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1.
Vet Res ; 52(1): 2, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397461

RESUMO

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos/virologia , Animais , Chlorocebus aethiops , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Fezes/química , Fezes/virologia , Imunoglobulina A/sangue , Imunoglobulina A/química , Imunoglobulina A/metabolismo , Imunoglobulina G/sangue , Mucosa Intestinal/metabolismo , RNA Viral , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/imunologia , Células Vero
2.
Nat Commun ; 12(1): 63, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397909

RESUMO

The SARS-CoV-2 pandemic poses the greatest global public health challenge in a century. Neutralizing antibody is a correlate of protection and data on kinetics of virus neutralizing antibody responses are needed. We tested 293 sera from an observational cohort of 195 reverse transcription polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections collected from 0 to 209 days after onset of symptoms. Of 115 sera collected ≥61 days after onset of illness tested using plaque reduction neutralization (PRNT) assays, 99.1% remained seropositive for both 90% (PRNT90) and 50% (PRNT50) neutralization endpoints. We estimate that it takes at least 372, 416 and 133 days for PRNT50 titres to drop to the detection limit of a titre of 1:10 for severe, mild and asymptomatic patients, respectively. At day 90 after onset of symptoms (or initial RT-PCR detection in asymptomatic infections), it took 69, 87 and 31 days for PRNT50 antibody titres to decrease by half (T1/2) in severe, mild and asymptomatic infections, respectively. Patients with severe disease had higher peak PRNT90 and PRNT50 antibody titres than patients with mild or asymptomatic infections. Age did not appear to compromise antibody responses, even after accounting for severity. We conclude that SARS-CoV-2 infection elicits robust neutralizing antibody titres in most individuals.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , /imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/sangue , /epidemiologia , Chlorocebus aethiops , Estudos de Coortes , Feminino , Hong Kong/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Células Vero , Adulto Jovem
3.
Cell ; 184(1): 76-91.e13, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33147444

RESUMO

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.


Assuntos
Infecções por Coronavirus/genética , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , /fisiologia , /metabolismo , Animais , /virologia , Linhagem Celular , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Coronavirus/classificação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Células Vero , Internalização do Vírus
4.
Antiviral Res ; 185: 104974, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33217430

RESUMO

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression to zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.


Assuntos
Antivirais/farmacologia , Cromossomos Artificiais Bacterianos , Replicon/efeitos dos fármacos , /genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Interferon-alfa/farmacologia , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Sofosbuvir/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacos
5.
Antiviral Res ; 185: 104988, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33248195

RESUMO

Pandemic spread of emerging human pathogenic viruses, such as the current SARS-CoV-2, poses both an immediate and future challenge to human health and society. Currently, effective treatment of infection with SARS-CoV-2 is limited and broad spectrum antiviral therapies to meet other emerging pandemics are absent leaving the World population largely unprotected. Here, we have identified distinct members of the family of polyether ionophore antibiotics with potent ability to inhibit SARS-CoV-2 replication and cytopathogenicity in cells. Several compounds from this class displayed more than 100-fold selectivity between viral-induced cytopathogenicity and inhibition of cell viability, however the compound X-206 displayed >500-fold selectivity and was furthermore able to inhibit viral replication even at sub-nM levels. The antiviral mechanism of the polyether ionophores is currently not understood in detail. We demonstrate, e.g. through unbiased bioactivity profiling, that their effects on the host cells differ from those of cationic amphiphiles such as hydroxychloroquine. Collectively, our data suggest that polyether ionophore antibiotics should be subject to further investigations as potential broad-spectrum antiviral agents.


Assuntos
Antibacterianos/farmacologia , Antivirais/farmacologia , Éteres Cíclicos/farmacologia , Ionóforos/farmacologia , /efeitos dos fármacos , Animais , Chlorocebus aethiops , Humanos , Células Vero , Replicação Viral/efeitos dos fármacos
6.
Antiviral Res ; 185: 104996, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33309540

RESUMO

Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a coronavirus (MERS-CoV). Since its emergence in 2012, nosocomial amplifications have led to its high epidemic potential and mortality rate of 34.5%. To date, there is an unmet need for vaccines and specific therapeutics for this disease. Available treatments are either supportive medications in use for other diseases or those lacking specificity requiring higher doses. The viral infection mode is initiated by the attachment of the viral spike glycoprotein to the human Dipeptidyl Peptidase IV (DPP4). Our attempts to screen antivirals against MERS led us to identify montelukast sodium hydrate (MSH), an FDA-approved anti-asthma drug, as an agent attenuating MERS-CoV infection. We showed that MSH directly binds to MERS-CoV-Receptor-Binding Domain (RBD) and inhibits its molecular interaction with DPP4 in a dose-dependent manner. Our cell-based inhibition assays using MERS pseudovirions demonstrated that viral infection was significantly inhibited by MSH and was further validated using infectious MERS-CoV culture. Thus, we propose MSH as a potential candidate for therapeutic developments against MERS-CoV infections.


Assuntos
Acetatos/farmacologia , Antivirais/farmacologia , Ciclopropanos/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Quinolinas/farmacologia , Sulfetos/farmacologia , Animais , Antiasmáticos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Indutores do Citocromo P-450 CYP1A2/farmacologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Antagonistas de Leucotrienos/farmacologia , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos
7.
Cell ; 184(1): 106-119.e14, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33333024

RESUMO

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.


Assuntos
/genética , Infecções por Coronavirus/genética , Coronavirus/fisiologia , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , /fisiologia , Células A549 , Animais , Vias Biossintéticas/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Colesterol/biossíntese , Colesterol/metabolismo , Análise por Conglomerados , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resfriado Comum/genética , Resfriado Comum/virologia , Coronavirus/classificação , Infecções por Coronavirus/virologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos , Fosfatidilinositóis/biossíntese , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral
8.
Nat Microbiol ; 6(1): 11-18, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33273742

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic is having a catastrophic impact on human health1. Widespread community transmission has triggered stringent distancing measures with severe socio-economic consequences. Gaining control of the pandemic will depend on the interruption of transmission chains until vaccine-induced or naturally acquired protective herd immunity arises. However, approved antiviral treatments such as remdesivir and reconvalescent serum cannot be delivered orally2,3, making them poorly suitable for transmission control. We previously reported the development of an orally efficacious ribonucleoside analogue inhibitor of influenza viruses, MK-4482/EIDD-2801 (refs. 4,5), that was repurposed for use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently in phase II/III clinical trials (NCT04405570 and NCT04405739). Here, we explored the efficacy of therapeutically administered MK-4482/EIDD-2801 to mitigate SARS-CoV-2 infection and block transmission in the ferret model, given that ferrets and related members of the weasel genus transmit the virus efficiently with minimal clinical signs6-9, which resembles the spread in the human young-adult population. We demonstrate high SARS-CoV-2 burden in nasal tissues and secretions, which coincided with efficient transmission through direct contact. Therapeutic treatment of infected animals with MK-4482/EIDD-2801 twice a day significantly reduced the SARS-CoV-2 load in the upper respiratory tract and completely suppressed spread to untreated contact animals. This study identified oral MK-4482/EIDD-2801 as a promising antiviral countermeasure to break SARS-CoV-2 community transmission chains.


Assuntos
Antivirais/farmacologia , /transmissão , Citidina/análogos & derivados , Hidroxilaminas/farmacologia , /efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Chlorocebus aethiops , Citidina/farmacologia , Citocinas/imunologia , Modelos Animais de Doenças , Transmissão de Doença Infecciosa/prevenção & controle , Feminino , Furões , Distribuição Aleatória , Células Vero
9.
Nat Chem Biol ; 17(1): 113-121, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33082574

RESUMO

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.


Assuntos
/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Anticorpos de Cadeia Única/química , Glicoproteína da Espícula de Coronavírus/química , /antagonistas & inibidores , /imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
10.
Bioorg Med Chem Lett ; 31: 127667, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33160024

RESUMO

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) continues to spread worldwide, with 25 million confirmed cases and 800 thousand deaths. Effective treatments to target SARS-CoV-2 are urgently needed. In the present study, we have identified a class of cyclic sulfonamide derivatives as novel SARS-CoV-2 inhibitors. Compound 13c of the synthesized compounds exhibited robust inhibitory activity (IC50 = 0.88 µM) against SARS-CoV-2 without cytotoxicity (CC50 > 25 µM), with a selectivity index (SI) of 30.7. In addition, compound 13c exhibited high oral bioavailability (77%) and metabolic stability with good safety profiles in hERG and cytotoxicity studies. The present study identified that cyclic sulfonamide derivatives are a promising new template for the development of anti-SARS-CoV-2 agents.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Sulfonamidas/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Cães , Relação Dose-Resposta a Droga , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
11.
Cell Res ; 31(1): 17-24, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262453

RESUMO

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic worldwide. Currently, however, no effective drug or vaccine is available to treat or prevent the resulting coronavirus disease 2019 (COVID-19). Here, we report our discovery of a promising anti-COVID-19 drug candidate, the lipoglycopeptide antibiotic dalbavancin, based on virtual screening of the FDA-approved peptide drug library combined with in vitro and in vivo functional antiviral assays. Our results showed that dalbavancin directly binds to human angiotensin-converting enzyme 2 (ACE2) with high affinity, thereby blocking its interaction with the SARS-CoV-2 spike protein. Furthermore, dalbavancin effectively prevents SARS-CoV-2 replication in Vero E6 cells with an EC50 of ~12 nM. In both mouse and rhesus macaque models, viral replication and histopathological injuries caused by SARS-CoV-2 infection are significantly inhibited by dalbavancin administration. Given its high safety and long plasma half-life (8-10 days) shown in previous clinical trials, our data indicate that dalbavancin is a promising anti-COVID-19 drug candidate.


Assuntos
/metabolismo , Antivirais , Glicoproteína da Espícula de Coronavírus/metabolismo , Teicoplanina/análogos & derivados , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Células CACO-2 , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Ligação Proteica/efeitos dos fármacos , Teicoplanina/farmacocinética , Teicoplanina/farmacologia , Células Vero
12.
Cell Res ; 31(1): 25-36, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262452

RESUMO

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , /prevenção & controle , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Anticorpos de Cadeia Única/farmacologia , Células Vero
13.
Eur J Clin Microbiol Infect Dis ; 40(1): 1-12, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33037944

RESUMO

Can a patient diagnosed with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) be infected again? This question is still unsolved. We tried to analyze local and literature cases with a positive respiratory swab after recovery. We collected data from symptomatic patients diagnosed with SARS-CoV-2 infection in the Italian Umbria Region that, after recovery, were again positive for SARS-CoV-2 in respiratory tract specimens. Samples were also assessed for infectivity in vitro. A systematic review of similar cases reported in the literature was performed. The study population was composed of 9 patients during a 4-month study period. Among the new positive samples, six were inoculated in Vero-E6 cells and showed no growth and negative molecular test in culture supernatants. All patients were positive for IgG against SARS-CoV-2 nucleoprotein and/or S protein. Conducting a review of the literature, 1350 similar cases have been found. The presumptive reactivation occurred in 34.5 days on average (standard deviation, SD, 18.7 days) after COVID-19 onset, when the 5.6% of patients presented fever and the 27.6% symptoms. The outcome was favorable in 96.7% of patients, while the 1.1% of them were still hospitalized at the time of data collection and the 2.1% died. Several hypotheses have been formulated to explain new positive respiratory samples after confirmed negativity. According to this study, the phenomenon seems to be due to the prolonged detection of SARS-CoV-2 RNA traces in respiratory samples of recovered patients. The failure of the virus to replicate in vitro suggests its inability to replicate in vivo.


Assuntos
/estatística & dados numéricos , /fisiopatologia , Adulto , Idoso , Animais , Chlorocebus aethiops , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/análise , Recidiva , Células Vero , Replicação Viral
15.
Biochem Pharmacol ; 183: 114302, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33121927

RESUMO

Baicalein is the main active compound of Scutellaria baicalensis Georgi, a medicinal herb with multiple pharmacological activities, including the broad anti-virus effects. In this paper, the preclinical study of baicalein on the treatment of COVID-19 was performed. Results showed that baicalein inhibited cell damage induced by SARS-CoV-2 and improved the morphology of Vero E6 cells at a concentration of 0.1 µM and above. The effective concentration could be reached after oral administration of 200 mg/kg crystal form ß of baicalein in rats. Furthermore, baicalein significantly inhibited the body weight loss, the replication of the virus, and relieved the lesions of lung tissue in hACE2 transgenic mice infected with SARS-CoV-2. In LPS-induced acute lung injury of mice, baicalein improved the respiratory function, inhibited inflammatory cell infiltration in the lung, and decreased the levels of IL-1ß and TNF-α in serum. In conclusion, oral administration of crystal form ß of baicalein could reach its effective concentration against SARS-CoV-2. Baicalein could inhibit SARS-CoV-2-induced injury both in vitro and in vivo. Therefore, baicalein might be a promising therapeutic drug for the treatment of COVID-19.


Assuntos
Antioxidantes/uso terapêutico , /patologia , Flavanonas/uso terapêutico , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Antioxidantes/farmacocinética , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Flavanonas/farmacocinética , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Células Vero
16.
Virus Res ; 292: 198246, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33249060

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-COV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally bioavailable compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine nucleotide biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS-COV-2 replication (EC50 range, 2.0-31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17 F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19.


Assuntos
Antivirais/farmacologia , Carbamatos/farmacologia , Carbazóis/farmacologia , Citocinas/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Síndrome da Liberação de Citocina/tratamento farmacológico , Citocinas/imunologia , Células HeLa , Humanos , Inflamação/tratamento farmacológico , Inflamação/virologia , Células Vero
17.
J Virol Methods ; 287: 113995, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33068703

RESUMO

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus (SARS-CoV), emerged in Wuhan, Hubei province of China, and has been responsible for coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2. The high economical and health impacts of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate, and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Antivirais/farmacologia , Testes de Neutralização/métodos , /imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Ensaios de Triagem em Larga Escala , Humanos , Células Vero , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
18.
Clin Microbiol Infect ; 27(1): 128.e1-128.e7, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32979576

RESUMO

OBJECTIVES: A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the current coronavirus disease 2019 global pandemic. Only a few laboratories routinely isolate the virus, which is because the current co-culture strategy is highly time-consuming and requires a biosafety level 3 laboratory. This work aimed to develop a new high-throughput isolation strategy using novel technologies for rapid and automated isolation of SARS-CoV-2. METHODS: We used an automated microscope based on high-content screening (HCS), and we applied specific image analysis algorithms targeting cytopathic effects of SARS-CoV-2 on Vero E6 cells. A randomized panel of 104 samples, including 72 that tested positive by RT-PCR and 32 that tested negative, were processed with our HCS strategy and were compared with the classical isolation procedure. RESULTS: The isolation rate was 43% (31/72) with both strategies on RT-PCR-positive samples and was correlated with the initial RNA viral load in the samples, in which we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with the HCS strategy, where 80% (25/31) of the positive samples were recovered by the third day of co-culture, compared with only 26% (8/30) with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive samples (31 with HCS versus 27 with classic strategy) after 1 week of co-culture. CONCLUSIONS: This system allows the rapid and automated screening of clinical samples with minimal operator workload, which reduces the risk of contamination and paves the way for future applications in clinical microbiology, such as large-scale drug susceptibility testing.


Assuntos
/métodos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , RNA Viral/análise , /isolamento & purificação , Animais , Automação Laboratorial , Biomarcadores/análise , Chlorocebus aethiops , Hospitalização , Humanos , Microscopia/métodos , Nasofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Manejo de Espécimes/métodos , Células Vero , Carga Viral
19.
ACS Appl Mater Interfaces ; 12(50): 55688-55695, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33267577

RESUMO

In the present study, we examined the inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by synthetic conjugated polymers and oligomers developed in our laboratories as antimicrobials for bacteria, fungi, and nonenveloped viruses. The results show highly effective light-induced inactivation with several of these oligomers and polymers including irradiation with near-UV and visible light. In the best case, one oligomer induced a 5-log reduction in pfu/mL within 10 min. In general, the oligomers are more active than the polymers; however, the polymers are active with longer wavelength visible irradiation. Although not studied quantitatively, the results show that in the presence of the agents at concentrations similar to those used in the light studies, there is essentially no dark inactivation of the virus. Because three of the five materials/compounds examined are quaternary ammonium derivatives, this study indicates that conventional quaternary ammonium antimicrobials may not be active against SARS-CoV-2. Our results suggest several applications involving the incorporation of these materials in wipes, sprays, masks, and clothing and other personal protection equipment that can be useful in preventing infections and the spreading of this deadly virus and future outbreaks from similar viruses.


Assuntos
/tratamento farmacológico , Polímeros/farmacologia , /efeitos dos fármacos , Animais , Chlorocebus aethiops , Humanos , Luz , Polímeros/efeitos da radiação , /efeitos da radiação , Raios Ultravioleta , Células Vero , Inativação de Vírus/efeitos dos fármacos , Inativação de Vírus/efeitos da radiação
20.
PLoS Comput Biol ; 16(12): e1008461, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33290397

RESUMO

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through 'repurposing' drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.


Assuntos
Catepsina B/química , Catepsina L/química , Serina Endopeptidases/química , Internalização do Vírus/efeitos dos fármacos , Animais , Chlorocebus aethiops , Reposicionamento de Medicamentos , Humanos , Modelos Teóricos , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírion
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