La leucémie T de l’adulte (ATL), caractérisée par une prolifération dérégulée de lymphocytesT CD4+ activés, se développe chez des individus infectés par le virus T-lymphotropehumain (HTLV-1). Une période asymptomatique de plusieurs décennies sépare l’infection del’apparition des symptômes cliniques de l’ATL, rendant complexe la compréhension des étapesinitiales de la leucémogenèse. Le modèle de la souris humanisée dans laquelle est reconstituéun système hémato-lymphoïde humain est pertinent pour étudier ces étapes, depuis l’infectionpar HTLV-1 jusqu’à l’apparition de la lymphoprolifération. Grâce à ce modèle, mes travaux dethèse ont aidé à mieux comprendre deux mécanismes importants. D’abord, le rôle du chimiotactisme dans le contact cellule infectée-cellule non infectée in vivo. En inhibant la sécrétion de leukotriène B4, un chimioattractant, dans des souris humanisées et infectées, la charge proviraleest plus faible que celle des souris témoins et la prolifération des CD4+ activés est égalementréduite, soulignant le rôle du leukotriène B4 dans la primo-infection. Ensuite, j’ai étudié l’implicationdes protéines PDZ dans le processus leucémogène in vivo. En effet, certaines de cesprotéines, dont Scribble, interagissent avec le PBM (PDZ-domain Binding Motif) de la protéinevirale Tax. Des souris humanisées ont été infectées avec un provirus soit sauvage soit muté auniveau du PBM et j’ai montré, dès 5 semaines après infection, que l’interaction de ce domaineavec des protéines PDZ augmente la prolifération des CD4+ activés et perturbe l’expression degènes impliqués dans la prolifération, l’organisation du cytosquelette et des voies de l’apoptose.Ces résultats attestent du rôle du PBM de Tax dans le soutien de la lymphoprolifération in vivo. / Human T-Cell Leukemia Virus 1 (HTLV-1) is the causative agent of Adult T-cell Leukemia(ATL) characterized by a deregulated proliferation of activated CD4+ T cells. An asymptomaticperiod of several decades separates the infection and the onset of the leukemia, complicating thestudy of the initial leukemogenic steps. Humanized mouse models are relevant to study thosesteps as these mice harbor human hemato-lymphoid cells that can be infected by HTLV-1.I used this animal model to better characterized two biological mecanisms during my thesis.First, the role of chemotaxis in infected-non infected cell contact in vivo. Inhibiting the secretionof leukotriene B4, a potent chemoattractant, in HTLV-1-infected humanized mice reduces theproviral load and the proliferation of activated CD4+ T cells. Those results establish the importanceof leukotriene B4 in HTLV-1 primo-infection. Next, I focused on the importance of thePDZ proteins in the leukemogenic process in vivo. The PDZ-domain Binding Motif (PBM) ofthe Tax viral protein interacts with several cellular PDZ proteins including Scribble. I infectedhumanized mice either with a wild-type or a PBM-mutated provirus of HTLV-1. I showed thatinteraction of the Tax PBM with PDZ proteins enhances activated CD4+ T cells proliferationfrom 5 weeks after infection and disrupts expression of genes implicated in cell proliferation,apoptotic processes and cytoskeleton organization. This study indicated that the PBM of Taxis important for sustaining the lymphoproliferation in vivo.

Le génome des rétrovirus existe sous deux formes différentes : sous forme d'ARN simple brin, qui est traduit ou encapsidé, ou sous forme d'ADN double brin intégré dans le génome de la cellule hôte infectée. Cette dernière forme, l'ADN proviral, est indispensable à la production de tous les ARNm viraux nécessaires à la synthèse des protéines virales, qui en retour agissent sur la région promotrice située au niveau du LTR 5'. Cependant, l'ADN proviral possède un second LTR à son extrémité 3', capable de réguler une transcription antisens, orientée dans la direction opposée à celle contrôlée par le LTR 5'. L'ADN proviral a donc deux brins codants, ce qui offre au virus un plus grand potentiel de synthèse protéique. Dans le cas du Virus de l'immunodéficience Humaine de type 1 (VIH-1), la transcription antisens permet la production d'une protéine, appelée ASP (Antisense Protein). Dans ce manuscrit, nous démontrons que cette activité transcriptionnelle antisens s'exprime préférentiellement dans les cellules d'origine monocytaire, en particulier les cellules dendritiques ; une localisation membranaire de la protéine ASP a par ailleurs été mise en évidence dans ce type cellulaire. Nos résultats suggèrent également que la transcription antisens du VIH-1 est indépendante de la protéine Tat, et que par ailleurs les deux types de transcriptions ne sont pas exprimés simultanément au sein d'une même cellule. En outre, nos données soulignent que la séquence codante de la protéine ASP est très fortement conservée parmi les différents isolats viraux. Sur la base de l'ensemble de ces résultats, notre hypothèse est que la protéine ASP du VIH-1 possède des fonctions cruciales dans le cycle réplicatif des rétrovirus, indépendantes de la production virale. / Genome of retroviruses exists in two different forms: as single-stranded RNA that is translated or packaged, or as double-stranded DNA integrated into the genome of the infected host cell. The latter form, the proviral DNA, is essential for the production of all viral mRNAs required for the synthesis of viral proteins, which in turn act on the promoter region located at the 5 '-LTR. However, the proviral DNA has a second LTR at its 3 '-end, capable of regulating antisense transcription oriented in the opposite direction to that controlled by the 5'-LTR. The proviral DNA has then two coding strands, which gives the virus a greater potential for protein synthesis. In the case of the Human Immunodeficiency Virus type 1 (HIV-1), antisense transcription allows the production of a protein called ASP (Antisense Protein). In this manuscript, we demonstrate that this antisense transcriptional activity is preferentially expressed in cells of the monocyte lineage, in particular dendritic cells; a membrane localization of the ASP protein was also observed in this cell type. Our results also suggest that the antisense transcription of HIV-1 is Tat-independent, and what's more that the two types of transcription are not expressed simultaneously within the same cell. In addition, our data highlight that the ASP protein coding sequence is highly conserved among different viral isolates. Based on these results, our hypothesis is that the ASP protein of HIV-1 has critical functions in the replicative cycle of retroviruses, distinct from viral production.

Le rétrovirus HTLV-1 (Human T-cell Leukemia virus type 1) est l'agent étiologique de la leucémie T de l'adulte (ATLL) et de maladies inflammatoires. Il infecte principalement les lymphocytes T-CD4+ et T-CD8+, et se réplique essentiellement via l'expansion clonale de sa cellule hôte selon deux mécanismes distincts : HTLV-1 induit une résistance à l'apoptose des cellules T-CD8+ alors qu'il favorise la prolifération des cellules T-CD4+. Au stade chronique de l'infection, en comparaison à leur contreparties T-CD8+, les cellules T-CD4+ infectées non transformées présentent des caractéristiques pré-leucémiques telles que des anormalités génomiques et des défauts d'activation de la télomérase, ce qui explique vraisemblablement pourquoi les cellules d'ATLL sont régulièrement de phénotype T-CD4+. L'infection par HTLV-1 s'accompagne de reprogrammations drastiques du transcriptome cellulaire. Parmi elles, les modifications d'expression de micro-ARN (miARN). Les miARN sont de petits ARN non-codants qui contrôlent négativement la traduction des ARNm, et qui ont été récemment mis en évidence dans les cellules transformées par le virus, et semblent participer au maintien du phénotype tumoral. Ces données posent la question de l'origine de ces perturbations et de leurs implications dans les processus d'expansion clonale et d'initiation de la transformation des cellules infectées. Pour adresser cette question, nous avons réalisé une étude intégrée de l'expression des miARN et des ARNm de cellules T, issues de patients infectés sans malignité / Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia / lymphoma (ATLL) hat regularly occurs after a prolonged period of viral latency. In vivo, HTLV-1 replication relies on the clonal expansion of its host CD4+ and CD8+ T-cells, yet the virus causes adult T-cell leukemia / lymphoma (ATLL) that is regularly of the CD4+ phenotype. Infected cells express Tax and HBZ viral oncoproteins. Tax is mainly expressed in untransformed cells, where it promotes cell proliferation, genetic instability, and miRNA dysregulation, whereas HBZ is expressed in both untransformed and malignant T-cells where it contributes to promote cell proliferation and to silence virus expression. Here, we show that an HBZ / miRNA axis promotes cell proliferation and genetic instability. MicroRNAs (miRNAs) are evolutionarily conserved, small (~21 nucleotides), noncoding RNAs that are encoded within the genomes of almost all eukaryotes from plants to mammals. In general, miRNAs, especially in animals, post-transcriptionally regulate protein synthesis by base pairing to partially complementary sequences in the 3’ untranslated regions (UTRs) of target mRNAs. Furthermore, whereas human lymphocyte subsets are known to possess specific miRNA signatures involved in T-cell differentiation and activation, little is known about the role of miRNA dysregulation in the clonal expansion of untransformed, infected CD4+ and CD8+ T-cells in vivo, including its role, if any, in viral persistence, inflammation, genetic instability, and early leukemogenesis. To our knowledge, no study to date has assessed the effects of HBZ on the biogenesis and activity of miRNAs. In order to assess the effect of HTLV-1 infection on the miRNA expression profiles of host cells in vivo, we performed an integrated analysis of miRNA- and mRNA-expression profiles of cloned CD4+ and CD8+ T-cells derived from infected individuals without malignancy

Le virus HTLV-1 (Human T-cell Leukemia Virus Type 1) est l’agent étiologique de la Leucémie T de l’adulte (ATL) qui est caractérisée par la prolifération de cellules T CD4+ activées. L’absence de modèle animal fiable reproduisant la leucémogénèse associée à l’infection a ralenti la compréhension des étapes précoces du processus leucémogène et le développement de stratégies thérapeutiques efficaces. Récemment l’amélioration des modèles de souris humanisées a permis la reconstitution d’un système immunitaire humain dans des souris. L’injection de cellules souches hématopoïétiques purifiées à partir de sang de cordon humain dans des souris nouveau-nées de la lignée Rag2-/-γc-/- conduit à la formation de novo de cellules dendritiques, B et T humaines. Ces dernières étant la cible de l’infection par HTLV-1, nous avons infecté des souris humanisées avec des cellules productrices de HTLV-1. Cette inoculation conduit à l’infection stable des cellules humaines dans la souris humanisée et la formation de lymphome ou de leucémie à cellules T humaines activées. Cette infection altère le développement des cellules T dans le thymus conduisant à un phénotype plus mature des thymocytes. Ce modèle animal reproduisant l’infection et la pathogénèse associée nous a permis de suivre l’évolution de la clonalité du virus au sein des différents organes lymphoïdes. Basées sur ces observations, des tests préliminaires ont permis d’étudier une nouvelle approche thérapeutique potentiellement applicable en clinique humaine. Ce travail nous a également permis d’affiner le protocole conduisant à l’humanisation des souris afin d’obtenir une meilleure reconstitution humaine dans ce modèle. / Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-γc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.

INTRODUÇÃO: O virus linfotrópico de células T humanas do tipo 1 (HTLV-1) é endêmico em várias partes do mundo e transmitido primariamente através de relações sexuais ou da mãe para o filho. MÉTODOS: Estes modos de transmissão foram investigados na coorte de pacientes com HTLV-1 acompanhados no Instituto de Infectologia Emílio Ribas de São Paulo comparando-se casais soroconcordantes e sorodiscordantes, para estudo da transmissão sexual, e binômios mãe-filho soroconcordantes e com filho soronegativo para estudo da transmissão vertical. Os dados foram consolidados e depois analisados utilizando o sistema RedCap (Research Electronic Data Capture) e o programa computacional estatístico Stata/IC 13.1. Resultados com valor de p < 0,05 foram considerados estatisticamente significativos. Variáveis com p < 0,2 na análise bivariada foram incluídos na análise multivariada. RESULTADOS: Entre janeiro de 2013 e maio de 2015, de 178 pacientes com HTLV-1 que se declararam casados, 107 (46 homens e 61 mulheres) tinham parceiro testado, resultando em 81 casais (26 homens e 26 mulheres já formavam pares entre si). Foram excluídos aqueles com HIV ou HTLV-2. A taxa de soroconcordância entre casais foi 46,9%. A carga proviral (PVL) de HTLV-1 foi comparada entre 19 casais soroconcordantes e 37 sorodiscordantes, e os casais soroconcordantes apresentaram cargas provirais mais elevadas (p = 0,03). Não houve diferença entre os grupos de acordo com idade, tempo de relacionamento, ter mãe ou irmão com HTLV-1, raça, local de nascimento, escolaridade, história de hemotransfusão, HAM/TSP, ATL ou soropositividade para hepatite C. Na análise multivariada, no entanto, o tempo de relacionamento (> 20 anos) manteve-se independentemente associado com a ocorrência de soroconcordância entre casais (p = 0,031). Por sua vez, no período de junho de 2006 a agosto de 2016 havia 192 mães com infecção pelo HTLV-1, resultando em 499 filhos expostos. Destes, 288 (57,7%) foram testados para HTLV-1, constituindo-se na amostra final para o estudo, juntamente com respectivas 134 mães. Entre os filhos testados, 41 foram positivos para HTLV-1, indicando taxa de transmissão vertical de 14,2%. Sete dos 134 núcleos familiares concentraram 20 (48,8%) do total de 41 filhos soropositivos e cinco apresentavam prole com três ou mais filhos soropositivos para HTLV-1. Estiveram associadas à soropositividade do filho: duração da amamentação >= 12 meses, PVL materna >= 100 cópias/104 PBMC, idade da mãe no parto > 26 anos, raça/etnia asiática, estado civil divorciada, filho com avó soropositiva para HTLV-1, ter irmão com HTLV-1. Na análise multivariada, amamentação >= 12 meses, PVL materna elevada e ter irmão com HTLV-1 mantiveram-se independentemente associados ao desfecho. CONCLUSÕES: Os resultados indicam que o HLTV-1 vem sendo transmitido ativamente na coorte, tanto por via sexual como materno infantil, com agregação familiar de casos e que o risco de transmissão entre casais persiste após décadas (mais de 20 anos) de sorodiscordância. Foi observada associação entre PVL e transmissão do vírus, tanto por via sexual como materno-infantil, mesmo depois de anos após o desfecho. PVL materna elevada e amamentação prolongada estiveram independentemente associados à transmissão vertical, sendo necessários outros estudos avaliando a influência de fatores genéticos. / INTRODUCTION: Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted primarily either through sexual intercourse or from mother to child. METHODS: The current study investigated sexual and vertical transmission among individuals diagnosed as HTLV-1-positive who have been followed up at the Institute of Infectious Diseases \"Emilio Ribas\". In order to study the sexual transmission and mother-to-child transmission, the selected individuals with their respective pairs (couples or mother and son, repectively) were classified into seroconcordant or serodiscordant groups according to serological findings. Data were collected and managed using Research Electronic Data Capture (REDCap) and Stata/IC 13.1 for Windows. p values < 0.05 were considered statistically significant. Variables with p < 0.2 in bivariate analysis were included in multivariate analysis. RESULTS: Between January 2013 and May 2015, 178 HTLV-1-positive patients had spouses, 107 of which (46 men and 61 women) had tested partners, thus forming the initial sample (81 couples). Individuals co-infected with HTLV-2 or human immunodeficiency virus were not included in the analysis. The rate of seroconcordance was 59.8%. The HTLV-1 proviral load was compared between 19 and 37 seroconcordant and serodiscondant couples, respectively, and the concordant couples showed higher proviral loads (p = 0.03). There were no differences between the groups according to age, relationship length, having a mother or sibling with HTLV-1, race, ethnicity, nationality, education, history of blood transfusion, HAM/TSP, ALT, or hepatitis C virus status. In multivariate analysis, relationship time (over 20 years) was shown associated with ocurrence of seroconcordance status (p = 0.031). In turn, between June 2006 and August 2016 there were 192 mothers with HTLV-1 infection, resulting in 499 exposed children. Of these, 288 (57.7%) were tested for HTLV-1, forming the final sample for the study with 134 respective mothers. Among the tested soons, 41 were positive for HTLV-1, indicating vertical transmission rate of 14.2%. Of 41 positive sons for HTLV-1, 40 (48.8%) were clustered in seven of 134 households, and five households had their offspring with three or more sons seropositive for HTLV-1. The following variables were associated with positive son for HTLV-1: breastfeeding duration >= 12 months, maternal PVL >= 100 copies/104 PBMC, mother\'s age at delivery > 26 years, asian race/ethnicity, divorced marital status, child with grandmother seropositive for HTLV-1, have brother with HTLV-1. In multivariate analysis, breastfeeding >= 12 months, higher maternal proviral load and have brother with HTLV-1 remained independently associated with the outcome. CONCLUSIONS: The results indicate that the HLTV-1 is actively transmitted in the cohort both sexually and vertically, with ocurrence of familial clustering. The sexual transmission risk persists among couples even after decades (over 20 years) of serodiscordance. PVL remained associated with the outcome even several years after the occurrence of the transmission both sexually and vertically. High maternal provirus load and breastfeeding beyond 12 months were independently associated with positive son for HTLV-1, but it is necessary further studies to evaluating the influence of genetic factors on the mother-to-child transmission.

La voie de signalisation NF-kB (nuclear factor kB) régule la transcription de gènes impliqués dans la réponse immune et l’inflammation. L’activation chronique de cette voie est fréquemment retrouvée associée à des désordres inflammatoires et des cancers. Les impacts fonctionnels de l’activation de la voie NF-kB ont été jusqu’à présent étudiés à l’échelle des promoteurs. Néanmoins, les études récentes de la distribution chromatinienne de NF-kB indiquent que la sous-unité NF-kB RelA se localise majoritairement dans les régions intragéniques, incluant des exons et des introns, où ses fonctions restent inconnues. Mes travaux ont consisté à adresser cette question dans le contexte de l’infection par le virus HTLV-1, un activateur chronique de la voie NF-kB, responsable de la leucémie T de l’adulte. Mes données montrent que l’activation de la voie NF-kB par l’oncogène viral Tax de HTLV-1 s’accompagne de modifications de l’épissage alternatif d’exons riches en GC qui coïncident avec le recrutement chromatinien de RelA à proximité de ces exons régulés. Les analyses intégratives des profils d’épissage et du remodelage de la chromatine, combinées à des essais de ciblage expérimental de la chromatine (TALE), démontrent que la fixation intragénique de RelA permet de recruter le régulateur d'épissage DDX17 pour moduler l’épissage alternatif de l’exon via son activité hélicase. Ces données révèlent que, outre ses fonctions transcriptionnelles, le facteur NF-kB RelA agit comme une ancre chromatinienne pour le facteur d’épissage DDX17 et fournit une spécificité de régulation d’épissage alternatif. Ces données revisitent nos connaissances des mécanismes physiopathologiques des maladies associées à HTLV-1 ainsi que d'autres désordres reliés à l’activation chronique de la voie NF-kB / The NF-kB (nuclear factor kB) signaling pathway regulates gene transcription of genes involved in immune response and inflammation. Chronic activation of NF-kB frequently associated with inflammatory disorders and cancer. The functional impacts of NF-kB have long been studied at the promoter level. Nevertheless, recent studies of the chromatin distribution of RelA indicate that this NF-kB subunit is predominantly localized in intragenic regions, including exons and introns, where its functions remain unknown. My work has addressed this question in the context of HTLV-1 infection, which is a constitutive activator of NF-kB, and the causative agent of the Adult T-cell Leukemia. The results show that the activation of NF-kB by the viral oncoprotein Tax results in changes in alternative splicing regulations of GC-rich exons that coincide with the chromatin recruitment of RelA in the vicinity of these exons. Integrative analysis of RNA splicing and chromatin occupancy, combined with experimental chromatin tethering assays (TALE) demonstrate that the intragenic binding of RelA leads to the recruitment of the splicing regulator DDX17, which modulates the inclusion rate of exon thanks to its helicase activity. Altogether, these data reveal that, besides its transcriptional role, NF-kB RelA acts as a chromatin anchor for the splicing factor DDX17 and provides alternative splicing specificity. These data revisit our knowledge of the physiopathologic mechanisms of HTLV-1 associated diseases , as well as other disorders related to the chronic activation of the NF-kB pathway

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

La paraparésie spastique tropicale ou la myélopathie associée à HTLV-1 (TSP/HAM) est une maladie neurologique chronique caractérisée par le développement de paralysies spastiques des membres inférieurs et de déficits sensoriels divers. Une infiltration périvasculaire souvent observée dans le système nerveux central des patients atteints de TSP/HAM correspondant essentiellement à des lymphocytes T CD4+, cibles préférentielles du HTLV-1 in vivo. Cependant, le facteur déclencheur du processus inflammatoire de la TSP/HAM est toujours méconnu. De ce fait, nous nous sommes intéressés à l'étude de l'implication d'une nouvelle population inflammatoire des T CD4+, les TH17, dans cette pathologie. Une quantification de l'expression de l'ARNm d'IL-17, la cytokine inflammatoire sécrétée par les TH17, a été menée sur les cellules du sang périphérique issues de patients infectés ainsi que sur des lignées cellulaires chroniquement infectées par HTLV-1. L'expression élevée de l'IL-17 détectée dans les lignées cellulaires est corrélée avec celle de la protéine transactivatrice du HTLV-1, Tax. Par ailleurs, Tax induit l'expression du régulateur transcriptionnel clé des TH17, RORγ, par l'intermédiaire de la cytokine pro-inflammatoire Ostéopontine. Finalement, nous avons pu montrer l'existence d'une relation dynamique entre l'expression de l'ARNm de Tax, OPN, RORγ, IL-17 et IL-22 chez les patients asymptomatiques et TSP/HAM avec une expression d'IL-17 et d'IL-22 plus élevée chez le groupe des TSP/HAM. Nos résultats suggèrent que l'infection par HTLV-1 in vivo induirait une réponse TH17 qui pourrait avoir un rôle majeur dans la pathogenèse de la TSP/HAM. / HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological inflammatory disease of the central nervous system characterized by a chronic, progressive inflammatory demyelinating myelopathy. It is thought that the pathogenesis of this disease involves a predominant infiltration of CD4+ T cells which are the main subset of in vivo infected cells with HTLV-1. However, until now, the identity of the triggering factor which promotes the inflammatory process in HAM/TSP remains unclear. Therefore, we investigated the implication of the new CD4+ T cells inflammatory lineage TH17 in this disease. We quantified the mRNA expression levels of IL-17, a cytokine associated with the TH17 response, in peripheral blood from 10 HAM/TSP patients, 6 healthy asymotomatic carriers (HCs) and 4 normal uninfected controls as well as in HTLV-1-infected T-cell lines. Elevated production of IL-17 observed in HTLV-1-infected T-cell lines was correlated with the expression of Tax, the major HTLV-1 regulatory protein. Thus, we established that Tax increases the expression of RORγ, the TH17-lineage specific transactivator, by inducing Osteopontin expression, an inflammatory cytokine known to promote TH17 response. Finally, we demonstrated a dynamic relationship between the expression of Tax, Osteopontin, RORγ, IL-17 and IL-22 mRNAs in HCs and HAM/TSP patients, where higher expression of IL-17 and IL-22 were observed in HAM/TSP cases. These findings suggest that in vivo infection by HTLV-1 may lead to a deleterious deviation of CD4+ T Helper response to TH17, that could play a major role in HAM/TSP pathogenesis.

A significativa prevalência da infecção pelo HTLV-1/2 no Brasil tornou compulsória a triagem sorológica em bancos de sangue desde 1993. A realização de um diagnóstico eficaz e seguro desta infecção tem importância no correto aconselhamento dos pacientes, bem como na adoção de medidas preventivas em relação à transmissão do HTLV-1/2. Entretanto, a ineficiência dos testes confirmatórios disponíveis somado ao alto custo, são fatores limitantes para a conclusão segura do status clínico do paciente. O objetivo deste trabalho foi desenvolver, padronizar e validar uma plataforma molecular (NAT) multiplex utilizando a metodologia de PCR em tempo real para o diagnóstico confirmatório e discriminatório da infecção pelo HTLV-1/2. Para tanto, foi padronizada a PCR em tempo real utilizando o sistema de sondas de hidrólise (TaqMan®) e a região gênica viral pol foi utilizada como alvo dessas reações. As reações foram otimizadas e validadas em amostras de DNA de controles positivos e negativos, amostras de DNA obtidas a partir do sangue total de indivíduos infectados pelo HTLV-1/2, candidatos à doação de sangue e de pacientes infectados por outras viroses (HIV, HBV e HCV). Após a padronização, a plataforma molecular foi validada em relação aos seguintes parâmetros: sensibilidade analítica, sensibilidade diagnóstica, especificidade analítica, especificidade diagnóstica, precisão e robustez. O limite de detecção do teste (LoD) foi de 3,85 cópias/reação para HTLV-1 e 10,88 cópias/reação para HTLV-2. Para a especificidade, não foi observada reação cruzada com os vírus HAV, HBV, HCV, HIV-1 e HIV-2 e B19V obtidos a partir de um painel de referência viral e nem com amostras de indivíduos portadores de HIV, HBV ou HCV. A sensibilidade diagnóstica foi de 94,6% para HTLV-1 e 78,6% para HTLV-2. O coeficiente de variação encontrado no parâmetro precisão foi de até 0,475%. A metodologia desenvolvida é uma ferramenta simples, de baixo custo, sensível e altamente específica, portanto, adequada para detecção e discriminação da infecção por este retrovírus. A padronização desta plataforma tem um importante impacto no fortalecimento da capacitação tecnológica nacional. Além disso, propõe seu uso no algoritmo diagnóstico, o qual permitirá a conclusão segura do diagnóstico do HTLV-1/2. / The significant prevalence of HTLV-1/2 infection in Brazil has turned the serological testing for this virus mandatory in national blood banks since 1993. The performance of efficient and safe diagnosis of this infection has importance on the correct counseling of the patients and the prevention of the transfusion/hemoderivatives transmitted HTLV-1/2 infection. However, the inefficiency of the available confirmatory tests combined with their high cost present limiting factors for adequate conclusions over the clinical status of the patient. A safe diagnosis of the HTLV-1/2 infection is of crucial importance for the correct counseling of the patients and prevention of the transmission of the infection by blood transfusions, breastfeeding and solid organ transplantations. Therefore, the objective of this study was to develop, optimize and validate a molecular multiplex platform (NAT) utilizing the real-time PCR methodology for confirmatory and discriminatory diagnosis of the HTLV-1/2 infection. DNA samples obtained from HTLV-1/2 positive patients, blood donor candidates, and HIV, HBV and HCV infected patients were used in this study. The real-time PCR was optimized using the TaqMan® system (hydrolysis probes) and the pol gene was a target for real-time amplification. After optimization, the molecular platform was validated by analysis of the analytic and diagnostic sensitivity, analytic and diagnostic specificity, precision, and robustness. The detection limit (LoD) of the test was 3.85 copies/reaction for HTLV-1 and 10.88 copies/reaction for HTLV-2. Evaluation of the specificity did not demonstrate cross reaction with human viruses like HAV, HBV, HCV, HIV-1, HIV-2 and B19V obtained from a diagnostic panel and also with samples obtained from HIV, HBV or HCV positive individuals. The diagnostic sensitivity was 94.6% for HTLV-1 and 78.6% for HTLV-2. The estimated variation coefficient of the precision analysis was not more than 0.475%. Therefore, this methodology presents simple, inexpensive, sensitive and highly specific test, and can be used adequately for the detection and discrimination of this retroviral infection. The optimization of this molecular platform have important impact on the improvement of the technologic capability and it can be used for the definition of a national diagnostic algorithm which will permit a safe conclusion of the HTLV-1/2 diagnosis.

O vírus linfotrópico das células T humanas tipo 1 (HTLV-1), é responsável por patologias como a mielopatia associada ao HTLV-1 ou paraparesia espástica tropical (HAM/TSP) e a leucemia/linfoma das células T do adulto (ATL) dentre outras. As vias de replicação até hoje demonstradas, não suportam a hipótese de um estado virêmico. Neste estudo, a detecção de partículas virais plasmáticas foi executada, por PCR em Tempo Real e Nested PCR em 190 amostras de pacientes infectados pelo HTLV-1(assintomáticos ou com HAM/TSP), em acompanhamento, no Instituto de Infectologia Emílio Ribas. 12 indivíduos (8%) testados por PCR em tempo real (n=150) e 6 indivíduos (18%) testados por Nested PCR (n=33, dado que sete amostras foram excluídas da análise) apresentaram RNA do HTLV-1 detectável no plasma. Em conclusão, foi possível identificar RNA plasmático do HTLV-1, tanto em pessoas assintomáticas quanto com HAM/TSP. Esta detecção abre novas possibilidades de discussão sobre a replicação do HTLV-1 e das vias de transmissão, sugerindo maiores investigações para elucidar o assunto. / The human T-cell lymphotropic virus type1 (HTLV-1) is responsible for some pathologies such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and Adult T-cell Leukemia/ Lymphoma (ATL) among others. Its ways of replication so far presented do not support the hypothesis of a viremic stage. In this study, the detection of the plasmatic viral load was performed by real time PCR and Nested PCR in 190 samples from HTLV-1 infected individuals (Either Asymptomatic or HAM/TSP cases) following up at Instituto de Infectologia Emílio Ribas. 12 individuals (8%) tested by Real time PCR (n= 150) and 6 individuals (18%) tested by Nested PCR (n= 33, given that 7 samples were excluded from the analysis) presented detectable HTLV-1 RNA in the plasma. In conclusion, it was possible to indentify HTLV-1 plasmatic RNA in asymptomatic carriers as well as in HAM/TSP cases. This detection opens new possibilities of discussion about HTLV-1 replication and transmission pathways, suggesting further investigation for clarifying this matter.