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1.
Recurso na Internet em Português | LIS - Localizador de Informação em Saúde | ID: lis-LISBR1.1-46977

RESUMO

Coronavírus é uma família de vírus que causam infecções respiratórias. O novo agente do coronavírus (nCoV-2019) foi descoberto em 31/12/19 após casos registrados na China. Os primeiros coronavírus humanos foram identificados em meados da década de 1960. A maioria das pessoas se infecta com os coronavírus comuns ao longo da vida, sendo as crianças pequenas mais propensas a se infectarem com o tipo mais comum do vírus. Os coronavírus mais comuns que infectam humanos são o alpha coronavírus 229E e NL63 e beta coronavírus OC43, HKU1.


Assuntos
Coronavirus , Coronavirus Humano NL63 , Coronavirus Humano 229E
2.
Recurso na Internet em Português | LIS - Localizador de Informação em Saúde | ID: lis-LISBR1.1-46945

RESUMO

Site do Ministério da Saúde. (Brasil). Tirando dúvidas sobre o corona Virús. O que é o Virús, suas causas, sintomas, tratamento, diagnostico é prevenção Saiba mais sobre o Coronavirus...


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Coronavirus Humano 229E/patogenicidade , Infecções por Coronavirus/prevenção & controle
3.
Internist (Berl) ; 60(11): 1136-1145, 2019 Nov.
Artigo em Alemão | MEDLINE | ID: mdl-31455974

RESUMO

BACKGROUND: There are six human pathogenic coronaviruses (CoV), which mainly cause infections of the respiratory system. In everyday clinical practice, it is helpful to know the relevance and characteristics of these pathogens. OBJECTIVE: To present the epidemiology, clinical picture and differences of human pathogenic CoV and to provide information on the diagnostics and treatment of patients suspected of having CoV infections. MATERIAL AND METHODS: Selective literature search, presentation of results and discussion of fundamental works and expert recommendations, including publications by the World Health Organization (WHO), the European Centre for Disease Prevention and Control (ECDC) and the Robert Koch Institute. RESULTS: The four endemic human CoVs (HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1) mainly cause mild respiratory tract infections. In addition to these four endemic HCoV, the two epidemic CoV, severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV can cause severe pneumonia. The SARS-CoV has not been detected in humans in the last 15 years and MERS-CoV has been circulating mainly on the Arabian Peninsula since 2012; however, neither a specific treatment nor approved vaccines exist for any of the six human pathogenic CoVs. CONCLUSION: All six human CoVs can be diagnosed using RT-PCR on respiratory specimens but this is rarely necessary for the four endemic strains. In current clinical practice SARS-CoV has no importance as it has not been detected in humans for 15 years; however, a possible MERS-CoV infection should be taken into account in patients with typical symptoms and travel history to endemic regions. In this case, rapid diagnostic and general hygiene practices are important to prevent further transmission.


Assuntos
Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/prevenção & controle , Coronavirus , Infecções Respiratórias , Síndrome Respiratória Aguda Grave/diagnóstico , Betacoronavirus , Coronavirus Humano 229E , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Coronavirus Humano NL63 , Coronavirus Humano OC43 , Humanos , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/virologia
4.
Viruses ; 11(1)2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30654597

RESUMO

Lipids play numerous indispensable cellular functions and are involved in multiple steps in the replication cycle of viruses. Infections by human-pathogenic coronaviruses result in diverse clinical outcomes, ranging from self-limiting flu-like symptoms to severe pneumonia with extrapulmonary manifestations. Understanding how cellular lipids may modulate the pathogenicity of human-pathogenic coronaviruses remains poor. To this end, we utilized the human coronavirus 229E (HCoV-229E) as a model coronavirus to comprehensively characterize the host cell lipid response upon coronavirus infection with an ultra-high performance liquid chromatography-mass spectrometry (UPLC⁻MS)-based lipidomics approach. Our results revealed that glycerophospholipids and fatty acids (FAs) were significantly elevated in the HCoV-229E-infected cells and the linoleic acid (LA) to arachidonic acid (AA) metabolism axis was markedly perturbed upon HCoV-229E infection. Interestingly, exogenous supplement of LA or AA in HCoV-229E-infected cells significantly suppressed HCoV-229E virus replication. Importantly, the inhibitory effect of LA and AA on virus replication was also conserved for the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV). Taken together, our study demonstrated that host lipid metabolic remodeling was significantly associated with human-pathogenic coronavirus propagation. Our data further suggested that lipid metabolism regulation would be a common and druggable target for coronavirus infections.


Assuntos
Coronavirus Humano 229E/fisiologia , Interações Hospedeiro-Patógeno , Metabolismo dos Lipídeos , Replicação Viral , Ácido Araquidônico/metabolismo , Linhagem Celular , Cromatografia Líquida , Glicerofosfolipídeos/metabolismo , Humanos , Ácido Linoleico/metabolismo , Espectrometria de Massas em Tandem
5.
Acta Crystallogr D Struct Biol ; 74(Pt 9): 841-851, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30198895

RESUMO

Human coronavirus 229E (HCoV-229E) usually causes mild upper respiratory infections in heathy adults, but may lead to severe complications or mortality in individuals with weakened immune systems. Virus entry of HCoV-229E is mediated by its spike (S) protein, where the S1 domain facilitates attachment to host cells and the S2 domain is involved in subsequent fusion of the virus and host membranes. During the fusion process, two heptad repeats, HR1 and HR2, in the S2 domain assemble into a six-helix membrane-fusion structure termed the fusion core. Here, the complete fusion-core structure of HCoV-229E has been determined at 1.86 Šresolution, representing the most complete post-fusion conformation thus far among published human alphacoronavirus (α-HCoV) fusion-core structures. The overall structure of the HCoV-229E fusion core is similar to those of SARS, MERS and HCoV-NL63, but the packing of its 3HR1 core differs from those of SARS and MERS in that it contains more noncanonical `x' and `da' layers. Side-by-side electrostatic surface comparisons reveal that the electrostatic surface potentials are opposite in α-HCoVs and ß-HCoVs at certain positions and that the HCoV-229E surface also appears to be the most hydrophobic among the various HCoVs. In addition to the highly conserved hydrophobic interactions between HR1 and HR2, some polar and electrostatic interactions are also well preserved across different HCoVs. This study adds to the structural profiling of HCoVs to aid in the structure-based design of pan-coronavirus small molecules or peptides to inhibit viral fusion.


Assuntos
Coronavirus Humano 229E/isolamento & purificação , Infecções por Coronavirus/virologia , Proteínas Recombinantes de Fusão/química , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Coronavirus Humano 229E/fisiologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência
6.
J Gen Virol ; 99(7): 908-912, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29786498

RESUMO

Human coronavirus 229E (HCoV-229E) is responsible for common colds. Like other coronaviruses, HCoV-229E exploits cellular proteases to activate fusion mediated by the spike protein. We analysed the proteolytic processing of the HCoV-229E spike protein by trypsin-like serine proteases leading to activation of the fusion process. Unlike in other coronaviruses, HCoV-229E fusion activation appears to be a one-step process. Indeed, cleavage of the S1/S2 interface does not seem to be a prerequisite, and the fusion activation is highly reliant on the S2' region, with arginine residue 683 acting as the recognition site.


Assuntos
Coronavirus Humano 229E/genética , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Proteínas do Core Viral/genética , Proteínas Virais de Fusão/química , Coronavirus Humano 229E/química , Infecções por Coronavirus , Células HEK293 , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética
7.
Biochem Biophys Res Commun ; 497(2): 705-712, 2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29458023

RESUMO

HCoV-229E spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This protein is composed of an N-terminal receptor-binding domain (S1) and a C-terminal trans-membrane fusion domain (S2). S2 contains a highly conserved heptad repeat 1 and 2 (HR1 and HR2). In this study, the HRs sequences were designed and connected with a flexible linker. The recombinant fusion core protein was crystallized and its structure was solved at a resolution of 2.45 Å. Then we characterized the binding of HR1s and HR2s via both sequence alignment and structural analysis. The overall structures, especially the residues in some positions of HR2 are highly conserved. Fourteen hydrophobic and three polar residues from each HR1 peptide are packed in layers at the coiled-coil interface. These core amino acids can be grouped into seven heptad repeats. Analysis of hydrophobic and hydrophilic interactions between HR2 helix and HR1 helices, shows that the HR1 and HR2 polypeptides are highly complementary in both shape and chemical properties. Furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting membrane fusion, a crucial step of HCoV-229E infection.


Assuntos
Coronavirus Humano 229E/química , Infecções por Coronavirus/virologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Clonagem Molecular , Coronavirus Humano 229E/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/genética
8.
Int J Mol Sci ; 19(2)2018 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-29415501

RESUMO

Human coronavirus 229E (HCoV-229E) infection in infants, elderly people, and immunocompromised patients can cause severe disease, thus calling for the development of effective and safe therapeutics to treat it. Here we reported the design, synthesis and characterization of two peptide-based membrane fusion inhibitors targeting HCoV-229E spike protein heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains, 229E-HR1P and 229E-HR2P, respectively. We found that 229E-HR1P and 229E-HR2P could interact to form a stable six-helix bundle and inhibit HCoV-229E spike protein-mediated cell-cell fusion with IC50 of 5.7 and 0.3 µM, respectively. 229E-HR2P effectively inhibited pseudotyped and live HCoV-229E infection with IC50 of 0.5 and 1.7 µM, respectively. In a mouse model, 229E-HR2P administered intranasally could widely distribute in the upper and lower respiratory tracts and maintain its fusion-inhibitory activity. Therefore, 229E-HR2P is a promising candidate for further development as an antiviral agent for the treatment and prevention of HCoV-229E infection.


Assuntos
Coronavirus Humano 229E/efeitos dos fármacos , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Inibidores de Proteínas Virais de Fusão/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Células Gigantes/virologia , Humanos , Fusão de Membrana , Camundongos , Peptídeos/química , Ligação Proteica , Inibidores de Proteínas Virais de Fusão/química
9.
Eur J Med Chem ; 146: 678-686, 2018 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-29407990

RESUMO

Several hitherto unknown (E)-but-2-enyl nucleoside phosphonoamidate analogs (ANPs) were prepared directed with nitrogen reagents by cross-metathesis in water-under ultrasound irradiation. Two diastereoisomers were formally identified by X-ray diffraction. These compounds were evaluated against a large spectrum of DNA and RNA viruses. Among them, the phosphonoamidate thymine analogue 19 emerged as the best prodrug against varicella-zoster virus (VZV) with EC50 values of 0.33 and 0.39 µM for wild-type and thymidine kinase deficient strains, respectively, and a selectivity index ≥200 µM. This breakthrough approach paves the way for new purine and pyrimidine (E)-but-2-enyl phosphonoamidate analogs.


Assuntos
Antivirais/farmacologia , Coronavirus Humano 229E/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 3/efeitos dos fármacos , Nucleosídeos/farmacologia , Antivirais/síntese química , Antivirais/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Relação Estrutura-Atividade
10.
Virology ; 517: 44-55, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29223446

RESUMO

Structure predictions suggest a partial conservation of RNA structure elements in coronavirus terminal genome regions. Here, we determined the structures of stem-loops (SL) 1 and 2 of two alphacoronaviruses, human coronavirus (HCoV) 229E and NL63, by RNA structure probing and studied the functional relevance of these putative cis-acting elements. HCoV-229E SL1 and SL2 mutants generated by reverse genetics were used to study the effects on viral replication of single-nucleotide substitutions predicted to destabilize the SL1 and SL2 structures. The data provide conclusive evidence for the critical role of SL1 and SL2 in HCoV-229E replication and, in some cases, revealed parallels with previously characterized betacoronavirus SL1 and SL2 elements. Also, we were able to rescue viable HCoV-229E mutants carrying replacements of SL2 with equivalent betacoronavirus structural elements. The data obtained in this study reveal a remarkable degree of structural and functional conservation of 5'-terminal RNA structural elements across coronavirus genus boundaries.


Assuntos
Coronavirus Humano 229E/genética , Coronavirus Humano NL63/genética , Genoma Viral , Sequências Reguladoras de Ácido Nucleico/fisiologia , Sequência de Bases , Linhagem Celular , Humanos , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Replicação Viral/fisiologia
11.
Eur J Clin Microbiol Infect Dis ; 37(2): 363-369, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29214503

RESUMO

Human coronaviruses (HCoV) OC43, 229E, NL63, and HKU1 are common respiratory viruses which cause various respiratory diseases, including pneumonia. There is a paucity of evidence on the epidemiology and clinical manifestations of these four HCoV strains worldwide. We collected 11,399 throat swabs from hospitalized children with acute respiratory tract infection from July 2009 to June 2016 in Guangzhou, China. These were tested for four strains of HCoV infection using real-time polymerase chain reaction (PCR). HCoV-positive patients were then tested for 11 other respiratory pathogens. 4.3% (489/11399) of patients were positive for HCoV, of which 3.0% were positive for OC43 (346/11399), 0.6% for 229E (65/11399), 0.5% for NL63 (60/11399), and 0.3% for HKU1 (38/11399). Patients aged 7-12 months had the highest prevalence of HCoV and OC43 when compared with other age groups (p < 0.001). The peak seasons of infection varied depending on the HCoV strain. Patients infected with a single strain of HCoV infection were less likely to present fever (≥ 38 °C) (p = 0.014) and more likely to present pulmonary rales (p = 0.043) than those co-infected with more than one HCoV strain or other respiratory pathogens. There were also significant differences in the prevalence of certain symptoms, including coughing (p = 0.032), pneumonia (p = 0.026), and abnormal pulmonary rales (p = 0.002) according to the strain of HCoV detected. This retrospective study of the prevalence of four HCoV strains and clinical signs among a large population of pediatric patients in a subtropical region of China provides further insight into the epidemiology and clinical features of HCoV.


Assuntos
Coronavirus Humano 229E/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Coronavirus Humano NL63/isolamento & purificação , Coronavirus Humano OC43/isolamento & purificação , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Adolescente , Criança , Pré-Escolar , China/epidemiologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/virologia , Estudos Retrospectivos
12.
Am J Infect Control ; 46(1): 105-107, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28893443

RESUMO

Frequently touched surfaces of a university classroom that is cleaned daily contained viable human coronavirus 229E (CoV-229E). Tests of a CoV-229E laboratory strain under conditions that simulated the ambient light, temperature, and relative humidity conditions of the classroom revealed that some of the virus remained viable on various surfaces for 7 days, suggesting CoV-229E is relatively stable in the environment. Our findings reinforce the notion that contact transmission may be possible for this virus.


Assuntos
Coronavirus Humano 229E/isolamento & purificação , Microbiologia Ambiental , Serviço de Limpeza , Universidades , Coronavirus Humano 229E/genética , Humanos , RNA Viral/isolamento & purificação , Propriedades de Superfície
13.
Nat Commun ; 8(1): 1735, 2017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29170370

RESUMO

RNA viruses are characterized by a high mutation rate, a buffer against environmental change. Nevertheless, the means by which random mutation improves viral fitness is not well characterized. Here we report the X-ray crystal structure of the receptor-binding domain (RBD) of the human coronavirus, HCoV-229E, in complex with the ectodomain of its receptor, aminopeptidase N (APN). Three extended loops are solely responsible for receptor binding and the evolution of HCoV-229E and its close relatives is accompanied by changing loop-receptor interactions. Phylogenetic analysis shows that the natural HCoV-229E receptor-binding loop variation observed defines six RBD classes whose viruses have successively replaced each other in the human population over the past 50 years. These RBD classes differ in their affinity for APN and their ability to bind an HCoV-229E neutralizing antibody. Together, our results provide a model for alphacoronavirus adaptation and evolution based on the use of extended loops for receptor binding.


Assuntos
Coronavirus Humano 229E/genética , Coronavirus Humano 229E/fisiologia , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Antígenos CD13/química , Antígenos CD13/metabolismo , Coronavirus Humano 229E/patogenicidade , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Evolução Molecular , Variação Genética , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Filogenia , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/química , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Ressonância de Plasmônio de Superfície
14.
PLoS Pathog ; 13(3): e1006286, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28355270

RESUMO

Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E)-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL)-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF) NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i) an unusual mechanism involving partial degradation of IKKß, NEMO and IκBα and (ii) upregulation of TNFAIP3 (A20), although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1). The data revealed that, in HCoV-infected (but not IL-1-treated) cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKß activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the host cellular chromatin landscape, thereby orchestrating the timely coordinated expression of genes involved in multiple signaling, immunoregulatory and metabolic processes.


Assuntos
Cromatina/fisiologia , Coronavirus Humano 229E , Infecções por Coronavirus/genética , NF-kappa B/metabolismo , Transcriptoma , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Humanos , Immunoblotting , Microdissecção e Captura a Laser , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
15.
J Virol ; 91(1)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27733646

RESUMO

Human coronavirus 229E (HCoV-229E), a causative agent of the common cold, enters host cells via two distinct pathways: one is mediated by cell surface proteases, particularly transmembrane protease serine 2 (TMPRSS2), and the other by endosomal cathepsin L. Thus, specific inhibitors of these proteases block virus infection. However, it is unclear which of these pathways is actually utilized by HCoV-229E in the human respiratory tract. Here, we examined the mechanism of cell entry used by a pseudotyped virus bearing the HCoV-229E spike (S) protein in the presence or absence of protease inhibitors. We found that, compared with a laboratory strain isolated in 1966 and passaged for a half century, clinical isolates of HCoV-229E were less likely to utilize cathepsin L; rather, they showed a preference for TMPRSS2. Two amino acid substitutions (R642M and N714K) in the S protein of HCoV-229E clinical isolates altered their sensitivity to a cathepsin L inhibitor, suggesting that these amino acids were responsible for cathepsin L use. After 20 passages in HeLa cells, the ability of the isolate to use cathepsin increased so that it was equal to that of the laboratory strain; this increase was caused by an amino acid substitution (I577S) in the S protein. The passaged virus showed a reduced ability to replicate in differentiated airway epithelial cells cultured at an air-liquid interface. These results suggest that the endosomal pathway is disadvantageous for HCoV-229E infection of human airway epithelial cells; therefore, clinical isolates are less able to use cathepsin. IMPORTANCE: Many enveloped viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease TMPRSS2 rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for coronavirus-mediated diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), should target cell surface TMPRSS2 rather than endosomal cathepsin.


Assuntos
Catepsina L/genética , Membrana Celular/virologia , Coronavirus Humano 229E/genética , Evasão da Resposta Imune , Serina Endopeptidases/genética , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus , Sequência de Aminoácidos , Substituição de Aminoácidos , Evolução Biológica , Catepsina L/antagonistas & inibidores , Catepsina L/imunologia , Membrana Celular/imunologia , Resfriado Comum/imunologia , Resfriado Comum/virologia , Coronavirus Humano 229E/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Endocitose , Endossomos/efeitos dos fármacos , Endossomos/imunologia , Endossomos/virologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Células HeLa , Humanos , Mutação , Inibidores de Proteases/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Alinhamento de Sequência , Serina Endopeptidases/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
16.
Int J Mol Sci ; 17(11)2016 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-27886052

RESUMO

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 10² copies for HCoV-OC43, and 3 × 10¹ copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 µL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples.


Assuntos
Coronavirus Humano 229E/isolamento & purificação , Coronavirus Humano NL63/isolamento & purificação , Coronavirus Humano OC43/isolamento & purificação , Coronavirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Respiratórias/diagnóstico , Doença Aguda , Coronavirus/genética , Coronavirus Humano 229E/genética , Coronavirus Humano NL63/genética , Coronavirus Humano OC43/genética , Primers do DNA/síntese química , Primers do DNA/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Diagnóstico Diferencial , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase Multiplex/normas , Desnaturação de Ácido Nucleico , Compostos Orgânicos/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Infecções Respiratórias/virologia
18.
Am J Trop Med Hyg ; 94(5): 1058-64, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-26928836

RESUMO

The human alphacoronaviruses HCoV-NL63 and HCoV-229E are commonly associated with upper respiratory tract infections (URTI). Information on their molecular epidemiology and evolutionary dynamics in the tropical region of southeast Asia however is limited. Here, we analyzed the phylogenetic, temporal distribution, population history, and clinical manifestations among patients infected with HCoV-NL63 and HCoV-229E. Nasopharyngeal swabs were collected from 2,060 consenting adults presented with acute URTI symptoms in Kuala Lumpur, Malaysia, between 2012 and 2013. The presence of HCoV-NL63 and HCoV-229E was detected using multiplex polymerase chain reaction (PCR). The spike glycoprotein, nucleocapsid, and 1a genes were sequenced for phylogenetic reconstruction and Bayesian coalescent inference. A total of 68/2,060 (3.3%) subjects were positive for human alphacoronavirus; HCoV-NL63 and HCoV-229E were detected in 45 (2.2%) and 23 (1.1%) patients, respectively. A peak in the number of HCoV-NL63 infections was recorded between June and October 2012. Phylogenetic inference revealed that 62.8% of HCoV-NL63 infections belonged to genotype B, 37.2% was genotype C, while all HCoV-229E sequences were clustered within group 4. Molecular dating analysis indicated that the origin of HCoV-NL63 was dated to 1921, before it diverged into genotype A (1975), genotype B (1996), and genotype C (2003). The root of the HCoV-229E tree was dated to 1955, before it diverged into groups 1-4 between the 1970s and 1990s. The study described the seasonality, molecular diversity, and evolutionary dynamics of human alphacoronavirus infections in a tropical region.


Assuntos
Resfriado Comum/epidemiologia , Resfriado Comum/virologia , Coronavirus Humano 229E/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/genética , Regulação Viral da Expressão Gênica , Humanos , Malásia/epidemiologia , Filogenia , Proteínas Virais/genética , Proteínas Virais/metabolismo
19.
Nucleic Acids Res ; 44(6): e52, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26657632

RESUMO

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.


Assuntos
Trifosfato de Adenosina/síntese química , Guanosina Trifosfato/síntese química , Marcação por Isótopo/métodos , Nucleotídeos/síntese química , Bacillus anthracis/química , Bacillus anthracis/genética , Isótopos de Carbono , Coronavirus Humano 229E/química , Coronavirus Humano 229E/genética , Creatina Quinase/química , Creatina Quinase/genética , Espectroscopia de Ressonância Magnética , Pentosiltransferases/química , Pentosiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Elementos de Resposta , Ribose/química , Ribose-Fosfato Pirofosfoquinase/química , Ribose-Fosfato Pirofosfoquinase/genética , Riboswitch , Transcrição Genética
20.
Viruses ; 7(12): 6689-706, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26694452

RESUMO

Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes-tumor necrosis factor alpha (TNF-α) and GTPase myxovirus resistance 1 (MX1)-in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E) and enterovirus 71 (EV71) infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH) sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP⁺ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.


Assuntos
Coronavirus Humano 229E/imunologia , Enterovirus Humano A/imunologia , Glucosefosfato Desidrogenase/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Resistência a Myxovirus/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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