RESUMO
Mayaro virus (MAYV) is mainly found in Central and South America and causes a febrile illness followed by debilitating arthritis and arthralgia similar to chikungunya virus (CHIKV). Infection leads to long-term sequelae with a direct impact on the patient's productive capacity, resulting in economic losses. Mayaro fever is a neglected disease due to the limited epidemiological data. In Brazil, it is considered a potential public health risk with the number of cases increasing every year. Most of our knowledge about MAYV biology is inferred from data obtained from other alphaviruses as well as more recent studies on MAYV. Here, we analyzed the kinetics of viral replication through standard growth curves, quantification of intracellular and extracellular particles, and RNA quantification. We compared transmission electron microscopy data during different stages of infection. This approach allowed us to establish a chronological order of events during MAYV replication and its respective timepoints including cell entry through clathrin-mediated endocytosis occurring at 15-30 min, genome replication at 2-3 h, morphogenesis at 4 hpi, and release at 4-6 hpi. We also present evidence of uncharacterized events such as ribosome reorganization as well as clusters of early viral precursors and release through exocytosis in giant forms. Our work sheds new and specific light on the MAYV replication cycle and may contribute to future studies on the field.
RESUMO
Cellular proteins often have multiple and diverse functions. This is illustrated with protein Spir-1 that is an actin nucleator, but, as shown here, also functions to enhance innate immune signalling downstream of RNA sensing by RIG-I/MDA-5. In human and mouse cells lacking Spir-1, IRF3 and NF-κB-dependent gene activation is impaired, whereas Spir-1 overexpression enhanced IRF3 activation. Furthermore, the infectious virus titres and sizes of plaques formed by two viruses that are sensed by RIG-I, vaccinia virus (VACV) and Zika virus, are increased in Spir-1 KO cells. These observations demonstrate the biological importance of Spir-1 in the response to virus infection. Like cellular proteins, viral proteins also have multiple and diverse functions. Here, we also show that VACV virulence factor K7 binds directly to Spir-1 and that a diphenylalanine motif of Spir-1 is needed for this interaction and for Spir-1-mediated enhancement of IRF3 activation. Thus, Spir-1 is a new virus restriction factor and is targeted directly by an immunomodulatory viral protein that enhances virus virulence and diminishes the host antiviral responses.
Assuntos
Infecção por Zika virus , Zika virus , Actinas/metabolismo , Animais , Imunidade Inata , Camundongos , Fenilalanina , Transdução de Sinais , Vaccinia virus/genética , Proteínas Virais/metabolismo , Zika virus/metabolismoRESUMO
The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (it was first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found infection cellular activities other than those usually described for the chikungunya virus replication cycle, i.e., we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area, and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages, as follows: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.
Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Vírus Chikungunya/ultraestrutura , Fenômenos Fisiológicos Virais , Replicação Viral , Animais , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral , Vacúolos/ultraestrutura , Células Vero , Liberação de VírusRESUMO
The recent introduction of Zika virus (ZIKV), the recurrence of dengue virus (DENV), and the lethality of yellow fever virus (YFV) have had a significant impact on Brazilian society and public health. Here, we targeted two cellular kinases implicated in cell proliferation and cancer that are also important for viral replication: mitogen-activated protein kinase kinase (MEK) and Src. We used two MEK inhibitors - trametinib and selumetinib - and two Src inhibitors - saracatinib and bosutinib - to inhibit ZIKV, DENV, and YFV replication in cell culture. The cytotoxicity of the four inhibitors was determined by the observation of abnormal morphology and quantification of adherent cells by crystal violet staining. The antiviral activity of these drugs was assessed based on the reduction of plaque-forming units in cell culture as evidence of the inhibition of the replication of the selected flaviviruses. All four inhibitors showed antiviral activity, but among them, trametinib was the safest and most efficacious against all of the viruses, inhibiting the replication of ZIKV and YFV by 1000-fold, and DENV2/3 by nearly 100-fold. This pan-antiviral effect shows that trametinib could be repurposed for the treatment of flaviviral infections.
Assuntos
Antivirais/farmacologia , Flavivirus/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Cricetinae , Flavivirus/classificação , Flavivirus/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Células Vero , Replicação Viral/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidoresRESUMO
Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 105 PFU of D2S20. The effectiveness of AZD6244 was observed even when the treatment of infected animals was initiated 1-2 days postinfection. This was also followed by a reduction in viral copy number in both the serum and the spleen. There was also an increase in IL-1ß and TNF-α levels in mice that were infected with D2S20 and treated with AZD6244 in comparison to infected mice that were treated with the vehicle only. These data demonstrate the potential of AZD6244 as a new therapeutic agent to treat DENV infection and possibly other flavivirus diseases.
Assuntos
Antivirais/uso terapêutico , Benzimidazóis/uso terapêutico , Vírus da Dengue/crescimento & desenvolvimento , Dengue Grave/prevenção & controle , Animais , Linhagem Celular , Cricetinae , Vírus da Dengue/efeitos dos fármacos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Interleucina-1beta/sangue , Camundongos , Dengue Grave/virologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/sangueRESUMO
Taterapox virus (TATV) is phylogenetically the closest related virus to variola-the etiological agent of smallpox. Despite the similarity, few studies have evaluated the virus. In vivo, TATV can infect several animals but produces an inapparent infection in wild-type mice; however, TATV does cause morbidity and mortality in some immunocompromised strains. We employed in vitro techniques to compare TATV to ectromelia (ECTV) and vaccinia (VACV) viruses. Both ECTV and TATV replicate efficiently in primate cell lines but TATV replicates poorly in murine cells lines. Furthermore, TATV induces cytopathic effects, but to a lesser extent than ECTV, and changes cytoskeletal networks differently than both ECTV and VACV. Bioinformatic studies revealed differences in several immunomodulator open reading frames that could contribute to the reduced virulence of TATV, which were supported by in vitro cytokine assays.
Assuntos
Orthopoxvirus/classificação , Orthopoxvirus/genética , Infecções por Poxviridae/virologia , Virulência/genética , Células A549 , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus da Varíola Bovina/genética , Vírus da Ectromelia/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fases de Leitura Aberta/genética , Orthopoxvirus/imunologia , Orthopoxvirus/isolamento & purificação , Filogenia , Análise de Sequência de Proteína , Baço/citologia , Baço/imunologia , Vaccinia virus/genética , Células VeroRESUMO
Evolution has equipped poxvirus genomes with the coding capacity for several virus-host interaction products which interfere with host cell gene expression and protein function, creating an adequate intracellular environment for a productive infection. We show here that Vaccinia virus (VACV) induces the expression of the cellular transcription factor EGR-1 (early growth response-1) in Mouse Embryonic Fibroblasts (MEFs) through the MEK (mitogen-activated protein kinase (MAPK)/ERK)/ERK (extracellular signal-regulated kinases) pathway, from 3 to 12 h post infection (h.p.i.). By using starved egr-1 knockout (egr-1-/-) MEFs, we demonstrate that VACV replication is reduced by ~1 log in this cell line. Although western blotting and electron microscopy analyses revealed no difference in VACV gene expression or morphogenesis, the specific infectivity of VACV propagated in egr-1-/- MEFs was lower than virus propagated in wild type (WT) cells. This lower infectivity was due to decreased VACV DNA replication during the next cycle of infection. Taken together, these results revealed that EGR-1 appears to facilitate VACV replication in starved fibroblasts by affecting viral particles infectivity.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vaccinia virus/fisiologia , Vacínia/genética , Vacínia/virologia , Animais , Linhagem Celular , Replicação do DNA , DNA Viral , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Deleção de Genes , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Fosforilação , Vacínia/metabolismo , Replicação ViralRESUMO
Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Vaccinia virus/fisiologia , Animais , Linhagem Celular , DNA Viral , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Fibroblastos/virologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase Quinases/genética , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosforilação , Proteínas Proto-Oncogênicas c-jun/genética , Replicação ViralRESUMO
The VACV replication cycle is remarkable in the sense that it is performed entirely in the cytoplasmic compartment of vertebrate cells, due to its capability to encode enzymes required either for regulating the macromolecular precursor pool or the biosynthetic processes. Although remarkable, this gene repertoire is not sufficient to confer the status of a free-living microorganism to the virus, and, consequently, the virus relies heavily on the host to successfully generate its progeny. During the complex virus-host interaction, viruses must deal not only with the host pathways to accomplish their temporal demands but also with pathways that counteract viral infection, including the inflammatory, innate and acquired immune responses. This review focuses on VACV and other DNA or RNA viruses that stimulate the MEK (MAPK - Mitogen Activated Protein Kinase)/ERK- Extracellular signal-Regulated Kinase) pathway as part of their replication cycle.
Assuntos
Sistema de Sinalização das MAP Quinases , Vaccinia virus/fisiologia , Vacínia/enzimologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Vírus de RNA/genética , Vírus de RNA/fisiologia , Vacínia/genética , Vacínia/virologia , Vaccinia virus/genética , Viroses/enzimologia , Viroses/genética , Viroses/virologia , Replicação ViralRESUMO
The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1(-/-) MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1(-/-) MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1(-/-) cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.
Assuntos
Endocitose , Interações Hospedeiro-Patógeno , Vaccinia virus/fisiologia , Internalização do Vírus , Quinases Ativadas por p21/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Varredura , Quinases Ativadas por p21/genéticaRESUMO
Vaccinia virus (VACV) is a poxvirus and encodes many proteins that modify the host cell metabolism or inhibit the host response to infection. For instance, it is known that VACV infection can activate the mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathway and inhibit activation of the pro-inflammatory transcription factor NF-κB. Since NF-κB and MAPK/AP-1 share common upstream activators we investigated whether six different VACV Bcl-2-like NF-κB inhibitors can also influence MAPK/AP-1 activation. Data presented show that proteins A52, B14 and K7 each contribute to AP-1 activation during VACV infection, and when expressed individually outwith infection. B14 induced the greatest stimulation of AP-1 and further investigation showed B14 activated mainly the MAPKs ERK (extracellular signal-regulated kinase) and JNK (Jun N-terminal kinase), and their substrate c-Jun (a component of AP-1). These data indicate that the same viral protein can have different effects on distinct signalling pathways, in blocking NF-κB activation whilst leading to MAPK/AP-1 activation.
Assuntos
Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Fator de Transcrição AP-1/metabolismo , Vaccinia virus/fisiologia , Proteínas Virais/metabolismo , Transdução de SinaisRESUMO
The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis where MEK2 was essential for the phosphorylation of MKK3/MKK6 and p38 MAPK that directly impacted on cyclin D1 expression. Importantly, the MEK1 inhibitor PD98059, like MEK1 silencing, induced an augment of cyclin D1 expression that correlated with an increase of MDA-MB-231 cell proliferation suggesting that MEK1 may play a regulatory role in these cells. In sum, the crucial role of MEK2 in MDA-MB-231 cell viability and the unknown relationship between MEK2 and MKK3/MKK6-p38 axis here revealed may open new therapeutic strategies for aggressive breast cancer.
Assuntos
Neoplasias da Mama/patologia , Ciclina D1/metabolismo , MAP Quinase Quinase 2/metabolismo , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 6/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Butadienos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Flavonoides/farmacologia , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Orthobunyaviruses are arboviruses in which at least 30 members are human pathogens. The members of group C orthobunyaviruses were first isolated in the Brazilian Amazon in 1950, since that time little information is accumulated about ecology and the medical impact of these virus groups in Brazil. Herein, we describe the evidence of Apeu virus (APEUV; an Orthobunyavirus member) infection in wild monkeys from the Brazilian Amazon forest. APEUV was detected by using a neutralizing antibody in serum and its RNA, suggesting past and acute infection of Amazonian monkeys by this virus. These results altogether represent an important contribution of orthobunyavirus ecology in the Amazon and an update about recent circulation and risk for humans with expansion of the cities to Amazon forest.
Assuntos
Alouatta , Animais Selvagens , Infecções por Bunyaviridae/veterinária , Cebus , Doenças dos Macacos/virologia , Orthobunyavirus/isolamento & purificação , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Infecções por Bunyaviridae/epidemiologia , Infecções por Bunyaviridae/virologia , Doenças dos Macacos/epidemiologia , RNA Viral/sangueRESUMO
Exploiting the inhibition of host signaling pathways aiming for discovery of potential antiflaviviral compounds is clearly a beneficial strategy for the control of life-threatening diseases caused by flaviviruses. Here we describe the antiviral activity of the MEK1/2 inhibitor U0126 against Yellow fever virus 17D vaccine strain (YFV-17D). Infection of VERO cells with YFV-17D stimulates ERK1/2 phosphorylation early during infection. Pharmacological inhibition of MEK1/2 through U0126 treatment of VERO cells blockades not only the YFV-stimulated ERK1/2 phosphorylation, but also inhibits YFV replication by â¼99%. U0126 was also effective against dengue virus (DENV-2 and -3) and Saint-Louis encephalitis virus (SLEV). Levels of NS4AB, as detected by immunofluorescence, are diminished upon treatment with the inhibitor, as well as the characteristic endoplasmic reticulum membrane invagination stimulated during the infection. Though not protective, treatment of YFV-infected, adult BALB/c mice with U0126 resulted in significant reduction of virus titers in brains. Collectively, our data suggest the potential targeting of the MEK1/2 kinase as a therapeutic tool against diseases caused by flaviviruses such as yellow fever, adverse events associated with yellow fever vaccination and dengue.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Replicação Viral , Febre Amarela/enzimologia , Vírus da Febre Amarela/fisiologia , Animais , Chlorocebus aethiops , Ativação Enzimática , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Células Vero , Febre Amarela/genética , Febre Amarela/virologia , Vírus da Febre Amarela/genéticaRESUMO
The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.
Assuntos
Movimento Celular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Fibrinolisina/imunologia , MAP Quinase Quinase Quinases/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Monócitos/imunologia , Receptor PAR-1/imunologia , Receptores CCR2/imunologia , Animais , Benzoxazinas/farmacologia , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , Cavidade Pleural/imunologia , Receptores CCR2/antagonistas & inibidores , Compostos de Espiro/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.
Assuntos
Humanos , Ensaio de Desvio de Mobilidade Eletroforética , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , /genética , Técnicas de Tipagem Bacteriana , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium tuberculosis/classificação , Micobactérias não Tuberculosas/classificação , Reação em Cadeia da PolimeraseRESUMO
The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.
Assuntos
Ensaio de Desvio de Mobilidade Eletroforética , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , RNA Ribossômico 16S/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Humanos , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium tuberculosis/classificação , Micobactérias não Tuberculosas/classificação , Reação em Cadeia da PolimeraseRESUMO
To investigate circulation of mimiviruses in the Amazon Region of Brazil, we surveyed 513 serum samples from domestic and wild mammals. Neutralizing antibodies were detected in 15 sample pools, and mimivirus DNA was detected in 9 pools of serum from capuchin monkeys and in 16 pools of serum from cattle.
Assuntos
Doenças dos Animais/epidemiologia , Doenças dos Animais/virologia , Viroses/veterinária , Sequência de Aminoácidos , Animais , Animais Domésticos , Animais Selvagens , Brasil/epidemiologia , DNA Viral , Geografia , Mamíferos , Mimiviridae , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Carga ViralRESUMO
Growing evidences suggest that Saccharomyces boulardii (SB) is efficacious against bacterial infections and inflammatory bowel diseases. This study investigated the effects of treatment with SB provided in a murine model of typhoid fever. Mice were divided into two groups: (1) control animals challenged with Salmonella Typhimurium (ST), and (2) animals receiving SB, and then challenged with ST. At days 0, 1, 5, 10 and 15 post-challenge, animals were euthanized and tissues collected to analyze bacterial translocation, cytokines, signaling pathways and histological analysis. Survival rate and animal weight were also evaluated. Treatment with SB increased survival rate and inhibited translocation of bacteria after ST challenge. Histological data showed that SB also protected mice against liver damage induced by ST. SB decreased levels of inflammatory cytokines and activation of mitogen-activated protein kinases (p38, JNK and ERK1/2), phospho-IκB, p65-RelA, phospho-jun and c-fos in the colon, signal pathways involved in the activation of inflammation induced by ST. Further experiments revealed that probiotic effects were due, at least in part, to the binding of ST to the yeast. Such binding diminishes ST translocation, resulting in decreased activation of signaling pathways which lead to intestinal inflammation in a murine model of typhoid fever.
Assuntos
Translocação Bacteriana/imunologia , Febre Paratifoide/imunologia , Saccharomyces/imunologia , Salmonella typhimurium/imunologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Histocitoquímica , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Camundongos , Análise de SobrevidaRESUMO
Viral manipulation of transduction pathways associated with key cellular functions such as survival, response to microbial infection, and cytoskeleton reorganization can provide the supportive milieu for a productive infection. Here, we demonstrate that vaccinia virus (VACV) infection leads to activation of the stress-activated protein kinase (SAPK)/extracellular signal-regulated kinase (ERK) 4/7 (MKK4/7)-c-Jun N-terminal protein kinase 1/2 (JNK1/2) pathway; further, the stimulation of this pathway requires postpenetration, prereplicative events in the viral replication cycle. Although the formation of intracellular mature virus (IMV) was not affected in MKK4/7- or JNK1/2-knockout (KO) cells, we did note an accentuated deregulation of microtubule and actin network organization in infected JNK1/2-KO cells. This was followed by deregulated viral trafficking to the periphery and enhanced enveloped particle release. Furthermore, VACV infection induced alterations in the cell contractility and morphology, and cell migration was reduced in the JNK-KO cells. In addition, phosphorylation of proteins implicated with early cell contractility and cell migration, such as microtubule-associated protein 1B and paxillin, respectively, was not detected in the VACV-infected KO cells. In sum, our findings uncover a regulatory role played by the MKK4/7-JNK1/2 pathway in cytoskeleton reorganization during VACV infection.