitro. In combination with its anti-inflammatory func-
tion, we predict that the drug has a good potential to
combat the disease. This possibility awaits confirmation by
clinical trials. We need to point out, although HCQ is less
toxic than CQ, prolonged and overdose usage can still
cause poisoning. And the relatively low SI of HCQ requires
careful designing and conducting of clinical trials to achieve
efficient and safe control of the SARS-CoV-2 infection.
Acknowledgements
We thank Professor Zhengli Shi and Dr. Xinglou Yang from Wuhan Institute of
Virology and Professor Fei Deng from National Virus Resource Center for
providing SARS-CoV-2 strain (nCoV-2019BetaCoV/Wuhan/WIV04/2019);
Professor Xiulian Sun for kind help in statistical analysis; Professor Zhenhua
Zheng for kindly providing the anti-LAMP1 rabbit polyclonal antibody; Prof.
Zhengli Shi for kindly providing the anti-NP polyclonal antibody; Beijing Savant
Biotechnology Co., ltd for kindly providing the anti-NP monoclonal antibody;
Min Zhou and Xijia Liu for their assistance with this study; Jia Wu, Jun Liu, Hao
Tang, and Tao Du from BSL-3 Laboratory and Dr. Ding Gao from the core
faculty of Wuhan Institute of Virology for their critical support; Professor
Gengfu Xiao, Professor Yanyi Wang and other colleagues of Wuhan Institute of
Virology and Wuhan National Biosafety Laboratory for their excellent
coordination; and Dr. Basil Arif for scientific editing of the manuscript. This
work was supported in part by grants from the National Science and
Technology Major Projects for “Major New Drugs Innovation and
Development”(2018ZX09711003 to W.Z.), the National Natural Science
Foundation of China (31621061 to Z.H.), and the Hubei Science and
Technology Project (2020FCA003 to Z.H.).
Author details
1
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety
Mega-Science, Chinese Academy of Sciences, 430071 Wuhan, China.
2
National
Engineering Research Center for the Emergency Drug, Beijing Institute of
Pharmacology and Toxicology, 100850 Beijing, China.
3
University of the
Chinese Academy of Sciences, 100049 Beijing, China
Author contributions
Z.H., M.W., and W.Z. conceived and designed the experiments and provided
the final approval of the manuscript. J.L., R.C., M.X., X.W., H.Z., H.H., and Y.L.
participated in multiple experiments; all the authors analyzed the data. M.W.,
R.C., J.L., and Z.H. wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies the paper at (https://doi.org/
10.1038/s41421-020-0156-0).
Received: 24 February 2020 Accepted: 4 March 2020
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