RESUMO
UNLABELLED: Coxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae. According to earlier studies, CVA9 binds to αVß3 and αVß6 integrins on the cell surface and utilizes ß2-microglobulin, dynamin, and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here, based on electron microscopy, that CVA9 is found in multivesicular structures 2 h postinfection (p.i.). A neutral red labeling assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicular bodies (MVBs) is crucial for promoting infection, as judged by the strong inhibitory effect of the wild-type form of Hrs and dominant negative form of VPS4 in CVA9 infection. CVA9 infection is dependent on phospholipase C at the start of infection, whereas Rac1 is especially important between 1 and 3 h p.i., when the virus is in endosomes. Several lines of evidence implicate that low pH does not play a role in CVA9 infection. The infection is not affected by Bafilomycin A1. In addition, CVA9 is not targeted to acidic late endosomes or lysosomes, and the MVBs accumulating CVA9 have a neutral pH. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger neutral MVBs, which are important for virus infection. IMPORTANCE: We demonstrate here that the enterovirus coxsackievirus A9 (CVA9) uses a nonclathrin and nonacidic pathway to infect cells. CVA9 does not accumulate in conventional late endosomes or lysosomes. We found that inhibitors of phospholipase C (PLC), Rac1, and the Na(+)/H(+) exchanger decreased CVA9 infection. The PLC inhibitor acts on early entry, the Rac1 inhibitor acts between 1 and 3 h, when the virus is in endosomes, and the Na(+)/H(+) exchange inhibitor acts during various steps during the virus life cycle. The infection depends on the formation of novel neutral multivesicular bodies (MVBs), which accumulate CVA9 during the first hours of entry. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger formation of neutral MVBs. The data show that these enteroviruses favor nonacidic conditions and complex MVBs to promote virus infection.
Assuntos
Enterovirus Humano B/fisiologia , Corpos Multivesiculares/química , Corpos Multivesiculares/virologia , Internalização do Vírus , Linhagem Celular , Células Epiteliais/virologia , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Corpos Multivesiculares/ultraestruturaRESUMO
Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in alpha2beta1. Furthermore, the mutation E336A did neither prevent EV1 induced and alpha2beta1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on alpha2beta1 clustering, but do not require the conformational regulation of the receptor.
Assuntos
Enterovirus Humano B/fisiologia , Enterovirus Humano B/patogenicidade , Integrina alfa2beta1/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , Integrina alfa2beta1/química , Integrina alfa2beta1/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Coxsackievirus A9 (CVA9) is an important pathogen of the Picornaviridae family. It utilizes cellular receptors from the integrin αv family for binding to its host cells prior to entry and genome release. Among the integrins tested, it has the highest affinity for αvß6, which recognizes the arginine-glycine-aspartic acid (RGD) loop present on the C terminus of viral capsid protein, VP1. As the atomic model of CVA9 lacks the RGD loop, we used surface plasmon resonance, electron cryo-microscopy, and image reconstruction to characterize the capsid-integrin interactions and the conformational changes on genome release. We show that the integrin binds to the capsid with nanomolar affinity and that the binding of integrin to the virion does not induce uncoating, thereby implying that further steps are required for release of the genome. Electron cryo-tomography and single-particle image reconstruction revealed variation in the number and conformation of the integrins bound to the capsid, with the integrin footprint mapping close to the predicted site for the exposed RGD loop on VP1. Comparison of empty and RNA-filled capsid reconstructions showed that the capsid undergoes conformational changes when the genome is released, so that the RNA-capsid interactions in the N termini of VP1 and VP4 are lost, VP4 is removed, and the capsid becomes more porous, as has been reported for poliovirus 1, human rhinovirus 2, enterovirus 71, and coxsackievirus A7. These results are important for understanding the structural basis of integrin binding to CVA9 and the molecular events leading to CVA9 cell entry and uncoating.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas do Capsídeo/metabolismo , Enterovirus Humano B/fisiologia , Integrinas/metabolismo , Modelos Moleculares , Ligação Viral , Desenvelopamento do Vírus/fisiologia , Antígenos de Neoplasias/química , Microscopia Crioeletrônica , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Integrinas/química , Ligação Proteica , Conformação Proteica , Ressonância de Plasmônio de Superfície , Desenvelopamento do Vírus/genéticaRESUMO
Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5' noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting.
Assuntos
Enterovirus/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Sondas de Oligonucleotídeos , Oligonucleotídeos , Infecções por Picornaviridae/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rhinovirus/isolamento & purificação , Enterovirus/genética , Humanos , Infecções por Picornaviridae/virologia , Rhinovirus/genética , Sensibilidade e EspecificidadeRESUMO
Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.
Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , Enterovirus/genética , Enterovirus/ultraestrutura , Internalização do Vírus , Microscopia Crioeletrônica , Enterovirus/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Modelos Biológicos , Dados de Sequência Molecular , Conformação Proteica , Análise de Sequência de DNARESUMO
Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.
Assuntos
Interferência de RNA , Análise de Célula Única/métodos , Viroses/genética , Teorema de Bayes , Microambiente Celular , Simulação por Computador , Genômica/métodos , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/métodos , Modelos Biológicos , RNA Interferente Pequeno , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , Biologia de Sistemas/métodos , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Viroses/metabolismo , Vírus/isolamento & purificação , Vírus/patogenicidadeRESUMO
Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize alphaV integrins, particularly alphaVbeta6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin beta6 subunit inhibited virus proliferation, confirming that alphaVbeta6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of beta2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin alphaVbeta6, beta2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Caveolina 1/fisiologia , Clatrina/fisiologia , Dinamina II/fisiologia , Enterovirus Humano B/fisiologia , Internalização do Vírus , Microglobulina beta-2/fisiologia , Fator 6 de Ribosilação do ADP , Amilorida/análogos & derivados , Amilorida/farmacologia , Cavéolas/fisiologia , Linhagem Celular Tumoral , Endocitose , Humanos , Pinocitose , RNA Interferente Pequeno/genéticaRESUMO
Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to alpha(V) integrins. The interaction of HPEV1 with alpha(V) integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins alpha(V)beta(3) and alpha(V)beta(6) to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, alpha(V)beta(6) bound more efficiently than alpha(V)beta(3) to immobilized HPEV1. Moreover, soluble alpha(V)beta(6), but not alpha(V)beta(3), blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.
Assuntos
Antígenos de Neoplasias/metabolismo , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Parechovirus/metabolismo , Infecções por Picornaviridae/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linhagem Celular , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/genética , Integrinas/química , Integrinas/genética , Conformação Molecular , Dados de Sequência Molecular , Parechovirus/química , Parechovirus/genética , Infecções por Picornaviridae/virologia , Ligação Proteica , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
During fall 2008, an outbreak of hand, foot, and mouth disease (HFMD) with onychomadesis (nail shedding) as a common feature occurred in Finland. We identified an unusual enterovirus type, coxsackievirus A6 (CVA6), as the causative agent. CVA6 infections may be emerging as a new and major cause of epidemic HFMD.
Assuntos
Doenças Transmissíveis Emergentes/virologia , Infecções por Coxsackievirus/virologia , Surtos de Doenças , Enterovirus , Doença de Mão, Pé e Boca/virologia , Adulto , Criança , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/patologia , Infecções por Coxsackievirus/epidemiologia , Infecções por Coxsackievirus/patologia , Primers do DNA , Enterovirus/classificação , Enterovirus/isolamento & purificação , Enterovirus/patogenicidade , Feminino , Finlândia/epidemiologia , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/patologia , Humanos , Masculino , Unhas Malformadas/virologia , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieRESUMO
Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.
Assuntos
Calpaína/metabolismo , Enterovirus Humano B/fisiologia , RNA Viral/biossíntese , Replicação Viral/fisiologia , Linhagem Celular , Vesículas Citoplasmáticas/química , Vesículas Citoplasmáticas/virologia , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Humanos , Microscopia Confocal , Microscopia Imunoeletrônica , Parechovirus/fisiologiaRESUMO
To analyze clinical characteristics and prevalence of rhinovirus infections in children in the hospital, we reviewed a retrospective dataset from a 20-year period, and conducted a short-term prospective study. Records of children and adolescents treated at our hospital during 1987-2006 with a documented rhinovirus infection were reviewed and compared with patients with other respiratory virus infections. Prospective study included all children >or=1 month of age admitted to pediatric infectious disease ward during an autumn period. Rhinoviruses were detected by reverse transcription-PCR and/or culture, and sequence analysis was used for virus typing. In the retrospective study, the median age of 580 children with rhinovirus infection was 1.9 years (interquartile range, 1.0-4.3 years), and 27% had an underlying medical condition. Eighty-four children (16% of in-patients) were treated at pediatric intensive care unit. Twenty-one children (4%) had a hospital-acquired rhinovirus infection. In the prospective study, rhinoviruses were detected in 28% of 163 hospital episodes. Acute wheezing illness was diagnosed in 61% of children with rhinovirus and in 31% of children with respiratory syncytial virus, enterovirus, or no study virus (P < 0.001). One-half of sequence-analyzed rhinovirus strains belonged to the newly identified C group. In conclusion, rhinovirus infections are a frequent cause of pediatric hospitalizations, and they are common also at the intensive care unit. Acute wheezing is the most frequent manifestation in hospital setting, but the range of clinical presentations is wide. Group C rhinoviruses may account for a large part of rhinovirus hospitalizations.
Assuntos
Infecções por Picornaviridae/epidemiologia , Rhinovirus/isolamento & purificação , Adolescente , Criança , Pré-Escolar , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/patologia , Infecção Hospitalar/fisiopatologia , Feminino , Genótipo , Hospitais , Humanos , Lactente , Masculino , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/fisiopatologia , Prevalência , Estudos Prospectivos , Estudos Retrospectivos , Rhinovirus/classificação , Rhinovirus/genéticaRESUMO
AIM: Except bacterial meningitis, the agents causing acute central nervous system (CNS) infections in children are disclosed in only approximately half of the cases, and even less in encephalitis. We studied the potential of modern microbiological assays to improve this poor situation. METHODS: In a prospective study during 3 years, all children attending hospital with suspected CNS infection were examined using a wide collection of microbiological tests using samples from the cerebrospinal fluid, serum, nasal swabs and stool. RESULTS: Among 213 patients, 66 (31%) cases suggested CNS infection and specific aetiology was identified in 56 patients. Of these microbiologically confirmed cases, viral meningitis/encephalitis was diagnosed in 25 (45%), bacterial meningitis in 21 (38%) and neuroborreliosis in 9 (16%) cases while 1 child had fungal infection. In meningitis patients, the causative agent was identified in 85% (35/41) cases and in encephalitis in 75% (12/16). The most common bacteria were Streptococcus agalactiae, Streptococcous pneumonie and Neisseria meningitidis, while the most frequently detected viruses were enteroviruses and varicella zoster virus. CONCLUSION: In 75% to 85% of paediatric CNS infections, specific microbiological diagnosis was obtained with modern laboratory techniques. The results pose a basis for prudent approach to these potentially serious diseases.
Assuntos
Infecções do Sistema Nervoso Central/diagnóstico , Técnicas Microbiológicas/métodos , Doença Aguda , Adolescente , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/líquido cefalorraquidiano , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Candidíase/diagnóstico , Candidíase/microbiologia , Infecções do Sistema Nervoso Central/microbiologia , Criança , Pré-Escolar , Diagnóstico Diferencial , Encefalite Viral/diagnóstico , Encefalite Viral/virologia , Paralisia Facial/etiologia , Fezes/microbiologia , Humanos , Lactente , Neuroborreliose de Lyme/complicações , Neuroborreliose de Lyme/diagnóstico , Neuroborreliose de Lyme/microbiologia , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Meningite Viral/diagnóstico , Meningite Viral/virologia , Reação em Cadeia da Polimerase , Estudos ProspectivosRESUMO
Rhinovirus is the major cause of common cold and frequently associates with acute wheezing, otitis media, sinusitis, and pneumonia. High prevalence of rhinovirus in hospitalized children and adults has been documented recently. We screened children > or =1 month of age, hospitalized for any infection, for the presence of rhinoviruses and recruited 24 families with > or =2 children for a 3-week follow-up study. Rhinovirus was detected in 46 (28%) of 163 hospitalizations by study children. Most rhinovirus-positive children (85%) had respiratory symptoms. During the follow-up, rhinoviruses were detected in virtually all children and in one-half of adults in families with a rhinovirus-positive index child, but commonly also in families with a rhinovirus-negative index child. Melting temperature and sequence analysis revealed the transmission routes of the viruses and showed that several virus types could circulate in the families simultaneously. Our studies corroborate the major contribution of rhinovirus to hospitalization of children, most often because of wheezing. Young children with respiratory symptoms are major spreaders of rhinovirus in family setting.
Assuntos
Resfriado Comum/patologia , Resfriado Comum/fisiopatologia , Rhinovirus/isolamento & purificação , Pré-Escolar , Resfriado Comum/transmissão , Resfriado Comum/virologia , Saúde da Família , Seguimentos , Genótipo , Hospitalização , Humanos , Lactente , Recém-Nascido , Epidemiologia Molecular , RNA Viral/genética , Infecções Respiratórias/patologia , Infecções Respiratórias/fisiopatologia , Infecções Respiratórias/virologia , Rhinovirus/classificação , Rhinovirus/genética , Análise de Sequência de DNA , Temperatura de TransiçãoRESUMO
BACKGROUND: Human bocavirus is a newly discovered parvovirus. It has been detected primarily in children with acute lower respiratory tract infection, but its occurrence, clinical profile, and role as a causative agent of respiratory tract disease are not clear. METHODS: We investigated the presence of human bocavirus by quantitative polymerase chain reaction of nasopharyngeal aspirate specimens and selected serum samples obtained from 259 children (median age, 1.6 years) who had been hospitalized for acute expiratory wheezing. The samples were analyzed for 16 respiratory viruses by polymerase chain reaction, virus culture, antigen detection, and serological assays. RESULTS: At least 1 potential etiologic agent was detected in 95% of children, and >1 agent was detected in 34% of children. Human bocavirus was detected in 49 children (19%). A large proportion of the cases were mixed infections with other viruses, but human bocavirus was the only virus detected in 12 children (5%). High viral loads of human bocavirus were noted mainly in the absence of other viral agents, suggesting a causative role for acute wheezing. In addition, infections that had uncertain clinical relevance and low viral loads were prevalent. Human bocavirus DNA was frequently detected in serum specimens obtained from patients with acute wheezing, suggesting systemic infection. CONCLUSIONS: Human bocavirus is prevalent among children with acute wheezing and can cause systemic infection. Results suggest a model for bocavirus infection in which high viral loads are potentially associated with respiratory symptoms and low viral loads indicate asymptomatic shedding. Therefore, quantitative polymerase chain reaction analysis may be important for additional studies of human bocavirus.
Assuntos
Bocavirus/isolamento & purificação , Infecções por Parvoviridae/diagnóstico , Sons Respiratórios , Infecções Respiratórias/virologia , Doença Aguda , Corticosteroides/uso terapêutico , Sequência de Bases , Criança , Pré-Escolar , DNA Viral/análise , Feminino , Finlândia , Seguimentos , Hospitalização , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Infecções por Parvoviridae/epidemiologia , Prevalência , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medição de Risco , Índice de Gravidade de DoençaRESUMO
Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the alpha2beta1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.
Assuntos
Dinamina II/química , Enterovirus Humano B/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Sítios de Ligação , Brefeldina A/farmacologia , Capsídeo/química , Linhagem Celular , Membrana Celular/metabolismo , Centrifugação com Gradiente de Concentração , Toxina da Cólera/metabolismo , Colesterol/metabolismo , Citoesqueleto/metabolismo , Eletroforese em Gel de Poliacrilamida , Endocitose , Genes Dominantes , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Hibridização In Situ , Hibridização in Situ Fluorescente , Cinética , Microdomínios da Membrana/virologia , Microscopia de Fluorescência , Microscopia de Vídeo , Fosforilação , Ligação Proteica , RNA Viral/metabolismo , Transdução de Sinais , Sacarose/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
Integrin alpha 2 beta 1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1- and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface alpha 2 beta 1 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of alpha 2 beta 1 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally alpha 2 beta 1 integrin was found inside caveolae and subsequently internalized into caveosome-like perinuclear structures. The internalization process, unlike cluster formation or lateral redistribution, was dependent on protein kinase C alpha activity. Caveolae are known to be highly immobile structures and alpha 2 beta 1 integrin clusters represent a previously unknown mechanism to activate endocytic trafficking via caveolae. The process was specific to alpha 2 beta 1 integrin, because the antibody-mediated formation of alpha V integrin clusters activated their internalization in coated vesicles and early endosomes. In addition to natural ligands human echovirus-1 (EV1) gains entry into the cell by binding to alpha 2 beta 1 and taking advantage of alpha 2 beta 1 internalization via caveolae.
Assuntos
Cavéolas/metabolismo , Estruturas da Membrana Celular/metabolismo , Membrana Celular/metabolismo , Integrina alfa2beta1/metabolismo , Proteína Quinase C/metabolismo , Citoesqueleto de Actina/metabolismo , Caveolina 1 , Caveolinas/metabolismo , Células Cultivadas , Vesículas Revestidas/metabolismo , Endossomos/metabolismo , Enterovirus Humano B/metabolismo , Humanos , Integrina alfaV/metabolismo , Microscopia Imunoeletrônica , Proteína Quinase C-alfaRESUMO
Baculovirus vectors have been shown to enter a variety of mammalian cell lines and gene transfer with wild-type baculovirus (WT) has been demonstrated both in vitro and in vivo. Different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. We have generated recombinant baculovirus vectors displaying an RGD-motif, recognized by alphaV integrin, on the viral surface. The RGD motifs within the C-terminus of coxsackie virus A9 and human parechovirus 1 VP1 proteins were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus. The recombinant RGD-presenting viruses bound more efficiently to the surface of human lung carcinoma cells (A549), known to contain alphaV integrins, as compared to WT baculovirus. In addition, the binding pattern of the RGD-displaying baculovirus showed extensive clustering. This most likely represents clustering of the integrin molecules on the cell surface, induced by binding of the RGD-displaying baculovirus. Finally, the transduction efficiency of an RGD-representing virus increased by almost three-fold as monitored by light emission measurements. In conclusion, these results suggest that the RGD-motif is functional on the surface of baculovirus and thereby these tropism-modified viruses bind more efficiently as well as enhance the transduction efficiency of human cancer cells expressing alphaV integrins.
Assuntos
Baculoviridae/genética , Oligopeptídeos/metabolismo , Transfecção/métodos , Animais , Baculoviridae/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Microscopia Confocal , Modelos Genéticos , Oligopeptídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
To determine the efficiency of reverse-transcriptase polymerase chain reaction (RT-PCR) assays currently used in diagnosing enterovirus and rhinovirus infections, we compared results obtained with RT-PCR methods, which detect both enteroviruses and rhinoviruses simultaneously, with results obtained by conventional virus isolation. Both tests were performed on 591 specimens: 38 samples (6%) had positive results by both RT-PCR and isolation, 90 samples (15%) had positive results by RT-PCR only, and 7 samples (1%) had positive results only by virus isolation. In conclusion, RT-PCR was superior in rapidity and sensitivity to virus isolation and is recommended as the primary diagnostic tool for enterovirus and rhinovirus infections.
Assuntos
Infecções por Enterovirus/diagnóstico , Enterovirus/isolamento & purificação , Infecções por Picornaviridae/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rhinovirus/isolamento & purificação , Humanos , Sensibilidade e EspecificidadeRESUMO
Childhood pneumonia is usually treated without determining its etiology. The causative organism can be isolated from specimens of blood, empyema fluid, or lung aspirate, but this is rarely done. The potential of transthoracic needle aspiration for identification of causative agents was tested with use of modern microbiological methods. Aspiration was performed for 34 children who had radiological signs compatible with community-acquired pneumonia and had alveolar consolidation. In addition to bacterial and viral cultures and viral antigen detection, nucleic acid detection for common respiratory pathogens was performed on aspirate specimens. Aspiration disclosed the etiology in 20 (59%) of 34 cases overall and in 18 (69%) of 26 patients from whom a representative specimen was obtained. Aspiration's advantages are high microbiological yield and a relatively low risk of a clinically significant adverse event. Aspiration should be used if identification of the causative agent outweighs the modest risk of the procedure.
Assuntos
Infecções Comunitárias Adquiridas/diagnóstico , Pneumonia/diagnóstico , Adolescente , Técnicas Bacteriológicas , Biópsia por Agulha , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/patologia , Feminino , Humanos , Lactente , Masculino , Pneumonia/microbiologia , Pneumonia/patologia , Reação em Cadeia da Polimerase , Estudos Prospectivos , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
INTRODUCTION: Viruses are known to induce apoptosis in their host cells. We studied whether cardiomyocyte apoptosis occurs upon coxsackievirus B3 (CVB3) infection and whether virus-associated apoptosis plays a role in the pathogenesis of experimental myocarditis. METHODS: BALB/c mice were infected with two variants of CVB3 causing either mild or severe myocarditis. Myocardial and serum samples were collected from Day 1 to Day 14 after virus inoculation. Apoptosis was detected in myocardial tissue sections using the terminal transferase-mediated DNA nick end labelling (TUNEL) assay and staining of active caspase 3, and compared with the presence of infectious CVB3 and viral proteins in cardiomyocytes. RESULTS: Compared with the noninfected control mice, infection with either CVB3 variant resulted in significantly increased cardiomyocyte apoptosis, which peaked on Day 5 after infection. At this time, the average percentages of apoptotic cardiomyocytes were 0.17% (SD 0.04; P=.03) and 0.77% (SD 0.11; P<.01) in mild and severe disease forms, respectively. The amount of apoptosis correlated with titers of infectious CVB3 in the heart muscle. Viral proteins were detected in the TUNEL-positive cells by immunohistochemistry. In the late stages of disease, apoptosis, together with inflammatory infiltrates persisted only in the severe disease form. CONCLUSIONS: CVB3-associated myocardial damage involves cardiomyocyte apoptosis. In the early stages of the disease, it appears to be triggered by viral replication in the cardiomyocytes.