No description available.

8 Abstract Human Herpes Virus 6 (HHV-6) consists of two closely related DNA viruses: HHV-6A and HHV-6B. The primoinfection proceeds at an early childhood usually as sixth exanthem disease or without any clinical symptoms. Both HHV-6 viruses are able to integrate to human genome using recombinant mechanisms which is unique compared to other human herpesviruses. The aim of the thesis is to study Ci-HHV-6 in a group of patients with malign disease and in the healthy population. We analysed 812 patients with malign disease and 420 healthy subjects from general population. The Ci-HHV-6 was assessed by real-time PCR, the specific localization of Ci-HHV-6 was determined using fluorescent in situ hybridization (FISH). Using comparative study, we did not identify significant difference between frequency of Ci-HHV-6 in patients with malign disease (1.11%) and healthy subjects (0.95%) (P-value 0.8). Consequently, we proved the heritability of Ci-HHV-6 in affected families. We determined the localization of Ci-HHV-6 to telomeric regions of chromosomes 2 and 18. We studied the production of viral proteins in the subjects with Ci-HHV-6. In this work, we conducted the first epidemiological study of Ci-HHV-6 in the Czech Republic. We also introduced novel methods which contribute to better characterization of this phenomenom.

A epidemia da Síndrome de Imunodeficiência Adquirida (AIDS) fez aumentar a incidência de sarcoma de Kaposi (SK) em todos os países e o SK passou a ser considerado doença definidora de AIDS. Desde a descoberta de seu agente etiológico, o herpesvírus humano tipo 8 (HHV-8), vários estudos vêm sendo realizados com o objetivo de caracterizar subtipos virais presentes em todas as formas de SK: clássica, endêmica, iatrogênica e epidêmica. Os sistemas rotineiramente usados na subtipagem do HHV-8 têm usado o seqüenciamento de um gene que contém regiões hipervariáveis e que codifica uma glicoproteína de membrana viral (ORF K1). O presente trabalho apresenta um sistema de subtipagem alternativo que se baseia na presença de sítios de restrição enzimática em um pequeno segmento do gene ORF K1, região hipervariável 1 (VR1) e que permite discriminar subtipos virais. A análise de 68 seqüências; 50 que pertenciam a 36 pacientes com sarcoma de Kaposi infectados e não infectados pelo HIV-1 de São Paulo e 18 a protótipos dos subtipos A a E, mostraram mapas de restrição enzimática característicos dos principais subtipos virais descritos até o momento. Tomando como base apenas as enzimas disponíveis comercialmente, foram selecionadas cinco que se mostraram úteis para a subtipagem de HHV-8: Taq I, Nsi I, Hinf I, Hae III e Mse I. Os resultados obtidos com a técnica de PCR-RFLP (reação em cadeia de polimerase associada à análise do polimorfismo de fragmentos de restrição enzimática) mostraram que de 48 espécimes brasileiros previamente classificados como sendo dos subtipos A, B e C por seqüenciamento gênico, todos foram corretamente subtipados pela técnica de PCR-RFLP. Três amostras (duas do subtipo A e uma do B) apresentaram mais um sítio de restrição enzimática além dos descritos como sendo os predominantes. Mais recentemente, outras 27 amostras de 18 casos de infecção por HHV-8 foram subtipadas pela PCR-RFLP. Houve detecção de oito isolados do subtipo A, sendo seis de variante predominante, um de variante minoritária conhecida e um de nova variante viral. Dois casos de infecção por HHV-8 do subtipo B e sete do subtipo C também foram identificados. Finalmente, um provável caso de infecção pelo subtipo E foi encontrado em paciente com SK-AIDS disseminado, resistente à quimioterapia. Na avaliação global, houve maior número de casos de infecção por HHV-8 dos subtipos A e C. Concluindo, devido à alta sensibilidade e especificidade, baixo custo, rapidez e facilidade de execução, a técnica de PCR-RFLP pode ser usada em larga escala para estudos de epidemiologia molecular, principalmente em países em desenvolvimento. / AIDS epidemic has increased the incidence rates of Kaposi’ s sarcoma (KS) in all countries, and KS has been considered an AIDS-defining illness. Since the discovery of the human herpesvirus 8 (HHV-8), the etiological agent of KS, several studies have been conducted in order to characterize HHV-8 in all forms of KS: classic, endemic, iatrogenic, and epidemic. The HHV-8 genome presents a hypervariable region termed ORF K1 useful for virus subtyping. The objectives of the present study were to describe an alternative method for subtyping HHV-8, to compare this new method with DNA sequencing, and to use this method for HHV-8 subtyping. After cloning and sequencing a segment of the ORF K1 (VR1) in 50 HHV-8/DNA isolates from 36 Brazilian KS-AIDS patients, we searched for restriction enzymatic sites in this segment of DNA, and compared them with 18 sequences reported in the literature. Then we constructed the enzymatic restriction maps useful for discriminating all HHV-8 subtypes described up to now, and standardized a PCR-RFLP (restriction fragment length polymorphism analysis) using five commercial enzymes: Taq I, Nsi I, Hinf I, Hae III e Mse I. After comparing the results obtained by the two methods, we used PCR-RFLP for HHV-8 subtyping in 27 new HHV-8/DNA isolates. The results obtained by DNA sequencing and PCR-RFLP showed 100% of concordance, and allowed the use of PCR-RFLP for HHV-8 subtyping. Indeed, we disclosed that among KS-AIDS patients from São Paulo, subtypes A and C are more prevalent than subtype B. Although additional restriction sites were detected in some Brazilian HHV-8 isolates, the majority of them belonged to the predominant strains described in the literature. Interestingly, one probable case of HHV-8 subtype E was detected in a patient who presented disseminated KS and resistance to chemotherapy. Because of its high sensitivity, specificity, low cost, and rapid execution, PCR-RFLP could be used on a large scale, mostly in countries with poor resources and where KS is endemic.

A epidemia da Síndrome de Imunodeficiência Adquirida (AIDS) fez aumentar a incidência de sarcoma de Kaposi (SK) em todos os países e o SK passou a ser considerado doença definidora de AIDS. Desde a descoberta de seu agente etiológico, o herpesvírus humano tipo 8 (HHV-8), vários estudos vêm sendo realizados com o objetivo de caracterizar subtipos virais presentes em todas as formas de SK: clássica, endêmica, iatrogênica e epidêmica. Os sistemas rotineiramente usados na subtipagem do HHV-8 têm usado o seqüenciamento de um gene que contém regiões hipervariáveis e que codifica uma glicoproteína de membrana viral (ORF K1). O presente trabalho apresenta um sistema de subtipagem alternativo que se baseia na presença de sítios de restrição enzimática em um pequeno segmento do gene ORF K1, região hipervariável 1 (VR1) e que permite discriminar subtipos virais. A análise de 68 seqüências; 50 que pertenciam a 36 pacientes com sarcoma de Kaposi infectados e não infectados pelo HIV-1 de São Paulo e 18 a protótipos dos subtipos A a E, mostraram mapas de restrição enzimática característicos dos principais subtipos virais descritos até o momento. Tomando como base apenas as enzimas disponíveis comercialmente, foram selecionadas cinco que se mostraram úteis para a subtipagem de HHV-8: Taq I, Nsi I, Hinf I, Hae III e Mse I. Os resultados obtidos com a técnica de PCR-RFLP (reação em cadeia de polimerase associada à análise do polimorfismo de fragmentos de restrição enzimática) mostraram que de 48 espécimes brasileiros previamente classificados como sendo dos subtipos A, B e C por seqüenciamento gênico, todos foram corretamente subtipados pela técnica de PCR-RFLP. Três amostras (duas do subtipo A e uma do B) apresentaram mais um sítio de restrição enzimática além dos descritos como sendo os predominantes. Mais recentemente, outras 27 amostras de 18 casos de infecção por HHV-8 foram subtipadas pela PCR-RFLP. Houve detecção de oito isolados do subtipo A, sendo seis de variante predominante, um de variante minoritária conhecida e um de nova variante viral. Dois casos de infecção por HHV-8 do subtipo B e sete do subtipo C também foram identificados. Finalmente, um provável caso de infecção pelo subtipo E foi encontrado em paciente com SK-AIDS disseminado, resistente à quimioterapia. Na avaliação global, houve maior número de casos de infecção por HHV-8 dos subtipos A e C. Concluindo, devido à alta sensibilidade e especificidade, baixo custo, rapidez e facilidade de execução, a técnica de PCR-RFLP pode ser usada em larga escala para estudos de epidemiologia molecular, principalmente em países em desenvolvimento. / AIDS epidemic has increased the incidence rates of Kaposi’ s sarcoma (KS) in all countries, and KS has been considered an AIDS-defining illness. Since the discovery of the human herpesvirus 8 (HHV-8), the etiological agent of KS, several studies have been conducted in order to characterize HHV-8 in all forms of KS: classic, endemic, iatrogenic, and epidemic. The HHV-8 genome presents a hypervariable region termed ORF K1 useful for virus subtyping. The objectives of the present study were to describe an alternative method for subtyping HHV-8, to compare this new method with DNA sequencing, and to use this method for HHV-8 subtyping. After cloning and sequencing a segment of the ORF K1 (VR1) in 50 HHV-8/DNA isolates from 36 Brazilian KS-AIDS patients, we searched for restriction enzymatic sites in this segment of DNA, and compared them with 18 sequences reported in the literature. Then we constructed the enzymatic restriction maps useful for discriminating all HHV-8 subtypes described up to now, and standardized a PCR-RFLP (restriction fragment length polymorphism analysis) using five commercial enzymes: Taq I, Nsi I, Hinf I, Hae III e Mse I. After comparing the results obtained by the two methods, we used PCR-RFLP for HHV-8 subtyping in 27 new HHV-8/DNA isolates. The results obtained by DNA sequencing and PCR-RFLP showed 100% of concordance, and allowed the use of PCR-RFLP for HHV-8 subtyping. Indeed, we disclosed that among KS-AIDS patients from São Paulo, subtypes A and C are more prevalent than subtype B. Although additional restriction sites were detected in some Brazilian HHV-8 isolates, the majority of them belonged to the predominant strains described in the literature. Interestingly, one probable case of HHV-8 subtype E was detected in a patient who presented disseminated KS and resistance to chemotherapy. Because of its high sensitivity, specificity, low cost, and rapid execution, PCR-RFLP could be used on a large scale, mostly in countries with poor resources and where KS is endemic.

No description available.

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Human herpesvirus type 6 (HHV-6) has been well described as an agent of meningoencephalitis in post-haematopoietic stem cell transplantation patients, but is a rare cause of meningoencephalitis in immunocompetent adults. We report an immunocompetent adult with HHV-6-associated meningoencephalitis. The patient was an elderly diabetic man who presented with fever and confusion, with cerebrospinal fluid (CSF) pleocytosis. HHV-6 DNA was amplified from CSF by polymerase chain reaction. In our review of the medical literature we examine clinical presentations, laboratory findings, neuroimaging studies, treatments and clinical outcomes in immunocompetent patients with HHV-6 meningoencephalitis.

L’herpèsvirus humain (HHV) 6 est un betaherpèsvirus largement répandu, associé à plusieurs maladies neuroinflammatoires, telles que des encéphalites ou la sclérose en plaques (SEP). Cependant, les mécanismes impliqués dans la neuropathologie induite par les deux espèces d’HHV-6, HHV-6A et HHV-B, sont peu connus. De plus, l’absence de modèle d’infection chez le petit animal a ralenti l’étude de la pathogénèse virale. Dans ce contexte, nous avons développé un modèle d’infection par HHV-6 chez des souris transgéniques, qui expriment la protéine CD46 humaine, identifiée comme récepteur cellulaire pour HHV-6. Nous avons pu démontrer une persistance de l’ADN viral d’HHV-6A, mais pas d’HHV-6B, dans le cerveau de souris transgéniques pendant plusieurs mois. De plus nos résultats montrent qu’HHV-6A induit la sécrétion de chimiokines pro-inflammatoires par les cellules neurales murines et provoque l’infiltration de cellules immunitaires dans le cerveau de souris infectées. Enfin, HHV-6A, mais pas HHV-6B, pourrait induire des réponses cellulaires chez les cellules murines via le récepteur de l’immunité innée TLR9 (toll-like receptor 9). En collaboration avec une équipe de Grenoble, nous avons ensuite montré que l’infection par HHV-6A induit l’expression de rétrovirus endogènes humains (HERV) dans des cellules mononuclées et des lignées neurales humaines. Ces HERV, en particulier leurs protéines d’enveloppe qui présentent des propriétés pro-inflammatoires, sont associés à diverses maladies autoimmunes dont la SEP. HHV-6A pourrait donc participer au développement de pathologies inflammatoires via l’induction de ces HERV. L’ensemble de ces travaux supporte ainsi l’existence d’un lien entre l’infection par HHV-6A et la neuroinflammation, et apporte de nouvelles pistes quant aux mécanismes potentiellement impliqués. / Human herpesvirus (HHV) 6 is a widely spread betaherpesvirus, which has been associated to several neuroinflammatory diseases, such as encephalitis or multiple sclerosis (MS). However, the mechanisms explaining the neuropathology induced by the two species of HHV-6, HHV-6A and HHV-6B, remain to be elucidated. Moreover, the lack of small animal model for HHV-6 infection has considerably hampered the study of viral pathogenesis. In this context, we have generated several lines of mice expressing the human CD46 protein, identified as a cellular receptor for HHV-6, and characterized the infection. We demonstrated that DNA of HHV-6A, but not HHV-6B, can persist in the brain of CD46 transgenic mice for several months after intracranial injection. Moreover our results show that HHV-6A induces chemokine secretion by in vitro cultured murine brain cells and provokes leucocyte infiltration in the brain of infected mice. Finally, HHV-6A, but not HHV-6B, could activate cellular responses in murine cells through binding to toll-like receptor 9. In collaboration with the team of P. Marche in Grenoble, we then showed that HHV-6A and HHV-6B infection induce the expression of envelope genes from human endogenous retrovirus W (HERV-W) in human blood mononucleated cells and human neural cell lines. Envelope proteins of HERV-W are known to exhibit strong pro-inflammatory properties and were associated to various autoimmune diseases, including multiple sclerosis. HHV-6A and HHV-6B could therefore participate in the development of inflammatory disorders via the activation of these HERV genes. Altogether this work supports the hypothesis of a link between HHV-6 infection neuroinflammation and opens new perspectives in the study of the mechanisms potentially involved.