RESUMO
Chikungunya virus (CHIKV) is a zoonotic arthropod-borne virus that has caused several outbreaks in tropical and subtropical areas worldwide during the last 50 years. The virus is known to target different human cell types throughout the course of infection including epithelial and endothelial cells, fibroblasts, primary monocytes and monocyte-derived macrophages (MDMs). The two latter are phagocytic cell populations of the innate immune system which are involved in some aspects of CHIKV pathogenesis. However, monocytes and macrophages also potentially contribute to the control of viral replication through the expression of different pattern recognition receptors sensing viral pathogens and subsequently, inducing an type I interferone (IFN-I)-dependent antiviral immune response. The aim of this study was to determine the modulation of the expression of Toll-like receptors (TLRs), cytokine secretion capabilities and antiviral factor production in monocytes and MDMs following infection with CHIKV. Moreover, we sought to determine the replication kinetics of CHIKV in these two cell populations. We found that the maximum peak of CHIKV replication was observed between 18- and 24-hours post-infection (hpi), while after that the is strongly reduced. Furthermore, CHIKV infection induced the pro-inflammatory cytokine production starting from the first 6 hpi in both monocytes and MDMs, with similar kinetics but different protein levels. In contrast, the kinetics of transcriptional expression of some TLRs were different between both cell types. In addition, IFN-I, 2',5'-oligoadenylate synthetase 1 (OAS1), and double-stranded RNA-activated protein kinase R (PKR) mRNA levels were detected in response to CHIKV infection of monocytes and MDMs, resulting the highest expression levels at 48 hpi. In conclusion, our data provides evidence that CHIKV infection activates the TLR pathways in primary monocytes and MDMs, which play a crucial role in CHIKV pathogenesis and/or host defense, differentially. However, additional studies are required to determine the functional role of TLRs in monocytes and MDMs.
Assuntos
Febre de Chikungunya/imunologia , Citocinas/biossíntese , Macrófagos/metabolismo , Monócitos/metabolismo , Animais , Febre de Chikungunya/virologia , Células Endoteliais/metabolismo , Fibroblastos , Humanos , Interferons/biossíntese , Replicação ViralRESUMO
Most previous studies of interferon-alpha/beta (IFN-α/ß) response antagonism by alphaviruses have focused upon interruption of IFN-α/ß induction and/or receptor signaling cascades. Infection of mice with Venezuelan equine encephalitis alphavirus (VEEV) or Sindbis virus (SINV) induces serum IFN-α/ß, that elicits a systemic antiviral state in uninfected cells successfully controlling SINV but not VEEV replication. Furthermore, VEEV replication is more resistant than that of SINV to a pre-existing antiviral state in vitro. While host macromolecular shutoff is proposed as a major antagonist of IFN-α/ß induction, the underlying mechanisms of alphavirus resistance to a pre-existing antiviral state are not fully defined, nor is the mechanism for the greater resistance of VEEV. Here, we have separated viral transcription and translation shutoff with multiple alphaviruses, identified the viral proteins that induce each activity, and demonstrated that VEEV nonstructural protein 2-induced translation shutoff is likely a critical factor in enhanced antiviral state resistance of this alphavirus.
Assuntos
Resistência à Doença , Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/virologia , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Proteínas não Estruturais Virais/metabolismo , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Encefalomielite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/mortalidade , Cavalos , Humanos , Interferons/biossíntese , Interferons/farmacologia , Camundongos , Mutação , Fenótipo , RNA Viral , Proteínas não Estruturais Virais/genéticaRESUMO
Dengue is the world's most common mosquito-borne viral infection and a leading cause of morbidity throughout the tropics and subtropics. Viruses are known to evade the establishment of an antiviral state by regulating the activation of interferon regulatory factor 3 (IRF3), a critical transcription factor in the alpha/beta interferon induction pathway. Here, we show that dengue virus (DENV) circumvents the induction of the retinoic acid-inducible gene I-like receptor (RLR) pathway during infection by blocking serine 386 phosphorylation and nuclear translocation of IRF3. This effect is associated with the expression of nonstructural 2B/3 protein (NS2B/3) protease in human cells. Using interaction assays, we found that NS2B/3 interacts with the cellular IκB kinase ε (IKKε). Docking computational analysis revealed that in this interaction, NS2B/3 masks the kinase domain of IKKε and potentially affects its functionality. This observation is supported by the DENV-associated inhibition of the kinase activity of IKKε. Our data identify IKKε as a novel target of DENV NS2B/3 protease.
Assuntos
Vírus da Dengue/imunologia , Quinase I-kappa B/antagonistas & inibidores , Evasão da Resposta Imune , Interferons/antagonistas & inibidores , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células Cultivadas , Humanos , Quinase I-kappa B/metabolismo , Interferons/biossíntese , Simulação de Acoplamento Molecular , Mapeamento de Interação de ProteínasRESUMO
BACKGROUND: Infection with dengue viruses (DENV) leads to a spectrum of disease outcomes. The pathophysiology of severe versus non-severe manifestations of DENV infection may be driven by host responses, which could be reflected in the transcriptional profiles of peripheral blood immune cells. METHODOLOGY/PRINCIPAL FINDINGS: We conducted genome-wide microarray analysis of whole blood RNA from 34 DENV-infected children in Nicaragua collected on days 3-6 of illness, with different disease manifestations. Gene expression analysis identified genes that are differentially regulated between clinical subgroups. The most striking transcriptional differences were observed between dengue patients with and without shock, especially in the expression of mitochondrial ribosomal proteins associated with protein biosynthesis. In the dengue hemorrhagic fever patients, one subset of differentially expressed genes encode neutrophil-derived anti-microbial peptides associated with innate immunity. By performing a meta-analysis of our dataset in conjunction with previously published datasets, we confirmed that DENV infection in vivo is associated with large changes to protein and nucleic acid metabolism. Additionally, whereas in vitro infection leads to an increased interferon signature, this was not consistently observed from in vivo patient samples, suggesting that the interferon response in vivo is relatively transient and was no longer observed by days 3-6 of illness. CONCLUSIONS/SIGNIFICANCE: These data highlight important differences between different manifestations of severity during DENV infection as well as identify some commonalities. Compilation of larger datasets in the future across multiple studies, as we have initiated in this report, may well lead to better prediction of disease manifestation via a systems biology approach.
Assuntos
Dengue/genética , Regulação da Expressão Gênica , Adolescente , Criança , Pré-Escolar , Análise por Conglomerados , Dengue/sangue , Dengue/metabolismo , Vírus da Dengue , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Lactente , Interferons/biossíntese , Interferons/genética , Masculino , Nicarágua , Reação em Cadeia da Polimerase , Transdução de SinaisRESUMO
Early experiments have resulted in the establishment of an efficient methodology for the production of a yellow fever vaccine in chicken embryo fibroblasts (CEF) using the 17DD virus strain [Freire, M.S., Mann, G.F., Marchevsky, R.S., Yamamura, A.M., Almeida, L.F., Jabor, A.V., Malachias, J.M., Coutinho, E.S., Galler, R., 2005. Production of yellow fever 17DD vaccine virus in primary culture of chicken embryo fibroblasts: yields, thermo and genetic stability, attenuation and immunogenicity. Vaccine 23, 2501-2512]. To investigate the role of the interferon system in vaccine virus yields, CEF cultures seeded at high and low cell densities and infected with the yellow fever 17DD virus were used. The supernatants of these cultures were tested for the presence of interferon by an assay based on the reduction of cytopathic effect of a challenge virus (Sindbis), for the enzymatic activity of the interferon-induced 2',5'-oligoadenylate synthetase and for the expression of 2',5'-oligoadenylate synthetase mRNA. The presence of interferon and its influence in the replication of yellow fever 17DD virus in CEF cultures was clearly demonstrated.
Assuntos
Fibroblastos/virologia , Interferons/biossíntese , Vacina contra Febre Amarela/biossíntese , Vírus da Febre Amarela/crescimento & desenvolvimento , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Chlorocebus aethiops , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Interferons/genética , Interferons/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sindbis virus/metabolismo , Células Vero , Replicação Viral/fisiologia , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologiaRESUMO
Human proteins are not routinely expressed at high levels in Escherichia coli for, among other reasons, different codon usage. Several purification procedures have been applied to recover recombinant proteins for further biological characterization. However, the vast majority involve costly chromatography procedures. In the present study, both (Hu)IFN(alpha 2b) (human interferon alpha 2b) and (Hu)IFN(alpha 8) were expressed efficiently in E. coli BL21-codonplus-RIL. Subsequently, both recombinant proteins were purified to homogeneity by passive elution from reverse-stained SDS/PAGE gels, a cost-effective purification procedure. After purification, both recovered proteins were biologically active. The (Hu)IFN(alpha 8) subtype induced 1.46-fold more antiviral activity than (Hu)IFN(alpha 2b) using Hep-2 human laryngeal carcinoma cell challenged with Mengo virus.
Assuntos
Antivirais/farmacologia , Interferon-alfa/farmacologia , Interferons/fisiologia , Mengovirus/efeitos dos fármacos , Mengovirus/imunologia , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Escherichia coli , Humanos , Interferon alfa-2 , Interferon-alfa/biossíntese , Interferon-alfa/genética , Interferons/biossíntese , Interferons/genética , Dados de Sequência Molecular , Proteínas RecombinantesRESUMO
The mechanisms of congenital transmission of Chagas disease remain largely unknown. To better understand the role of maternal immunology during pregnancy in congenital Chagas transmission, we studied the cytokine production and the parasitic load in three groups of mothers: infected mothers who transmitted the disease to their babies (M+B+-), infected mothers who did not transmit the disease to their babies (M+B-) and not infected mothers as a control group (M-B-). M+B+ mothers produced less IFNgamma and more IL-10 than the M+B- mothers, and they are not able to produce IL-2. M+B+ mothers showed a higher parasitic load. These results, indicated that the congenital Chagas transmission is associated with an immunological imbalance and a high parasitic load in the M+B+ mothers.
Assuntos
Doença de Chagas/imunologia , Doença de Chagas/transmissão , Citocinas/biossíntese , Transmissão Vertical de Doenças Infecciosas , Complicações Infecciosas na Gravidez/imunologia , Trypanosoma cruzi/fisiologia , Animais , Portador Sadio/imunologia , Doença de Chagas/parasitologia , Citocinas/imunologia , Feminino , Humanos , Imunidade Celular , Interferon gama/biossíntese , Interferons/biossíntese , GravidezRESUMO
The mechanisms of congenital transmission of Chagas disease remain largely unknown. To better understand the role of maternal immunology during pregnancy in congenital Chagas transmission, we studied the cytokine production and the parasitic load in three groups of mothers: infected mothers who transmitted the disease to their babies (M+B+-), infected mothers who did not transmit the disease to their babies (M+B-) and not infected mothers as a control group (M-B-). M+B+ mothers produced less IFNgamma and more IL-10 than the M+B- mothers, and they are not able to produce IL-2. M+B+ mothers showed a higher parasitic load. These results, indicated that the congenital Chagas transmission is associated with an immunological imbalance and a high parasitic load in the M+B+ mothers.
Assuntos
Animais , Feminino , Humanos , Gravidez , Citocinas/biossíntese , Complicações Infecciosas na Gravidez/imunologia , Doença de Chagas/imunologia , Doença de Chagas/transmissão , Transmissão Vertical de Doenças Infecciosas , Trypanosoma cruzi/fisiologia , Citocinas/imunologia , Doença de Chagas/parasitologia , Imunidade Celular , Interferon gama/biossíntese , Interferons/biossíntese , Portador Sadio/imunologiaRESUMO
High-level expression from one particular heterologous gene in Escherichia coli generally requires the optimization of codon usage. Genes encoding for Hepatitis C virus core protein (HCcAg), human interferon alpha2 and 8 subtypes (HUIFNalpha2 and HUIFNalpha8) show a high content of AGA/AGG codons. These are encoded by the product of the dnaY gene in E. coli. The proteins used in this work have a high therapeutic value and were used as models for studying the effects of these rare codons on the efficiency of heterologous gene expression in E. coli. Expression plasmids were constructed to express any of these proteins and the dnaY gene product simultaneously in E. coli. After dnaY gene expression, HCcAg, and HUIFNalpha2 expression levels increased 5 and 3 times, respectively. However, HUIFNalpha8 expression was barely detected either supplying or not the additional dnaY gene product. These results suggest that the high frequency of AGA/AGG codons present in the HCcAg and HUIFNalpha2 genes could be one of the factors limiting its expression in E. coli. Nevertheless, for HUIFNalpha8 it seems that other factors prevail upon the lack of dnaY product. Data presented here for HCcAg and HUIFNalpha2 expressions proved the value of this approach to obtain therapeutic proteins in E. coli.
Assuntos
Antivirais/metabolismo , Escherichia coli/genética , Interferon-alfa/genética , Interferons/genética , Proteínas do Core Viral/genética , Sequência de Bases , Códon , Expressão Gênica , Humanos , Interferon-alfa/biossíntese , Interferons/biossíntese , Plasmídeos , RNA de Transferência/genética , Transformação Bacteriana , Proteínas do Core Viral/biossínteseRESUMO
Humoral and cellular immune responses were analyzed with Fuenzalida-Palacios rabies vaccine associated with pGPL-Mc, polar glycopeptidolipids extracted from Mycobacterium chelonae, aiming at its use as adjuvant. These results were compared to those obtained with BCG, a well-known immunostimulator, under the same conditions. Rabies vaccine plus pGPL-Mc (2.5 mg/kg) induced a significant increase in serum neutralizing activity, in vitro lymphocyte proliferation (spontaneous, specific and mitogen stimulation) and delayed type hypersensibility. In addition, pGPL-Mc, as well as BCG, enhanced the vaccine potency. Our results support further studies to encourage the use of pGPL-Mc as an immunostimulator of veterinary vaccines, before consideration for human vaccines.