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1.
Nat Commun ; 15(1): 5112, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38879641

RESUMO

Virus infectivity is traditionally determined by endpoint titration in cell cultures, and requires complex processing steps and human annotation. Here we developed an artificial intelligence (AI)-powered automated framework for ready detection of virus-induced cytopathic effect (DVICE). DVICE uses the convolutional neural network EfficientNet-B0 and transmitted light microscopy images of infected cell cultures, including coronavirus, influenza virus, rhinovirus, herpes simplex virus, vaccinia virus, and adenovirus. DVICE robustly measures virus-induced cytopathic effects (CPE), as shown by class activation mapping. Leave-one-out cross-validation in different cell types demonstrates high accuracy for different viruses, including SARS-CoV-2 in human saliva. Strikingly, DVICE exhibits virus class specificity, as shown with adenovirus, herpesvirus, rhinovirus, vaccinia virus, and SARS-CoV-2. In sum, DVICE provides unbiased infectivity scores of infectious agents causing CPE, and can be adapted to laboratory diagnostics, drug screening, serum neutralization or clinical samples.


Assuntos
Inteligência Artificial , Efeito Citopatogênico Viral , Microscopia , SARS-CoV-2 , Humanos , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Microscopia/métodos , COVID-19/virologia , Redes Neurais de Computação , Animais , Vaccinia virus/fisiologia , Vaccinia virus/patogenicidade , Saliva/virologia , Chlorocebus aethiops , Células Vero , Rhinovirus/patogenicidade , Rhinovirus/fisiologia , Linhagem Celular
2.
J Gen Virol ; 104(11)2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37909282

RESUMO

Enterovirus 71 (EV-A71) is a major public health problem, causing a range of illnesses from hand-foot-and-mouth disease to severe neurological manifestations. EV-A71 strains have been phylogenetically classified into eight genogroups (A to H), based on their capsid-coding genomic region. Genogroups B and C have caused large outbreaks worldwide and represent the two canonical circulating EV-A71 subtypes. Little is known about the antigenic diversity of new genogroups as compared to the canonical ones. Here, we compared the antigenic features of EV-A71 strains that belong to the canonical B and C genogroups and to genogroups E and F, which circulate in Africa. Analysis of the peptide sequences of EV-A71 strains belonging to different genogroups revealed a high level of conservation of the capsid residues involved in known linear and conformational neutralization antigenic sites. Using a published crystal structure of the EV-A71 capsid as a model, we found that most of the residues that are seemingly specific to some genogroups were mapped outside known antigenic sites or external loops. These observations suggest a cross-neutralization activity of anti-genogroup B or C antibodies against strains of genogroups E and F. Neutralization assays were performed with diverse rabbit and mouse anti-EV-A71 sera, anti-EV-A71 human standards and a monoclonal neutralizing antibody. All the batches of antibodies that were tested successfully neutralized all available isolates, indicating an overall broad cross-neutralization between the canonical genogroups B and C and genogroups E and F. A panel constituted of more than 80 individual human serum samples from Cambodia with neutralizing antibodies against EV-A71 subgenogroup C4 showed quite similar cross-neutralization activities between isolates of genogroups C4, E and F. Our results thus indicate that the genetic drift underlying the separation of EV-A71 strains into genogroups A, B, C, E and F does not correlate with the emergence of antigenically distinct variants.


Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Doença de Mão, Pé e Boca , Humanos , Camundongos , Animais , Coelhos , Enterovirus Humano A/genética , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Genótipo , Anticorpos Monoclonais
3.
Curr Res Microb Sci ; 3: 100158, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35935678

RESUMO

Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (Weiss et al., 2021, Murer et al., 2022). Our results here not only reinforce the broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 d, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data emphasize the potential of repurposable drugs against COVID-19.

4.
Viruses ; 14(7)2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35891387

RESUMO

Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAs.


Assuntos
COVID-19 , Interferon Tipo I , MicroRNAs , Antivirais/farmacologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Interferon Tipo I/genética , RNA de Cadeia Dupla , SARS-CoV-2
5.
Curr Res Virol Sci ; 3: 100019, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35072124

RESUMO

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the ß-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

6.
Front Microbiol ; 11: 1907, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922374

RESUMO

Enterovirus A71 (EV-A71) is a leading cause of hand-foot-and-mouth disease (HFMD) and can be associated with severe neurological complications. EV-A71 strains can be classified into seven genogroups, A-H, on the basis of the VP1 capsid protein gene sequence. Genogroup A includes the prototype strain; genogroups B and C are responsible of major outbreaks worldwide, but little is known about the others, particularly genogroups E and F, which have been recently identified in Africa and Madagascar, respectively. The circulation of EV-A71 in the African region is poorly known and probably underestimated. A rapid and specific assay for detecting all genogroups of EV-A71 is required. In this study, we developed a real-time RT-PCR assay with a competitive internal control (IC). The primers and TaqMan probe specifically target the genomic region encoding the VP1 capsid protein. Diverse EV-A71 RNAs were successfully amplified from the genogroups A, B, C, D, E, and F, with similar sensitivity and robust reproducibility. Neither cross reaction with other EVs nor major interference with the competitive IC was detected. Experimentally spiked stool and plasma specimens provided consistent and reproducible results, and validated the usefulness of the IC for demonstrating the presence of PCR inhibitors in samples. The analysis in an African laboratories network of 1889 untyped enterovirus isolates detected 15 EV-A71 of different genogroups. This specific real-time RT-PCR assay provides a robust and sensitive method for the detection of EV-A71 in biological specimens and for the epidemiological monitoring of EV-A71 including its recently discovered genogroups.

7.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30602612

RESUMO

Human enteroviruses of species A (EV-A) are the leading cause of hand-foot-and-mouth disease (HFMD). EV-A71 is frequently implicated in HFMD outbreaks and can also cause severe neurological manifestations. We investigated the molecular epidemiological processes at work and the contribution of genetic recombination to the evolutionary history of EV-A in Madagascar, focusing on the recently described EV-A71 genogroup F in particular. Twenty-three EV-A isolates, collected mostly in 2011 from healthy children living in various districts of Madagascar, were characterized by whole-genome sequencing. Eight different types were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent recent intra- and intertypic genetic exchanges between the noncapsid sequences of Madagascan EV-A isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination, with one isolate displaying a mosaic genome resulting from recent genetic exchanges with Madagascan coxsackieviruses A7 and possibly A5 and A10 or common ancestors. The engineering and characterization of recombinants generated from progenitors belonging to different EV-A types or EV-A71 genogroups with distantly related nonstructural sequences indicated a high level of permissiveness for intertypic genetic exchange in EV-A. This permissiveness suggests that the primary viral functions associated with the nonstructural sequences have been highly conserved through the diversification and evolution of the EV-A species. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify surveillance of EV-A circulation and HFMD cases to prevent possible outbreaks due to emerging strains.IMPORTANCE Human enteroviruses of species A (EV-A), including EV-A71, are the leading cause of hand-foot-and-mouth disease (HFMD) and may also cause severe neurological manifestations. We investigated the circulation and molecular evolution of EV-A in Madagascar, focusing particularly on the recently described EV-A71 genogroup F. Eight different types, collected mostly in 2011, were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent genetic exchanges between the different types of isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination. The engineering and characterization of recombinants involving progenitors belonging to different EV-A types indicated a high degree of permissiveness for genetic exchange in EV-A. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify the surveillance of EV-A circulation to prevent possible HFMD outbreaks due to emerging strains.


Assuntos
Enterovirus Humano A/genética , Recombinação Genética/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Pré-Escolar , Chlorocebus aethiops , Surtos de Doenças , Infecções por Enterovirus/virologia , Evolução Molecular , Genoma Viral/genética , Genótipo , Células HEK293 , Doença de Mão, Pé e Boca/genética , Doença de Mão, Pé e Boca/virologia , Humanos , Madagáscar , Epidemiologia Molecular , Permissividade , Filogenia , Células Vero , Sequenciamento Completo do Genoma/métodos
8.
Emerg Infect Dis ; 24(4): 754-757, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29553325

RESUMO

We analyzed whole-genome sequences of 8 enterovirus A71 isolates (EV-A71). We confirm the circulation of genogroup C and the new genogroup E in West Africa. Our analysis demonstrates wide geographic circulation and describes genetic exchanges between EV-A71 and autochthonous EV-A that might contribute to the emergence of pathogenic lineages.


Assuntos
Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Variação Genética , Genoma Viral , Genótipo , Humanos , Filogenia , Recombinação Genética
9.
Front Microbiol ; 7: 1294, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27617004

RESUMO

Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C) consists of more than 20 types, among which the three serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions. A simple method was developed to quickly sequence the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to being sequenced by a high-throughput technique. The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures. By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses.

10.
J Virol ; 90(21): 9683-9692, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27535047

RESUMO

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCE: RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.


Assuntos
Antivirais/farmacologia , Poliaminas/metabolismo , Vírus de RNA/efeitos dos fármacos , Acetiltransferases/metabolismo , Animais , Linhagem Celular , Febre de Chikungunya/tratamento farmacológico , Febre de Chikungunya/virologia , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/metabolismo , Surtos de Doenças , Ebolavirus/efeitos dos fármacos , Ebolavirus/metabolismo , Eflornitina/farmacologia , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Espermina/análogos & derivados , Espermina/farmacologia , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/virologia
11.
Diagn Microbiol Infect Dis ; 84(2): 116-22, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26643063

RESUMO

Acute enterovirus (EV) meningitis is a frequent cause of hospitalisation, and over 100 EV serotypes may be involved. A total of 215 patients of all ages with meningitis signs were investigated in 2 Tunisian hospitals. Their cerebrospinal fluid (CSF) was analysed retrospectively for EVs with a TaqMan real-time RT-qPCR. The virus strains were typed, and their evolutionary relationships were determined by Bayesian phylogenetic methods. An EV genome was detected in 21/215 patients (9.8%). The CSF viral loads ranged from 3.27 to 5.63 log10 genome copies/mL. The strains were identified in 13/21 patients and assigned to EV-B types. Viruses identified in Tunisian patients were genetically related to variants detected in France. The viral loads were similar in Tunisian and French patients for most EV types. The phylogenetic data and viral loads determined in Tunisian and French patients suggest that close EV variants were involved in aseptic meningitis in the 2 countries over a same period.


Assuntos
Infecções por Enterovirus/epidemiologia , Enterovirus/classificação , Enterovirus/isolamento & purificação , Meningite Viral/epidemiologia , Meningite Viral/virologia , Adolescente , Adulto , Líquido Cefalorraquidiano/virologia , Criança , Pré-Escolar , Feminino , França/epidemiologia , Variação Genética , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Epidemiologia Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tunísia/epidemiologia , Carga Viral
12.
J Gen Virol ; 96(Pt 7): 1682-95, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25711966

RESUMO

Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.


Assuntos
Barreira Hematoencefálica/virologia , Células Endoteliais/fisiologia , Células Endoteliais/virologia , Enterovirus/fisiologia , Antígenos Virais/análise , Apoptose , Linhagem Celular , Enterovirus/crescimento & desenvolvimento , Humanos , Modelos Biológicos , Permeabilidade , Sorogrupo
13.
J Infect Dis ; 210(4): 576-84, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24652491

RESUMO

BACKGROUND: Acute enterovirus (EV) meningitis is a major cause of hospitalization among adults and children. It is caused by multiple EV genotypes assigned to 4 species (EV-A, EV-B, EV-C, and EV-D). METHODS: We determined viral loads in the cerebrospinal fluid (CSF) of 156 patients of all ages with EV meningitis during a 5-year observational prospective study. The virus strains were genotyped, and their time origin was determined with Bayesian phylogenetic methods. RESULTS: The CSF viral loads ranged between 3.4 and 7.5 log10 copies/mL (median, 4.9 log10 copies/mL). They were higher in neonates than in infants and children (P = .02) but were comparable in adults. Viral loads were associated with EV genotypes (P < .001). The EV strains were identified in 152 of 156 patients and assigned to 23 genotypes within the EV-A and EV-B species. The most frequent genotypes, echoviruses 6 and 30, were associated with different viral loads (P < .001). The highest viral loads were in meningitis cases caused by coxsackievirus A9, B4, and B5 genotypes. Most patients infected by a same genotype were infected by a major virus variant of recent emergence. CONCLUSIONS: The variations in CSF viral loads in patients at the onset of EV meningitis are related to genotypic differences in the virus strains involved.


Assuntos
Infecções por Enterovirus/líquido cefalorraquidiano , Infecções por Enterovirus/virologia , Enterovirus/isolamento & purificação , Meningite Viral/líquido cefalorraquidiano , Meningite Viral/virologia , Adolescente , Adulto , Criança , Pré-Escolar , Enterovirus/genética , Genótipo , Hospitalização , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Filogenia , Estudos Prospectivos , Carga Viral , Adulto Jovem
14.
J Virol Methods ; 185(1): 142-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22766179

RESUMO

Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections.


Assuntos
Infecções por Enterovirus/diagnóstico , Enterovirus/isolamento & purificação , Meningite Asséptica/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Líquido Cefalorraquidiano/virologia , Infecções por Enterovirus/virologia , Humanos , Meningite Asséptica/virologia , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga Viral/normas
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