RESUMEN
Nas Américas, uma rede de laboratórios de 22 países contribui para a detecção de variantes do SARS-CoV-2. Até agora, 37 países e territórios confirmaram a presença de uma ou mais das quatro variantes de preocupação. Monitorá-las é a chave para detectar quaisquer mudanças incomuns ou inesperadas que possam afetar as medidas de controle, incluindo as vacinas
Asunto(s)
Betacoronavirus/crecimiento & desarrollo , Infecciones por Coronavirus , Vigilancia en Desastres , Virus/crecimiento & desarrollo , Especialización/tendenciasRESUMEN
Em comunicado técnico publicado nesta quinta-feira (4/3) pelo Observatório Covid-19 Fiocruz, pesquisadores alertam para a dispersão geográfica no território de ‘variantes de preocupação’, assim como sua alta prevalência nas três regiões do Brasil avaliadas (Sul, Sudeste e Nordeste).
Asunto(s)
Infecciones por Coronavirus , Mutación , Betacoronavirus/crecimiento & desarrollo , PandemiasRESUMEN
Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2) [1-3]. Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in the nasal turbinates, lung and brain, with high lethality, and cytokine/chemokine production. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the nasal turbinates and lung, and no clinical signs of infection. The K18-hACE2 model provides a stringent model for testing vaccines and antivirals, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.
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Betacoronavirus/crecimiento & desarrollo , Infecciones por Coronavirus/patología , Peptidil-Dipeptidasa A/genética , Neumonía Viral/patología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/crecimiento & desarrollo , Replicación Viral/genética , Células A549 , Adenoviridae/genética , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/metabolismo , COVID-19 , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Pandemias , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , SARS-CoV-2 , Células Vero , Acoplamiento ViralRESUMEN
SARS-CoV-2, the causative agent of COVID-19, has been responsible for a million deaths worldwide as of September 2020. At the time of this writing, there are no available US FDA-approved therapeutics for the treatment of SARS-CoV-2 infection. Here, we describe a detailed protocol to generate recombinant (r)SARS-CoV-2 using reverse-genetics approaches based on the use of a bacterial artificial chromosome (BAC). This method will allow the production of mutant rSARS-CoV-2-which is necessary for understanding the function of viral proteins, viral pathogenesis and/or transmission, and interactions at the virus-host interface-and attenuated SARS-CoV-2 to facilitate the discovery of effective countermeasures to control the ongoing SARS-CoV-2 pandemic. © 2020 Wiley Periodicals LLC. Basic Protocol: Generation of recombinant SARS-CoV-2 using a bacterial artificial chromosome Support Protocol: Validation and characterization of rSARS-CoV-2.
Asunto(s)
Betacoronavirus/genética , Cromosomas Artificiales Bacterianos/genética , Animales , Betacoronavirus/crecimiento & desarrollo , Chlorocebus aethiops , Contención de Riesgos Biológicos , SARS-CoV-2 , Transfección , Células VeroRESUMEN
Early in the course of the coronavirus infection disease 2019 (COVID-19) pandemic in South Africa, the Department of Health implemented a policy of community screening and testing (CST). This was based on a community-orientated primary care approach and was a key strategy in limiting the spread of the pandemic, but it struggled with long turnaround times (TATs) for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) reverse transcriptase polymerase chain reaction test. The local experience at Symphony Way Community Day Centre (Delft, Cape Town), highlighted these challenges. The first positive tests had a median TAT of 4.5 days, peaking at 29 days in mid-May 2020. Issues that contributed to long TATs were unavailability of viral transport medium, sample delivery and storage difficulties, staffing problems, scarcity of testing supplies and other samples prioritised over CST samples. At Symphony Way, many patients who tested COVID-19 positive had abandoned their self-isolation because of the delay in results. Employers were unhappy with prolonged sick leave whilst waiting for results and patients were concerned about not getting paid or job loss. The CST policy relies on a rapid TAT to be successful. Once the TAT is delayed, the process of contacting patients, and tracing and quarantining contacts becomes ineffective. With hindsight, other countries' difficulties in upscaling testing should have served as warning. Community screening and testing was scaled back from 18 May 2020, and testing policy was changed to only include high-risk patients from 29 May 2020. The delayed TATs meant that the CST policy had no beneficial impact at local level.
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Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Accesibilidad a los Servicios de Salud , Tamizaje Masivo , Neumonía Viral/diagnóstico , Políticas , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/métodos , Coronavirus , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Humanos , Tamizaje Masivo/métodos , Pandemias , Neumonía Viral/epidemiología , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , SARS-CoV-2 , Síndrome Respiratorio Agudo Grave , Sudáfrica , Factores de TiempoRESUMEN
Patients and physicians worldwide are facing tremendous health care hazards that are caused by the ongoing severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) pandemic. Remdesivir (GS-5734) is the first approved treatment for severe coronavirus disease 2019 (COVID-19). It is a novel nucleoside analog with a broad antiviral activity spectrum among RNA viruses, including ebolavirus (EBOV) and the respiratory pathogens Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and SARS-CoV-2. First described in 2016, the drug was derived from an antiviral library of small molecules intended to target emerging pathogenic RNA viruses. In vivo, remdesivir showed therapeutic and prophylactic effects in animal models of EBOV, MERS-CoV, SARS-CoV, and SARS-CoV-2 infection. However, the substance failed in a clinical trial on ebolavirus disease (EVD), where it was inferior to investigational monoclonal antibodies in an interim analysis. As there was no placebo control in this study, no conclusions on its efficacy in EVD can be made. In contrast, data from a placebo-controlled trial show beneficial effects for patients with COVID-19. Remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. Although this is an important milestone in the fight against COVID-19, approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. Further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Adenosina Monofosfato/farmacocinética , Adenosina Monofosfato/farmacología , Alanina/farmacocinética , Alanina/farmacología , Antivirales/farmacocinética , Betacoronavirus/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , COVID-19 , Ensayos Clínicos como Asunto , Ensayos de Uso Compasivo/métodos , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Esquema de Medicación , Ebolavirus/efectos de los fármacos , Ebolavirus/crecimiento & desarrollo , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/mortalidad , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/virología , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/crecimiento & desarrollo , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Pandemias , Seguridad del Paciente , Neumonía Viral/mortalidad , Neumonía Viral/patología , Neumonía Viral/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/crecimiento & desarrollo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , SARS-CoV-2 , Síndrome Respiratorio Agudo Grave/mortalidad , Síndrome Respiratorio Agudo Grave/patología , Síndrome Respiratorio Agudo Grave/virología , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). Here, we quantify SARS-CoV-2 viral load from participants with a diverse range of COVID-19 disease severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. We detected SARS-CoV-2 plasma RNA in 27% of hospitalized participants, and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, we report that a higher prevalence of detectable SARS-CoV-2 plasma viral load is associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, are associated with increased risk of mortality. Our data show that SARS-CoV-2 viral loads may aid in the risk stratification of patients with COVID-19, and therefore its role in disease pathogenesis should be further explored.
Asunto(s)
Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Adulto , Anciano , Anticuerpos Antivirales/sangre , Betacoronavirus/genética , Betacoronavirus/crecimiento & desarrollo , Biomarcadores/sangre , Proteína C-Reactiva , COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/patología , Femenino , Hospitalización , Humanos , Inflamación/sangre , Inflamación/virología , Interleucina-6/sangre , Estudios Longitudinales , Massachusetts/epidemiología , Persona de Mediana Edad , Pandemias , Neumonía Viral/sangre , Neumonía Viral/mortalidad , Neumonía Viral/patología , ARN Viral/sangre , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Carga Viral , Viremia/sangre , Viremia/virologíaRESUMEN
The spread of SARS-CoV-2 in Beijing before May, 2020 resulted from transmission following both domestic and global importation of cases. Here we present genomic surveillance data on 102 imported cases, which account for 17.2% of the total cases in Beijing. Our data suggest that all of the cases in Beijing can be broadly classified into one of three groups: Wuhan exposure, local transmission and overseas imports. We classify all sequenced genomes into seven clusters based on representative high-frequency single nucleotide polymorphisms (SNPs). Genomic comparisons reveal higher genomic diversity in the imported group compared to both the Wuhan exposure and local transmission groups, indicating continuous genomic evolution during global transmission. The imported group show region-specific SNPs, while the intra-host single nucleotide variations present as random features, and show no significant differences among groups. Epidemiological data suggest that detection of cases at immigration with mandatory quarantine may be an effective way to prevent recurring outbreaks triggered by imported cases. Notably, we also identify a set of novel indels. Our data imply that SARS-CoV-2 genomes may have high mutational tolerance.
Asunto(s)
Betacoronavirus/crecimiento & desarrollo , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Adulto , Beijing/epidemiología , COVID-19 , Infecciones por Coronavirus/epidemiología , Femenino , Genoma Viral , Genómica , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pandemias , Filogenia , Neumonía Viral/epidemiología , Polimorfismo de Nucleótido Simple , SARS-CoV-2 , Viaje , Adulto JovenRESUMEN
OBJECTIVES: We performed an environmental sampling study to investigate the environmental contamination of SARS-CoV-2 by COVID-19 patients with prolonged PCR positive status of clinical samples. METHODS: We sampled the air from rooms for nine COVID-19 patients with illness or positive PCR > 30 days, before and after nasopharyngeal/oropharyngeal swabbing and before and after nebulization treatment. We also sampled patients' surroundings and healthcare workers' personal protection equipment (PPE) in a non-ICU ward. SARS-CoV-2 was detected by PCR. RESULTS: Eighty-eight samples were collected from high-touch surfaces and floors in patient rooms and toilets, with only the bedsheets of two patients and one toilet positive for SARS-CoV-2. All air samples (n = 34) were negative for SARS-CoV-2. Fifty-five samples collected from PPE were all negative. CONCLUSION: Contamination of near-patient surroundings was uncommon for COVID-19 patients with prolonged PCR positive status if environmental cleaning/disinfection were performed rigorously. Airborne transmission of SARS-CoV-2 was unlikely in these non-ICU settings.
Asunto(s)
Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/virología , Equipo de Protección Personal , Neumonía Viral/virología , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Desinfección/métodos , Microbiología Ambiental , Monitoreo del Ambiente/métodos , Personal de Salud , Hospitales , Humanos , Pandemias/prevención & control , Habitaciones de Pacientes , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa , ARN Viral/aislamiento & purificación , SARS-CoV-2Asunto(s)
Alquenos/farmacología , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Polifenoles/farmacología , Subunidades de Proteína/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Internalización del Virus/efectos de los fármacos , Alquenos/química , Enzima Convertidora de Angiotensina 2 , Animales , Antivirales/química , Betacoronavirus/genética , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/metabolismo , Sitios de Unión , Bioensayo , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/tratamiento farmacológico , Descubrimiento de Drogas , Expresión Génica , Células HEK293 , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Pandemias , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/tratamiento farmacológico , Polifenoles/química , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroAsunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Mesilato de Imatinib/farmacología , Niacinamida/análogos & derivados , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , COVID-19 , Células CACO-2 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Reposicionamiento de Medicamentos , Humanos , Concentración 50 Inhibidora , Niacinamida/farmacología , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Ensayos Clínicos Controlados Aleatorios como Asunto , SARS-CoV-2 , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
COVID-19 has become a global pandemic caused by the novel coronavirus SARS-CoV-2. Understanding the origins of SARS-CoV-2 is critical for deterring future zoonosis, discovering new drugs, and developing a vaccine. We show evidence of strong purifying selection around the receptor binding motif (RBM) in the spike and other genes among bat, pangolin, and human coronaviruses, suggesting similar evolutionary constraints in different host species. We also demonstrate that SARS-CoV-2's entire RBM was introduced through recombination with coronaviruses from pangolins, possibly a critical step in the evolution of SARS-CoV-2's ability to infect humans. Similar purifying selection in different host species, together with frequent recombination among coronaviruses, suggests a common evolutionary mechanism that could lead to new emerging human coronaviruses.
Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/patología , Neumonía Viral/patología , Recombinación Genética , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/clasificación , Betacoronavirus/crecimiento & desarrollo , Sitios de Unión , COVID-19 , Quirópteros/virología , Infecciones por Coronavirus/virología , Evolución Molecular , Genoma Viral , Humanos , Pandemias , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Filogenia , Neumonía Viral/virología , Estructura Terciaria de Proteína , SARS-CoV-2 , Alineación de SecuenciaRESUMEN
To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell transcriptomics across various healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Intestinal goblet cells, enterocytes, and kidney proximal tubule cells appear highly permissive to SARS-CoV-2, consistent with clinical data. Our analysis also predicts non-canonical entry paths for lung and brain infections. Spermatogonial cells and prostate endocrine cells also appear to be permissive to SARS-CoV-2 infection, suggesting male-specific vulnerabilities. Both pro- and anti-viral factors are highly expressed within the nasal epithelium, with potential age-dependent variation, predicting an important battleground for coronavirus infection. Our analysis also suggests that early embryonic and placental development are at moderate risk of infection. Lastly, SCARF expression appears broadly conserved across a subset of primate organs examined. Our study establishes a resource for investigations of coronavirus biology and pathology.
Asunto(s)
Infecciones por Coronavirus/patología , Mucosa Nasal/metabolismo , Neumonía Viral/patología , Receptores Virales/genética , Tropismo Viral/genética , Internalización del Virus , Células A549 , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Línea Celular , Chlorocebus aethiops , Enterocitos/metabolismo , Perfilación de la Expresión Génica , Células Caliciformes/metabolismo , Células HEK293 , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Mucosa Nasal/virología , Pandemias , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2 , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Análisis de la Célula Individual , Células VeroRESUMEN
There is a vast practice of using antimalarial drugs, RAS inhibitors, serine protease inhibitors, inhibitors of the RNA-dependent RNA polymerase of the virus and immunosuppressants for the treatment of the severe form of COVID-19, which often occurs in patients with chronic diseases and older persons. Currently, the clinical efficacy of these drugs for COVID-19 has not been proven yet. Side effects of antimalarial drugs can worsen the condition of patients and increase the likelihood of death. Peptides, given their physiological mechanism of action, have virtually no side effects. Many of them are geroprotectors and can be used in patients with chronic diseases. Peptides may be able to prevent the development of the pathological process during COVID-19 by inhibiting SARS-CoV-2 virus proteins, thereby having immuno- and bronchoprotective effects on lung cells, and normalizing the state of the hemostasis system. Immunomodulators (RKDVY, EW, KE, AEDG), possessing a physiological mechanism of action at low concentrations, appear to be the most promising group among the peptides. They normalize the cytokines' synthesis and have an anti-inflammatory effect, thereby preventing the development of disseminated intravascular coagulation, acute respiratory distress syndrome and multiple organ failure.
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Antiinflamatorios/uso terapéutico , Antivirales/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Péptidos/uso terapéutico , Neumonía Viral/tratamiento farmacológico , Fármacos del Sistema Respiratorio/uso terapéutico , Enfermedad Aguda , Antiinflamatorios/síntesis química , Antivirales/síntesis química , Betacoronavirus/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Síndrome de Liberación de Citoquinas/complicaciones , Síndrome de Liberación de Citoquinas/diagnóstico , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Síndrome de Liberación de Citoquinas/virología , Coagulación Intravascular Diseminada/complicaciones , Coagulación Intravascular Diseminada/diagnóstico , Coagulación Intravascular Diseminada/tratamiento farmacológico , Coagulación Intravascular Diseminada/virología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Factores Inmunológicos/síntesis química , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/virología , Pandemias , Péptidos/síntesis química , Neumonía Viral/complicaciones , Neumonía Viral/diagnóstico , Neumonía Viral/virología , Insuficiencia Respiratoria/complicaciones , Insuficiencia Respiratoria/diagnóstico , Insuficiencia Respiratoria/prevención & control , Insuficiencia Respiratoria/virología , Fármacos del Sistema Respiratorio/síntesis química , SARS-CoV-2 , Relación Estructura-ActividadRESUMEN
The world is in the midst of a pandemic caused by a novel coronavirus and is desperately searching for possible treatments. The antiviral remdesivir has shown some effectiveness against SARS-CoV-2 in vitro and in a recent animal study. We use data from a study of remdesivir in rhesus macaques to fit a viral kinetics model in an effort to determine the most appropriate mathematical descripton of the effect of remdesivir. We find statistically significant differences in the viral decay rate and use this to inform a possible mathematical formulation of the effect of remdesivir. Unfortunately, this model formulation suggests that the application of remdesivir will lengthen SARS-CoV-2 infections, putting into question its potential clinical benefit.
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Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacocinética , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Modelos Estadísticos , Neumonía Viral/tratamiento farmacológico , Adenosina Monofosfato/sangre , Adenosina Monofosfato/farmacocinética , Alanina/sangre , Alanina/farmacocinética , Animales , Antivirales/sangre , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Esquema de Medicación , Humanos , Inflamación , Macaca mulatta , Pandemias , Neumonía Viral/sangre , Neumonía Viral/virología , SARS-CoV-2 , Carga Viral , Replicación ViralRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , Técnicas de Cultivo de Célula/métodos , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidad , COVID-19 , Pandemias , ARN Viral/análisis , ARN Viral/aislamiento & purificación , SARS-CoV-2 , Replicación Viral/efectos de los fármacosRESUMEN
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mainly affects the lungs. Sarcoidosis is an autoinflammatory disease characterized by the diffusion of granulomas in the lungs and other organs. Here, we discuss how the two diseases might involve some common mechanistic cellular pathways around the regulation of autophagy.
Asunto(s)
Autofagia/efectos de los fármacos , Betacoronavirus/patogenicidad , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Edema Pulmonar/tratamiento farmacológico , Sarcoidosis/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Autofagia/genética , Azitromicina/uso terapéutico , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Cloroquina/uso terapéutico , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/virología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Isoniazida/uso terapéutico , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/virología , Pandemias , Neumonía Viral/epidemiología , Neumonía Viral/genética , Neumonía Viral/virología , Edema Pulmonar/epidemiología , Edema Pulmonar/genética , Edema Pulmonar/virología , Rifampin/uso terapéutico , SARS-CoV-2 , Sarcoidosis/epidemiología , Sarcoidosis/genética , Sarcoidosis/virología , Síndrome Respiratorio Agudo Grave/epidemiología , Síndrome Respiratorio Agudo Grave/genética , Síndrome Respiratorio Agudo Grave/virología , Índice de Severidad de la EnfermedadRESUMEN
Coronavirus disease 2019 (COVID-19), which is caused by a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has recently emerged as a global health threat. To address this health emergency, various therapeutic approaches are currently under investigation. There is limited evidence on the effectiveness of hydroxychloroquine (HCQ) and chloroquine (CQ) as COVID-19 therapies, and thus World Health Organization (WHO) mentioned that "Current data shows that this drug does not reduce deaths among hospitalized COVID-19 patients, nor help people with mild or moderate disease." CQ and HCQ are typically used for the treatment of malaria but have been recognized for certain beneficial effects in COVID-19 patients based on some clinical outcomes from the clinical treatment of COVID-19. A standard dose of HCQ has been proven effective and less toxic than CQ in COVID-19 patients; however, a comprehensive understanding of a patient's clinical condition is necessary. Based on several hospital findings, the Food and Drug Administration (FDA) has officially cancelled the emergency use authorization for HCQ and CQ for treating hospitalized COVID-19 patients on June 15, 2020. In this review, we highlight both pros and cons of the clinical use of CQ and HCQ in COVID-19 patients.
Asunto(s)
Antiinfecciosos/administración & dosificación , Betacoronavirus/efectos de los fármacos , Cloroquina/administración & dosificación , Infecciones por Coronavirus/tratamiento farmacológico , Hidroxicloroquina/administración & dosificación , Factores Inmunológicos/administración & dosificación , Neumonía Viral/tratamiento farmacológico , Antiinfecciosos/efectos adversos , Azitromicina/uso terapéutico , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , COVID-19 , Cloroquina/efectos adversos , Ensayos Clínicos como Asunto , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Sinergismo Farmacológico , Humanos , Hidroxicloroquina/efectos adversos , Factores Inmunológicos/efectos adversos , Pandemias , Neumonía Viral/patología , Neumonía Viral/virología , SARS-CoV-2 , Resultado del TratamientoRESUMEN
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.