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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.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Human Herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are ubiquitous betaherpesviruses. Both viruses are associated with a variety of adult disorders including neurological disorder, such as multiple sclerosis and chronic fatigue syndrome. HHV-6 viruses are capable of establishing latency by integration into the telomeres of the host chromosome and are transmitted in a Mendelian manner in approximately one percent of the population. To date little is known about the immunological and neurological consequences of HHV-6 inheritance. This study focused on a unique population of individuals that inherited HHV-6 and present with chronic fatigue-like symptoms, including hypersomnia, generalized fatigue, headache, and short term and long term memory impairment. The central hypothesis of this study was that active replication of HHV-6 correlates with patient symptoms. To address this aim we first looked at the reactivation of integrated HHV-6 in vitro by inducing viral replication with epigenetic modifiers trichostatin A (TSA), valproic acid, sodium butyrate, and carbamazepine, and found TSA to be an effective method of inducing reactivation of HHV-6 from its integrated form. Additionally, a reactivated HHV-6A virus isolated from a patient with inherited HHV-6 was fully sequenced and the nucleotide and amino acid sequence was compared to that of fully sequenced HHV-6 laboratory strains, as well as the inherited virus. The reactivated virus was found to be very similar to the HHV-6A GS strain; however, there was some divergence at the right end of the viral genome and regions of the genome that do not contain herpesvirus core genes. Interestingly, the sequenced reactivated virus was found to differ from the HHV-6 virus which was inherited. Finally, HHV-6 replication was assessed by performing reverse transcriptase PCR assay for the viral glycoprotein U100 in patients receiving antiviral treatment. Results indicated that short term antiviral treatment was insufficient to abrogate viral replication, while treatment of six weeks or longer eliminated viral mRNA in patient blood samples. Furthermore, sequencing of the viral mRNA and inherited viral DNA indicate that the source of the mRNA detected in patient blood samples was an exogenously acquired HHV-6 virus, as the U100 glycoprotein sequences were not identical. Together these studies indicate that although HHV-6 can be reactivated from its integrated form, individuals in this unique population harbored an exogenous HHV-6 virus, in addition to the inherited virus; we termed this condition inherited herpesvirus syndrome. The fact that these individuals are able to acquire exogenous HHV-6 viruses suggest that there may be some level of immune tolerance or immune dysfunction; we suggest that further studies focus on uncovering the immune response to HHV-6 in individuals with an inherited form of the virus.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22359号 / 医博第4600号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長尾 美紀, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

While the latent genome of most Herpesviruses persists as a nuclear circular episome, previous research has suggested that Human Herpesvirus 6 (HHV-6) may integrate into host cell chromosomes, and be vertically transmitted in the germ-line. Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DRR) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DRL), we established a hypothesis that during latency, the HHV-6A and HHV-6B genome integrates into the telomeres of human chromosomes through homologous recombination with the n(TTAGGG) viral repeats, and the integrated virus can be induced to lytic replication. We sought, first, to definitively illustrate the in vitro and in vivo integration of HHV-6A and HHV-6B. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6A and Molt3 cell line by HHV-6B, the virus integrated into telomere of chromosomes. Next, peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per ml of blood. FISH confirmed that HHV-6 DNA co-localized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, while the integration site was identical among members of the same family. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome through the germ-line [inherited HHV-6 (iHHV-6)]. Amplification and sequencing of the HHV-6A and more recently HHV-6B viral-chromosome junction identified DRR integrated into the telomere directly adjacent to the subtelomere of the chromosome. After mapping the DRR of iHHV-6, we subsequently focused on determining if the DRL was present in the integrated genome and whether the remaining telomere sequence of the chromosome was extended beyond the DRL. Southern hybridization of PCR amplified HHV-6 integrated cell lines and iHHV-6 patients PBMCs indicate the presence of DRL within the integrated viral genome. Therefore, the genomic structure of the iHHV-6 is as follows: chromosome-subtelomere-(TTAGGG)5-41-DRR-U-DRL-(TTAGGG)n. During latent integration, no circular episomes were detected even by PCR. However, trichostatin-A treatment of PBMCs and in vitro integrated HEK-293 cells induced the reactivation of iHHV-6 from its latent integrated state. We demonstrated the induction of integrated iHHV-6 with trichostatin-A lead to the excision of the integrated genome and generation of the U-DR-U junction which signifies circularization and/or concatemer formation of the viral genome through rolling-circle replication. Taken together, the data suggests that HHV-6A and HHV-6B are unique among human herpesviruses: they specifically and efficiently integrate into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

Herpesviruses have co-speciated with several vertebrate and invertebrate animals throughout the history of evolution. In the immunocompetent human host, primary infection is usually benign, whereafter the virus is brought into life-long latency. Viral reactivation can however cause severe disease in immunocompromised, and rarely also in immunocompetent, patients. The overall aim of this thesis was to study the immunologic effects of cytomegalovirus (CMV) and herpes simplex type 1 (HSV-1) infection in neurocognitive disorders. CMV is known to promote T-cell differentiation towards a more effector-oriented phenotype, similar to what is seen in the elderly. We have addressed the frequency of CMV-specific CD8+ T-cells in Alzheimer's disease (AD). Furthermore, we have investigated whether AD patients present with a different CMV-specific immune profile, overall CD8 phenotype or inflammatory cytokine response to anti-CD3/CD28 beads, CMV pp65 and amyloid beta. Subjects with AD presented with a lower proportion of CMV-specific CD8+ T-cells compared to non-demented (ND) controls, but no differences in overall CD8 differentiation were seen. Overall, AD subjects presented with a more pro-inflammatory peripheral blood mononuclear cell (PBMC) phenotype. When PBMCs were challenged with CD3/CD28-stimulation, CMV seropositive AD subjects presented with more IFN-γ release than both CMV seronegative AD subjects and CMV seropositive ND controls. For effective screening of humoral herpesvirus immunity, both in research and in clinical practice, efficient immunoassays are needed. We have addressed the methodology of multiplex herpesvirus immunoassays and related bioinformatics and investigated antibody levels in AD patients and ND controls. Subjects with AD presented with lower levels of human herpesvirus 6 (HHV-6) IgG. However, there was no difference in HHV-6 DNA levels in PBMCs between the groups. Herpes simplex encephalitis (HSE) is a devastating disease, where antiviral treatment has greatly decreased mortality but not eliminated the associated long-term neurocognitive morbidity. We have investigated the correlation between N-Methyl-D-Aspartate Receptor (NMDAR) autoimmunity and recovery of neurocognitive functions after HSE. Approximately one quarter of all HSE cases developed NMDAR autoantibodies within 3 months after onset of disease. Antibody development was associated with an impaired neurocognitive recovery during the two year follow-up and could become an important therapy guiding factor in the future.