Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38.036
Filtrar
1.
Euro Surveill ; 25(28)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32700671

RESUMO

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Animais , Betacoronavirus/genética , Western Blotting , Células COS , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Genoma Viral , Camundongos , Pandemias , Peptidil Dipeptidase A/imunologia , Plasmídeos , Pneumonia Viral/genética , Proteínas Recombinantes/imunologia , Vírus da SARS/genética , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Transfecção , Células Vero , Integração Viral
2.
J Exp Med ; 217(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32692348

RESUMO

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Imunoensaio/métodos , Pneumonia Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Linhagem Celular , Quimera/genética , Quimera/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Células HEK293 , HIV-1/genética , HIV-1/imunologia , Humanos , Testes de Neutralização/métodos , Pandemias , Pneumonia Viral/virologia , Recombinação Genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
3.
Proc Natl Acad Sci U S A ; 117(28): 16587-16595, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32571934

RESUMO

At the end of 2019, a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was detected in Wuhan, China, that spread rapidly around the world, with severe consequences for human health and the global economy. Here, we assessed the replicative ability and pathogenesis of SARS-CoV-2 isolates in Syrian hamsters. SARS-CoV-2 isolates replicated efficiently in the lungs of hamsters, causing severe pathological lung lesions following intranasal infection. In addition, microcomputed tomographic imaging revealed severe lung injury that shared characteristics with SARS-CoV-2-infected human lung, including severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation. SARS-CoV-2-infected hamsters mounted neutralizing antibody responses and were protected against subsequent rechallenge with SARS-CoV-2. Moreover, passive transfer of convalescent serum to naïve hamsters efficiently suppressed the replication of the virus in the lungs even when the serum was administrated 2 d postinfection of the serum-treated hamsters. Collectively, these findings demonstrate that this Syrian hamster model will be useful for understanding SARS-CoV-2 pathogenesis and testing vaccines and antiviral drugs.


Assuntos
Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Pulmão/patologia , Pneumonia Viral/virologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/terapia , Cricetinae , Humanos , Imunização Passiva , Pulmão/diagnóstico por imagem , Pulmão/virologia , Mesocricetus , Pandemias , Pneumonia Viral/patologia , Ribonucleoproteínas/química , Células Vero , Proteínas Virais/química , Replicação Viral
4.
Cell ; 182(2): 429-446.e14, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32526206

RESUMO

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Sistema Respiratório/virologia , Genética Reversa/métodos , Idoso , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Fibrose Cística/patologia , DNA Recombinante , Feminino , Furina/metabolismo , Humanos , Imunização Passiva , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Sistema Respiratório/patologia , Serina Endopeptidases/metabolismo , Células Vero , Virulência , Replicação Viral
5.
Curr Protoc Microbiol ; 57(1): ecpmc105, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32475066

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID50 ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.


Assuntos
Betacoronavirus/isolamento & purificação , Protocolos Clínicos , Ensaio de Placa Viral/métodos , Animais , Chlorocebus aethiops , Humanos , Coloração e Rotulagem , Células Vero
6.
Viruses ; 12(6)2020 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-32517266

RESUMO

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Ensaio de Placa Viral/métodos , Inativação de Vírus , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/patogenicidade , Celulose , Chlorocebus aethiops , Meios de Cultura/química , Formaldeído , Guanidinas/farmacologia , Células HEK293 , Humanos , Pandemias , Fenóis/farmacologia , Propiolactona/farmacologia , Sefarose , Células Vero
7.
Viruses ; 12(6)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32521706

RESUMO

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Inativação de Vírus/efeitos dos fármacos , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Técnicas de Cultura de Células/métodos , Chlorocebus aethiops , Contenção de Riscos Biológicos/métodos , Contenção de Riscos Biológicos/normas , Humanos , Nasofaringe/virologia , Pandemias , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos , Células Vero , Carga Viral/métodos
8.
Viruses ; 12(6)2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32532085

RESUMO

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) signals an urgent need for an expansion in treatment options. In this study, we investigated the anti-SARS-CoV-2 activities of 22 antiviral agents with known broad-spectrum antiviral activities against coronaviruses and/or other viruses. They were first evaluated in our primary screening in VeroE6 cells and then the most potent anti-SARS-CoV-2 antiviral agents were further evaluated using viral antigen expression, viral load reduction, and plaque reduction assays. In addition to remdesivir, lopinavir, and chloroquine, our primary screening additionally identified types I and II recombinant interferons, 25-hydroxycholesterol, and AM580 as the most potent anti-SARS-CoV-2 agents among the 22 antiviral agents. Betaferon (interferon-ß1b) exhibited the most potent anti-SARS-CoV-2 activity in viral antigen expression, viral load reduction, and plaque reduction assays among the recombinant interferons. The lipogenesis modulators 25-hydroxycholesterol and AM580 exhibited EC50 at low micromolar levels and selectivity indices of >10.0. Combinational use of these host-based antiviral agents with virus-based antivirals to target different processes of the SARS-CoV-2 replication cycle should be evaluated in animal models and/or clinical trials.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Animais , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Humanos , Interferons/metabolismo , Lipogênese/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Transdução de Sinais/efeitos dos fármacos , Células Vero , Carga Viral/efeitos dos fármacos , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
9.
BMC Infect Dis ; 20(1): 421, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32552663

RESUMO

BACKGROUND: The number of human Q fever cases in South Korea has been rapidly increasing since 2015. We report the first isolation of Coxiella burnetii in Korea in two patients who initially presented with non-specific febrile illness and were finally diagnosed with acute Q fever in South Korea. CASE PRESENTATION: Two adult patients with fever had serologic tests against C. burnetii initially negative, and polymerase chain reaction against 16S rRNA using whole blood was also negative. After bacterial amplification of C. burnetii in immune-depressed mice, we isolated C. burnetii from patients with acute Q fever. The isolates KZQ2 and KZQ3 were confirmed by polymerase chain reaction, nucleotide sequence analysis, and morphologic observation using a transmission electron microscope. CONCLUSIONS: These results can help us understand the clinical and epidemiologic features of Q fever in South Korea.


Assuntos
Coxiella burnetii/isolamento & purificação , Febre/microbiologia , Febre Q/diagnóstico , Febre Q/epidemiologia , Adulto , Idoso , Animais , Chlorocebus aethiops , Coxiella burnetii/genética , Coxiella burnetii/imunologia , Humanos , Incidência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Febre Q/microbiologia , RNA Ribossômico 16S/genética , República da Coreia/epidemiologia , Testes Sorológicos , Células Vero
10.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1113-1125, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597061

RESUMO

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.


Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Proteínas Virais , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Citoplasma/virologia , Vírus da Diarreia Epidêmica Suína/genética , Domínios Proteicos , Suínos , Células Vero , Proteínas Virais/química , Proteínas Virais/metabolismo
11.
Viruses ; 12(6)2020 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-32545799

RESUMO

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Testes de Neutralização/métodos , Pneumonia Viral/tratamento farmacológico , Amodiaquina/farmacologia , Animais , Células CACO-2 , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/terapia , Quimioterapia Combinada , Emetina/farmacologia , Células HEK293 , Células HT29 , Mepesuccinato de Omacetaxina/farmacologia , Humanos , Soros Imunes/imunologia , Imunização Passiva/métodos , Nelfinavir/farmacologia , Pandemias , Piranos/farmacologia , Pirróis/farmacologia , Células Vero
12.
PLoS One ; 15(6): e0235106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574196

RESUMO

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, has devastated health infrastructure around the world. Both ACE2 (an entry receptor) and TMPRSS2 (used by the virus for spike protein priming) are key proteins to SARS-CoV-2 cell entry, enabling progression to COVID-19 in humans. Comparative genomic research into critical ACE2 binding sites, associated with the spike receptor binding domain, has suggested that African and Asian primates may also be susceptible to disease from SARS-CoV-2 infection. Savanna monkeys (Chlorocebus spp.) are a widespread non-human primate with well-established potential as a bi-directional zoonotic/anthroponotic agent due to high levels of human interaction throughout their range in sub-Saharan Africa and the Caribbean. To characterize potential functional variation in savanna monkey ACE2 and TMPRSS2, we inspected recently published genomic data from 245 savanna monkeys, including 163 wild monkeys from Africa and the Caribbean and 82 captive monkeys from the Vervet Research Colony (VRC). We found several missense variants. One missense variant in ACE2 (X:14,077,550; Asp30Gly), common in Ch. sabaeus, causes a change in amino acid residue that has been inferred to reduce binding efficiency of SARS-CoV-2, suggesting potentially reduced susceptibility. The remaining populations appear as susceptible as humans, based on these criteria for receptor usage. All missense variants observed in wild Ch. sabaeus populations are also present in the VRC, along with two splice acceptor variants (at X:14,065,076) not observed in the wild sample that are potentially disruptive to ACE2 function. The presence of these variants in the VRC suggests a promising model for SARS-CoV-2 infection and vaccine and therapy development. In keeping with a One Health approach, characterizing actual susceptibility and potential for bi-directional zoonotic/anthroponotic transfer in savanna monkey populations may be an important consideration for controlling COVID-19 epidemics in communities with frequent human/non-human primate interactions that, in many cases, may have limited health infrastructure.


Assuntos
Chlorocebus aethiops , Infecções por Coronavirus/veterinária , Pandemias/veterinária , Peptidil Dipeptidase A/genética , Pneumonia Viral/veterinária , Doenças dos Primatas/genética , Serina Endopeptidases/genética , Animais , Betacoronavirus/metabolismo , Infecções por Coronavirus/genética , Infecções por Coronavirus/transmissão , Suscetibilidade a Doenças , Pneumonia Viral/genética , Pneumonia Viral/transmissão , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequenciamento Completo do Genoma , Zoonoses/transmissão
13.
Emerg Microbes Infect ; 9(1): 1457-1466, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32543353

RESUMO

Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Genoma Viral , Pneumonia Viral/virologia , Animais , Betacoronavirus/classificação , Betacoronavirus/isolamento & purificação , Linhagem Celular , Chlorocebus aethiops , Haemophilus parainfluenzae/isolamento & purificação , Humanos , Oriente Médio , Fases de Leitura Aberta , Pandemias , Filogenia , RNA Viral , Deleção de Sequência , Taiwan , Viagem , Células Vero , Cultura de Vírus , Sequenciamento Completo do Genoma
15.
Viruses ; 12(6)2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32486283

RESUMO

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas/métodos , Genes Reporter/fisiologia , Vírus de RNA/efeitos dos fármacos , Replicon/fisiologia , Animais , Antivirais/química , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Vírus de RNA/genética , Transfecção , Células Vero
16.
Viruses ; 12(6)2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32532094

RESUMO

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.


Assuntos
Anticoagulantes/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Guanidinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Betacoronavirus/metabolismo , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Gabexato/análogos & derivados , Gabexato/farmacologia , Células HEK293 , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
17.
Arch Virol ; 165(8): 1791-1801, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32474686

RESUMO

In this study, we compared the characteristics of two strains of Zika virus (ZIKV) isolated in Thailand, one isolated from a febrile patient and one isolated from tissues of a fetus medically terminated due to congenital Zika syndrome (CZS). Replication profiles showed that the isolate from the fetal tissues replicated significantly more slowly than the fever-associated isolate in human lung A549 cells during the first 24 hours postinfection but showed a similar growth profile over longer-term infection. A much smaller difference was observed in Aedes albopictus C6/36 cells. In a quasispecies analysis, a high proportion (approximately 20%) of nonfunctional genomes was identified, caused by an adenine insertion in the prM gene. This insertion was found to be present in two Thai fever strains and as such may represent a common feature of Thai endemic ZIKV. Comparison between viral RNA copy number and viral titer showed that the isolate from fetal tissues was produced more efficiently than the fever-associated isolate. Together, these results suggest that different ZIKV isolates differ in their replication capacity, and this might contribute to the fetotropic potential of a particular strain.


Assuntos
Vírus Satélites/genética , Infecção por Zika virus/virologia , Zika virus/genética , Células A549 , Aedes/virologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Feto/virologia , Humanos , Masculino , RNA Viral/genética , Tailândia , Células Vero , Carga Viral/genética , Replicação Viral/genética
18.
PLoS Biol ; 18(6): e3000715, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511245

RESUMO

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.


Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Coronavirus/classificação , Coronavirus/fisiologia , Retículo Endoplasmático/patologia , Retículo Endoplasmático/virologia , Replicação Viral , Animais , Betacoronavirus/genética , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Células Vero
19.
Int J Mol Sci ; 21(12)2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32575728

RESUMO

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.


Assuntos
Técnicas de Laboratório Clínico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Betacoronavirus/genética , Células CACO-2 , Chlorocebus aethiops , Técnicas de Laboratório Clínico/economia , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/economia , Custos e Análise de Custo , Humanos , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Células Vero , Proteínas da Matriz Viral/genética
20.
Curr Protoc Microbiol ; 58(1): e108, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32585083

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan, Hubei Province, China, in late 2019. Since then, the virus has spread globally and caused a pandemic. Assays that can measure the antiviral activity of antibodies or antiviral compounds are needed for SARS-CoV-2 vaccine and drug development. Here, we describe in detail a microneutralization assay, which can be used to assess in a quantitative manner if antibodies or drugs can block entry and/or replication of SARS-CoV-2 in vitro. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Microneutralization assay to test inhibition of virus by antibodies (purified antibodies or serum/plasma) Basic Protocol 2: Screening of anti-SARS-CoV-2 compounds in vitro Support Protocol: SARS-CoV-2 propagation.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Neutralização/métodos , Animais , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Camundongos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA