Rational design of a 'two-in-one' immunogen DAM drives potent immune response against mpox virus.

The global outbreak of the mpox virus (MPXV) in 2022 highlights the urgent need for safer and more accessible new-generation vaccines. Here, we used a structure-guided multi-antigen fusion strategy to design a 'two-in-one' immunogen based on the single-chain dimeric MPXV extracellular enveloped virus antigen A35 bivalently fused with the intracellular mature virus antigen M1, called DAM. DAM preserved the natural epitope configuration of both components and showed stronger A35-specific and M1-specific antibody responses and in vivo protective efficacy against vaccinia virus (VACV) compared to co-immunization strategies. The MPXV-specific neutralizing antibodies elicited by DAM were 28 times higher than those induced by live VACV vaccine. Aluminum-adjuvanted DAM vaccines protected mice from a lethal VACV challenge with a safety profile, and pilot-scale production confirmed the high yield and purity of DAM. Thus, our study provides innovative insights and an immunogen candidate for the development of alternative vaccines against MPXV and other orthopoxviruses.

Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.

Surveillance of SARS-CoV-2 at the Huanan Seafood Market.

SARS-CoV-2, the causative agent of COVID-19, emerged in December 2019. Its origins remain uncertain. It has been reported that a number of the early human cases had a history of contact with the Huanan Seafood Market. Here we present the results of surveillance for SARS-CoV-2 within the market. From January 1st 2020, after closure of the market, 923 samples were collected from the environment. From 18th January, 457 samples were collected from 18 species of animals, comprising of unsold contents of refrigerators and freezers, swabs from stray animals, and the contents of a fish tank. Using RT-qPCR, SARS-CoV-2 was detected in 73 environmental samples, but none of the animal samples. Three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.99% to 100% with the human isolate HCoV-19/Wuhan/IVDC-HB-01/2019. SARS-CoV-2 lineage A (8782T and 28144C) was found in an environmental sample. RNA-seq analysis of SARS-CoV-2 positive and negative environmental samples showed an abundance of different vertebrate genera at the market. In summary, this study provides information about the distribution and prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stages of the COVID-19 outbreak.

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy.

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

A Novel Coronavirus from Patients with Pneumonia in China, 2019.

In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).

Neutralizing antibody levels associated with injectable and aerosolized Ad5-nCoV boosters and BA.2 infection.

BACKGROUND: Several COVID-19 vaccines are in widespread use in China. Few data exist on comparative immunogenicity of different COVID-19 vaccines given as booster doses. We aimed to assess neutralizing antibody levels raised by injectable and inhaled aerosolized recombinant adenovirus type 5 (Ad5)-vectored COVID-19 vaccine as a heterologous booster after an inactivated COVID-19 vaccine two-dose primary series. METHODS: Using an open-label prospective cohort design, we recruited 136 individuals who had received inactivated vaccine primary series followed by either injectable or inhaled Ad5-vectored vaccine and measured neutralizing antibody titers against ancestral SARS-CoV-2 virus and Omicron BA.1 and BA.5 variants. We also measured neutralizing antibody levels in convalescent sera from 39 patients who recovered from Omicron BA.2 infection. RESULTS: Six months after primary series vaccination, neutralizing immunity against ancestral SARS-CoV-2 was low and neutralizing immunity against Omicron (B.1.1.529) was lower. Boosting with Ad5-vectored vaccines induced a high immune response against ancestral SARS-CoV-2. Neutralizing responses against Omicron BA.5 were ≥ 80% lower than against ancestral SARS-CoV-2 in sera from prime-boost subjects and in convalescent sera from survivors of Omicron BA.2 infection. Inhaled aerosolized Ad5-vectored vaccine was associated with greater neutralizing titers than injectable Ad5-vectored vaccine against ancestral and Omicron SARS-CoV-2 variants. CONCLUSIONS: These findings support the current strategy of heterologous boosting with injectable or inhaled Ad5-vectored SARS-CoV-2 vaccination of individuals primed with inactivated COVID-19 vaccine.

Comparative viromes of Culicoides and mosquitoes reveal their consistency and diversity in viral profiles.

The genus Culicoides includes biting midges, some of which are vectors for viruses that cause diseases in humans and animals. Knowledge of the roles of Culicoides in viral ecology is inadequate. We collected ~300 000 samples of Culicoides and mosquitoes in 15 representative regions within Yunnan, China. Using mosquitoes as reference vectors, we designed a comparative virome strategy to study the viral composition, diversity, hosts and spatiotemporal distribution of Culicoides. A map of viromes in Culicoides and mosquitoes in Yunan province, China, was constructed. At the same locations, Culicoides and mosquitoes usually share a similar viral diversity. At least 10 important pathogenic viruses were detected from Culicoides. Many novel viruses were discovered, including 21 segmented viruses of Flaviviridae, 180 viruses of Monjiviricetes and 130 viruses of Bunyavirales. The findings demonstrate that Culicoides is an important part of viral ecology and should be studied and monitored for potentially emerging viruses.

The First Human Infection Case of Macacine Alphaherpesvirus 1 Virus in China.

BACKGROUND: Macacine alphaherpesvirus 1 (BV) was first reported in the 1930s and only about 60 cases have been diagnosed since then. METHODS: A 53-year-old male who worked as a veterinary surgeon, developed a fever with nausea and vomiting in April 2021 in Beijing, China. Real-time polymerase chain reaction (PCR) and metagenomics Next Generation Sequencing (mNGS) were used for diagnosis. RESULTS: BV DNA was confirmed by mNGS and PCR. The case died 51 days after onset, due to the damage to the brain and spinal cord caused by a viral infection and hypoxic-ischemic encephalopathy. The typical BV inclusion bodies in the brain were found for the first time. CONCLUSIONS: Here we reported the first human infection case of BV in China. This fatal case highlights the potential threat of BV to occupational workers and the essential role of surveillance.

Pseudotyped Virus for Flaviviridae.

Members of Flaviviridae are enveloped single positive-stranded RNA viruses including hepacivirus, pestivirus, pegivirus, and mosquito-transmitted flavivirus, which are important pathogens of infectious diseases and pose serious threats to human health. Pseudotyped virus is an artificially constructed virus-like particle, which could infect host cells similar to a live virus but cannot produce infectious progeny virus. Therefore, pseudotyped virus has the advantages of a wide host range, high transfection efficiency, low biosafety risk, and accurate and objective quantification. It has been widely used in biological characteristics, drug screening, detection methods, and vaccine evaluation of Flaviviridae viruses like hepatitis C virus, Japanese encephalitis virus, dengue virus, and Zika virus.

Immunogenicity and Safety of a 3-Dose Regimen of a SARS-CoV-2 Inactivated Vaccine in Adults: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

BACKGROUND: Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic needs effective vaccines. METHODS: In a phase 2 randomized, double-blind, placebo-controlled trial, 500 adults aged 18-59 years or ≥60 years were randomized in 2:2:1 ratio to receive 3 doses of 5 µg or 10 µg of a SARS-CoV-2 inactivated vaccine, or placebo separated by 28 days. Adverse events (AEs) were recorded through day 28 after each dosing. Live virus or pseudovirus neutralizing antibodies, and receptor binding domain immunoglobulin G (RBD-IgG) antibody were tested after the second and third doses. RESULTS: Two doses of the vaccine elicited geometric mean titers (GMTs) of 102-119, 170-176, and 1449-1617 for the 3 antibodies in younger adults. Pseudovirus neutralizing and RBD-IgG GMTs were similar between older and younger adults. The third dose slightly (<1.5 fold) increased GMTs. Seroconversion percentages were 94% or more after 2 doses, which were generally similar after 3 doses. The predominant AEs were injection-site pain. All the AEs were grade 1 or 2 in intensity. No serious AE was deemed related to study vaccination. CONCLUSIONS: Two doses of this vaccine induced robust immune response and had good safety profile. A third dose given 28 days after the second dose elicited limited boosting antibody response.

Enterovirus D68 Protease 2Apro Targets TRAF3 To Subvert Host Innate Immune Responses.

Human enterovirus D68 (EV-D68) has received considerable attention recently as a global reemergent pathogen because it causes severe respiratory tract infections and acute flaccid myelitis (AFM). The nonstructural protein 2A protease (2Apro) of EVs, which functions in the cleavage of host proteins, comprises a pivotal part of the viral immune evasion process. However, the pathogenic mechanism of EV-D68 is not fully understood. In this study, we found that EV-D68 inhibited antiviral type I interferon responses by cleaving tumor necrosis factor receptor-associated factor 3 (TRAF3), which is the key factor for type I interferon production. EV-D68 inhibited Sendai virus (SEV)-induced interferon regulatory factor 3 (IRF3) activation and beta interferon (IFN-ß) expression in HeLa and HEK293T cells. Furthermore, we demonstrated that EV-D68 and 2Apro were able to cleave the C-terminal region of TRAF3 in HeLa and HEK293T cells, respectively. A cysteine-to-alanine substitution at amino acid 107 (C107A) in the 2Apro protease resulted in the loss of cleavage activity to TRAF3, and mutation of glycine at amino acid 462 to alanine (G462A) in TRAF3 conferred resistance to 2Apro These results suggest that control of TRAF3 by 2Apro may be a mechanism EV-D68 utilizes to subvert host innate immune responses.IMPORTANCE Human enterovirus 68 (EV-D68) has received considerable attention recently as a global reemergent pathogen because it causes severe respiratory tract infections and acute flaccid myelitis. The nonstructural protein 2A protease (2Apro) of EV, which functions in cleavage of host proteins, comprises an essential part of the viral immune evasion process. However, the pathogenic mechanism of EV-D68 is not fully understood. Here, we show for the first time that EV-D68 inhibited antiviral type I interferon responses by cleaving tumor necrosis factor receptor-associated factor 3 (TRAF3). Furthermore, we identified the key cleavage site in TRAF3. Our study may suggest a new mechanism by which the 2Apro of EV facilitates subversion of host innate immune responses. These findings increase our understanding of EV-D68 infection and may help identify new antiviral targets against EV-D68.

One Hundred Days of Coronavirus Disease 2019 Prevention and Control in China.

The epidemic of novel coronavirus disease was first reported in China in late December 2019 and was brought under control after some 2 months in China. However, it has become a global pandemic, and the number of cases and deaths continues to increase outside of China. We describe the emergence of the pandemic, detail the first 100 days of China's response as a phase 1 containment strategy followed by phase 2 containment, and briefly highlight areas of focus for the future. Specific, simple, and pragmatic strategies used in China for risk assessment, prioritization, and deployment of resources are described. Details of implementation, at different risk levels, of the traditional public health interventions are shared. Involvement of society in mounting a whole country response and challenges experienced with logistics and supply chains are described. Finally, the methods China is employing to cautiously restart social life and economic activity are outlined.

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.

BACKGROUND: In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. METHODS: We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus. FINDINGS: The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues. INTERPRETATION: 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. FUNDING: National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.

Increased Pathogenicity and Virulence of Middle East Respiratory Syndrome Coronavirus Clade B In Vitro and In Vivo.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. We isolated the clade B MERS-CoV ChinaGD01 strain from a patient infected during the South Korean MERS outbreak in 2015 and compared the phylogenetics and pathogenicity of MERS-CoV EMC/2012 (clade A) and ChinaGD01 (clade B) in vitro and in vivo Genome alignment analysis showed that most clade-specific mutations occurred in the orf1ab gene, including mutations that were predicted to be potential glycosylation sites. Minor differences in viral growth but no significant differences in plaque size or sensitivity to beta interferon (IFN-ß) were detected between these two viruses in vitro ChinaGD01 virus infection induced more weight loss and inflammatory cytokine production in human DPP4-transduced mice. Viral titers were higher in the lungs of ChinaGD01-infected mice than with EMC/2012 infection. Decreased virus-specific CD4+ and CD8+ T cell numbers were detected in the lungs of ChinaGD01-infected mice. In conclusion, MERS-CoV evolution induced changes to reshape its pathogenicity and virulence in vitro and in vivo and to evade adaptive immune response to hinder viral clearance.IMPORTANCE MERS-CoV is an important emerging pathogen and causes severe respiratory infection in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. In this study, we showed that a clade B virus ChinaGD01 strain caused more severe disease in mice, with delayed viral clearance, increased inflammatory cytokines, and decreased antiviral T cell responses, than the early clade A virus EMC/2012. Given the differences in pathogenicity of different clades of MERS-CoV, periodic assessment of currently circulating MERS-CoV is needed to monitor potential severity of zoonotic disease.

Sr9Mg4.45(1)Bi9 and Sr9Mg4.42(1)Sb9: Mg-Containing Zintl Phases with Low Thermal Conductivity.

Zintl phases with nominal 9-4-9 formulas are very interesting for their potential applications as thermoelectric materials. However, the formation of such phases usually requires divalent transition metals, for example, Zn, Mn, and Cd, which are covalently bonded to the pnictogen atoms. In this report, for the first time, two Mg-containing compounds with such structures as Sr9Mg4.45(1)Bi9 and Sr9Mg4.42(1)Sb9 were synthesized and their structures were determined by the single-crystal X-ray diffraction method. Both title compounds crystallize in the orthorhombic space group Pnma and are isostructural with Ca9Mn4.41(1)Sb9, which features complex polyanion structures compared to the classical 9-4-9 phases. For Sr9Mg4.45(1)Bi9, its low thermal conductivity, combined with its high electrical conductivity and moderate Seebeck coefficient, leads to a decent figure of merit of 0.57 at 773 K, which obviously prevails in the unoptimized 9-4-9 phases. The discovery of such Mg-containing 9-4-9 phases is very significant, as the discovery not only enriches the structure map of the well-known 9-4-9 family but also provides very valuable thermoelectric candidates surely deserving of more in-depth investigation.

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro.

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CLpro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CLpro activity in vitro with IC50's of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC50's of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CLpro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CLpro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.

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).

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first broke out in 2019 and subsequently spread worldwide. Chloroquine has been sporadically used in treating SARS-CoV-2 infection. Hydroxychloroquine shares the same mechanism of action as chloroquine, but its more tolerable safety profile makes it the preferred drug to treat malaria and autoimmune conditions. We propose that the immunomodulatory effect of hydroxychloroquine also may be useful in controlling the cytokine storm that occurs late phase in critically ill patients with SARS-CoV-2. Currently, there is no evidence to support the use of hydroxychloroquine in SARS-CoV-2 infection. METHODS: The pharmacological activity of chloroquine and hydroxychloroquine was tested using SARS-CoV-2-infected Vero cells. Physiologically based pharmacokinetic (PBPK) models were implemented for both drugs separately by integrating their in vitro data. Using the PBPK models, hydroxychloroquine concentrations in lung fluid were simulated under 5 different dosing regimens to explore the most effective regimen while considering the drug's safety profile. RESULTS: Hydroxychloroquine (EC50 = 0.72 µM) was found to be more potent than chloroquine (EC50 = 5.47 µM) in vitro. Based on PBPK models results, a loading dose of 400 mg twice daily of hydroxychloroquine sulfate given orally, followed by a maintenance dose of 200 mg given twice daily for 4 days is recommended for SARS-CoV-2 infection, as it reached 3 times the potency of chloroquine phosphate when given 500 mg twice daily 5 days in advance. CONCLUSIONS: Hydroxychloroquine was found to be more potent than chloroquine to inhibit SARS-CoV-2 in vitro.