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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.
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
3.
Viruses ; 11(1)2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30634419

RESUMO

Known human coronaviruses are believed to have originated in animals and made use of intermediate hosts for transmission to humans. The intermediate hosts of most of the human coronaviruses are known, but not for HCoV-NL63. This study aims to assess the possible role of some major domestic livestock species as intermediate hosts of HCoV-NL63. We developed a testing algorithm for high throughput screening of livestock sera with ELISA and confirmation with recombinant immunofluorescence assay testing for antibodies against HCoV-NL63 in livestock. Optimization of the ELISA showed a capability of the assay to significantly distinguish HCoV-NL63 from HCoV-229E (U = 27.50, p < 0.001) and HCoV-OC43 (U = 55.50, p < 0.001) in coronavirus-characterized sera. Evaluation of the assay with collected human samples showed no significant difference in mean optical density values of immunofluorescence-classified HCoV-NL63-positive and HCoV-NL63-negative samples (F (1, 215) = 0.437, p = 0.509). All the top 5% (n = 8) most reactive human samples tested by ELISA were HCoV-NL63 positive by immunofluorescence testing. In comparison, only a proportion (84%, n = 42) of the top 25% were positive by immunofluorescence testing, indicating an increased probability of the highly ELISA reactive samples testing positive by the immunofluorescence assay. None of the top 5% most ELISA reactive livestock samples were positive for HCoV-NL63-related viruses by immunofluorescence confirmation. Ghanaian domestic livestock are not likely intermediate hosts of HCoV-NL63-related coronaviruses.


Assuntos
Doenças dos Bovinos/virologia , Infecções por Coronavirus/veterinária , Coronavirus Humano NL63/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Adolescente , Adulto , Idoso , Algoritmos , Animais , Anticorpos Antivirais/sangue , Bovinos , Fazendeiros , Feminino , Imunofluorescência , Gana/epidemiologia , Ensaios de Triagem em Larga Escala , Humanos , Gado/virologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
PLoS One ; 13(9): e0203489, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30183777

RESUMO

Virus like particles (VLPs) produced by the expression of viral structural proteins can serve as versatile nanovectors or potential vaccine candidates. In this study we describe for the first time the generation of HCoV-NL63 VLPs using baculovirus system. Major structural proteins of HCoV-NL63 have been expressed in tagged or native form, and their assembly to form VLPs was evaluated. Additionally, a novel procedure for chromatography purification of HCoV-NL63 VLPs was developed. Interestingly, we show that these nanoparticles may deliver cargo and selectively transduce cells expressing the ACE2 protein such as ciliated cells of the respiratory tract. Production of a specific delivery vector is a major challenge for research concerning targeting molecules. The obtained results show that HCoV-NL63 VLPs may be efficiently produced, purified, modified and serve as a delivery platform. This study constitutes an important basis for further development of a promising viral vector displaying narrow tissue tropism.


Assuntos
Coronavirus Humano NL63 , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Peptidil Dipeptidase A/farmacologia , Mucosa Respiratória/metabolismo , Vacinas de Partículas Semelhantes a Vírus , Animais , Linhagem Celular , Humanos , Macaca mulatta , Mucosa Respiratória/citologia , Spodoptera , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/farmacologia
5.
Emerg Infect Dis ; 24(10): 1964-1966, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30226169

RESUMO

We describe an outbreak of severe respiratory illness associated with human coronavirus NL63 in a long-term care facility in Louisiana in November 2017. Six of 20 case-patients were hospitalized with pneumonia, and 3 of 20 died. Clinicians should consider human coronavirus NL63 for patients in similar settings with respiratory disease.


Assuntos
Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Coronavirus Humano NL63 , Infecção Hospitalar , Instalações de Saúde , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Idoso , Idoso de 80 Anos ou mais , Infecções por Coronavirus/diagnóstico , Surtos de Doenças , Feminino , Humanos , Assistência de Longa Duração , Louisiana/epidemiologia , Masculino , Reação em Cadeia da Polimerase , Vigilância em Saúde Pública , RNA Viral , Infecções Respiratórias/diagnóstico
6.
J Infect Dis ; 217(11): 1728-1739, 2018 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-29741740

RESUMO

Background: Human coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime. Methods: Nasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing. Results: HCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching. Conclusions: In this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.


Assuntos
Infecções por Coronavirus/epidemiologia , Coronavirus Humano NL63/genética , Adolescente , Adulto , Evolução Biológica , Criança , Pré-Escolar , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Feminino , Hospitalização , Humanos , Lactente , Recém-Nascido , Quênia/epidemiologia , Masculino , Epidemiologia Molecular , Filogenia , Prevalência , Estudos Prospectivos , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Adulto Jovem
7.
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
8.
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
9.
J Virol ; 92(3)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29142129

RESUMO

The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed.IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavirus Humano NL63/fisiologia , Endocitose , Internalização do Vírus , Linhagem Celular , Clatrina/metabolismo , Endossomos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/metabolismo
10.
Mater Sci Eng C Mater Biol Appl ; 76: 735-742, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28482585

RESUMO

A novel biopolymeric material in the form of nano/microspheres was developed which was capable of adsorbing coronaviruses. The biopolymer was obtained by crosslinking of chitosan (CHIT) with genipin, a nontoxic compound of plant origin, in inverted emulsion and reacting the chitosan nano/microspheres obtained (CHIT-NS/MS) with glycidyltrimethyl-ammonium chloride (GTMAC). As a result the nano/microspheres of N-(2-hydroxypropyl)-3-trimethyl chitosan (HTCC-NS/MS) were obtained. HTCC-NS/MS were studied as the adsorbents of human coronavirus NL63 (HCoV-NL63), mouse hepatitis virus (MHV), and human coronavirus HCoV-OC43 particles in aqueous virus suspensions. By studying cytopathic effect (CPE) caused by these viruses and performing PCR analyses it was found HTCC-NS/MS strongly adsorb the particles of HCoV-NL63 virus, moderately adsorb mouse hepatitis virus (MHV) particles, but do not adsorb HCoV-OC43 coronavirus. The adsorption capacity of HTCC-NS/MS well correlated with the antiviral activity of soluble HTCC against a given virus. Importantly, it was shown that HCoV-NL63 particles could be desorbed from the HTCC-NS/MS surface with a salt solution of high ionic strength with retention of virus virulence. The obtained material may be applied for the removal of coronaviruses, purification and concentration of virus samples obtained from biological matrices and for purification of water from pathogenic coronaviruses.


Assuntos
Nanosferas , Adsorção , Animais , Antivirais , Infecções por Coronavirus , Coronavirus Humano NL63 , Humanos , Camundongos , Microesferas
11.
J Virol ; 91(11)2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28331093

RESUMO

Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.


Assuntos
Coronavirus Humano NL63/química , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Coronavirus Humano NL63/fisiologia , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , RNA Viral/metabolismo , Montagem de Vírus , Replicação Viral
12.
Pediatr Infect Dis J ; 36(2): 231-233, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28081049

RESUMO

Human coronavirus NL63 (HCoV-NL63) primarily infects the upper respiratory tract. However, it may cause severe lower respiratory tract infection, and the clinical course may be severe in immunocompromised patients. To our knowledge, child death due to HCoV-NL63 has not been reported. We present a fatal lower respiratory tract disease associated with HCoV-NL63 in a 7-month-old malnourished infant.


Assuntos
Infecções por Coronavirus , Coronavirus Humano NL63 , Infecções Respiratórias , Evolução Fatal , Humanos , Lactente , Masculino
13.
J Virol ; 91(5)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28077633

RESUMO

Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5' and 3' ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination "hot spot" in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances.IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential.


Assuntos
Quirópteros/virologia , Infecções por Coronavirus/veterinária , Infecções Respiratórias/veterinária , Sequência de Aminoácidos , Animais , Sequência Conservada , Infecções por Coronavirus/epidemiologia , Coronavirus Humano NL63 , Monitoramento Epidemiológico , Evolução Molecular , Variação Genética , Genoma Viral , Quênia/epidemiologia , Filogenia , Filogeografia , Prevalência , Recombinação Genética , Infecções Respiratórias/epidemiologia , Análise de Sequência de DNA , Proteínas Virais/química , Proteínas Virais/genética
14.
J Microbiol Immunol Infect ; 50(6): 763-770, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26746130

RESUMO

BACKGROUND/PURPOSE: Human coronavirus (HCoV) NL63 is recognized in association with upper or lower respiratory tract illnesses in children. This study surveyed the prevalence of HCoV-NL63 and influenza viruses in patients with influenza-like illness in Taiwan during 2010-2011. METHODS: Throat samples from 107 hospitalized patients with pneumonia and 175 outpatients with influenza-like illness were examined using real-time polymerase chain reaction assays with virus-specific primers, and then virus-positive specimens were confirmed by sequencing the polymerase chain reaction products. RESULTS: HCoV-NL63 infection was identified in 8.4% (9/107) of hospitalized patients with pneumonia, but not found in outpatients with influenza-like illness. Age distribution of HCoV-NL63 infection in hospitalized patients with pneumonia indicated that the group aged 16-25 years (20%) had the highest positive rate compared with the other groups, and exhibited a similar age-specific pattern to influenza A/H1N1 infection, but not influenza A/H3N2 and B infections in hospitalized patients. Seasonal prevalence of HCoV-NL63 infection was late winter, overlapping the highest peak of the influenza A/H1N1 epidemic during December 2010 to March 2011 in Taiwan. Co-infection of HCoV-NL63 and influenza A/H1N1 was detected in three hospitalized patients. Clinical manifestation analysis indicated that the main symptoms for HCoV-NL63 infection included fever (88.9%), cough (77.8%), and pneumonia (100%). Co-infection caused significantly higher rates of breathing difficulties, cough, and sore throat than those of single infection with HCoV-NL63 and influenza A/H1N1. Phylogenetic analysis indicated a low level of heterogeneity between Taiwan and global HCoV-NL63 strains. CONCLUSION: Understanding epidemiology of HCoV-NL63 in Taiwan provides an insight for worldwide surveillance of HCoV-NL63 infection.


Assuntos
Infecções por Coronavirus/epidemiologia , Coronavirus Humano NL63/classificação , Influenza Humana/epidemiologia , Pneumonia/epidemiologia , Adolescente , Adulto , Idoso , Sequência de Bases , Coinfecção/patologia , Coinfecção/virologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/isolamento & purificação , Coronavirus Humano NL63/patogenicidade , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/patologia , Influenza Humana/virologia , Pessoa de Meia-Idade , Filogenia , Pneumonia/diagnóstico , Pneumonia/patologia , Pneumonia/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Análise de Sequência de RNA , Taiwan/epidemiologia , Adulto Jovem
15.
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
16.
Nat Struct Mol Biol ; 23(10): 899-905, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27617430

RESUMO

The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.


Assuntos
Infecções por Coronavirus/virologia , Coronavirus Humano NL63/química , Epitopos/química , Polissacarídeos/análise , Glicoproteína da Espícula de Coronavírus/química , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Coronavirus Humano NL63/imunologia , Microscopia Crioeletrônica , Drosophila , Epitopos/imunologia , Humanos , Modelos Moleculares , Polissacarídeos/imunologia , Conformação Proteica , Multimerização Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/ultraestrutura
17.
Bing Du Xue Bao ; 32(1): 56-61, 2016 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-27295884

RESUMO

A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63.


Assuntos
Colorimetria/métodos , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Coronavirus/diagnóstico , Coronavirus Humano NL63/genética , Primers do DNA/genética , Humanos , Transcrição Reversa , Sensibilidade e Especificidade
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.
Sci Rep ; 6: 22677, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26948040

RESUMO

First identified in The Netherlands in 2004, human coronavirus NL63 (HCoV-NL63) was found to cause worldwide infections. Patients infected by HCoV-NL63 are typically young children with upper and lower respiratory tract infection, presenting with symptoms including croup, bronchiolitis, and pneumonia. Unfortunately, there are currently no effective antiviral therapy to contain HCoV-NL63 infection. CoV genomes encode an integral viral component, main protease (M(pro)), which is essential for viral replication through proteolytic processing of RNA replicase machinery. Due to the sequence and structural conservation among all CoVs, M(pro) has been recognized as an attractive molecular target for rational anti-CoV drug design. Here we present the crystal structure of HCoV-NL63 M(pro) in complex with a Michael acceptor inhibitor N3. Structural analysis, consistent with biochemical inhibition results, reveals the molecular mechanism of enzyme inhibition at the highly conservative substrate-recognition pocket. We show such molecular target remains unchanged across 30 clinical isolates of HCoV-NL63 strains. Through comparative study with M(pro)s from other human CoVs (including the deadly SARS-CoV and MERS-CoV) and their related zoonotic CoVs, our structure of HCoV-NL63 M(pro) provides critical insight into rational development of wide spectrum antiviral therapeutics to treat infections caused by human CoVs.


Assuntos
Antivirais/isolamento & purificação , Coronavirus Humano NL63/enzimologia , Peptídeo Hidrolases/química , Antivirais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Drogas , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Conformação Proteica
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