Potential dual therapeutic approach against SARS-CoV-2/COVID-19 with

Nitazoxanide and Hydroxychloroquine

By

Srivatsan Padmanabhan M.D., Ph.D.

Email: psrivats@outlook.com
Abstract:

Coronaviruses are ubiquitous in nature and infects diverse species with frequent cross-species
transmission. There have been multiple events of animal to human transmission throughout history
including SARS, MERS and most recently SARS-CoV-2(COVID-19) that is responsible for the current
pandemic. The rapid spread of SARS-CoV-2/COVID-19 has overwhelmed the capabilities of major nations
due to the sheer number of patients with severe disease and critically ill patients requiring intensive care.
The high molecular and clinical similarity between SARS and SARS-CoV-2 enable us to draw some parallels
between them and utilize nearly two decades of existing research from SARS. Herein I describe a Novel
synergistic therapeutic approach against SARS-CoV-2/COVID-19 by repurposing Nitazoxanide and
Hydroxychloroquine to target viral and host factors for controlling this disease. Both these drugs are
readily available, expected to be highly potent and can be administered at the population level to ensure
infected patients do not advance to the point of needing prolonged hospitalization and critical care
thereby changing the course of this disease. The proposed treatment regimen would be expected to
diminish the severity of illness by reducing viral titers and rescuing the innate-immune system
dysregulation brought about by the viral infection that underlies the high mortality in the older/vulnerable
populations thereby allowing our health-care system a more controlled disease response.

Introduction:

SARS-CoV-2 is an enveloped, single-stranded, positive sense RNA virus belonging to the genera
Betacoronaviridae. Coronaviruses are ubiquitous in nature and are known to infect diverse species with
frequent cross-species transmission. Coronaviruses derive their name from the spike proteins that are
embedded on their envelope and these proteins determine the viral host/cell type specificity. Human
coronaviruses including the HCoV-229E and OC43 are responsible for causing around 30% cases of
common cold. Molecular evolution and epidemiological studies(Lau et al. 2011) have indicated multiple
events of animal to human transmission throughout history. This includes SARS (2003), MERS (2013) and
SARS-CoV-2(COVID-19), the most recent iteration of a zoonotic coronavirus entering the human
population(Hofmann et al. 2005).

The SARS epidemic in 2003 resulted in over 8000 people being infected with a CFR of 10% and the more
recent MERS outbreak lead to about 2500 clinical cases with a much higher CFR of 35%. However, SARS-
CoV-2 infection has reached pandemic status with over 100 countries being infected and the number of
cases worldwide rising (134,471 as of 03/12/20) with an estimated CFR ranging from 2% to 3% depending
on the data source (Porcheddu et al. 2020). SARS-CoV-2 has already surpassed the number of cases of
SARS and MERS combined within a short time since its emergence in human population just 4 months
ago. The rapidity and the modes of transmission of SARS-CoV-2 (not yet fully understood), the prolonged
incubation period during which the person is infectious together with the lack of herd immunity and high
CFR (Z. Wu and McGoogan 2020) especially in older populations can severely strain the resources of any
country. Ongoing transmission is likely during the initial 4-14 days (Xu et al. 2020)of relatively
asymptomatic incubation and by people with mild to minimal symptoms (Woelfel et al. 2020). Although
the duration of viral shedding was anywhere from 8-37 days, the median duration of viral shedding was
19 days in severe cases and 24 days with critical disease status (Zhou et al., n.d.). Reducing viral
shedding/load can significantly reduce the R0 of the infection, in tandem with other epidemiological
measures including quarantine.
 Given the current spread and estimated CFR of COVID-19, it has the potential to impact a significant
percent of the world population. The first step in mitigating the effect of COVID-19 lies in understanding
the virus that causes it -SARS-CoV-2. The in-depth molecular analysis and study of coronaviruses like SARS,
MERS, HCoV-229E, OC43, NL63, HKU1 and beta-coronaviruses like MHV-1 help us in understanding the
parallels between these viruses in replication, immune evasion and pathogenicity. SARS and SARS-CoV-2
being almost 85% identical at protein level (Tang et al. 2020)allow us to extrapolate some key steps of
viral replication and pathogenicity. Also, the explosion of genomic sequence data and in-vitro studies using
anti-viral compounds have given us additional tools in addressing this pathogen (Wang et al. 2020).

Current treatment:

The most promising drug candidate against SARS-CoV-2 presently is Gilead's experimental drug-
Remdesivir, an adenosine analogue that was initially developed against Ebola.

Several candidate compounds were identified in an invitro screen by Wang et al., including Remdesivir,
chloroquine and others (Wang et al. 2020). China and South Korea have reported efficacy for Chloroquine;
although significant variation in dosing regimens may dilute the potential efficacy of treatment (Gao, Tian,
and Yang, n.d.).

Other treatments include Ritonavir/Lopinavir with or without interferon and several other combinations.
Unfortunately we are unaware if any of these treatments have had a significant impact on the overall
disease landscape with the exception of maybe Remdesivir. The only published data is from a case-report
of first patient in WA who was treated with Remdesivir and showed rapid improvement in lung infiltrates
and clinical symptoms(Holshue et al. 2020). Clinical trials are underway at this time. However, its current
experimental status, route of administration(iv) and accessibility limit its wide community use.

Some estimates place that upto 80% (Guan et al. 2020) of COVID-19 infections are mild to moderate and
do not require hospitalization. However, among 20% of patients who worsen and require hospitalization,
there is significant morbidity and mortality as follows: the median duration of hospitalization was 12 days
(Guan et al. 2020) for all patients and tended to be higher with severity of illness; time from dyspnea to
intubation was about 10 days, with ICU stay of 7-8 days; the median time of illness to discharge was 22
days ; to death was 18.5 days; (Zhou et al., n.d.). This clinical data suggests that there is a reasonable time-
frame available for therapies to be instituted that can make a difference in the overall outcomes,
especially if initiated early on during the time-course of the disease.

We are now at a critical juncture to identify anti-viral treatments from readily available medications that
can be administered at the population level to ensure infected patients do not advance to the point of
needing prolonged hospitalization and critical care.

Designing drug therapies based on viral biology, host immune responses and the interplay between them
will enable a targeted approach against SARS-CoV-2. An ideal drug regimen should:

- target and reduce viral replication,

- upregulate host anti-viral responses

- down-regulate the virus induced immune-dysregulation (that results in lung damage and cytokine storm
involved in the pathogenesis of ARDS and multi-organ failure)

Reducing viral load/shedding may reduce R0, thereby mitigating spread of the disease. Most importantly,
due to the rapidity in spread of this disease, these drugs should be readily available. Current US FDA-
approved medications that have activity against the SARS-CoV-2, based on in-vitro data, include
Chloroquine (Wang et al. 2020; Gao, Tian, and Yang, n.d.), Hydroxychloroquine (Yao et al. 2020) and
Nitazoxanide (Wang et al. 2020).

Targeting viral infection and replication with Hydroxychloroquine:

The spike (S) proteins of SARS and SARS-CoV-2 utilizes the ACE2 receptor, a surface carboxypeptidase for
cellular entry. The SARS-CoV-2 RBD (Receptor Binding Domain) has a much higher affinity (15 fold)
compared to SARS RBD likely resulting in increased infectivity (Wrapp et al. 2020) (Hoffmann, Kleine-
Weber, Krüger, et al. 2020, 2). Both SARS and SARS-CoV-2 require an obligate proteolysis by TMPRSS2 for
the S-protein to bind ACE2 (Hoffmann, Kleine-Weber, Krüger, et al. 2020; Hoffmann, Kleine-Weber,
Schroeder, et al. 2020). TMPRSS2 inhibitors such as Camostat and Bromhexine are therefore promising
therapeutic targets. (Phillips 2014, 2)(Hoffmann, Kleine-Weber, Schroeder, et al. 2020, 2).

Chloroquine and Hydroxychloroquine have been shown to have in-vitro anti-viral efficacy in SARS and
SARS-CoV-2 when pre-treated (prophylactic) and also post-viral entry (treatment) – indicating multiple
mechanisms of action (Yao et al. 2020; Vincent et al. 2005). Chloroquine was shown in-vitro to increase
endosomal pH, prevent virus/cell fusion, interfere with the glycosylation of the ACE2 receptor and thus
interfering with the S protein binding. However, chloroquine was effective even after 2-5h post infection
suggesting antiviral effect independent of viral entry (Vincent et al. 2005; Wang et al. 2020) . The
EC50/EC90 for Chloroquine was 1.1/6.9 mM based on Wang-et al. However, Yao et al. had a significantly
higher EC50 for chloroquine of 23.90 mM at 24h reducing to 5.47 mM with 48h incubation. Interestingly,
Yao et al. showed that hydroxychloroquine has an even better activity against SARS-CoV-2 with an EC50
of 6 at 24h and 0.72 mM at 48h – an almost 7.5-fold increased activity compared with chloroquine in-
vitro. Gao et al. report encouraging data with chloroquine use in an multicenter clinical trial in China to
treat SARS-CoV-2/COVID-19. In a series of 100 patients they found improvement in pneumonia, imaging
findings and shortening the duration of disease(Gao, Tian, and Yang, n.d.).

Given that hydroxychloroquine is safer, more potent with direct anti-viral activity and easily available, this
would be another obvious choice for a drug candidate. Yao et al. further characterized a dosing regimen
based on physiologically based pharmacokinetic models to achieve tissue concentrations that is adequate
for anti-viral efficacy(Yao et al. 2020). In addition to direct anti-viral effect, hydroxychloroquine also has
immune-modulatory properties. It has been shown to down-regulate pro-inflammatory cytokines
including IL6 levels(Willis et al. 2012) and thereby potentially down-regulate and minimize cytokine
release syndrome/shock/ARDS that could be beneficial in reducing the intensity of this disease. Yao et al.
recommend 400mg twice a day for 2 days followed by 200mg twice a day for 3 more days.
Targeting host anti-viral response with Nitazoxanide:

Viral infection and replication in host cells triggers the interferon response, a critical step to control viral
infection. Indeed SARS , MERS and likely SARS-CoV-2 based on homology (A. Wu et al. 2020) all have
mechanisms to down-regulate the interferon pathway that is critical for the pathogenicity of these
viruses.

SARS virus encodes multiple protein products including NSP1 (degrades IFN mRNA), ORF6 (antagonizes
STAT-1), Nsp15 and N proteins that blocks key aspects of the interferon pathway. Infact, SARS infected
cells do not show upregulation of IFN despite large amounts of ds-RNA(Frieman and Baric 2008). Similar
to SARS, SARS-CoV-2/COVID-19 shows higher mortality with age. The intensity of the innate immune
response is known to be higher in the young as compared to older individuals, a phenomenon known as
immune senescence (Frieman and Baric 2008; Frieman et al. 2007; Baas et al. 2008). The age-related
increased susceptibility for SARS and SARS-CoV-2 have been suggested to correspond to the reduced
intensity of innate immune response. Children in particular have milder symptoms, a phenotype that is
recapitulated in the SARS mouse models(Chen et al. 2010; Deng et al. 2017).

The Senescent BALB/c mice model shows features reminiscent of human SARS. Upon viral challenge, this
mouse model shows a biphasic inflammatory response; an early response, by day 2, of inflammatory
cytokines including TNF-α, IL-6, associated chemokines (CXCL10, CCL2, CCL3, and CCL5) that recruits
inflammatory cells (CD4+, NK cells, macrophages) associated with peak viral titers. This was followed by a
second peak on day 7 that correlated with T-cell infiltration and elevated levels of T-cell associated
cytokines IL-6, IFN-γ, IL-2, and IL-5 that is paralleled by pneumonitis and diffuse alveolar damage (Chen et
al. 2010). In another mouse model of SARS infection, dsRNA inducer (poly I:C – interferon inducer)
significantly reduced viral titers by upregulating the innate immune response (Barnard et al. 2006). Taken
together in older individuals the dysfunctional innate immune response predisposes to high viral titers
and ensuing eventual CD8+ T-cell response likely results in cytokine storm/diffuse lung damage that is the
hallmark of SARS(Chen et al. 2010) and possibly a similar mechanism is involved in the pathogenesis of
SARS-CoV-2 lung injury.

In MERS, differences in innate immune response also corelate with case fatality. In a patient who died
from MERS - lower expression levels of RIG-1, MDA-5(ds-RNA sensors that activate interferon pathway)
correlated with reduced IRF3 and IRF7 was found when compared to survivors (Josset et al. 2013).

An elegant study by Deng et al. with mutant-nsp15 SARS virus furthers our understanding of the
importance of the interferon pathway in disease pathogenesis. nsp15 mutant defective in down-
regulating the host dsRNA response, is non-pathogenic in mouse models. Indeed, 100% of wild-type mice
survived a lethal dose challenge by mutant nsp15 viruses. In contrast, 100% of mice lacking IFN signaling
succumbed to the mutant virus indicating clearly that the interferon response by itself is sufficient to
protect against lethal infection (Deng et al. 2017). Indeed, the nsp15 protein is 90% similar between SARS
and SARS-CoV-2 indicating conserved host innate immune evasion mechanisms between these related
viruses.

An excellent drug candidate that has been completely under-represented in trials for SARS-CoV-2/COVID-
19 is Nitazoxanide. Data from Wang et al. suggests that the EC50 for Nitazoxanide was 2mM; the sharp
slope of the curve suggests a specific anti-viral effect with a EC90 of around 5-6 mM (Wang et al. 2020).
Nitazoxanide is an antiprotozoal that is currently FDA approved for treating Cryptosporidium and Giardia.
It was discovered in the 1980s and has an excellent safety record for a variety of indications. It was
subsequently shown to have broad anti-viral activity (Rossignol 2014) and has been approved in Brazil for
treating Noro and Rota viruses.

Nitazoxanide upregulates the innate anti-viral mechanisms by broadly amplifying cytoplasmic RNA sensing
and type I IFN pathways(Jasenosky et al. 2019). Nitazoxanide interferes with the viral infection by
upregulating the precise host mechanisms that viruses target to bypass host cellular defenses (Frieman
and Baric 2008)(Jasenosky et al. 2019). Nitazoxanide has shown varying activities in different viral
infections and has been therapeutically used for several viruses including hepatitis B, C, Rotavirus and
Norovirus (Rossignol 2014; Korba et al. 2008; Rossignol 2016). Multiple unbiased approaches have
repeatedly demonstrated that Coronaviridae are especially sensitive to Nitazoxanide (in the low
micromolar range) (Cao, Forrest, and Zhang 2015; Wang et al. 2020).

The remarkably low IC50 for all coronaviruses hints that the interferon blocking mechanisms of these
viruses lie upstream or are exquisitely sensitive to Nitazoxanide thereby effectively bypassing the innate-
immune system dysregulation fraught by these viruses. Nitazoxanide enhances MDA5, MAVS and IFN-
.(Jasenosky et al. 2019). The IC50 for MERS, SARS and SARS-CoV-2 are all around 1-2 g/ml which is
significantly lower than the plasma levels that can be achieved by appropriate dosing of this medication.

Indeed, Nitazoxanide has been used to treat a related beta-coronavirus HCoV-OC43. In a controlled clinical
study (2018) of 92 patients with common cold caused by OC43, duration of symptoms were reduced by
an average of 3 days (Personal communication Rossignol). In this study the dose used was 600 mg twice
a day of NT-300 (sustained release) tablets. More frequent dosing of immediate release tablets should
also mirror a similar efficacy.

Proposed drug dosing for SARS-CoV-2/COVID-19 - Dual targeted therapy – (Figure-1)

The combination of Hydroxychloroquine and Nitazoxanide is likely to have synergistic activity against the
SARS-CoV2/COVID-19 virus due to multitude of action as follows: Hydroxychloroquine mediates reduced
viral replication, reduced Viral entry (by interfering with ACE2 glycosylation and increasing endosomal pH)
and immunomodulatory activities to down-regulate IL6 mediated host damage/cytokine storm. In
addition, Nitazoxanide upregulates innate immune response to prevent ongoing viral replication in
infected cells and potentially prevent viral infection of bystander uninfected cells to reduce overall viral
load. The combination of these two activities is hypothesized to synergistically reduce the disease
intensity and potentially reduce viral shedding to reduce transmission.

Proposed dosing regimen:

1) Hydroxychloroquine 400 mg twice a day for 2-3 days (loading) then 200mg twice a day for 4 days
; based on (Yao et al. 2020)
2) Nitazoxanide (600mg -SR tabs) twice a day; if unavailable, using the immediate release
formulation 500mg three times a day for 7 days (absorption better with food) should achieve
adequate trough levels that easily exceeds the EC50 of SARS-CoV2. The duration of treatment can
likely be adjusted based on clinical response.
Conclusion:

The rapid spread of the SARS-CoV-2/COVID-19 has overwhelmed the capabilities of major nations due to
the sheer number of patients with severe disease and critically ill patients requiring intensive care. This
disease has a high mortality and morbidity in older populations (CFR OF 15% in > age 80 and CFR of 49%
in critically ill patients). In addition, older patients are also more likely to be super-spreaders (disease
severity corelates with increased viral load/shedding) causing significant spread of this pandemic. The
proposed treatment regimen would be expected to reduce the severity of illness by reducing viral titers
and rescuing the innate-immune system dysregulation brought upon by the viral infection that underlies
the high mortality in the older/vulnerable populations. Reducing the severity of this disease could mean
the difference between collapse of the health-care system to a more controlled disease response.

We are in the process of initiating a clinical trial with this combination of drugs in SARS-CoV-2/COVID-19
patients.

Acknowledgements:

Appreciate Ms.Sandhya Pande for critical inputs, a big thanks to Ms. Kavitha Padmanabhan for extensive
editing and critical inputs to the manuscript.

References:

Baas, Tracey, Anjeanette Roberts, Thomas H. Teal, Leatrice Vogel, Jun Chen, Terrence M. Tumpey, Michael
G. Katze, and Kanta Subbarao. 2008. “Genomic Analysis Reveals Age-Dependent Innate Immune
Responses to Severe Acute Respiratory Syndrome Coronavirus.” Journal of Virology 82 (19): 9465–
76. https://doi.org/10.1128/JVI.00489-08.
Barnard, Dale L, Craig W Day, Kevin Bailey, Matthew Heiner, Robert Montgomery, Larry Lauridsen, Paul
KS Chan, and Robert W Sidwell. 2006. “Evaluation of Immunomodulators, Interferons and Known
in Vitro SARS-CoV Inhibitors for Inhibition of SARS-Cov Replication in BALB/c Mice.” Antiviral
Chemistry and Chemotherapy 17 (5): 275–84. https://doi.org/10.1177/095632020601700505.
Cao, Jianzhong, J. Craig Forrest, and Xuming Zhang. 2015. “A Screen of the NIH Clinical Collection Small
Molecule Library Identifies Potential Anti-Coronavirus Drugs.” Antiviral Research 114 (February):
1–10. https://doi.org/10.1016/j.antiviral.2014.11.010.
Chen, Jun, Yuk Fai Lau, Elaine W. Lamirande, Christopher D. Paddock, Jeanine H. Bartlett, Sherif R. Zaki,
and Kanta Subbarao. 2010. “Cellular Immune Responses to Severe Acute Respiratory Syndrome
Coronavirus (SARS-CoV) Infection in Senescent BALB/c Mice: CD4+ T Cells Are Important in Control
of SARS-CoV Infection.” Journal of Virology 84 (3): 1289–1301.
https://doi.org/10.1128/JVI.01281-09.
Deng, Xufang, Matthew Hackbart, Robert C. Mettelman, Amornrat O’Brien, Anna M. Mielech, Guanghui
Yi, C. Cheng Kao, and Susan C. Baker. 2017. “Coronavirus Nonstructural Protein 15 Mediates
Evasion of DsRNA Sensors and Limits Apoptosis in Macrophages.” Proceedings of the National
Academy of Sciences of the United States of America 114 (21): E4251–60.
https://doi.org/10.1073/pnas.1618310114.
Frieman, Matthew, and Ralph Baric. 2008. “Mechanisms of Severe Acute Respiratory Syndrome
Pathogenesis and Innate Immunomodulation.” Microbiology and Molecular Biology Reviews 72
(4): 672–85. https://doi.org/10.1128/MMBR.00015-08.
Frieman, Matthew, Boyd Yount, Mark Heise, Sarah A. Kopecky-Bromberg, Peter Palese, and Ralph S. Baric.
2007. “Severe Acute Respiratory Syndrome Coronavirus ORF6 Antagonizes STAT1 Function by
Sequestering Nuclear Import Factors on the Rough Endoplasmic Reticulum/Golgi Membrane.”
Journal of Virology 81 (18): 9812–24. https://doi.org/10.1128/JVI.01012-07.
Gao, Jianjun, Zhenxue Tian, and Xu Yang. n.d. “Breakthrough: Chloroquine Phosphate Has Shown
Apparent Efficacy in Treatment of COVID-19 Associated Pneumonia in Clinical Studies,” 2.
Guan, Wei-jie, Zheng-yi Ni, Yu Hu, Wen-hua Liang, Chun-quan Ou, Jian-xing He, Lei Liu, et al. 2020. “Clinical
Characteristics of Coronavirus Disease 2019 in China.” New England Journal of Medicine 0 (0): null.
https://doi.org/10.1056/NEJMoa2002032.
Hoffmann, Markus, Hannah Kleine-Weber, Nadine Krüger, Marcel Müller, Christian Drosten, and Stefan
Pöhlmann. 2020. “The Novel Coronavirus 2019 (2019-NCoV) Uses the SARS-Coronavirus Receptor
ACE2 and the Cellular Protease TMPRSS2 for Entry into Target Cells.” BioRxiv, January,
2020.01.31.929042. https://doi.org/10.1101/2020.01.31.929042.
Hoffmann, Markus, Hannah Kleine-Weber, Simon Schroeder, Nadine Krüger, Tanja Herrler, Sandra
Erichsen, Tobias S. Schiergens, et al. 2020. “SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2
and Is Blocked by a Clinically Proven Protease Inhibitor.” Cell 0 (0).
https://doi.org/10.1016/j.cell.2020.02.052.
Hofmann, Heike, Krzysztof Pyrc, Lia van der Hoek, Martina Geier, Ben Berkhout, and Stefan Pöhlmann.
2005. “Human Coronavirus NL63 Employs the Severe Acute Respiratory Syndrome Coronavirus
Receptor for Cellular Entry.” Proceedings of the National Academy of Sciences of the United States
of America 102 (22): 7988–93. https://doi.org/10.1073/pnas.0409465102.
Holshue, Michelle L., Chas DeBolt, Scott Lindquist, Kathy H. Lofy, John Wiesman, Hollianne Bruce,
Christopher Spitters, et al. 2020. “First Case of 2019 Novel Coronavirus in the United States.” New
England Journal of Medicine 382 (10): 929–36. https://doi.org/10.1056/NEJMoa2001191.
Jasenosky, Luke D., Cristhian Cadena, Chad E. Mire, Viktoriya Borisevich, Viraga Haridas, Shahin Ranjbar,
Aya Nambu, et al. 2019. “The FDA-Approved Oral Drug Nitazoxanide Amplifies Host Antiviral
Responses and Inhibits Ebola Virus.” IScience 19 (September): 1279–90.
https://doi.org/10.1016/j.isci.2019.07.003.
Josset, Laurence, Vineet D. Menachery, Lisa E. Gralinski, Sudhakar Agnihothram, Pavel Sova, Victoria S.
Carter, Boyd L. Yount, Rachel L. Graham, Ralph S. Baric, and Michael G. Katze. 2013. “Cell Host
Response to Infection with Novel Human Coronavirus EMC Predicts Potential Antivirals and
Important Differences with SARS Coronavirus.” MBio 4 (3). https://doi.org/10.1128/mBio.00165-
13.
Korba, Brent E., Abigail B. Montero, Kristine Farrar, Karen Gaye, Sampa Mukerjee, Marc S. Ayers, and Jean-
François Rossignol. 2008. “Nitazoxanide, Tizoxanide and Other Thiazolides Are Potent Inhibitors
of Hepatitis B Virus and Hepatitis C Virus Replication.” Antiviral Research 77 (1): 56–63.
https://doi.org/10.1016/j.antiviral.2007.08.005.
Lau, Susanna K. P., Paul Lee, Alan K. L. Tsang, Cyril C. Y. Yip, Herman Tse, Rodney A. Lee, Lok-Yee So, et al.
2011. “Molecular Epidemiology of Human Coronavirus OC43 Reveals Evolution of Different
Genotypes over Time and Recent Emergence of a Novel Genotype Due to Natural
Recombination.” Journal of Virology 85 (21): 11325. https://doi.org/10.1128/JVI.05512-11.
Phillips, Robert. 2014. “TMPRSS2 Promotes Metastasis through Proteolysis.” Nature Reviews Urology 11
(10): 546–546. https://doi.org/10.1038/nrurol.2014.238.
Porcheddu, Rossella, Caterina Serra, David Kelvin, Nikki Kelvin, and Salvatore Rubino. 2020. “Similarity in
Case Fatality Rates (CFR) of COVID-19/SARS-COV-2 in Italy and China.” The Journal of Infection in
Developing Countries 14 (02): 125–28. https://doi.org/10.3855/jidc.12600.
Rossignol, Jean-François. 2014. “Nitazoxanide: A First-in-Class Broad-Spectrum Antiviral Agent.” Antiviral
Research 110 (October): 94–103. https://doi.org/10.1016/j.antiviral.2014.07.014.
———. 2016. “Nitazoxanide, a New Drug Candidate for the Treatment of Middle East Respiratory
Syndrome Coronavirus.” Journal of Infection and Public Health 9 (3): 227–30.
https://doi.org/10.1016/j.jiph.2016.04.001.
Tang, Xiaolu, Changcheng Wu, Xiang Li, Yuhe Song, Xinmin Yao, Xinkai Wu, Yuange Duan, et al. 2020. “On
the Origin and Continuing Evolution of SARS-CoV-2.” National Science Review, March, nwaa036.
https://doi.org/10.1093/nsr/nwaa036.
Vincent, Martin J., Eric Bergeron, Suzanne Benjannet, Bobbie R. Erickson, Pierre E. Rollin, Thomas G.
Ksiazek, Nabil G. Seidah, and Stuart T. Nichol. 2005. “Chloroquine Is a Potent Inhibitor of SARS
Coronavirus Infection and Spread.” Virology Journal 2 (1): 69. https://doi.org/10.1186/1743-
422X-2-69.
Wang, Manli, Ruiyuan Cao, Leike Zhang, Xinglou Yang, Jia Liu, Mingyue Xu, Zhengli Shi, Zhihong Hu, Wu
Zhong, and Gengfu Xiao. 2020. “Remdesivir and Chloroquine Effectively Inhibit the Recently
Emerged Novel Coronavirus (2019-NCoV) in Vitro.” Cell Research 30 (3): 269–71.
https://doi.org/10.1038/s41422-020-0282-0.
Willis, R, AM Seif, G McGwin, LA Martinez-Martinez, EB González, N Dang, E Papalardo, et al. 2012. “Effect
of Hydroxychloroquine Treatment on Pro-Inflammatory Cytokines and Disease Activity in SLE
Patients: Data from LUMINA (LXXV), a Multiethnic US Cohort.” Lupus 21 (8).
https://doi.org/10.1177/0961203312437270.
Woelfel, Roman, Victor Max Corman, Wolfgang Guggemos, Michael Seilmaier, Sabine Zange, Marcel A
Mueller, Daniela Niemeyer, et al. 2020. “Clinical Presentation and Virological Assessment of
Hospitalized Cases of Coronavirus Disease 2019 in a Travel-Associated Transmission Cluster.”
Preprint. Infectious Diseases (except HIV/AIDS). https://doi.org/10.1101/2020.03.05.20030502.
Wrapp, Daniel, Nianshuang Wang, Kizzmekia S. Corbett, Jory A. Goldsmith, Ching-Lin Hsieh, Olubukola
Abiona, Barney S. Graham, and Jason S. McLellan. 2020. “Cryo-EM Structure of the 2019-NCoV
Spike in the Prefusion Conformation.” Science, February.
https://doi.org/10.1126/science.abb2507.
Wu, Aiping, Yousong Peng, Baoying Huang, Xiao Ding, Xianyue Wang, Peihua Niu, Jing Meng, et al. 2020.
“Genome Composition and Divergence of the Novel Coronavirus (2019-NCoV) Originating in
China.” Cell Host & Microbe 27 (3): 325–28. https://doi.org/10.1016/j.chom.2020.02.001.
Wu, Zunyou, and Jennifer M. McGoogan. 2020. “Characteristics of and Important Lessons From the
Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases
From the Chinese Center for Disease Control and Prevention.” JAMA, February.
https://doi.org/10.1001/jama.2020.2648.
Xu, Jiabao, Shizhe Zhao, Tieshan Teng, Abualgasim Elgaili Abdalla, Wan Zhu, Longxiang Xie, Yunlong Wang,
and Xiangqian Guo. 2020. “Systematic Comparison of Two Animal-to-Human Transmitted Human
Coronaviruses: SARS-CoV-2 and SARS-CoV.” Viruses 12 (2): 244.
https://doi.org/10.3390/v12020244.
Yao, Xueting, Fei Ye, Miao Zhang, Cheng Cui, Baoying Huang, Peihua Niu, Xu Liu, et al. 2020. “In Vitro
Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the
Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (Sars-Cov-2).” Clinical Infectious
Diseases: An Official Publication of the Infectious Diseases Society of America, March.
https://doi.org/10.1093/cid/ciaa237.
Zhou, Fei, Ting Yu, Ronghui Du, Guohui Fan, Ying Liu, Zhibo Liu, Jie Xiang, et al. n.d. “Clinical Course and
Risk Factors for Mortality of Adult Inpatients with COVID-19 in Wuhan, China: A Retrospective
Cohort Study,” 9.
Figure 1.