RESUMO
BACKGROUND: Plasmodium ovale is one of the five malaria species infecting humans. Recent data have shown that the name of this neglected species masks two distinct genotypes also called curtisi and wallikeri. Some authors show that these species could be sympatric. These two subspecies are not differentiated by microscopy techniques and malaria rapid diagnostic tests. This diagnostic defect is the result of low parasitaemia, antigenic polymorphism and absence of antibodies performance and requires the use of sequencing techniques. An accurate and easy discrimination detection method is necessary. METHODS: A new molecular assay was developed to easily identify the two genotypes of P. ovale. This tool allowed the study of 90 blood samples containing P. ovale, confirmed by molecular biology techniques, which were obtained from patients with imported malaria. RESULTS: The new marker was validated on well genotyped samples. The genotype of 90 P. ovale samples mainly imported from the Ivory Coast and the Comoros Islands was easily and quickly realized. The distribution of the two subspecies was described with a significant number of samples and showed that the two genotypes were present in the studied countries. CONCLUSION: This work confirms the presence of the two species in the same country for the first time, in the Ivory Coast and the Comoros Islands. A better genotyping of P. ovale types may improve a better characterization of the clinical pathophysiology for each.
Assuntos
Malária/parasitologia , Parasitologia/métodos , Plasmodium ovale/classificação , Plasmodium ovale/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Comores , Côte d'Ivoire , Humanos , Plasmodium ovale/isolamento & purificaçãoRESUMO
BACKGROUND: Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites. METHODS: For this purpose, salivary gland proteins 6 (SG6) and 5'nucleotidases (5'nuc) from An. gambiae (gSG6 and g-5'nuc) and An. funestus (fSG6 and f-5'nuc) were selected and produced in recombinant form. The specificity of the IgG response against these salivary proteins was tested using an ELISA with sera from individuals living in three Senegalese villages (NDiop, n = 50; Dielmo, n = 38; and Diama, n = 46) that had been exposed to distinct densities and proportions of the Anopheles species. Individuals who had not been exposed to these tropical mosquitoes were used as controls (Marseille, n = 45). RESULTS: The IgG responses against SG6 recombinant proteins from these two Anopheles species and against g-5'nucleotidase from An. gambiae, were significantly higher in Senegalese individuals compared with controls who were not exposed to specific Anopheles species. Conversely, an association was observed between the level of An. funestus exposure and the serological immune response levels against the f-5'nucleotidase protein. CONCLUSION: This study revealed an Anopheles salivary antigenic protein that could be considered to be a promising antigenic marker to distinguish malaria vector exposure at the species level. The epidemiological interest of such species-specific antigenic markers is discussed.
Assuntos
Anopheles/imunologia , Antígenos/imunologia , Mordeduras e Picadas de Insetos/imunologia , Mordeduras e Picadas de Insetos/parasitologia , Proteínas de Insetos/imunologia , Malária/imunologia , Malária/transmissão , Proteínas e Peptídeos Salivares/imunologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/imunologia , Adulto , Sequência de Aminoácidos , Animais , Anopheles/genética , Anopheles/parasitologia , Antígenos/genética , Biomarcadores , Estudos de Casos e Controles , Reações Cruzadas , Feminino , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunoglobulina G/sangue , Proteínas de Insetos/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas e Peptídeos Salivares/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We developed a new Neisseria meningitidis multiplex PCR to determine six serogroups, including X-specific primers, and to allow direct W135/Y discrimination. This assay offers a simple and low-cost method for serogrouping N. meningitidis from cerebrospinal fluid that could be useful in Africa.
Assuntos
Meningite Meningocócica/diagnóstico , Meningite Meningocócica/microbiologia , Neisseria meningitidis/classificação , Neisseria meningitidis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , África , Técnicas de Tipagem Bacteriana/métodos , Líquido Cefalorraquidiano/microbiologia , Primers do DNA/genética , DNA Bacteriano/genética , Genes Bacterianos , Genótipo , Humanos , Neisseria meningitidis/genética , Sorotipagem/métodosRESUMO
There are very few previous reports of expression of native full-length maedi visna virus (MVV) Env gp150 protein in the literature. Therefore the use of different plasmid and viral expression vectors to obtain full-length gp150 was investigated. A mammalian expression plasmid, pN3-Env, was constructed containing the MVV env gene encoding the precursor protein gp150 Env. The functionality of the recombinant plasmid was tested for expression in HEK293 cells. A recombinant modified vaccinia Ankara virus, MVA-Env, with expression detected in avian cells was also made. The expression of the MVV gp150 Env precursor protein was shown for the first time upon transfection of the eukaryotic HEK293 cells by the pN3-Env plasmid DNA as demonstrated by Western blot analysis. These plasmid or viral expression vectors are of potential use in MVV vaccines.
Assuntos
Produtos do Gene env/biossíntese , Genes env , Vetores Genéticos , Precursores de Proteínas/biossíntese , Vírus Visna-Maedi/genética , Animais , Linhagem Celular , Produtos do Gene env/genética , Humanos , Plasmídeos , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Vacinas de DNA , Vacinas Virais , Visna/virologia , Vírus Visna-Maedi/imunologiaRESUMO
The recent increase of West Nile neuroinvasive disease (WNND) incidence in southern Europe made this change in epidemiology a major concern for public health. The lack of a vaccine or specific treatment against human WNV infection imposes the need to discover biological markers associated with disease severity for diagnostic and/or therapeutic purposes. Recently, using a brain proteomic study from a mouse model of West Nile virus (WNV) infection with neuronal involvement, we reported the kinetic up-regulation of high-mobility group box-1 (HMGB1) and peroxiredoxin-6 (PRDX6), before and after onset of clinical symptoms, respectively. To evaluate whether these proteins could be useful biomarkers for the distinction of WNV disease severity in humans, HMBG1 and PRDX6 concentrations in serum from WNV-infected patients (n=49) diagnosed for either WNF (n=22) or WNND (n=27), were measured by ELISA and compared to concentrations in serum from uninfected healthy individuals (n=30). HMGB1 concentrations were significantly higher in WNND than in either WNF patients (p<0.05) or healthy individuals (p<0.001). In contrast, PRDX6 levels were significantly higher in healthy individuals compared with WNV-infected patients (p<0.001), regardless of clinical symptoms. The present study highlighted the deregulation of HMGB1 and PRDX6 serum level in WNV-infected patients and provided HMGB1 as candidate biomarker distinguishing disease severity. Further investigation in larger cohorts could confirm HMGB1 and PRDX6 as auxiliary biomarkers in confirmed cases of WNV infection and validate the usefulness of measuring HMBG1 for prediction of detrimental clinical outcome.
Assuntos
Biomarcadores/sangue , Proteína HMGB1/sangue , Índice de Gravidade de Doença , Febre do Nilo Ocidental/diagnóstico , Febre do Nilo Ocidental/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Peroxirredoxina VI/sangue , Prognóstico , Adulto JovemRESUMO
During the last decade, the epidemiology of WNV in humans has changed in the southern regions of Europe, with high incidence of West Nile fever (WNF) cases, but also of West Nile neuroinvasive disease (WNND). The lack of human vaccine or specific treatment against WNV infection imparts a pressing need to characterize indicators associated with neurological involvement. By its intimacy with central nervous system (CNS) structures, modifications in the cerebrospinal fluid (CSF) composition could accurately reflect CNS pathological process. Until now, few studies investigated the association between imbalance of CSF elements and severity of WNV infection. The aim of the present study was to apply the iTRAQ technology in order to identify the CSF proteins whose abundances are modified in patients with WNND. Forty-seven proteins were found modified in the CSF of WNND patients as compared to control groups, and most of them are reported for the first time in the context of WNND. On the basis of their known biological functions, several of these proteins were associated with inflammatory response. Among them, Defensin-1 alpha (DEFA1), a protein reported with anti-viral effects, presented the highest increasing fold-change (FC>12). The augmentation of DEFA1 abundance in patients with WNND was confirmed at the CSF, but also in serum, compared to the control individual groups. Furthermore, the DEFA1 serum level was significantly elevated in WNND patients compared to subjects diagnosed for WNF. The present study provided the first insight into the potential CSF biomarkers associated with WNV neuroinvasion. Further investigation in larger cohorts with kinetic sampling could determine the usefulness of measuring DEFA1 as diagnostic or prognostic biomarker of detrimental WNND evolution.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Doenças do Sistema Nervoso Central/líquido cefalorraquidiano , Febre do Nilo Ocidental/líquido cefalorraquidiano , Doenças do Sistema Nervoso Central/patologia , Ensaio de Imunoadsorção Enzimática , Humanos , Febre do Nilo Ocidental/patologiaRESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus responsible for hemorrhagic manifestations and multiple organ failure, with a high mortality rate. In infected humans, damage to endothelial cells and vascular leakage may be a direct result of virus infection or an immune response-mediated indirect effect. The main target cells are mononuclear phagocytes, endothelial cells and hepatocytes; the liver being a key target for the virus, which was described as susceptible to interferon host response and to induce apoptosis. To better understand the early liver cell alterations due to virus infection, the protein profile of in vitro CCHFV-infected HepG2 cells was analyzed using two quantitative proteomic approaches, 2D-DIGE and iTRAQ. A set of 243 differentially expressed proteins was identified. Bioinformatics analysis (Ingenuity Pathways Analysis) revealed multiple host cell pathways and functions altered after CCHFV infection, with notably 106 proteins related to cell death, including 79 associated with apoptosis. Different protein networks emerged with associated pathways involved in inflammation, oxidative stress and apoptosis, ubiquitination/sumoylation, regulation of the nucleo-cytoplasmic transport, and virus entry. Collectively, this study revealed host liver protein abundances that were modified at the early stages of CCHFV infection, offering an unparalleled opportunity of the description of the potential pathogenesis processes and of possible targets for antiviral research.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/metabolismo , Febre Hemorrágica da Crimeia/virologia , Hepatócitos/virologia , Animais , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/genética , Hepatócitos/química , Hepatócitos/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , ProteômicaRESUMO
Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV infection in the nervous system, a kinetic analysis on the host proteome modifications in the brain of CHIKV-infected mice sampled before and after the onset of clinical symptoms was performed. The combination of 2D-DIGE and iTRAQ proteomic approaches, followed by mass spectrometry protein identification revealed 177 significantly differentially expressed proteins. This kinetic analysis revealed a dramatic down-regulation of proteins before the appearance of the clinical symptoms followed by the increased expression of most of these proteins in the acute symptomatic phase. Bioinformatic analyses of the protein datasets enabled the identification of the major biological processes that were altered during the time course of CHIKV infection, such as integrin signaling and cytoskeleton dynamics, endosome machinery and receptor recycling related to virus transport and synapse function, regulation of gene expression, and the ubiquitin-proteasome pathway. These results reveal the putative mechanisms associated with severe CHIKV infection-mediated neurological disease and highlight the potential markers or targets that can be used to develop diagnostic and/or antiviral tools.
Assuntos
Encéfalo/metabolismo , Febre de Chikungunya/metabolismo , Vírus Chikungunya , Proteoma , Proteômica , Animais , Antígenos Virais/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Expressão Gênica , Redes Reguladoras de Genes , Cinética , Camundongos , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
BACKGROUND: The recent West Nile virus (WNV) outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-)emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical symptoms. METHODOLOGY/PRINCIPAL FINDINGS: To this end, 2D-DIGE and gel-free iTRAQ labeling approaches were combined, followed by protein identification by mass spectrometry. Using these quantitative proteomic approaches, a set of 148 proteins with modified abundance was identified. The bioinformatics analysis (Ingenuity Pathway Analysis) of each protein dataset originating from the different time-point comparisons revealed that four major functions were altered during the course of WNV-infection in mouse brain tissue: i) modification of cytoskeleton maintenance associated with virus circulation; ii) deregulation of the protein ubiquitination pathway; iii) modulation of the inflammatory response; and iv) alteration of neurological development and neuronal cell death. The differential regulation of selected host protein candidates as being representative of these biological processes were validated by western blotting using an original fluorescence-based method. CONCLUSION/SIGNIFICANCE: This study provides novel insights into the in vivo kinetic host reactions against WNV infection and the pathophysiologic processes involved, according to clinical symptoms. This work offers useful clues for anti-viral research and further evaluation of early biomarkers for the diagnosis and prevention of severe neurological disease caused by WNV.
Assuntos
Redes e Vias Metabólicas/fisiologia , Doenças dos Roedores/metabolismo , Febre do Nilo Ocidental/metabolismo , Animais , Encéfalo/virologia , Chlorocebus aethiops , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Doenças dos Roedores/imunologia , Doenças dos Roedores/patologia , Índice de Gravidade de Doença , Eletroforese em Gel Diferencial Bidimensional , Células Vero , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/patologia , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/isolamento & purificação , Vírus do Nilo Ocidental/fisiologiaRESUMO
The aim of this study was the development of gag and pol dual labelled probe real-time PCR and RT PCR assays to quantify the proviral load and the transcripts of the British Visna/maedi virus EV1 strain. Primers and probes were chosen based on the consensus sequences of gag and pol clones representative of EV1 genetic variants. Both PCRs had a detection limit of 3 copies of target gene, with a linearity over 6 orders of magnitude. The performances of the two PCRs in vivo were evaluated and compared on a panel of DNAs extracted from blood of sheep infected experimentally with EV1. The pol assay detected in most cases lower numbers of viral molecules than gag assay, yielding some false negative results. The gag real-time RT PCR had a detection limit of 100 RNA molecules with a linearity over 5 orders of magnitude. This did not result in a lower performance of the RT PCR compared to the PCR in cells permissive for virus replication, which contain higher numbers of viral transcripts than proviral genomes. The real-time assays developed in this study, particularly the gag assay, provide a sensitive tool which can be used to quantify the viral load in experimental infections.