Dissertations / Theses on the topic 'Virologie médicale'

Le virus de l’hépatite delta (HDV), est satellite du virus de l’hépatite B. La variabilité génétique de HDV a conduit à la définition de génotypes viraux, présentant une répartition géographique spécifique. Les travaux antérieurs du laboratoire ont permis l’identification de 7 génotypes distincts. L’activité « Centre National de Référence du Virus de l’Hépatite Delta » de notre laboratoire nous a permis de caractériser 606 isolats provenant de patients suivis en France et d’identifier un 8ème génotype (HDV-8). Environ 78% des patients présentaient le génotype majoritaire et ubiquitaire HDV-1, 0. 1% HDV-2, 16. 3% HDV-5, 1. 2% HDV-6, 3. 6% HDV-7 et 0. 9% à HDV-8. La totalité des patients infectés par les virus de types -5, -6, -7 et -8 était d’origine africaine. Une grande diversité génétique a de plus été mise en évidence au sein du génotype HDV-1, conduisant à l’individualisation de 3 sous-types : HDV-1AB (isolats originaires d’Europe/Asie), HDV-1C1 et 1C2 (Afrique). Une étude menée en collaboration avec l’Université d’Istanbul nous a permis de retrouver cette diversité des virus HDV-1 au sein des souches circulant en Turquie. L’ensemble de nos résultats nous a conduit à émettre l’hypothèse que HDV aurait pour origine l’Afrique d’où il aurait suivi le cours des migrations humaines via le Moyen-Orient. Nous avons de plus mis au point un test permettant la quantification plasmatique de l’ARN HDV quel que soit le génotype. Ce test, utilisé en routine au laboratoire, a été utilisé dans 2 études pilotes dans le cadre de collaborations avec d’une part l’hôpital Beaujon (France) et d’autre part l’hôpital Hippokration (Athènes, Grèce). Nos travaux ont permis de montrer la variabilité génétique de HDV. Celle-ci doit être prise en compte dans l’élaboration des tests diagnostics. Des études multicentriques prospectives permettront d’apprécier son retentissement sur le pouvoir pathogène du virus et la prise en charge thérapeutique
Hepatitis Delta Virus (HDV) is satellite of hepatitis B virus. The genetic variability of HDV has led to the definition of viral genotypes, presenting specific geographic distribution. Previous studies in our laboratory have allowed to identify 7 distinct genotypes. Our laboratory is a national reference centre for HDV. This has allowed us to characterize 606 isolates from patients followed in France, and to identify an 8th genotype (HDV-8). Approximately 78% patients were infected by HDV-1, which is the most common genotype. 0. 1% were infected by HDV-2, 16. 3% by HDV-5, 1. 2% by HDV-6, 3. 6% by HDV-7 and 0. 9% by HDV-8. All patients infected by viruses HDV-5, -6, -7 and -8 were of African origin. An important genetic diversity was evidenced among HDV-1 viruses, leading to the individualisation of 3 subtypes : HDV-1AB (isolates from Europe/Asia), HDV-1C1 and 1C2 (Africa). A collaborative study with the University of Istanbul allowed us to confirm this diversity of HDV-1 viruses among isolates circulating in Turkey. Taken together, our results tend to indicate that HDV might have originated from Africa and have followed the course of human migrations via Middle-East. We have developed a test to quantify HDV RNA in plasma whatever the viral genotype. This test, employed for routine diagnosis in our laboratory, was used in 2 pilot studies within the context of collaborations with Beaujon Hospital (France) and Hippokration Hospital (Athens, Greece). We have evidenced and characterized the genetic variability of HDV. This variability should be taken into account for the elaboration of diagnostic tests. Further multicenter prospective studies should allow to estimate the impact of the genetic variability on the course of the disease and on the treatment

The development of cervical cancer depends on high-risk human papillomavirus (HR-HPV) persistent infection in the cervix. The transformation process leading to invasive cancer can take many years even decades and provides ample opportunity to detect, prevent and cure true precursor lesions. Although cervical cancer is widely preventable, it is the fourth most common cancer among women throughout the world, being a real public health issue, especially in developing countries, as 85 % of deaths occur in low and middle-income countries. The situation in Bolivia is particularly alarming, as the cervical cancer incidence is around 38.5 per 100,000 women, which is estimated to be the highest incidence in Latin America. Prevention of cervical cancer in Bolivia is mainly based on the cytological examination of a Papanicolaou smear (Pap) and more recently on visual inspection after application of acetic acid (VIA). However, many economic, sociocultural, and geographic barriers impair this prevention program being successful, as reflected by the low coverage obtained with these screening tests.In order to reduce the cervical cancer incidence and mortality in the department of Cochabamba in Bolivia, we aimed to assess a low-cost HPV test applicable on self-samples. We believe that this strategy could improve the poor screening programs developed in our country. An evaluation was first made to knowledge of Bolivian women about human papillomavirus (HPV) and cervical cancer. As expected, Bolivian women, from rural, peri-urban and urban areas, knew little or nothing about those. Secondly, their degree of acceptability and confidence towards HPV self-sampling was assessed. Most of the women found self-sampling easier to perform (86.9 % to 93.2 %) and more comfortable (79.4 % to 83.3 %) compared to physician sampling. However, accuracy to detect cervical pre-cancer was higher in their point of view when it was performed on specimens taken by a physician (35.1% to 63.5%). Accordingly, the campaign of vaginal HPV self-sampling in a peri-urban area increased screening coverage, reaching in three months the annual rate average. Finally, the determination of accuracy to detect preneoplastic lesions was assessed for three screening methods, in 469 women, divided in two groups. The first group included 362 women that underwent three consecutively primary screening tests: self-collected sampling for HR-HPV detection, conventional cervical cytology and visual inspection under acetic acid (VIA). The second group included 107 women referred with a positive HR-HPV test that underwent were triage by conventional cervical cytology and VIA. The presence of high-grade intraepithelial neoplasia or invasive cancer (CIN 2+) was verified by colposcopy and biopsy.Among primary screening tests, the sensitivity of the HR-HPV test to detect CIN 2+ lesions was the highest (76 %). In HR-HPV positive women, the sensitivity of the VIA and cytology to detect CIN 2+ lesions were 100% and 81%, respectively.In conclusion, the knowledge about cervical cancer and HPV infection is poor in Bolivia. Despite greater acceptance of the vaginal self-sampling in all areas, women kept greater confidence in the screening performed by the gynecologist, although HPV self-sampling improved coverage rate. Finally, HPV testing on self- samples was the most sensitive screening test and VIA was the most sensitive method for the triage.
Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)
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HIV-1 infections can be treated but not cured by the current antiretroviral therapy regimens. One of the major barriers to HIV-1 eradication is the persistence of the virus in treated HIV+ individuals under the form of reservoirs. A continuum of molecular mechanisms, at the epigenetic, transcriptional and post-transcriptional levels maintains HIV-1 gene expression silent in its reservoirs. A better understanding of HIV-1 molecular mechanisms of persistence thus allows to devise novel therapeutic approaches to eradicate the virus. In this context, our thesis aimed at characterizing the molecular mechanisms of HIV-1 persistence in its T-lymphoid and myeloid reservoirs. More specifically, we have studied the epigenetic and transcriptional mechanisms of HIV-1 persistence at three different levels. First, we have investigated the DNA methylation-mediated mechanisms underlying the heterogeneity of the DNA methylation inhibitor 5-aza-2’-deoxycytidine potency in the reactivation of HIV-1 gene expression from latently-infected CD4+ T cells. Second, we have studied the contribution of the intragenic binding sites for the cellular PU.1 transcription factor in the specific regulation of HIV-1 gene expression in myeloid lineages. Finally, in a third study, we have designed a new tool to study the transcriptional landscape of HIV-1 in LTRs-suppressed proviruses. Collectively, our work has offered individual insights into the molecular mechanisms underlying the heterogeneity of HIV-1 T-lymphoid and myeloid reservoirs, with the ultimate goal of developing new HIV-1 curative strategies and improving the quality of life of HIV+ individuals.
Doctorat en Sciences
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Les gastro-entérites aiguës virales constituent un problème de santé publique mondial avec une morbidité et une mortalité importantes chez le jeune enfant, surtout dans les pays en voie de développement. Cette étude constitue la première investigation virologique, épidémiologique et clinique des cinq principaux virus responsables de gastro-entérites en Tunisie. Nos résultats montrent que les rotavirus du groupe A et les norovirus étaient les plus fréquemment détectés dans respectivement 213 (27%) et 128 (16,2%) cas. Les rotavirus du groupe A, les norovirus et les virus Aichi étaient significativement plus fréquents chez les enfants hospitalisés que chez les externes. Chez les enfants hospitalisés, contrairement à d’autres études, aucune différence significative de la fréquence et de la sévérité de la diarrhée entre les rotavirus et les norovirus n’a été détectée. Le typage moléculaire a montré que les souches prédominantes dans les selles diarrhéiques étaient le rotavirus A de type G3P[8] et le norovirus de type GII. 4, variant Hunter. Une donnée intéressante est que ce variant a été retrouvé chez les enfants tunisiens dès janvier 2003, alors qu’il n’a été détecté pour la première fois qu’en février 2004, en Europe. Dans les prélèvements d’eaux usées, les rotavirus ont été détectés dans 80 (32 %) prélèvements, les norovirus dans 11 (4,4 %), les virus Aichi dans 15 (6 %) et les adénovirus 40/41 dans 1 (0,4 %) seul prélèvement. Une corrélation entre les souches détectées dans les eaux usées et les coquillages et celles retrouvées dans les selles des patients a été démontrée. Ceci suggère qu’il existe une relation entre la contamination hydrique et les diarrhées infantiles
Viral acute gastroenteritis is a worldwide problem of public health with an important morbidity and mortality in young children, especially in developing countries. This study constitutes the first viral, epidemiological and clinical investigation of the five main viruses responsible for gastroenteritis in Tunisia. Our results show that Group A rotaviruses and noroviruses were the most frequently detected with 213 (27%) and 128 (16. 2%) cases, respectively. Group A rotaviruses, noroviruses and Aichi viruses were significantly more frequent in hospitalized children than in outpatients. In hospitalized children, contrary to other studies, no significant difference was observed between rotavirus and norovirus infections with regard to the incidence and the clinical severity of the disease. The molecular typing showed the predominant strains as rotavirus type G3P[8] and norovirus genotype GGII. 4, variant Hunter. One interesting finding is that this variant was detected in Tunisian children as soon as January 2003, whereas it was described for the first time in Europe in February 2004. In the sewage samples, rotaviruses were detected in 80 (32%) cases, noroviruses in 11 (4. 4%) cases, Aichi viruses in 15 (6%) cases and adenoviruses type 40/41 in 1 (0. 4%) sample. A correlation between the strains detected in the sewage and the shellfish and the strains detected in the human stools has been shown. This suggests the existence of a relation between water contamination and pediatric diarrheas. This study provide very interesting data that permit a better understanding of the molecular and clinical epidemiology of enteric viruses in Tunisia and in Africa for which data are very rare

Le diagnostic virologique est un sujet d’actualité particulièrement du fait des récentes épidémiesou pandémies telles que la pandémie d’influenza A(H1N1) en 2009 ou la diffusion du virus Zika dansles Amériques et la région du Pacifique entre 2014 et 2017, associée à des cas de microcéphalie et dessyndromes de Guillain Barré. Encore plus récemment, en août 2018, le ministre de la santé de laRépublique Démocratique du Congo annonçait la 10e épidémie de virus Ebola dans le pays et endécembre 2019, le coronavirus SARS-CoV-2 est à l’origine d’une pandémie au départ de la Chine. Avecle nombre croissant de migrants et de voyageurs favorisant la dissémination des maladies virales, leslaboratoires diagnostiques doivent être parés à la fois pour l’identification des virus communs maisaussi de ceux importés.Les techniques les plus anciennes de diagnostic virologique tendent à devenir obsolètes suite audéveloppement rapide des techniques moléculaires depuis les années 90. Cependant, nous utilisonstoujours un mélange de techniques moléculaires et non moléculaires au sein de notre laboratoire.Les objectifs de ce travail sont de passer en revue les différentes techniques communémentutilisées pour la détection directe des virus avec leurs avantages et leurs inconvénients et de fournirune réflexion sur la place de chaque technique, en 2020, dans un laboratoire diagnostique.Nous aborderons tout d’abord les cultures cellulaires et nous insisterons sur leur polyvalence quipermet parfois de mettre en évidence des micro-organismes que l’on ne suspectait pas. Nousillustrerons ce point par un article relatant la mise en évidence de Chlamydia trachomatis du serovar Lresponsables de la lymphogranulomatose vénérienne dans des prélèvements envoyés pour suspiciond’infection herpétique.Le travail se focalisera ensuite plus particulièrement sur le diagnostic des infections viralesrespiratoires. Nous verrons les principes des tests de détection antigéniques et discuterons de leurslimites en se basant sur un article qui traite du diagnostic des virus influenza A et B par 3 différentstests immunochromatographiques. Cet article montre que la sensibilité des tests varie en fonction dela charge virale dans le prélèvement ainsi que du sous-type de virus.Nous poursuivrons avec les tests d’amplification d’acides nucléiques (tests moléculaires) enexpliquant la technique de PCR (Polymerase Chain Reaction) et une technique d’amplificationisothermique (Nicking Enzyme Amplification Reaction - NEAR). Nous illustrerons par un article portantsur l’évaluation du test Alere i influenza A&B (technique NEAR) en comparaison du test Sofia influenzaA+B (immunochromatographie). Cet article montre un gain de sensibilité de l’Alere i par rapport auSofia pour le diagnostic de l’influenza A mais pas pour l’influenza B. Il constitue également un travailpréliminaire sur l’appréciation de l’utilité d’une technique PCR rapide dans la prise en charge despatients. La conclusion est qu’il pourrait y avoir un apport de ce type de technique pour la diminutiondes hospitalisations, de la prescription des examens complémentaires et des antibiotiques. Celapermettrait également une prescription plus adéquate de l’oseltamivir pour le traitement de la grippe.Le point important est que l’impact du résultat est d’autant plus grand qu’il est délivré précocementdans la prise en charge des patients, idéalement lorsqu’ils sont encore aux urgences.Suite au travail sur l’Alere i, nous avons entrepris d’évaluer un test PCR multiplex (FilmArrayRespiratory Panel) pour le diagnostic des virus afin de voir si la détection d’un plus grand nombre depathogènes pourrait avoir un impact plus grand sur la prise en charge des patients. Cette évaluation adonné lieu à deux articles. Le premier détaille les avantages et inconvénients des différents outils dediagnostic pour la détection des virus respiratoires et sert d’état des lieux sur les tests utilisésactuellement dans les laboratoires de virologie. Le deuxième article porte plus particulièrement surl’apport du FilmArray dans la prise en charge des patients. La conclusion est que ce n’est pas le résultatdu test qui a un impact sur cette prise en charge mais plutôt d’autres facteurs notamment l’âge ou desmarqueurs inflammatoires biologiques.Nous terminerons ce travail par un aperçu des techniques de séquençage qui seront sans aucundoute de plus en plus utilisées pour le diagnostic en virologie.
Doctorat en Sciences médicales (Médecine)
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Les relations entre la délétion Δ32 sur le gène codant pour le récepteur CCR5 aux β­ chemokines et la progression de la maladie VIH ont été étudiées chez des patients infectés par le VIH-1 suivis dans plusieurs cohortes prospectives françaises ou étrangères. Environ 17% des patients à contage daté de la cohorte SEROCO étaient porteurs de la délétion sur un des deux allèles du gène ; ces patients progressaient moins vite depuis la contamination vers le SIDA et le décès que les autres patients. Cet effet a été retrouvé lors d'une collaboration avec la cohorte d'Amsterdam d'hommes homosexuels, indépendamment de 2 autres mutations ultérieurement décrites sur les gènes codant pour le corécepteur CCR2 et pour le ligand SDF-1 du corécepteur CXCR4. La charge virale précoce sérique était plus basse d'environ 0,25 log chez les hétérozygotes pour la délétion que chez les autres patients. Cette charge virale plus basse expliquait dans l'analyse multivariée une partie de l'effet protecteur conféré par la délétion. Par ailleurs, notre étude a permis de décrire le deuxième cas d'homozygote infecté par le VIH, confirmant que la protection conférée par la délétion à l'état homozygote n'est pas totale. Enfin, les liens entre la délétion Δ32 et la survenue de certaines infections opportunistes ont été étudiés en réunissant les données de 3 cohortes, SEROCO, HEMOCO et SEROGEST. La toxoplasmose survenait significativement moins souvent comme pathologie inaugurale de SIDA chez les hétérozygotes que chez les autres patients, y compris après prise en compte de l'âge, du taux de lymphocytes CD4 et de la prescription d'une prophylaxie primaire spécifique. Grâce au suivi des patients actuellement traités, ce travail va être poursuivi en étudiant la relation entre délétion Δ32 et la réponse aux traitements antirétroviraux. Par ailleurs la physiopathologie de la primo-infection VIH et ses liens avec la délétion vont être étudiés dans la cohorte PRIMO de patients récemment infectés
The role of the Δ32 deletion on the gene coding for the CCR5 receptor for beta­ chemokines on HIV-1 disease progression was studied in HIV-infected patients followed in several prospective multicenter cohorts. Around 17% of patients with a known date of infection from the SEROCO cohort were heterozygous for the deletion : these patients progressed less rapidly since infection to AlDS or death than the other patients. Ln a collaborative study with the Amsterdam cohort study, this protective effect was observed independently of two other mutations on genes coding for the CCR2 receptor and the SDF-1 ligand. Early serum viral load was 0. 25 log lower in Δ32 heterozygous patients than in wild-type patients; this lower viral load explained partiy. The protective effect of the deletion in the Cox multivariate analysis. This study allowed us to describe an HIV-infected subject who was homozygous for the deletion, which confirms that homozygous patients are not totally protected from HIV infection. The relationship between the Δ32 deletion and the occurrence of several opportunistic infections was studied in 1657 patients followed in the SEROCO, HEMOCO and SEROGEST cohorts. The risk of toxoplasmosis as a first AIDS-defining illness since inclusion was significantly reduced in heterozygous patients, even after adjustment for age, CD4 cell count and primary specifie prophylaxis. Since most patients who are still followed in these cohorts are now treated by highly active antiretroviral therapy, we are going to study whether the deletion affects the response treatment. The relationship between pathophysiology of primary HIV-1 infection and the Δ32 deletion will be studied in the PRIMO cohort which has recruited since 1996 recently infected patients

Ce travail rend compte des recherches sur la leucémie aiguë menées par Jean Bernard et ses collaborateurs de 1940 à 1970 et les replace dans les transformations globales de la recherche médicale au cours de cette période. Cette maladie passa de l'incurabilité à une certaine curabilité, grâce à l'association d'agents chimiothérapeutiques. Parallèlement, l'exsanguino-transfusion et la greffe de moelle osseuse furent testées puis abandonnées. Les travaux de laboratoire portèrent principalement sur la cytologie, sur les virus des leucémies murines et sur l'immunologie des leucémies humaines et animales. Quant à l'évolution générale de la recherche médicale, elle fut caractérisée par une molécularisation du biologique et du médical, une standardisation des matériaux et des méthodes, un renforcement des liens avec l'industrie et des échanges scientifiques, en particulier franco-américains, ainsi qu'une augmentation des financements publics et privés, dans un nouveau cadre institutionnel.

L’épidémiologie moléculaire des virus VIH-1 en France est caractérisée par une augmentation constante de la diversité virologique et de la fréquence des virus de sous-types non-B chez les patients en primo-infection. Entre 1997 et 2007, 28.4% des 591 patients suivis étaient infectés par des virus de sous-types non-B. De plus, 49 patients (8.3%) étaient infectés par des souches de sous-types différents sur les gènes pol et env, témoignant d’évènements de recombinaisons entre ces gènes. Ces virus recombinants étaient isolés à la fois chez des patients originaires d’Afrique sub-saharienne (28.3%) et des sujets caucasiens (6.3%). Ces résultats témoignent des échanges de souches virales entre les populations d'origine africaine et caucasienne, contribuant encore à augmenter la diversité virologique dans ces deux populations. Parmi 131 virus de sous-types non-B, 12.2% étaient classés comme ayant un tropisme CXCR4 par méthode génotypique, mais seulement 0.8% par méthode phénotypique, indiquant d’une part la faible proportion de virus non-B de tropisme CXCR4 en primo-infection en France, et d’autre part le manque de spécificité des méthodes génotypiques de détermination du tropisme pour ces sous-types, rendant nécessaire la mise au point d’autres algorithmes spécifiques pour ces virus.L’analyse de 987 virus isolés dans la cohorte entre 1999 et 2010 a mis en évidence que 12.7% d’entre eux étaient regroupés en "clusters" de transmission. Les patients en primo-infection contribuent donc de façon significative à la propagation de l’épidémie de VIH en France, particulièrement les hommes homo/bi-sexuels, avec une fréquence augmentant au cours de la période récente (2006-2010).La comparaison des quasi-espèces virales circulant concomitamment chez 8 patients en primo-infection (« receveurs ») et leurs 8 partenaires sexuels respectifs (« donneurs ») a révélé dans tous les cas la transmission d’un virus unique, présent de façon minoritaire parmi les sous-populations virales du donneur. La transmission virale muqueuse implique donc une sélection génétique drastique
High genetic diversity is a major characteristics of HIV-1. In France, although subtype B strains are still predominant, the proportion of non-B viruses isolated in patients at the time of primary HIV-1 (PHI) infection increases over time. Between 1997 and 2007, 28.4% of patients were infected with non-B subtypes strains. Forty-nine viruses showed different phylogenies between the pol and env genes, indicating that recombinations have occurred in 8.3% of cases. These recombinants were isolated both in patients from Sub-Saharan Africa (28.3%) and in white subjects (6.3%).The phenotypic analysis of viral tropism of 131 non-B strains showed a very low (0.8%) proportion of CXCR4-tropic strains (X4 strains) at the time of PHI. Compared to phenotypic tests, genotypic predictions can overestimate (12.2% versus 0.8%) the proportion of X4 strains in non-B subtypes.The phylogenetic analysis of 987 strains isolated in 1999-2010 showed that 12.7% of PHI cosegregated into 56 transmission chains. PHIs are a significant source of onward transmission, especially in men having sex with men, with increasing frequency during the recent years (10.2% in 1999-2006 versus 15.2% in 2006-2010, p=0.02).The comparison of the viral quasispecies isolated in plasma and PBMC samples from 8 patients at the time of PHI ("recipients") and their transmitting partners ("donors") suggested that a severe genetic bottleneck occurrs during HIV-1 heterosexual and homosexual transmission. Indeed, we observed in all cases the transmission of a single variant, which was derived from an infrequent variant population within the blood of the donor. The proportion of X4 quasispecies in donors were higher in case of X4 versus CCR5-tropic viral transmission, suggesting that X4 transmission may be associated with a threshold of X4 circulating quasispecies in donors

CCR5 et CXCR4 sont les corécepteurs d'entrée du VIH utilisés par le virus in vivo en plus du récepteur principal CD4 pour infecter les cellules. Au début et tout le long de l'infection, on retrouve chez les patients infectés par le VIH, des virions R5 utilisant le corécepteur CCR5 pour infecter les cellules. Dans les stades tardifs de l'infection et chez environ la moitié des personnes infectées par le VIH, on observe en plus de ces souches R5, l'apparition de souches X4, utilisant le corécepteur CXCR4 pour infecter les cellules. Cette apparition de souches X4 est un facteur d'aggravation de la maladie. Les causes de cette commutation de R5 vers X4 sont mal définies. Le but de mon travail de thèse a été de trouver de nouvelles stratégies thérapeutiques visant l'un ou l'autre de ces corécepteurs. La première partie de mon travail compare les deux isoformes de CXCR4 en tant que corécepteurs du VIH. Ces deux isoformes, CXCR4-A et CXCR4-B, diffèrent de 9 acides aminés en NH2 terminal suite à un épissage alternatif. Nous avons montré que l'isoforme CXCR4-B est la plus performante en tant que corécepteur du VIH mais que ces deux variants sont équivalents pour la migration vers leur ligand commun SDF-1. Ainsi, nous proposons qu'en ciblant exclusivement l'isoforme B qui est la plus favorable à l'infection, via par exemple des siRNA, il serait possible de limiter les infections par des souches X4 tout en gardant une partie des fonctions essentielles de ce récepteur dans l'organisme, assurées par l'isoforme A. Nos résultats suggèrent également que l'infection par des souches R5 augmente le ratio en ARNm CXCR4-B / CXCR4-A dans des PBMC, et que ce ratio est en partie responsable de la commutation de R5 vers X4 associée à une aggravation de la maladie. Cibler cette isoforme CXCR4-B pourrait donc se révéler bénéfique. La deuxième partie de cette thèse étudie la modulation de la fonction de corécepteur du VIH de CCR5 par S1P1, un autre membre de la famille des récepteurs couplés aux protéines G qui permet la remise en circulation des lymphocytes après leur séjour dans les organes lymphoïdes secondaires par chimiotactisme vers son ligand S1P, abondant dans le sang. Nous montrons que S1P1 interagit physiquement avec CCR5 et gêne l'entrée des virus R5 dans la cellule-hôte. A l'inverse, S1P1 active les étapes post-entrée du cycle viral, notamment l'expression génique virale. La résultante de ces effets opposés est une augmentation de la production virale par des cellules infectées in vitro. Ce projet a également montré que l'utilisation de FTY720, un antagoniste fonctionnel de S1P1, diminue l'infection de cellules dendritiques par des virus HIV-R5 in vitro, ainsi que la virémie dans un modèle de souris SCID infectées après reconstitution immunologique. La mise en évidence des interactions entre CCR5 et S1P1 ouvre donc des perspectives thérapeutiques
CCR5 and CXCR4 are the two HIV entry coreceptors used by the virus in addition to the main receptor CD4 in vivo to infect cells. R5 virions, that use CCR5 as a coreceptor to infect cells, are detected in most HIV patients. At late stages of infection and in about half of HIV infected persons, there is an emergence of X4 virions that use CXCR4 as a coreceptor, in addition to R5 virions. This emergence is associated with an increase in disease progression. The reasons for this R5 to X4 switch are poorly understood. The goal of my PhD work was to find new therapeutic strategies that target these coreceptors.The first part of this work compares the two CXCR4 isoforms as HIV coreceptors. Those two isoforms, CXCR4-A and CXCR4-B, differ by 9 amino acids at their NH2 terminal extremity as a consequence of an alternative splicing. We have shown that CXCR4-B isoform is more efficient as an HIV coreceptor but that those two variants are equivalent in terms of chemotaxis toward their common ligand SDF-1. Thus, we propose that by targeting specifically the B isoform that supports infection, via siRNA by example, it is possible to limit X4 development while keeping essential functions of this receptor. Our results also suggest that R5 infection increases CXCR4-B / CXCR4-A mRNA ratio in PBMC and that this ratio is in part responsible for R5 to X4 switch. Thus, targeting CXCR4-B isoform could be beneficial.The second part of this PhD thesis studies the effect on CCR5 coreceptor function of S1P1, another G protein-coupled receptor that enables lymphocytes egress from lymph nodes by chemotaxis toward its ligand S1P that is abundant in blood. We have shown that S1P1 physically interacts with CCR5 and blocks R5 virus entry. On the other hand, S1P1 activates post-entry steps of the viral cycle, in particular gene expression. The resulting effect is an increase in viral production by infected cells in vitro. We also showed that the use of FTY720, a S1P1 functional antagonist, decreases dendritic cell infection by R5 viruses in vitro, and in vivo infection in a SCID mouse model. The emphasis of CCR5 and S1P1 interactions opens new therapeutic strategies

La viroterapia oncolítica es un tratamiento emergente basado en el uso de virus replicativos que de manera selectiva infectan células tumorales y provocan su muerte. Hasta la fecha, se han producido grandes avances en dotar a los virus de selectividad tumoral. Sin embargo, el principal desafío actual es potenciar la eficacia de este abordaje a fin de poder implementarlo en la práctica clínica habitual. Una de las estrategias más prometedoras para dotar de mayor potencia antitumoral a los virus oncolíticos, pasa por incorporar genes terapéuticos en el genoma viral, a fin de que se expresen en el microambiente tumoral y aporten beneficios adicionales a la oncolisis. No obstante, se ha observado que, en ocasiones, la expresión de una proteína terapéutica puede afectar negativamente al fitness viral, haciendo que la capacidad replicativa del virus disminuya de manera considerable. Estudios previos indican que el adenovirus utiliza la desviación en su uso de codones como mecanismo para optimizar el reparto de los recursos traduccionales de la célula. En base a este hecho, hemos hipotetizado que la expresión de transgenes terapéuticos podría afectar al equilibrio en el 5 uso de codones del genoma adenoviral, y conducir a un fenómeno de competición intergénica por dichos recursos. A fin de determinar el impacto del uso de codones de los transgenes armados en el genoma adenoviral, hemos estudiado cómo un mismo transgén con distintos patrones de uso de codones afecta a la expresión de las proteínas virales y a la producción de nuevos viriones. Nuestros datos sugieren que los transgenes, cuando se expresan en la fase tardía pos- infección, compiten con los genes virales por los recursos traduccionales, afectando el fitness viral de manera dependiente al uso de codones. Transgenes altamente optimizados, secuestrarían los recursos traduccionales de la célula en detrimento de los genes virales, conduciendo así a una replicación viral deficiente. No obstante, hemos demostrado que es posible rescatar la actividad viral mediante la modulación del grado de optimización de los transgenes. Estos resultados se han evidenciado en adenovirus armados con genes codificantes para la proteína reportera verde fluorescente o GFP, así como con dos transgenes terapéuticos, la enzima hialuronidasa (que degrada la matriz celular y reduce la desmoplasia tumoral) y la enzima LmPDT (que cataliza la conversión de prodrogas en bases púricas citotóxicas). En conjunto, concluimos que adaptar el uso de codones de los transgenes armados en el genoma adenoviral es un parámetro crítico que debería considerarse en el diseño de adenovirus oncolíticos armados a fin maximizar el beneficio terapéutico de los mismos.

O Epstein-Barr vírus (EBV) é a única espécie humana pertencente ao gênero Lymphocryptovirus. A transmissão ocorre através da saliva contaminada e geralmente ainda na infância. Nosso estudo analisou 165 amostras clínicas de pacientes, portadores de HIV, em tratamento com antiretrovirais, atendidos no Sistema Hospitalar do Sistema Penitenciário do Estado de São Paulo. Nosso enfoque foi pesquisar o EBV nas células mononucleares do sangue periférico, através das técnicas de PCR, Nested-PCR e seqüenciamento de nucleotídeos. Os resultados obtidos, indicaram que 11,51% (19) das amostras analisadas, apresentaram-se positivas para o EBV. Essas 19 amostras, foram seqüenciadas com primers específicos para a região da EBNA-1 (Epstein Barr Nuclear Antigen 1). As amostras foram alinhadas com o auxílio do DNASTAR. Ao alinharmos as amostras, encontramos uma troca de base (de G para A) em 7 amostras e essa troca não alterou a conformação da proteína EBNA-1. Na análise filogenética de nossas sequências com as depositadas no GenBank, foi possível observar dois grupos, que representam tipo 1 e o tipo 2 do EBV. 100% das amostras estudadas por nós foram identificadas como pertencentes ao grupo que caracteriza o tipo 2. Sendo assim, as 7 amostras que apresentaram a troca sugerem a origem um novo subtipo.
The Epstein-Barr Virus (EBV) is the only species to the genus Lymphocriptovirus that infects humans. One of the possible route for its transmission thought by contamined saliva and usually occurs in the childhood. This study analysed 165 clinical samples from HIV infected patients, treated by HARRT, attended in the Hospitalar System in the Penitentiary System from Sao Paulo State. The aim of this study was to search EBV in peripheral blood mononuclear cells by PCR, Nested-PCR and sequencing analysis. The results showed 11,51% of the analysed samples, positive for EBV. This samples, was sequenced with specifics primers from the EBNA-1 (Epstein Barr Nuclear Antigen 1) region. The samples were aligned by DNASTAR program. The aligned sequences showed the base conversion G to A in seven samples. This conversion caused no alteration in the EBNA-1 protein conformation. In the phylogenetic analysis the studied sequences with the sequences from GenBank was possible to observe two groups represented with type 1 and type 2 from EBV. 100% the samples studied was identified with the group characterized by the type 2 to EBV. So the seven samples showed the conversion, suggesting the origin of the one new subtype.

Novel human astrovirus (HAstV) are enteric virus belonging to the Astroviridae family and were discovered 10 years ago by high-throughput sequencing. They are divided in two different clades, the HAstV-MLB including 3 genotypes (MLB1-3) and HAstV-VA including 5 genotypes (VA1-5). While their role during gastroenteritis is debated, they have been reported as the sole agent identified in many cases of severe central nervous system infection, mainly in immunocompromised patients. This suggests that these emerging and highly divergent viruses can be associated with serious clinical manifestations, requiring additional basic and epidemiological studies to better understand their pathogenesis, prevalence and clinical correlation. We implemented several cell culture systems allowing the propagation of two distinct genotypes of novel HAstV from clinical stool samples, namely HAstV-MLB1 and HAstV-MLB2. Both strains could efficiently replicate in human HuH-7 hepatoma and A549 respiratory cell lines. In addition, both strains could establish a persistent infection over several cell passages in both cell lines, and HAstV-MLB1 could also establish a persistent infection in HuH-7.5 cells. In the latter, electron microscopy revealed a high production of capsid arrays and significant intracellular reorganization. Immunofluorescence assays revealed only a low proportion (5-10%) of infected cells. We explored the innate immune response to HAstV-MLB infection and observed that IFN expression was either abolished or delayed and diminished, depending on the cell line, during acute infection. During persistent infection, IFN expression was abolished in all cases, while when co-stimulated with poly I:C, IFN expression remained inhibited in a cell and genotype-dependent manner. Addition of exogenous IFN led to the cure (IFN-β) and relative inhibition (IFN-λ) of HAstV-MLB infection in HuH-7 cell line, while there was no effect in A549 infected cell line. At the epidemiological level, using a sensitive and specific real-time RT-PCR assay, we found that novel HAstVs could be identified in 6-10% of cases of undiagnosed gastroenteritis in Spanish pediatric children of < 5 years old. Together with a Japanese study, our prevalence is the highest observed to date. Children under 2 years old had a higher prevalence rate, compared to older ones. HAstV-MLB1 and HAstV-VA1 accounted for 31% and 26% of all novel HAstV observed in our cohort, while no HAstV-MLB3 were detected. Nevertheless, we found a high rate of co-infection with other enteric viruses (66%), precluding to assess a firm association between the presence of novel HAstV and the occurrence of gastroenteritis in such cases. We could not identify differences in the mean Cq values between mono- and co-infection episodes. We then performed a case-control study to assess the role of novel HAstV in gastroenteritis. We found a prevalence of 6.3% and 4% in symptomatic and asymptomatic children, respectively, without statistical difference between groups. However, we found that asymptomatic children had statistically significant higher HAstV-MLB viral load (median 6.52 log10 genomes/ml, IQR 4.52-6.84) compared to symptomatic children (median 2.35 log10 genomes/ml, IQR 2.13-3.76). Similarly, in symptomatic patients, we observed a higher viral load when novel HAstVs were found in coproculture-positive (median 5.19 log10 genomes/ml, IQR 4.24-6.22) compared to coproculture-negative (median 2.31 log10 genomes/ml, IQR 2.11-3.32) stool samples. These findings suggest that novel HAstV are not associated with gastroenteritis, but could modulate the gut microbiome and may confer protection to invading pathogens, although the mechanism remains to be elucidated.
Los astrovirus humanos (HAstV) no clásicos son virus entéricos emergentes que pertenecen a la familia de los Astroviridae, la cual incluye virus asociados a gastroenteritis principalmente en la población pediátrica. Se descubrieron por primera vez hace 10 años mediante secuenciación masiva, y hoy se dividen en dos grupos filogenéticos distintos: los HAstV-MLB (MLB1-3), y los HAstV-VA (VA1-5). Su asociación con gastroenteritis no está del todo confirmada, y también han sido identificados en casos de meningoencefalitis en pacientes inmunodeprimidos. En nuestro trabajo hemos implementado varios sistemas de cultivo celular permisivos para la replicación de dos genotipos de HAstV-MLB, HAstV-MLB1 y HAstV-MLB2, utilizando muestras clínicas. Ambos genotipos pueden replicar en las líneas celulares humanas HuH-7 y A549, de hepatoma y tejido respiratorio, respectivamente. Además, ambos pueden establecer una infección persistente en el cultivo, detectándose señal positiva por inmunofluorescencia en 5-10% de las células. La microscopía electrónica identifica una gran cantidad de cápsides víricas dentro de las células infectadas, y una importante reorganización intracelular. En los cultivos persistentemente infectados no se detecta inducción de la respuesta interferón (IFN), y la capacidad de los virus para bloquear la expresión de IFN inducida por poliI:C es distinta para cada tipo celular. La sensibilidad frente a un tratamiento exógeno tanto con IFN-β como con IFN-λ, es efectivo en las células HuH-7 pero nulo en las células A549. En nuestros estudios epidemiológicos en niños menores de 5 años con gastroenteritis no diagnosticada, se detectaron HAstV no clásicos en 6-10% de los casos. MLB1 y HAstV-VA1 representaron el 31% y el 26% de todos ellos, respectivamente. Se detectó co-infección con algún otro virus entérico en 66% de las muestras positivas, y no se observaron diferencias en los valores Cq entre casos de mono- y co-infección. En un estudio de casos y controles, su prevalencia fue similar en ambos grupos (6.3% versus 4%, respectivamente). No obstante, se observó que el promedio de carga vírica en los casos asintomáticos fue significativamente superior que en los niños enfermos, y en pacientes sintomáticos, se observó una carga viral mayor en aquellas heces que eran positivas para coprocultivo en comparación con las negativas.

O CMV é o principal agente de infeção congénita, atingindo cerca de 0,2 a 2,2% de todos os recém-nascidos. Este agente exibe uma imensa variabilidade genética principalmente nos genes que codificam glicoproteínas estruturais do invólucro. A mais relevante é a glicoproteína B (gB) do vírus, codificada pelo gene UL55, sendo um importante alvo do sistema imunitário do hospedeiro humano. Com base nas variações de sequências deste gene, o vírus pode ser classificado em pelo menos, 4 genótipos distintos (gB1-4). O objetivo do presente estudo foi determinar quais os genótipos para o UL55, presentes em amostras de casos de infeção congénita e/ou perinatal utilizando a técnica de PCR em tempo real. A confirmação dos resultados foi realizada por técnicas de sequenciação (Sanger e Next-Generation Sequencing) e por análise do polimorfismo do tamanho dos fragmentos de restrição (RFLP). No total, foram analisadas 36 amostras de urina e 20 amostras de líquido amniótico (LA), colhidos entre 2009 e 2016. Das 35 amostras de urina genotipadas, em 29 foi detetado um genótipo (13 gB1; 7 gB2; 6 gB4; 3 gB3), sendo em 6 detetado mais do que um genótipo. Das 19 amostras de LA, em 17 foi detetado um genótipo (5 gB1; 5 gB2; 5 gB3; 2 gB4) e em 2 foi detetado mais do que um genótipo. Não houve amplificação em 2 amostras (1 urina e 1 LA). Recorrendo às diferentes técnicas de confirmação, não foi possível confirmar a presença de infeções mistas nem de 5 gB4 detetados em amostras de urina. A nível nacional, o gB1 parece ser o genótipo mais frequente na infeção congénita, em concordância com o descrito na literatura. Corroborámos também a ideia de que todos os genótipos estão envolvidos neste tipo de infeção. Por PCR em tempo real foi possível assinalar a existência de infeções mistas, no entanto, estes dados devem ser alvo de melhor análise, dadas as contradições, principalmente em comparação com a sequenciação.
Cytomegalovirus (CMV) is the main congenital infection agent, affecting about 0.2 to 2.2% of all newborns. This pathogen exhibits extensive genetic variability mainly in structural genes encoding envelope glycoproteins. The most relevant is the CMV glycoprotein B (gB), encoded by the UL55 gene, is an important target of the imune system of the human host. On the basis of sequence variation of this gene, the virus can be classified, at least into 4 gB genotypes (types 1-4). The aim of this study was to determine the genotypes for UL55, present in samples from cases of congenital and/or perinatal infection using real-time PCR. Confirmation of the results was performed by sequencing techniques (Sanger and Next-Generation Sequencing) and restriction fragment length polymorphism(RFLP). Overall, we analyzed 36 urines and 20 amniotic fluids (LAs), collected from 2009 to 2016.Thirty-five urine samples could be assigned a gB genotype, in 29 was detected single genotype (13 gB1; 7 gB2; 6 gB4; 3 gB3). The presence of mixed genotypes was also detected in 6 urines. Of the 19 LAs, 17 has detected a single genotype (5 gB1; gB2 5, 5 gB3; two gB4). In 2 was detected mixed genotypes. No amplification was detected in other 2 samples (1 urine and 1 LA). Resorting to differenttechniques of confirmation was not possible to confirm the presence of mixed infections, neither 5 gB4 detected in urine samples. At the national level, the gB1 seems to be the most common genotype in congenital infection, consistent with that described in the literature. Also we corroborate the notion that all genotypes can be involved in this type of infection.For real-time PCR was also possible to note the existence of mixed infections, however, these date should be subjected to further analysis, given the apparent contradiction, in comparison to sequencing techniques.

L'une des principales barrières à la prise en charge optimale des patients sous traitement antirétroviral est l'accès limité aux tests de charge virale (CV) et de génotypage particulièrement en milieu décentralisé. Ces tests ne sont généralement disponibles qu'au niveau des structures sanitaires centrales de grandes villes et le plasma en est l'échantillon de référence. Or, son transfert des régions périphériques vers les laboratoires de références est difficile, voire impossible. Pour rapprocher les patients du laboratoire, nous avons démontré la possibilité d'assurer un suivi virologique complet (CV et génotypage) à partir des DBS collectés et acheminés dans des conditions de terrain. Nous avons également pour la première fois, documenté la réponse virologique au traitement antirétroviral et la diversité génétique du VIH-1 chez des patients adultes suivis en milieux décentralisés au Sénégal, au Mali et en Guinée Conakry. Globalement, malgré les défauts d'observance au traitement souligné, les résultats de nos travaux ne montrent pas de différences significatives dans la survenue de l'échec virologique entre patients suivis dans les structures sanitaires centrales et périphériques, ceci quelque soit le pays considéré. Au Sénégal, chez les enfants nés de mères séropositives, la résistance vis à vis des INNTI était plus prépondérante, probablement du fait de l'utilisation systématique de la Névirapine durant la PTME. Par ailleurs, aucune mutation de résistance aux inhibiteurs d'intégrase n'a été observée malgré des taux de résistance élevés chez des patients en échec de première et deuxième ligne de traitement. Nos travaux confirment également une grande diversité génétique des sous-types viraux avec cependant la prédominance du CRF02_AG dans la sous région Ouest Africaine. Ces travaux de thèse mettent en évidence la faisabilité et la pertinence du DBS comme support pour le suivi virologique des patients en milieux décentralisés. Son utilisation a permis de montrer d'autre part des taux d'échecs virologiques élevés indiquant la nécessité de renforcer l'adhérence au traitement. Enfin, nos résultats soulignent l'utilité de prendre davantage en considération les profils de résistance pour initier un traitement de relais
One of the major barriers to the optimal care of patients undergoing antiretroviral therapy is the limited access to viral load (VL) and genotyping tests, especially in remote areas. These technologies are usually available only at central health facilities in larger cities and plasma is the reference sample. However, plasma or whole blood samples shipment from remote areas to reference lab faces several constraints or even impossible. In order to bring closer patients to reference lab, we have demonstrated the ability of DBS (Dried Blood Spots) collected and shipped in field conditions to provide complete virological monitoring (VL and genotyping). We also documented for the first time, virological outcome of ART and HIV-1 genetic diversity in adult patients followed up in decentralized settings in Senegal, Mali and Guinea Conakry. Overall, despite the low treatment adherence noted sometimes, our findings show no significant differences in the occurrence of virological failure among patients followed up in the central and peripheral health facilities, whatever the country. In Senegal, no integrase inhibitors associated DRM has been found despite the high rate of resistance in patients failing first and second-line treatment. Furthermore, among children born to HIV infected mothers, NNRTI-associated drug resistant mutations (DRM) were more predominant, probably because of systematic use of Nevirapine in MTCT. Our studies also confirm the high genetic diversity of viral subtypes, with the dominance of CRF02_AG in West Africa. This work presented here highlights the feasibility and relevance of DBS as support for the virological monitoring of patients in decentralized settings in West Africa. Furthermore, its use showed high rate of virological failure indicating the need to reinforce adherence to treatment. Finally, our results highlight the utility to considering carefully drug resistance patterns before switching to another ART regimen

O vírus citomegálico humano (CMV) é o principal agente de infeção congénita. Em Portugal, os estudos publicados apontam para uma prevalência desta infeção entre 0,7% e 1,1%. A importância do rastreio desta infeção é reconhecida desde há vários anos, mas as condições para a sua realização, de forma que seja técnica e economicamente viável, ainda não estão reunidas. A metodologia de pools de urina descrita por uma equipa portuguesa, revelou uma correlação total com os resultados obtidos pelo método de referência, a cultura celular, e permite uma redução bastante significativa, quer nos tempos de execução quer nos custos em reagentes, abrindo assim a possibilidade efetiva de utilizar esta técnica para o rastreio da infeção congénita. Este estudo tem como primeiro objetivo rastrear recém-nascidos do Hospital da Luz e Maternidade Alfredo da Costa num determinado período de tempo, no sentido de determinar a prevalência da infeção congénita por CMV nessa população. O rastreio tem como base a utilização de pools (20 urinas) e a deteção de DNA viral por PCR em tempo real. As 20 urinas de cada pool positiva são posteriormente testadas individualmente. O segundo objetivo deste trabalho foi a deteção de carga viral de CMV, por PCR em tempo real, a partir de uma fralda com urina CMV positiva. Como resultados, obtiveram-se 45 pools, 4 delas positivas com uma urina positiva em cada pool, sendo a prevalência deste rastreio 0,44%. Esta metodologia confirmou a sua utilidade para um rastreio universal de infeção congénita por CMV, no entanto verificaram-se dificuldades na aplicação da mesma, o que deverá ser tomado em consideração na implementação de um eventual programa de rastreio. Os resultados obtidos na extração de urina CMV positiva através de fraldas de recém-nascidos foram muito promissores, o que abre a possibilidade da sua utilização para o diagnóstico da infeção congénita.
The human cytomegalovirus (CMV) is the main agent of congenital infection. In Portugal, this infection has shown a prevalence of 0.7 to 1.1%. The importance of the screening of this infection is well recognized for several years now, but the economic viability of its usage is not yet assured. The urine pool technique, described by a Portuguese team, revealed a total correlation with the results obtained by the gold standard method (cell culture) and allows a very significant reduction of the time and cost of the procedure. Therefore, this technique could be used for congenital CMV screening. The main purpose of this study was the screening of congenital CMV infection in newborns from Hospital da Luz and Maternidade Alfredo da Costa in a specific period of time and the determination of its prevalence. The screening is based on the analysis of pools made of 20 urines and viral DNA detection by real time PCR. In the positive pools, the 20 samples were then individually tested to determine which one was positive. The second purpose of this study was the detection and quantification of viral CMV DNA by real time PCR from a diaper with positive urine for CMV. In our screening, we analysed 45 pools of which 4 were positive with one positive urine each and we concluded that the prevalence of congenital CMV infection was 0,44%. With these results, we were able to show that this technique can be used for universal screening but we also verified some difficulties in its application which should be considered in future studies. The results obtained from the extraction of the urines collected from the diapers were very promising, showing that this technique could be used for congenital CMV infection diagnosis.

O vírus da hepatite delta (HDV) é o agente patogénico responsável por uma das formas mais severas de hepatite viral. O genoma consiste numa molécula circular de RNA de cadeia simples de polaridade negativa e apresenta uma única proteína viral, o antigénio delta pequeno (S-HDAg). Na sequência de um mecanismo de editing outra proteína viral é traduzida, o antigénio delta grande (L-HDAg). Apesar de partilharem grande parte da sua sequência, as duas proteínas desempenham funções distintas. O S-HDAg é essencial para a acumulação de RNAs virais enquanto o L-HDAg inibe a replicação viral e é necessário para o empacotamento. O HDV depende extensivamente de factores do hospedeiro para completar o seu ciclo de replicação. Pensa-se que a polymerase II (pol II) do hospedeiro é redireccionada para transcrever o RNA viral. No presente trabalho procurou-se caracterizar o S-HDAg e clarificar o seu papel no ciclo de replicação do HDV. Observamos que quando o S-HDAg é expresso na presença de replicação do RNA viral, o antigénio co-localiza com a pol II. Contudo, a co-localização com pol II verifica-se mesmo na presença de RNA viral incapaz de ser replicado e na ocorrência de inibição da replicação viral, sugerindo que o S-HDAg não participa directamente na transcrição do RNA viral. Assim, propomos que o S-HDAg é essencial para acumulação de RNAs virais protegendo ou estabilizando os RNAs. Observamos ainda que na ausência de RNA viral o S-HDAg co-localiza com a nucleolina nos nucléolos. Contudo, na presença de RNA incapaz de ser replicado, o antigénio desloca-se para o nucleoplasma mantendo-se a nucleolina nos nucléolos, sugerindo que o S-HDAg não interage directamente com a nucleolina. Ao estudarmos as características estruturais do S-HDAg verificámos, utilizando um preditor de desordem intrínseca, que apresenta um elevado grau de desordem. A previsão foi confirmada in vitro por dicroísmo circular observando-se que apenas 30% dos amino ácidos adoptam uma conformação de hélice . A ausência de uma estrutura rígida pode conferir ao antigénio flexibilidade para se adaptar a diferentes parceiros e participar em vários passos do ciclo de replicação viral. A multimerização do S-HDAg foi analisada por dispersão de luz dinâmica. Os resultados indicam que o antigénio recombinante purificado é capaz de formar multímeros de 12 moléculas. Adicionalmente, foram observados multímeros de seis a oito moléculas em gel de poliacrilamida desnaturante, após cross-linking. Os mesmos multímeros foram observados para S-HDAg presente em partículas virais sugerindo que a multimerização do antigénio ocorre in vivo. Finalmente, estudamos a capacidade do S-HDAg interagir com ácidos nucleicos. Verificamos que multímeros e monómeros de S-HDAg são capazes de interagir in vitro com RNA e DNA. A falta de especificidade observada pode dever-se apenas a interacções electrostáticas entre o S-HDAg de carga positiva (+12) e ácidos nucleicos de carga negativa. Propomos que, in vivo, a fosforilação extensiva do S-HDAg reduza a carga positiva contribuindo para que a interacção seja específica para os RNAs virais.
Hepatitis delta virus (HDV) is the causative agent of one of the most severe forms of viral hepatitis. It has a small single-stranded circular RNA genome of negative polarity and only one viral protein, the small delta antigen (S-HDAg). Following site-specific RNA editing, a second longer protein is translated, the large delta antigen (L-HDAg). Although these viral proteins share most of their sequence they play distinct roles. S-HDAg is essential for the accumulation of HDV RNAs whereas L-HDAg inhibits HDV replication and is necessary for viral assembly. With such a limited coding capacity HDV must rely extensively on host cell components to complete its replication cycle. The host DNA-directed RNA polymerase II (pol II) is thought to be re-directed to transcribe HDV RNAs. The objective of this study was to further characterize S-HDAg and clarify its role(s) during the HDV replication cycle. We observed that when S-HDAg was expressed in vivo along with replicating HDV RNA it co-located with host pol II. However, such co-localization was also observed in the presence of non-replicating HDV RNAs or when replication was inhibited by specific doses of -amanitin. Thus, we propose that S-HDAg is essential for HDV RNA accumulation by stabilizing or protecting the viral RNAs rather than acting as a direct player in HDV RNA transcription. Additionally, we observed that S-HDAg located in nucleolus when expressed in the absence of HDV RNA, and co-located with host nucleolin. However, in the presence of non-replicating HDV RNAs, S-HDAg moved to the nucleoplasm whereas nucleolin was unchanged. This suggests that S-HDAg is not interacting directly with nucleolin. In our examination of S-HDAg‟s structural features we applied a meta-predictor of intrinsic disorder, PONDR-FIT. It predicted that full-length S-HDAg has extensive intrinsic disorder. This result was confirmed in vitro by circular dichroism measurements that indicated no more than 30% of S-HDAg amino acids adopted an -helical structure. Such a lack of a well-defined rigid structure is expected to grant flexibility to the antigen allowing it to interact with several partners and perform distinct roles during the HDV replication cycle. Protein multimerization was studied by dynamic light scattering. Data analysis indicated that purified recombinant S-HDAg was able to assemble into homomultimers as high as dodecamers. Similarly, denaturing polyacrylamide gel electrophoresis with prior cross-linking indicated formation of at least hexamers and octamers. Similar multimers were observed for S-HDAg present in virus-like particles indicating that S-HDAg multimerization also occurs in vivo.Finally we examined the ability of S-HDAg to bind nucleic acids in vitro. Both multimers and monomers bound to conformations of both RNA and DNA. Such a lack of specificity was probably due to electrostatic interactions between the positively-charged S-HDAg (+12) and negatively-charged nucleic acids. We propose that in vivo, extensive post-translational phosphorylation of S-HDAg reduces the positive charge, thereby contributing to interactions more specific for HDV RNAs and possibly dependent upon protein multimerization. Despite our observations presented here, some issues relating to our aims remain unresolved.

A resistência aos antibióticos em bactérias Gram-negativas pode ser aumentada pela extrusão de antibióticos através de sistemas de efluxo. Em Escherichia coli, o principal sistema de efluxo é o AcrAB-TolC o qual tem como principal fonte energética a força proto-motriz. Este trabalho pretendeu estudar alguns aspectos essenciais da bioenergética na actividade de efluxo de E. coli usando três estirpes bem caracterizadas genotipica e fenotipicamente. Foi utilizado um método fluorimétrico semi-automático no qual a fluorescência do fluorocromo brometo de etídeo, substrato de bombas de efluxo foi seguida, permitindo a medição em tempo real da actividade de efluxo e acumulação de fluorocromo (inibição do efluxo). A utilização de brometo de etídeo é particularmente vantajosa pois emite baixa fluorescência no exterior da célula bacteriana tornando-se extremamente fluorescente no seu interior. Este método é uma nova aplicação do termociclador em tempo real RotorGeneTM 3000 que permite o cálculo da cinética de transporte reflectindo o balanço entre acumulação de substrato por difusão passiva através da membrana e a sua extrusão/efluxo, proporcionando uma detecção rápida e económica de inibidores de efluxo. Os resultados obtidos mostram, para todas as estirpes, que a GLU e o pH afectam a acumulação e o efluxo do brometo de etídeo. De todos os inibidores de vias biossintéticas testados, o ortovanadato de sódio, foi o que demonstrou maior actividade inibitória, a qual é revertida na presença de GLU. Em conclusão, este estudo mostra que a actividade de efluxo de E. coli depende não só da fosforilação oxidativa por via da força proto-motriz mas também da energia proveniente da hidrólise de ATP pelas ATPases. O ortovanadato de sódio tem potencial para ser um novo inibidor de bombas de efluxo de largo espectro. A tecnologia utilizada neste trabalho demonstrou ser apropriada para a caracterização bioenergética da actividade de bombas de efluxo e permite a selecção de novos inibidores de bombas de efluxo em bactérias.
Antibiotic resistance in Gram-negative bacteria can be increased by extrusion of the antibiotic through efflux systems. In Escherichia coli, the major efflux pump system is the AcrAB-TolC which is mainly driven by energy coming from the proton motive force. In this work was studied basic aspects of the bioenergetics of the efflux activity of E. coli using three genotipically and phenotipically well characterized strains. It was used the semi-automated fluorimetric method that utilizes the fluorochrome ethidium bromide, a known fluorescent efflux pump substrate, which allows the real time measurement of efflux activity and efflux inhibition (accumulation). Ethidium bromide has been shown to be particularly suitable to be used as a probe because it emits weak fluorescence outside the bacterial cell and becomes strongly fluorescent inside the cell. This method is a new application of the RotorGeneTM 3000 real-time thermocycler and provides the sum of transport kinetics reflecting the balance between accumulation of substrate via passive diffusion through the membrane permeability barrier and extrusion via efflux, thereby offers a rapid and inexpensive screening of inhibitors. The results obtained show, for all strains, that GLU and pH affects the accumulation and efflux of ethidium bromide. From all the inhibitors of energy biosynthetic pathways tested, sodium orthovanadate was the one that revealed the highest inhibitory activity and this inhibitory effect was reversed by the presence of GLU in the medium. In conclusion, this study shows the dependence of the efflux activity of E. coli on energy from the hydrolysis of ATP by the ATPases, besides the already known dependence on the oxidative phosphorylation, to maintain the proton motive force of the cells. Sodium orthovanadate has potential to be a new broad range efflux-pump inhibitor. The technology used in this work showed to be suitable for the characterization of the bioenergetic requirements of the bacterial efflux pump activity, allowing the screening of new efflux pump inhibitors.

A potential strategy to produce safer and broadly protective influenza vaccines is to co-express, in the same cell host, multiple hemagglutinins (HA) with a matrix protein (M1) which self-assemble in virus-like particles (VLPs). This study demonstrates the suitability of combining stable expression and the baculovirus-expression vector system (BEVs) in insect Hi5 cells for production of such multi-HA Influenza VLPs. Stable pools of Hi5 cells expressing two HAs were generated and later infected with a M1-encoding baculovirus at two cell concentrations (CCIs; 2×106 cells/mL and 3×106 cells/mL). The HA concentration in culture supernatant was followed over time, with more productive infections observed at higher CCIs. To extend the culture time, a re-feed strategy was implemented based on the identification of key nutrients which were exhausted during cell growth. Afterwards, supplemented cultures infected at a CCI of 4×106 cells/mL allowed a 4-fold increase in HA concentration, at harvest, when compared to cultures infected at a CCI of 2×106 cells/mL. The production of multi-HA influenza VLPs using the aforementioned strategy could be successfully scaled-up to 2L bioreactor cultures with even higher volumetric (1.5-fold) HA yields. To surpass the unpredictability of gene expression promoted by the random integration strategy mentioned above, the recombinase-mediated cassette exchange (RMCE) technology was explored. The feasibility of having two cassettes flanked by distinct pairs of flippase recognition target sites (FRTs) was evaluated. Unfortunately, significant cross-interaction was observed between the selected pairs. To circumvent this bottleneck, a backup strategy consisting in the co-expression of two genes from the same locus after implementation of one cassette system, in insect Sf9 cells, was attempted. However, the isolated clones showed low expression of both M1 and HA proteins. Ongoing work focuses on the isolation of clones tagged in high expression loci by fluorescence activated cell sorter technology. This work demonstrates how the versatility of insect cell expression technology can be explored to produce Influenza VLPs as vaccine candidates.
A co-expressão de várias hemaglutininas (HA) e proteína da matriz (M1), no mesmo hospedeiro, formando partículas semelhantes a vírus (VLPs), constitui uma importante estratégia para desenvolver vacinas contra o vírus da gripe. Este trabalho mostra a combinação de uma linha celular estável de células de insecto com o sistema de expressão mediada por baculovírus para a produção deste tipo de VLPs. Foram estabelecidas duas populações de células de insecto Hi5, expressando duas HAs, posteriormente infectadas com um baculovírus contendo a proteína M1, a duas concentrações celulares diferentes (CCI; 2 e 3×106 cells/mL) sendo que a mais elevada demostrou ser a mais produtiva. De seguida, implementou-se uma estratégia baseada na adição de nutrientes específicos para prolongar o tempo de cultura. As culturas previamente suplementadas e infectadas a uma CCI de 4×106 células/mL produziram 4x mais HA comparativamente às culturas infectadas a uma CCI de 2×106 células/mL, não suplementadas. Esta estratégia foi também aplicada num biorreactor de 2L permitindo 1,5x mais produção, volumétrica, de HA comparativamente a experiências em pequena escala. De forma a ultrapassar a imprevisibilidade de uma integração aleatória, foi explorado o sistema de troca de cassete mediado por recombinase (RMCE). A viabilidade de um sistema com duas cassetes integradas flanqueadas por diferentes locais de reconhecimento (FRTs) foi avaliada, tendo sido observada a interação entre ambos os pares selecionados. Como segunda estratégia, foi implementado um sistema com uma cassete para co-expressão de dois genes em simultâneo, em células de insecto Sf9. Porém, os clones isolados mostram fraca expressão de M1 e HA, pelo que uma estratégia de isolamento de clones com expressão génica mais forte está em desenvolvimento utilizando uma tecnologia de sorteamento. Assim, este trabalho demonstra a versatilidade da tecnologia aplicada em células de insecto, que pode ser explorada para produzir VLPs multivalentes, com potencial para se tornar a próxima geração de vacinas para o vírus da gripe.

A maioria dos métodos utilizados na caracterização genética do HIV-1 baseia-se na análise de regiões específicas do genoma viral, fornecendo informação parcial sobre o mesmo e, por consequência, revelando-se inadequados para a identificação de vírus recombinantes. O único método que permite uma caracterização integral do genoma viral passa pela sua sequenciação completa. No entanto, este é um método dispendioso, laborioso e de difícil implementação quando se pretende a análise de elevados números de amostras. Como alternativa a este último, o conjunto de métodos genericamente designados de MHA (Multiple Region Hybridization Assay) baseiam-se na amplificação, por PCR em tempo-real, de várias regiões ao longo do genoma viral e na sua caracterização com sondas específicas (TaqMan). Tendo este modelo por base, o objectivo deste estudo foi o desenvolvimento de um ensaio de hibridação múltipla (MHABG0214) passível de ser aplicado ao estudo de um elevado número de amostras. Este método foi desenvolvido tendo como objectivo a genotipagem as estirpes circulantes dominantes na epidemia Portuguesa, nomeadamente os subtipos B, G e formas genéticas recombinantes CRF02_AG e CRF14_BG. Com base em alinhamentos de sequências de referência de genoma completo, delinearam-se primers universais e subtipo-específicos para a amplificação de diversas regiões codificantes distribuídas ao longo do genoma do HIV-1 (Gag, Protease, Transcriptase Reversa, Integrase, Rev, Gp120 e Gp41). A optimização foi efectuada, inicialmente, para um conjunto de amostras de referência e seguidamente avaliada num conjunto de 50 amostras clínicas. O MHABG0214 foi implementado numa estratégia de PCR em tempo-real, numa detecção dependente de SYBR® Green I para todas as regiões ou, como alternativa, usando sondas TaqMan (Gp41). Apresentamos ainda uma estratégia em que a análise de resultados se baseia, simplesmente, numa abordagem usando PCR/gel de agarose convencional. Estas abordagens constituem ferramentas úteis na identificação das estirpes de HIV-1 em Portugal.
Since most methods used for the genetic characterization of HIV-1 are based on the analysis of singular regions of the viral genome, they only provide a fragmented view of the later, and often fail to identify recombinant viruses. The most efficient method for HIV-1 genetic characterization involves full-genome sequencing, but the associated costs and low throughput preclude it from being routinely used for the analysis of large numbers of viral strains. One of the alternatives for a consistent genetic analysis of large numbers of viral strains are the methods generally known as Multi Region Hybridization Assays (MHA). MHA relies on the amplification by real-time PCR of several regions scattered along the HIV-1 genome, and their characterization subtype-specific TaqMan probes. In this context, the aim of our study was the development of a new multi-region hybridization assay (MHABG0214) for genotyping of the major HIV-1 forms circulating in Portugal, subtypes B, G and CRF02_AG and CRF14_BG circulating recombinant viruses. Based on full alignments of representative HIV-1 reference sequences, we designed universal and subtype-specific primers/probes for the amplification of several different coding regions of the viral genome (Gag, Protease, Reverse Transcriptase, Integrase, Rev, Gp120 and Gp41). Optimization of reaction conditions, established using 7 HIV-1 references, served as a starting point for the analysis of 45 HIV-1 strains circulating in Portugal. MHABG0214 was implemented using a real-time PCR-based approach, with detection dependent on the use of either SYBR® Green I (all regions) or a TaqMan probe (Gp41). Alternatively, a technically less demanding strategy based on conventional PCR and agarose gel analysis of reaction products was also developed. Regardless of the strategies used and the results obtained, these approaches are useful tools to identify strains of HIV-1 in Portugal.

Os jovens são um grupo onde a prevalência e a incidência das Infecções Sexualmente Transmissíveis (IST) é cada vez maior. Este facto parece dever-se a um grupo de factores determinantes, entre os quais se destaca o comportamento de utilização do preservativo, enquanto único método capaz de evitar a transmissão das IST. A resolução do problema passa pela implementação de programas de prevenção e controlo ajustados à população juvenil. No entanto, a investigação dos factores determinantes das IST que estão na base destes programas tem sido escassa em Portugal. O presente estudo do tipo observacional, analítico e transversal incidiu sobre a totalidade dos 1101 alunos de uma escola da periferia de Lisboa e teve como objectivos: descrever alguns conhecimentos e factores sociodemográficos, escolares e comportamentais determinantes das IST; averiguar se existem diferenças atribuíveis à idade e ao sexo nestes factores determinantes; averiguar se estes factores determinantes se encontram associados à utilização do preservativo. Na colheita dos dados foi utilizado um questionário de auto-preenchimento, desenvolvido para o efeito, e no tratamento dos mesmos recorreu-se à estatística descritiva e analítica. Os resultados obtidos no presente estudo demonstram que a principal fonte de informação sobre sexualidade dos alunos são os amigos, mas estes preferiam que fossem os profissionais de saúde. Para além dos vírus da imunodeficiência humana e do herpes genital, a identificação de outras IST foi insuficiente. Os conhecimentos sobre a transmissão sexual das IST foram satisfatórios, mas não se repercutem na utilização do preservativo pelos alunos durante as relações não coitais. O sexo e a idade dos alunos encontram-se significativamente associados a fontes de informação sobre sexualidade e a algumas dimensões do conhecimento sobre as IST, do comportamento sexual e da procura de tratamento médico. Todavia, a maioria destes factores determinantes não está associada à utilização do preservativo pelos alunos.
The prevalence and incidence of sexually transmitted infections (STI) is increasing in young people. This seems to happen as a consequence of multiple factors, among which is condom use behaviour, was the only safe method to prevent STI transmission. One of the solutions for the problem is the implementation of prevention and control programs youth oriented. Yet, research on STI determinants, the rational for these programs, has been scarce in Portugal. This observational, analytical and cross sectional study, surveyed all the 1101 students at a school in the suburban Lisbon area. It aimed at: describing knowledge, demographic, scholar and behavioural determinant factors of STI; to determine differences between age group and gender and which of the factors are associated with condom use. Data was collected using a structured questionnaire developed for this study. Data analysis was conducted using both descriptive and analytical statistics. The results indicated that the main source of information on sexuality are student’s friends, but they would rather prefer to be informed by health professionals. Apart from the human immunodeficiency virus and genital herpes, knowledge of other STI was insufficient. Knowledge about SIT sexual transmission was satisfactory, but does not lead to condom use during non-coital relations. Sex and age of the students are significantly related to different sources of information on sexuality and to some dimensions of STI knowledge, sexual behaviour and the seeking for medical treatment. Nevertheless, the majority of these determinant factors are not related to condom use by these students.

Orientador: Marilia de Freitas Calmon
Coorientador: Paula Rahal
Coorientador: Laura Sichero
Banca: Isabel Maria Vicente Guedes de Carvalho-Mello
Banca: Enrique Mario Boccardo Pierulivo
Resumo: A Papilomatose Respiratória Recorrente (PRR) é uma doença caracterizada pela presença de tumores benignos no trato respiratório superior, sendo a laringe o sítio de lesão mais comum. Esta doença tem uma distribuição de idade bimodal, permitindo sua classificação em papilomatose juvenil ou adulta. O principal agente etiológico da PRR é o Papilomavírus Humano (HPV), um grupo de vírus de DNA, dos quais mais de 150 tipos já foram identificados. HPV-6 e HPV-11 são os tipos mais encontrados em PRR. Há poucos estudos sobre a distribuição das variantes moleculares de HPV de baixo risco. Desta maneira, o objetivo deste estudo foi avaliar a variabilidade genética do gene E6 entre isolados de HPV-6 e HPV-11 detectados em amostras de papilomatose respiratória recorrente (PRR) obtidas em uma coorte de pacientes brasileiros. A fim de comparar as sequências de nucleotídeos identificados em nosso estudo com isolados previamente reportados provenientes de outras partes do mundo, e de diferentes sítios anatômicos (papilomatose de laringe, verrugas genitais, câncer cervical e esfregaço anal), foi realizada a análise filogenética para determinar as relações filogenéticas das variantes detectadas no Brasil com as variantes isoladas em outras regiões do mundo. A região codificante completa do gene E6 de 25 amostras foi clonada e sequenciada. Em 18 isolados foi detectado o DNA do HPV-6 (72%), e em 7 isolados o DNA do HPV-11 (28%). Um total de quatro variantes genômicas diferentes de HPV-6 e duas variantes genômicas de HPV-11 foram identificadas e nenhuma variante pode ser associada com o quadro clínico do paciente. Para a reconstrução filogenética foram utilizadas as sequências de E6 detectadas neste estudo adicionalmente às sequências anteriormente publicadas originárias da Eslovênia e da África do Sul. Devido ao pequeno... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Recurrent respiratory papillomatosis (RRP) is a disease characterized by benign neoplasms and can occur anywhere within the upper respiratory tract, but the most common lesion site is the larynx. This disease has a bimodal age distribution which forms the basis of its classification as juvenile or adult. The main etiological agent of RRP is Human Papillomavirus virus (HPV), a group of DNA virus with more than 150 identified types. HPV-6 and 11 are the most common types identified in RRP. There are few studies about the distribution of natural molecular variants of low-risk HPVs. So, the aim of this study was to evaluate the E6 early gene variability among HPV-6 and HPV-11 isolates detected in recurrent respiratory papillomatosis (RRP) samples obtained in a cohort of Brazilian patients. In order to compare nucleotide sequences identified in our study with previously reported sequences isolates from different anatomic sites (laryngeal papillomas, genital warts, cervical cancer and anal swabs) obtained from other parts of the world was performed phylogenetic analysis to determine the phylogenetic relationships of variants detected in Brazil with variants isolated in others regions of world. The complete coding region of the E6 gene of 25 samples were cloned and sequenced. HPV-6 DNA was detect in 18 isolates (72%) and HPV-11 DNA in 7 isolates (28%). A total of four different HPV-6 genomic variants and two HPV-11 genomic variants were identified and any variant could not be associated with the clinical outcome. Phylogenetic trees for both HPV types were reconstructed enclosing E6 sequences detected in our study in addition to formerly published sequences from Slovenia and South Africa. The small number of samples analyzed prevents the evaluation of the association between specific molecular variants and the... (Complete abstract click electronic access below)
Mestre

Ciências biomédicas
O vírus GBV-C é um dos potenciais membros da família Flaviviridae sendo, essencialmente, transmitido por via parentérica. Apesar de inicialmente descrito como agente de hepatites, dados recentes parecem demonstrar a não patogenicidade deste vírus. Curiosamente, o GBV-C parece exercer um efeito benéfico nos indivíduos co-infectados com HIV, facto que estimulou a realização de inúmeros estudos sobre a biologia deste vírus. A escassez de informação de carácter genético/molecular relativa aos vírus GBV-C circulantes em Portugal, motivou a caracterização genética das estirpes virais circulantes na Grande Lisboa. Como corolário da abordagem utilizada, foi ainda possível avaliar a prevalência do GBV-C nesta área geográfica, tendo a análise efectuada recaído sobre num grupo indivíduos, na sua maioria utilizadores de drogas injectáveis co-infectados com HIV e/ou HCV. A aplicação de um método para análise semi-quantitativa da carga viral de GBV-C constituiu, também, um dos propósitos deste trabalho. Foram analisadas 214 amostras do soro humano nas quais o RNA de GBV-C foi detectado por uma reacção de transcrição reversa seguida de amplificação da 5´UTR por nested-PCR. A taxa de virémia na população estudada foi de 40,65%, tendo esta sido estatisticamente associada ao consumo de drogas e idade. As monoinfecções por GBV-C foram detectadas em 22,99% das amostras analisadas enquanto que a taxa de infecções duplas GBV-C/HIV ou GBV-C/HCV foram de 19,54% e 16,09%, respectivamente. Finalmente, as infecções triplas (GBVC/HIV/HCV) foram registadas em 41,38% das amostras analisadas. A caracterização de duas regiões distintas do genoma viral (E1/E2 e NS5A/NS5B) incluiu, entre outras, a análise filogenética das respectivas sequências nucleotídicas, tendo por base três modelos de reconstrução filogenética. A análise da região E1/E2 de 45 sequências virais, evidenciou a segregação de 10 destas com referências do genótipo 1 e 34 com referências do genótipo 2. Nestas últimas, um grupo de 16 sequências formou um putativo novo subtipo associado à área geográfica estudada, denominado G2*, e corroborado pela análise da região NS5A/NS5B. A descoberta de novas variantes genómicas, a compreensão da sua distribuição, elucidação das suas características biológicas e interacção destas com outros vírus, dão ênfase à necessidade de continuidade dos estudos relacionados envolvendo o GBV-C.
The GB virus C (GBV-C) is a tentative member of the Flaviviridae family, readily transmitted by parenteral routes. Although initially regarded as a potential cause of hepatitis, more recent studies have disclosed no evidence for an association between GBV-C and human disease. With the demonstration of a positive effect of an active GBV-C infection on the outcome of HIV infection, the research on GBV-C has been recently reboosted. Due to the scarcity of information regarding GBV-C infection in Portugal, we decided to carry out a genetic characterization of the viral strains circulating in the Greater Lisbon. An immediate consequence of this study was the evaluation of the prevalence of GBV-C infection in a population sample including a large number of IDUs, also characterized by high HIV and/or HCV seroprevalence. Furthermore, in order to quantify the GBV-C viral load, a semiquantitative Real-Time-PCR analysis was also executed. GBV-C viremia was assessed by nested-PCR amplification of a section of the conserved 5’- untranslated region, using total RNA extracted from 214 plasma samples. The overall prevalence of GBV-C infection was 40.6%. GBV-C viremia was found to be statistically associated with the age of the infected individuals or the use of intravenous drugs. Among those with GBV-C viremia, mono-infection was detected only in 22.99% of them, while co-infection with HIV, HCV, or HIV/HCV was revealed in 19.54%, 16.09%, and 41.38% of the population, respectively. The genetic characterization of GBV-C of two distinct genomic regions (E1/E2 e NS5A/NS5B), was carried out by different approaches, including phylogenetic analysis of nucleotide sequences, based on three different methods. Of 45 viral strains comprising the E1/E2 genomic region, 10 showed segregation with genotype 1 and 34 with genotype 2 GBV-C references. However, 16 of the strains assigned to genotype 2 were shown to form a separate cluster (designated G2*), also confirmed by NS5A/NS5B genomic region analysis. The discovery of newly genomic variants, the understanding of their geographical distribution, elucidation of biological characteristics and their interaction with other viruses, emphasize the need to keep on scientific research concerning GBV-C.

O vírus sincicial respiratório humano (VSRH) é o agente viral mais freqüentemente relacionado a doenças do trato respiratório inferior em crianças abaixo de um ano de idade. Analíse da varibilidade antigênica e gênica mostraram que o VSRH pode ser divido em dois grupos: A e B. O vírus é um membro do gênero Pneumovirus pertencente a família Paramyxoviridea, e possui três principais proteínas que são: glicoproteina F (fusão), glicoproteina G (adesão), glicoproteina SH (pequena proteína hidrofóbica). A proteína F é responsável pela fusão da célula ao vírus, enquanto a proteína G tem papel fundamental na replicação do vírus, porém a função da proteína SH, ainda não está bem definida, estudos recentes mostram-na como responsável por inibir a sinalização do fator de necrose tumoral alfa (TNF-a). Neste estudo foram colhidas amostras de 965 crianças, entre os anos de 2004 e 2005, dentre as quais 424 foram positivas. 117 amostras foram seqüenciadas a proteína SH e G e comparadas com amostras que circularam mundialmente. A analíse filogenética mostrou uma baixa variabilidade entre os genótipos estudados tanto do grupo A quanto do B.
The human respiratory syncytial virus (HRSV) is the major cause of lawer respiratory tract infections in infantis, young children and elderly. Analysis of the antigenic and genetic variability has shown that there are two groups of the virus HRSV, A and B. The virus (HRSV) is a member of the genus pneumovirus in the paramyxoviridae family. The virus encodes three membrane-bound glicoproteins, namely the fusion (F) attachment (G) and small hydrophobic (SH) proteins. The F mediates fusion of the virus and cell membranes and the G proteins is involved in virus attachment. The biological properties of the F and G glicoproteins and role that they play during virus replication relatively well understood, however the functional significance of the SH protein during replication remains unclear, although recent study shown that it can inhibit TNF-alpha. In this study, HRSV strains were isolated from nasopharyngeal aspirates collected from 965 children between 2004 and 2005, yielding 424 positive samples. We sequenced the small hydrophobic protein (SH) gene and protein (G) of 117 samples and compared them with other viruses identified worldwide. The phylogenetic analysis showed a low genetic variably among the isolates but allowed us to classify the viruses into different genotypes for the A and B HRSV strains.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) é considerado um agente patogénico animal exclusivo e Streptococcus pyogenes (GAS) um agente patogénico humano exclusivo. Recentemente foram encontrados fatores de virulência fágicos de GAS em estirpes de SDSD de origem bovina e casos de infeção humana associada a SDSD têm vindo a ser reportados. Em consequência, o potencial zoonótico de SDSD foi sugerido, contudo o papel destes fatores de virulência na patogénese de SDSD não foi comprovado. Um dos objetivos desta tese foi detetar a presença e expressão de fatores de virulência de GAS, entre isolados de SDSD contemporâneos de origem portuguesa, isolados de amostras de leite de bovinos disgnosticados com mastite em herdades leiteiras portuguesas entre 2011-13 e comparar estes dados com os reportados de uma coleção portuguesa de SDSD previamente estudada de 2002-03. O potencial de infeção in vitro e in vivo foi também avaliado e comparado entre coleções. Determinantes genéticos de GAS (os genes de virulência speB, speC, speF, speH, speK, speL, speM, smeZ, spd1, sdn e o elemento quimérico Tn1207.3/Φ10394.4) foram pesquisados por PCR e a sua expressão averiguada por PCR após síntese de cDNA. A produção de DNases extracelulares foi avaliada e correlacionada com o perfil genotipico dos genes spd1 e sdn. Para estudar o potencial de infeção, in vitro, foram utilizadas linhas celulares repiratórias normais e tumorais humanas (BTEC e Detroit 562, respetivamente) e in vivo, o modelo animal zebrafish. Os resultados sugerem que os fatores de virulência pesquisados são característicos de SDSD de origem bovina e a produção de DNases extracelulares é independente dos genes spd1 e sdn. Os estudos de infeção in vitro e in vivo revelam que os potenciais de infeção de SDSD são específicos de estirpe e independentes dos genes de virulência pesquisados. O potencial zoonótico de SDSD é novamente sugerido uma vez que estirpes de origem bovina foram capazes de infetar linhas celulares humanas e o zebrafish.
Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is considered an exclusively animal pathogen and Streptococcus pyogenes (GAS) a strictly human pathogen. GAS phage virulence determinants were recently found in SDSD strains of bovine origin, and cases of human infection associated with SDSD have been recently reported. The SDSD zoonotic potential has been therefore suggested, however the role of those virulence genes in the pathogenesis of the bovine SDSD has not been proved. One of the objectives of this thesis was to detect the presence and expression of GAS virulence determinants, among contemporary SDSD strains, isolated from milk samples of bovines diagnosed with mastitis in Portuguese dairy herds between 2011-13 and compare the data with the one previously reported of a study of a Portuguese SDSD collection of 2002-03. In vitro and in vivo infection potential was also evaluated and compared between both collections. GAS genetic determinants (virulence genes speB, speC, speF, speH, speK, speL, speM, smeZ, spd1, sdn and the chimeric element Tn1207.3/Φ10394.4) were screened by PCR and their expression was assessed by PCR after cDNA synthesis. Extracellular DNase production was assessed and correlated with spd1 and sdn genotypic profile. To study the infection potential, in vitro, human normal and tumoral respiratory cell lines (BTEC and Detroit 562, respectively) were used, and in vivo, the zebrafish animal model was chosen. Results suggested that the virulence determinants screened are characteristic of SDSD of bovine origin and that extracellular DNase production was independent on the spd1 and sdn genes. In vitro and in vivo infection studies revealed that the infection potentials of SDSD are strain-specific and independent on the virulence genes screened. Zoonotic potential of SDSD is further suggested, as strains from bovine origin were able to infect human cell lines, as well as the zebrafish.

A Adesão à Terapêutica Anti-retroviral (TARV) pertence ao grupo das maiores preocupações de saúde pública na área do VIH. O único factor de possibilidade de controlo de sucesso da TARV por parte do paciente VIH+ é o comportamento de adesão ao tratamento. As características do próprio paciente, o enquadramento sócio-cultural do VIH na comunidade e no país, a qualidade da relação com os profissionais de saúde e as próprias dificuldades com o tratamento, são factores amplamente estudados nas últimas décadas, e que medeiam o sucesso da adesão à TARV. A partir de uma abordagem bottom-up, desenvolveu-se um estudo exploratório com o objectivo de explorar o modo como pacientes TARV em Maputo experienciam estes factores e o impacto dessa experiência na adesão à TARV. A investigação contou com a colaboração de 602 utentes em TARV na Cidade de Maputo com idades entre os 21 e os 56 anos. Os resultados obtidos ao longo dos quatro estudos realizados indicam que mais homens revelaram ter interrompido o tratamento que as mulheres, e maioritariamente por mal-estar físico ou por indicação médica. Ao contrário dos homens, as mulheres são quem mais revela ter dificuldades com os profissionais de saúde, assim como, interromper o tratamento por faltas às consultas. A adesão à TARV é mais reduzida quando na presença das seguintes características: género masculino; medos relacionados com estigma e discriminação, com falhar a toma da TARV ou com não sentir melhoras com o tratamento; ter tido dificuldades com os profissionais de saúde, nomeadamente, o paciente sentir que não tem as suas dúvidas esclarecidas, que o médico só se preocupa com a medicação, que o médico não o compreende e que não cria espaço para que o paciente aborde outros aspectos relacionados com a sua saúde. A investigação apela ao desenvolvimento de intervenções que atendam às diferenças de género, nomeadamente no relacionamento com os profissionais de saúde, família e activistas, que possibilitem o desenvolvimento de estratégias de coping com a TARV mais suportadas socialmente. Encoraja-se a continuação do desenvolvimento de actividades formativas da sociedade em geral, sobre o VIH e a TARV. A necessidade de diminuição do estigma é uma das maiores preocupações levantadas por este trabalho.
Antiretroviral treatment (ART) adherence is one of the main concerns of Public Health on HIV field. The behavior of adherence to the treatment is the only factor that HIV+ patient can have under personal control. Patients own characteristics,the HIV socio-cultural outcomes in the community as well as in the country, the quality of the relationship with health professionals and the difficulties on dealing with the treatment, are factors greatly studied during the last decades, and that play a crucial role on ART treatment adherence. From a bottom-upperspective, an exploratory study was developed with the goal to explore how the mentioned factors were experienced by the ART patients in Maputo, and the impact of that experience on ART adherence.The research counted with the collaboration of 602 ART patients in Maputo City, with ages between 21 and 56 years old. The results from the four studies indicate that more men than women revealed having had interrupted the treatment, and in majority due to physical discomfort and medical prescription. Women have revealed having more difficulties with health professionals than men, and treatment interruption due to missing clinical appointments. Male, fears related to stigma and discrimination, to being afraid of missing ART schedules, fear of not feeling health improvement with ART, as well as having had difficulties with health professionals, not having their questions about ART being clarified, feeling that the doctor only cares about medication, not feeling understood by the doctor and feeling that the doctor doesn’t make room to talk about other health issues; are items that have an impact on ART adherence, reducing it. The present study encourages the development of interventions that attempt to gender differences, especially on the relationship with health professionals, with family and with activists, which can promote the development of ART coping strategies with more social support. It is encouraged to keep the development of training and educationalactivities for general society about HIV and TARV. Stigma reduction is one of the main concerns of this research results.

La maladie pied-main-bouche (PMB) et l’herpangine sont deux maladies pédiatriques bénignes causées par les entérovirus (EV), en particulier les sérotypes de l’espèce A (EV-A). Le sérotype EV-A71 fait l’objet d’une surveillance dans les pays du Sud Est de l’Asie car il est associé à des atteintes neurologiques sévères chez les très jeunes enfants, parfois mortelles (défaillance cardio-pulmonaire). Les infections causées par les autres EV-A tel que le coxsackievirus A16 (CV-A16) provoquent rarement des atteintes sévères. En Europe, les cas de maladie PMB causés par l’EV-A71 ne font pas l’objet d’une déclaration obligatoire, car ce virus ne cause pas d’épidémies de grande ampleur. L’objectif général de la thèse était d’étudier l’épidémiologie des EV-A en Europe et nous avons utilisé une approche phylogénétique bayésienne pour analyser un échantillon de 500 souches. Nous montrons la circulation discontinue de l’EV-A71 de deux populations virales principales (sous génogroupes C1 et C2), ce qui explique la rareté des épidémies en Europe. L’épidémiologie de ce virus est aussi caractérisée par des transports de souches entre les pays Européens et sporadiquement entre l’Europe et l’Asie (sous génogroupes B5 et C4). La recombinaison génétique intertypique survient rarement parmi les populations d’EV-A71 en circulation et ne contribue pas significativement à leur diversité génétique. Cependant, ce mécanisme génétique est relié à l’émergence d’un sous génogroupe CV-A16 qui circule en France depuis 2011. Comparés à l’EV-A71, les sérotypes CV-A2, CV-A4, CV-A6 sont plus fréquemment sujets à des événements de recombinaison intertypiques. L’analyse de la sélection à l’échelle moléculaire indique que la fixation des mutations dans les protéines de capside de l’EV-A71 est lente, probablement à cause des contraintes structurales et fonctionnelles. La surveillance des infections à EV-A71 en Europe devrait être renforcée à cause de la neurovirulence de ce virus, de l’introduction récente et répétée de souches variantes « asiatiques » et de l’existence d’une grande diversité de génogroupes en Afrique et en Inde encore peu explorée
Hand-Foot and Mouth Disease (HFMD) and Herpangina are two benign pediatric diseases caused by Enteroviruses (EV), especially enterovirus A species serotypes (EV-A). Infections caused by the EV-A71 serotype are monitored in countries of South East Asia because they are associated with severe neurological symptoms in young children and may be fatal (cardiopulmonary failure). Infections caused by the other EV-A serotypes, e.g. coxsackievirus A16 (CV-A16), rarely induce severe symptoms. In Europe, EV-A71 HFMD cases are not notifiable because this virus does not cause large-scale epidemics. The overall objective of this thesis was to study the EV-A epidemiology in Europe and we used a Bayesian phylogenetic approach to analyze 500 viral strains. We show a discontinued circulation of two EV-A71 populations (C1 and C2 subgenogroups), which explains the rare outbreaks in Europe. The epidemiology of this virus is characterized by transportation events of viral strains between European countries and sporadically between Europe and Asia (C4 and B5 subgenogroups). Intertypic genetic recombination occur rarely among circulating EV-A71 populations and does not contribute significantly to their genetic diversity. We found that genetic mechanism was related to the emergence of a new CV-A16 subgenogroup, which is circulating in France since 2011. In comparison with EV-A71, a number of serotypes (CV-A2, CV-A4, and CV-A6) are more frequently involved in intertypic recombination events. The structural and functional constraints are possible factors involved in the slow mutation fixation in the EV-A71 capsid proteins as determined by analyses of molecular selection. Neurovirulence, the recent and repeated introductions of variants “Asian” strains, and the diversity of genogroups in Africa and India call for strengthened surveillance of EV-A71 infections among European countries

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A Papilomatose Respiratória Recorrente (PRR) é uma doença caracterizada pela presença de tumores benignos no trato respiratório superior, sendo a laringe o sítio de lesão mais comum. Esta doença tem uma distribuição de idade bimodal, permitindo sua classificação em papilomatose juvenil ou adulta. O principal agente etiológico da PRR é o Papilomavírus Humano (HPV), um grupo de vírus de DNA, dos quais mais de 150 tipos já foram identificados. HPV-6 e HPV-11 são os tipos mais encontrados em PRR. Há poucos estudos sobre a distribuição das variantes moleculares de HPV de baixo risco. Desta maneira, o objetivo deste estudo foi avaliar a variabilidade genética do gene E6 entre isolados de HPV-6 e HPV-11 detectados em amostras de papilomatose respiratória recorrente (PRR) obtidas em uma coorte de pacientes brasileiros. A fim de comparar as sequências de nucleotídeos identificados em nosso estudo com isolados previamente reportados provenientes de outras partes do mundo, e de diferentes sítios anatômicos (papilomatose de laringe, verrugas genitais, câncer cervical e esfregaço anal), foi realizada a análise filogenética para determinar as relações filogenéticas das variantes detectadas no Brasil com as variantes isoladas em outras regiões do mundo. A região codificante completa do gene E6 de 25 amostras foi clonada e sequenciada. Em 18 isolados foi detectado o DNA do HPV-6 (72%), e em 7 isolados o DNA do HPV-11 (28%). Um total de quatro variantes genômicas diferentes de HPV-6 e duas variantes genômicas de HPV-11 foram identificadas e nenhuma variante pode ser associada com o quadro clínico do paciente. Para a reconstrução filogenética foram utilizadas as sequências de E6 detectadas neste estudo adicionalmente às sequências anteriormente publicadas originárias da Eslovênia e da África do Sul. Devido ao pequeno...
Recurrent respiratory papillomatosis (RRP) is a disease characterized by benign neoplasms and can occur anywhere within the upper respiratory tract, but the most common lesion site is the larynx. This disease has a bimodal age distribution which forms the basis of its classification as juvenile or adult. The main etiological agent of RRP is Human Papillomavirus virus (HPV), a group of DNA virus with more than 150 identified types. HPV-6 and 11 are the most common types identified in RRP. There are few studies about the distribution of natural molecular variants of low-risk HPVs. So, the aim of this study was to evaluate the E6 early gene variability among HPV-6 and HPV-11 isolates detected in recurrent respiratory papillomatosis (RRP) samples obtained in a cohort of Brazilian patients. In order to compare nucleotide sequences identified in our study with previously reported sequences isolates from different anatomic sites (laryngeal papillomas, genital warts, cervical cancer and anal swabs) obtained from other parts of the world was performed phylogenetic analysis to determine the phylogenetic relationships of variants detected in Brazil with variants isolated in others regions of world. The complete coding region of the E6 gene of 25 samples were cloned and sequenced. HPV-6 DNA was detect in 18 isolates (72%) and HPV-11 DNA in 7 isolates (28%). A total of four different HPV-6 genomic variants and two HPV-11 genomic variants were identified and any variant could not be associated with the clinical outcome. Phylogenetic trees for both HPV types were reconstructed enclosing E6 sequences detected in our study in addition to formerly published sequences from Slovenia and South Africa. The small number of samples analyzed prevents the evaluation of the association between specific molecular variants and the... (Complete abstract click electronic access below)

Globalmente, os ixodídeos são considerados o segundo vetor artrópode com maior importância em Saúde Pública, sendo considerável o aumento na incidência e distribuição geográfica de vírus transmitidos por estes. O número crescente de estudos direcionados para a identificação de novos vírus transmitidos por carraças, tem vindo a ser motivado por descobertas recentes de vírus patogénicos e altamente virulentos transmitidos por estes artrópodes, sendo destes exemplos, os vírus Bourbon, Several Fever with Thrombocytopenia Syndrome e Heartland. O facto de diversas espécies de carraças terem demonstrado albergar uma série de potenciais novos vírus desconhecidos, aliado à limitação no conhecimento relativo à distribuição de vírus transmitidos por carraças em Portugal, motivou o presente trabalho, que incluiu a identificação e caracterização preliminar de vírus presentes em carraças colhidas no nosso país. Os exemplares em estudo foram coletados da vegetação em vários locais dos distritos de Lisboa e Setúbal, e analisados utilizando duas abordagens distintas, uma delas dependente e a outra independente de um conhecimento prévio das sequências que viríamos a amplificar. As abordagens de heminested-, e multiplex-PCR, utilizando primers degenerados, foram dirigidas para dois géneros distintos Thogotovirus e Phlebovirus, cuja circulação em Portugal já fora anteriormente comprovada. Adicionalmente, foi efetuada em macerados de carraças uma abordagem de amplificação não-dirigida utilizando a técnica Sequence - Independent Single Primer Amplification. Das várias abordagens utilizadas neste estudo, a técnica de multiplex- PCR dirigida para o segmento L do genoma viral de Phlebovirus, foi notoriamente a que apresentou melhores resultados, tendo um grande número de pools revelado a presença dessas sequências virais. A análise filogenética das sequências virais obtidas revelou a segregação aparente em duas linhagens distintas de flebovírus. Os vírus detetados, sob as condições utilizadas, não conseguiram replicar em células Vero E6 e DH82. O isolamento e caracterização mais aprofundada destes vírus, bem como o seu eventual impacto na saúde humana/animal justificam, futuramente novos estudos.
On a global scale, ticks are the second most important vectors responsible for transmiting infectious agents to humans, and have indisputable impact on human health, especially given the rise in the incidence and geographical distribution, in recent years, of some of the diseases these agents cause. The number of studies that describe new tick-borne viruses has been motivated by the recent discovery of some which display considerable pathogenicity towards humans, such as the Bourbon, Several Fever with Thrombocytopenia Syndrome e Heartland viruses. This work has been motivated by the fact that not only ticks seem to harbor a plethora of new viruses, but also because the distribution of most of them in Portugal is unknown. Our objectives included the identification, and preliminary characterization, of viruses associated with ticks collected in Portugal. The specimens we have studied were collected from the vegetation in southern Portugal, in the districts of Lisbon and Setubal, and they were tentatively detected using molecular methods, defined as sequence-dependent and sequence-independent approaches. Therefore, we have used heminested- and multiplex-PCR protocols using primers that were designed to specifically target sequences of viruses classified as Thogotovirus and Phlebovirus, the circulation of which has already been described in Portugal. Additionally, the presence of these viruses in tick macerates was also attempted using Sequence - Independent Single Primer Amplification which allows for viral detection without a priori knowledge of the targeted sequences. Of all the techniques used, the specific amplification of phleboviruses L segment-sequences using a multiplex-PCR protocol was revealed as the one allowing the detection of new viral sequences in a large number of tick macerates. Their analysis using phylogenetic inference disclosed segregation into two distinct genetic lineages. We have also shown that, under the experimental conditions used, the detected viruses were not able to readily replicate in Vero E6 or DH82 cells. A more thorough characterization of these viruses, including the assessment of their potential impact on human health should be pursued in future studies.

A proteína Nef do vírus da imunodeficiência humana tipo 1 (VIH-1) é uma proteína miristilada, de 27 kDa, expressada em níveis elevados imediatamente após a infecção. Embora seja dispensável para a replicação viral in vitro, Nef parece desempenhar um papel fundamental ao nível da patogénese viral in vivo. Entre as inúmeras funções biológicas que lhe são atribuídas, contam-se a estimulação da replicação viral, a indução do aumento da infecciosidade dos viriões, a modulação negativa da expressão superficial do receptor celular CD4 e do complexo principal de histocompatibilidade de classe I (MHC I), a modulação de inúmeras vias de sinalização celular/transdução de sinal em linfocitos T e a interferência no processo de indução da morte celular programada, protegendo da apoptose as células infectadas e induzindo-a nas células vizinhas não infectadas, incluindo linfocitos T CD8+ específicos para o VIH-1. Devido ao seu papel fundamental ao nível do ciclo replicativo viral, a proteína Nef foi considerada como um potencial alvo terapêutico e vacinal. Neste trabalho, o gene nef do VIH-1 foi amplificado por PCR a partir da linha celular 8E5/LAV (HIV-1) e clonado no sistema de expressão “transportador-adjuvante” pVUB3 (Cote-Sierra et al., 1998), baseado na lipoproteína OprI da membrana externa de Pseudomonas aeruginosa. Desta clonagem resultou a construção do plasmídio recombinante pVUB3nef8E5, de 4958 pb, a partir do qual se induziu a expressão da proteína de fusão OprI-Nef ao nível da membrana externa de Escherichia coli. A produção da proteína de fusão foi demonstrada por visualização de extractos proteicos de membrana externa em gel de poliacrilamida/SDS e confirmada por experiências de imunodetecção com anticorpos anti-OprI e anti-Nef. Com o objectivo de obter extractos da proteína de fusão com um grau de pureza elevado, um oligonucleótido 6xHis foi introduzido a jusante da construção híbrida oprI-nef de pVUB3nef8E5, no que resultou a formação do novo vector de expressão pVUB3nefB-6xHis (4944 pb). Após um processo de optimização das condições experimentais relativas à purificação, sob condições desnaturantes, da proteína de fusão OprI-Nef-6xHis por cromatografia de afinidade e um passo adicional de ultrafiltração, foi possível a obtenção de extractos proteicos finais com um grau de pureza relativamente elevado (análise em gel de poliacrilamida/SDS e por imunodetecção). Estes apresentaram uma concentração proteica aproximada de 4 μg/μl, correspondendo a um rendimento global do processo de purificação de cerca de 1,5 mg de proteína por litro de cultura bacteriana, e uma quantidade de lipopolissacáridos (endotoxinas) adequada à realização de experiências de imunização. A proteína de fusão OprI-Nef-6xHis foi utilizada como imunogénio em experiências de imunização em modelo murino, com o objectivo de se caracterizar a componente humoral da resposta imune anti-Nef, por comparação com a administração da proteína Nef em tampão PBS (rNef) e em adjuvante de Freund (rNef/AF). A imunização com a proteína de fusão induziu uma resposta humoral anti-Nef caracterizada por um título de diluição limite de anticorpos da classe IgG muito elevado (1:37300), significativamente superior ao obtido no grupo rNef, sem que a sua administração tivesse aparentemente qualquer tipo de efeitos secundários nos animais. Por outro lado, comparativamente aos grupos rNef e rNef/AF, observou-se um desvio da resposta imune no sentido Th1, facto relevante no caso da infecção pelo VIH. Este fenómeno, bem com a indução de um título elevado de anticorpos anti-Nef da classe IgG, dever-se-á, muito provavelmente, à presença da componente lipídica na proteína de fusão. Finalmente, a utilização da proteína de fusão OprI-Nef-6xHis como antigénio em experiências de ELISA permitiu detectar anticorpos anti-Nef em 77% dos soros testados de indivíduos infectados com o VIH, indicando que a proteína híbrida obtida, embora purificada de um modo desnaturante, manterá epitopos de Nef sob uma forma imunologicamente relevante. Os resultados obtidos apontam ainda para um reconhecimento imune alargado destes epitopos por anticorpos naturais anti-Nef produzidos contra diferentes genótipos do VIH-1, e, muito provavelmente, mesmo contra o VIH-2, o que parece substanciar a utilização desta proteína de fusão como potencial imunogénio anti-VIH. Em conclusão, o sistema de expressão pVUB3 parece constituir um modelo promissor para a produção heteróloga, em bactérias Gram-negativas, de lipoproteínas de fusão contendo determinantes antigénicos de proteínas dos vírus da imunodeficiência humana, a serem eventualmente incluídas como agentes de imunização em esquemas experimentais de vacinação anti-VIH.
The human immunodeficiency virus type 1 (HIV-1) Nef protein is a 27 kDa myristylated early protein, expressed immediately after infection at relatively high levels. Although not necessary for viral replication in vitro, it has been shown to be a major determinant for AIDS pathogenesis. Nef can exert several effects at the cellular level: enhancement of viral replication and virion infectivity, downregulation of the cell-surface expression of CD4 and MHC class I molecules, modulation of T cell signal transduction pathways and regulation of programmed cell death (protecting infected cells from apoptosis and leading to bystander cell killing, including of HIV-specific cytotoxic T lymphocytes). Therefore, Nef has been considered a valuable target for the development of novel antiviral therapies and/or vaccines. In this work, HIV-1 nef gene from 8E5/LAV (HIV-1) cell line was cloned into the carrier-adjuvant plasmid system pVUB3 (Cote-Sierra et al., 1998), based on the major lipoprotein (OprI) from the outer membrane of Pseudomonas aeruginosa. This resulted in the construction of the 4958-bp recombinant plasmid pVUB3nef8E5, which allowed the inducible production of an outer membrane-bound OprI-Nef fusion protein in Escherichia coli. The expression of OprI-Nef was firstly demonstrated by SDS-PAGE and further confirmed by Western blotting with anti-OprI and anti-Nef antibodies. In order to obtain high purity OprI-Nef extracts, a 6xHis tag was introduced downstream of oprI-nef in pVUB3nef8E5, which resulted in the construction of the expression vector pVUB3nefB-6xHis. The expression of the new inducible fusion protein (OprI-Nef-6xHis) was confirmed by SDS-PAGE and immunoblotting, being the protein purified under denaturing conditions by immobilised metal affinity chromatography, followed by ultrafiltration. The purified final extracts had a protein concentration of 4 μg/μl, corresponding to a global purification yield of 1,5 mg of protein/litre of bacterial culture, and low levels of endotoxins, adequate for immunization experiments. Aiming at the characterization of humoral immune responses induced by immunization with Nef as a fusion protein with OprI, three groups of mice were immunized, respectively, with OprI-Nef-6xHis, Nef in the presence of Freund’s adjuvant or Nef alone in PBS. Mice immunized with the fusion protein developed high levels of IgG anti-Nef antibodies (endpoint titre of 1:37,300), which was significantly higher than in mice immunized with the protein alone, with no visible adverse side effects for the inoculated animals. On the other hand, the analysis of the isotypic patterns of anti-Nef antibodies showed that immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies, indicating a predominant Th2 immune response, whereas OprI-Nef-6xHis immunization biased the humoral response towards IgG2a production, indicating the preferential induction of a Th1 immune response. As a whole, these results most probably reflect the in-built adjuvanticity and immune response modulation capacity of OprI-Nef-6xHis by virtue of its lipid moiety. Finally, an ELISA-based method for detection of anti-Nef antibodies in sera from HIV infected individuals was developed, using the fusion protein OprI-Nef-6xHis as coating antigen. On the overall, 77% of the tested sera were considered positive, irrespective of HIV-1 env genotypes and, probably, even of HIV type. This result shows that, despite of OprI-Nef-6xHis biochemical purification under denaturing conditions, there is a broad immune recognition of Nef epitopes in the fusion protein by natural anti-Nef antibodies, what seems to support its future use as an anti-HIV immunogen. In conclusion, the data obtained in this work indicate that pVUB3 constitutes an appropriate expression system for the heterologous production of bacterial fusion lipoproteins containing HIV antigenic determinants, to be potentially included in experimental anti-HIV immunization protocols.

Mundialmente, estima-se que 40 milhões de pessoas estejam infectadas com VIH, 5 milhões das quais cronicamente infectadas com VHC. A co-infecção com VIH aumenta a taxa de persistência do VHC, acelera a velocidade de progressão da doença hepática e reduz significativamente a resposta à terapia do VHC. O VHC possui extensa diversidade genética, sendo classificado em seis genótipos e cerca de 90 subtipos com padrões epidemiológicos e resposta à terapia distintos. Tendo isto presente e devido a serem limitados os dados relativos aos genótipos e subtipos do VHC circulantes em Portugal e ainda ao facto de os utilizadores de drogas injectáveis (UDIs) serem um grupo de risco importante para a co-infecção por VIH e VHC, realizámos um estudo retrospectivo para determinar a prevalência da infecção por VHC e a distribuição de subtipos deste vírus num grupo de UDIs infectados com VIH. Amostras de plasma de 66 indivíduos (1998-2001) foram testadas para anticorpos anti-VHC (ensaio imunoenzimático) e RNA do VHC (amplificação da 5’UTR por RT-PCR). Para identificar os subtipos de VHC e detectar recombinantes, as amostras com RNA viral detectável foram sujeitas a amplificação, sequenciação e análise filogenética de sequências nucleotídicas parciais para C/E1 e NS5B. Encontrámos que 86,4% dos indivíduos possuíam anticorpos anti-VHC, 93,0% dos quais com infecção activa. Todas as amostras, exceptuando duas, com RNA viral detectável originaram amplicões para C/E1 e NS5B. A análise filogenética permitiu incluir as estirpes de VHC nos subtipos 1a (43,8%), 1b (3,6%), 2a (1,8%), 3a (21,1%), 4a (8,8%) e 4d (15,8%), revelando um padrão epidemiológico semelhante ao dos UDIs de outros países do Sul da Europa. Apenas uma amostra apresentou discordância entre os subtipos das duas regiões (4d para C/E1 e 4a para NS5B) sugerindo um potencial recombinante intragenótipo. No total, os subtipos 1a, 4a e 4d do VHC são responsáveis por 68,4% das infecções nos UDIs infectados com VIH analisados. Considerando que apenas 20-30% dos doentes positivos para VIH e co-infectados com os genótipos 1 e 4 respondem à terapia do VHC e que ocorre transmissão do VHC dos UDIs para a população em geral, são prioritários estudos alargados de vigilância epidemiológica e implementação de estratégias de prevenção para controlar ambos os vírus neste grupo de risco.
Among the estimated 40 million persons infected with HIV worldwide, about 5 million are chronically infected with HCV. Co-infection with HIV increases HCV persistence, accelerates HCV-related liver disease, and dramatically reduces HCV treatment response rates. Based on its genetic variability, HCV has been classified into six major genotypes and about 90 subtypeswith distinct epidemiological patterns and response to therapy. With this in mind and given that the HCV genotypes and subtypes circulating in Portugal is largely unknown and that injection drug users (IDUs) are an important risk group for co-infection with HIV and HCV, we conducted a retrospectivestudy to determine the prevalence of HCV infection and HCV subtype distribution in a group of IDUs infected with HIV.Plasma samples, collected between 1998 and 2001 from 66 IDUs, were tested for anti-HCV antibodies (immunoenzymatic test) and for viral RNA (RT-PCR amplification of the 5’UTR region). To identify HCV subtypes and detect potential recombinants, samples positive for HCV RNA were further subjected to amplification, sequencing and phylogenetic analysisof partial C/E1 and NS5B sequences.We found 86.4% of the individuals with antibodies anti-HCV, 93.0% of whom showed active infection. All but two samples with detectable HCV RNA were amplified for C/E1 and NS5B regions. Phylogenetic analysis allowed us to classify the HCV strains as subtypes 1a (43.8%), 1b (3.6%), 2a (1.8%), 3a (21.1%), 4a (8.8%), and 4d (15.8%), in agreement with the epidemiologic pattern described for IDUs from Southern European countries. Globally, C/E1 and NS5B based trees demonstrated similar topologies. However, one sample presented discordant subtypes for C/E1 (4d) and NS5B (4a), suggesting infection by a potential intragenotype recombinant of HCV. Overall, HCV subtypes 1a, 4a, and 4d account for 68.4% of the infections in the group of HIV-infected IDUs under study. Considering that only 20-30% of the HIV-positive patients infected with genotypes 1 and 4 respond to current HCV therapy and the strong evidence that IDUs spread HCV to the general population, there is urgent need of further studies on HCV and HIV epidemiologic surveillance and effective preventive strategies to control both viruses in this risk group.

Introdução – O estudo dos vírus torna-se fundamental porque, para além de estarem em grande proporção no meio ambiente e fazerem parte do corpo humano, pois cada célula humana contém DNA viral, são agentes patogénicos que infectam os seus hospedeiros e podem provocar doenças. Além disso, o estudo dos vírus permite aprofundar o modo como uma infecção viral induz mecanismos de reprogramação celular, tornando possível usar o genoma viral como veículo na transferência de genes para células e organismos para fins científicos e medicinais (Flint et alii, 2008). Objectivo - Neste trabalho de revisão bibliográfica irá ser analisado o impacto da investigação em virologia na atribuição do Prémio Nobel. O principal objectivo deste trabalho consiste em numerar alguns investigadores com maior influência no estudo da área da virologia, de entre os que receberam o Prémio Nobel. Serão, também, abordadas, sempre que possível, as seguintes características relativas a cada investigador: o tema da descoberta que levou o investigador ao reconhecimento internacional e o ano em que recebeu o prémio, qual o vírus estudado; determinar quanto tempo leva entre a publicação do artigo referente à descoberta e a atribuição do Prémio Nobel; determinar quanto tempo decorre entre a publicação do artigo que diz respeito à descoberta e a sua aplicação clínica; qual a formação académica do investigador, a sua idade quando publicaram o primeiro artigo e outros aspectos biográficos. Material e métodos - Iniciou-se esta pesquisa acedendo ao site do Prémio Nobel com o intuito de tomar conhecimento sobre a quem tinha sido atribuído o Prémio Nobel da Medicina. De seguida, seleccionou-se os investigadores com descobertas mais relevantes na área da virologia e realizou-se a sua pesquisa biográfica recorrendo, também, ao site do Prémio Nobel e, posteriormente, uma bibliográfica nas bases de dados como Google Scholar e Pubmed de artigos publicados pelos mesmos. Resultado e discussão - Os investigadores com maior influência no estudo da área da virologia, de entre os que receberam o Prémio Nobel, são os que revolucionaram o conhecimento científico sobre os vírus (Nobel Media, 2019). Alguns dos investigadores mais influentes são: Harald Zur Hausen, Stanley Prusiner, Peter Doherty, Michael Bishop, David Baltimore, Max Delbrück, Peyton Rous, François Jacob, Max Theiler Conclusão - Com a realização deste trabalho, e após a análise dos investigadores laureados com o Prémio Nobel em resultado da sua investigação na área dos vírus, observamos que o perfil apresentado inclui, principalmente, as seguintes características: género masculino, nacionalidade americana, publicação do artigo referente ao Prémio, apartir de investigação na Escola de Medicina de Harvard ou no Instituto Pasteur, com idade compreendida entre os 26 e os 35 anos, e atribuição do Prémio 5 a 20 anos mais tarde, já na faixa etária entre os 51 e os 60 anos. Relativamente às primeiras publicações como investigador, foram feitas com idades compreendidas entre os 26 e os 35 anos, no Instituto Nacional de Saúde, na Universidade Michigan, ou no Instituto Pasteur.
Introduction – The study of viruses is essential because, besides being in great proportion in the environment and are part of human body, as every human cell contains viral DNA, are pathogens that infect their hosts and can cause diseases. Furthermore, analyze víruses allows to know more about the way how a viral induces cell reprogramming mechanisms, making it possible to use viral genome as a vehicle in gene transfer to cells and organisms for scientific and medicinal purposes. Objectives – In this bibliographic review will be analyzed the impact of virology research in awarding the Nobel Prize. The main purpose of this paper is to name some of the most influential researchers in the study of virology, among those who won the Nobel Prize. Whenever possible, will also name the following characteristics relative to each researcher: the theme of the discovery that led the researcher to international recognition and the year they received the award; what virus they studied; to determine how long it takes between the publication of the discovery article and the attribution of Nobel prize; to determine how long elapses between the publication of the article concerning the discovery and its clinical application; what is the academic path, age when published their first article and other bibliographic aspects. Material and methods – This research began by accessing the Nobel prize website in order to know who had been awarded the Nobel prize of medicine. Then, selected the researchers with the most relevant findings in the field of virology and carried out their biographical research also using the Nobel prize website and later, a bibliographic in databases such as Google Scholar e Pubmed of articles published by them. Results and discussion – The most influential researchers in the study of virology among the Nobel laureates are the ones who revolutionized scientific knowledge about viroses. Some of the most influential researchers are: Harald Zur Hausen, Stanley Prusiner, Peter Doherty, Michael Bishop, David Baltimore, Max Delbrück, Peyton Rous, François Jacob, Max Theiler. Conclusion – With the completion of this work and after the analysis of the researchers awarded the Nobel Prize as a result of their research in the field of viruses, we observed that the profile presented mainly includes the following characteristics: male gender, American nationality, publication of the article referring to the award, from research at Harvard School of Medicine or at the Pasteur Institute, aged between 26 and 35, and awarding the prize 5 to 20 years later, already aged between 51 and 60 years. Relatively to the first publications as a researcher were made with ages between 26 and 35 years old, at the National Institute of Health, University of Michigan or Pasteur Institute.

O presente relatório é sobre o Estágio no Servicio de Enfermedades Infecciosas e Laboratório de Virologia no Hospital Carlos III. A infecção por VIH/SIDA continua a ser uma importante prioridade em saúde global. Embora se tenha alcançado um importante progresso na prevenção de novas infecções e na diminuição de mortes relacionadas com SIDA por ano, o número de pessoas que vivem com VIH continua a aumentar, trazendo consequências clínicas, psicológicas e sociais para os indivíduos e para as comunidades. O estágio teve como objectivos principais aprofundar os conhecimentos sobre o VIH/SIDA e as alterações hematológicas associadas, sobre Medicina Tropical e do viajante, principalmente sobre imunizações, riscos de viagem e a aplicação destes conhecimentos nos doentes infectados por VIH. Também teve como objectivo aprofundar os conhecimentos sobre as opções terapêuticas e sobre o VIH 2 e co-infectados por VIH 1 e 2. O estágio teve a duração de 3 meses e foi realizado no Serviço de Doenças de Infecciosas, Unidade de VIH e Laboratórios de virologia do Hospital Carlos III. A parte clínica foi realizada nas enfermarias nas consultas externas na Unidade VIH e na Unidade de Medicina Tropical e Viajantes, e a componente laboratorial nos Laboratórios de Parasitologia e Biologia Molecular. Concluí que a TARV, quando indicada, é fundamental para a qualidade de vida do doente infectado por VIH/SIDA. Com Tratamento adequado e assistência médica os doentes infectados por VIH vivem com qualidade de vida e longevidade. Para garantir a qualidade e se preparar para o futuro é necessário incentivar a pesquisa e o desenvolvimento de novos, melhores e mais baratos instrumentos de prevenção e tratamento, incluindo a vacinação. Os conhecimentos adquiridos foram importantes para contribuir para a melhoria qualidade nos serviços de saúde prestados ao meu país de origem, Cabo Verde.
This report is about the internship at the Department of Infectious Diseases and Virology Laboratories at Hospital Carlos III. Infection by HIV / AIDS remains a major global health priority. Although it has achieved significant progress in preventing new infections and reduction of AIDS-related deaths per year, the number of people living with HIV continues to increase, bringing clinical, psychological and social consequences for individuals and for communities. The stage was designed primarily to increase knowledge of HIV / AIDS and associated hematological changes, Tropical Medicine and traveler, mostly about immunizations, trip hazards and the application of such knowledge in HIV infected patients. It also aimed to increase knowledge of the treatment options and about 2 and HIV co-infected by HIV 1 and 2. The internship lasted three months and was conducted at the Infectious Diseases Unit, HIV and virology laboratories, Hospital Carlos III. The clinical part was performed in outpatient wards in the HIV Unit and Unit of Tropical Medicine and Travelers, and the laboratory component of Parasitology and Molecular Biology Laboratories. I Concluded that HAART, when necessary, is critical to the quality of life of patients infected by HIV / AIDS. With proper medical care and treatment of HIV infected patients living with quality of life and longevity. To ensure the quality and prepare for the future is necessary to encourage research and development of new, better and cheaper tools for prevention and treatment, including vaccination. The knowledge gained is important to contribute to improving the quality of health services rendered to my country, Cape Verde.

A virologia ambiental é uma área ainda pouco desenvolvida da virologia, embora nos últimos anos, tem-se assistido a um crescente interesse e preocupação pelos assuntos relacionados com a transmissão de vírus através de água e alimentos contaminados. Os microrganismos patogénicos transmitidos por águas residuais são posteriormente libertados no ambiente, afetando a qualidade da água e consequentemente, a saúde humana. A gastroenterite viral é uma das doenças humanas mais comuns em todo o mundo, contraída pelo consumo e uso de águas contaminas por vírus entéricos. Desta forma epidemiologicamente relevante documentar a presença dos agentes etiológicos dessas doenças em águas ambientais, bem como as características que levam à sua transmissão. O presente trabalho foca-se na importância da pesquisa de vírus nas águas de consumo e em alimentos por via a melhorar a saúde das populações envolvidas, descrevendo os principais patogénios encontrados, bem como as principais doenças e métodos de tratamento. Posteriormente são referidos os principais modos de prevenção destas ocorrências e quais os obstáculos que precisam de ser ultrapassados para evitar a sua atual persistência.
Environmental virology is still an undeveloped area of virology, although in recent years, there has been a growing interest and concern in matters related to the transmission of viruses through contaminated water and food. Pathogenic microorganisms transmitted by wastewater are subsequently released into the environment, affecting water quality and, consequently, human health. Viral gastroenteritis is one of the most common human diseases worldwide, contracted by the consumption and use of contaminated water by enteric viruses. Therefore, it is epidemiologically relevant to document the presence of the etiological agents of these diseases in environmental waters, as well as the characteristics that lead to their transmission. The present work focuses on the importance of researching viruses in drinking water and food to improve the health of the populations involved, describing the main pathogens found, as well as the main diseases and treatment methods. Subsequently, the main ways of preventing these occurrences and the obstacles that need to be overcome to avoid their current persistence are mentioned.

Relatório de estágio de mestrado, Análises Clínicas, Universidade de Lisboa, Faculdade de Farmácia, 2015
Este relatório de estágio pretende descrever as atividades realizadas durante o estágio curricular do Mestrado em Análises Clínicas da Faculdade de Farmácia da Universidade de Lisboa. O estágio decorreu no Instituto Português de Oncologia (IPO) de Lisboa Francisco Gentil, nas áreas de Bioquímica, Imunologia e Virologia. São descritos as metodologias e equipamento utilizados, bem como os parâmetros analisados e o controlo de qualidade.
This internship report aims to describe the activities performed during the curricular traineeship in Masters of Clinical Analysis of the Faculty of Pharmacy of the Lisbon University. The internship was held at Portuguese Institute of Oncology – Lisbon, in Biochemistry, Immunology and Virology. We describe the methodology and equipment used, as well as the parameters analysed and the quality control.
As infeções fúngicas são infeções muito frequentes nos doentes HIV/SIDA. A imunodeficiência causada pela infeção crónica por HIV aumenta o risco de co-infeção por patogéneos que são controlados pela resposta imunitária inata e adaptativa. A administração terapêutica HAART nem sempre restaura a resposta imune patogéneo-específica aos níveis normais. As principais infeções fúngicas nos doentes HIV/SIDA são a candidose, a criptococose, a peniciliose, a pneumocistose, a histoplasmose e a coccidioidomicose. As infeções por Candida e Cryptococcus são as mais comuns. A maioria dos casos de histoplasmose e coccidioidomicose ocorrem em regiões onde os microrganismos são endémicos. Os objetivos gerais deste trabalho foram, através de uma revisão bibliográfica, que incluiu pesquisa de artigos, livros e documentação importante da OMS e do CDC, realçar a importância das infeção fúngicas nos doentes infetados pelo HIV/SIDA e analisar as características de cada uma delas.
Fungal infections are very common infections in HIV/AIDS patients. The immunodeficiency caused by HIV chronic infection increases the risk of co-infection by pathogens that are controlled by the innate and adaptive immune response. The HAART therapeutical administration does not always restore the pathogen-specific immune response to normal levels. The main fungal infections in HIV/AIDS patients are candidiasis, cryptococcosis, penicilliosis, pneumocystosis, histoplasmosis and coccidioidomycosis. Candida and Cryptococcus infections are the most common. Most cases of histoplasmosis and coccidioidomycosis occur in areas where microorganisms are endemic. The aims of this study were, through a literature review, which included the research of articles, books and important documents from the WHO and the CDC, to emphasize the importance of fungal infection in patients infected by HIV/AIDS and to analyse the characteristics of each one of them.

No contexto da pandemia por SARS-CoV-2, em 2020 foram implementadas medidas para uma gestão controlada da propagação de contágio, minimizando ao máximo o risco de transmissão do vírus. A Direção Geral da Saúde legislou normas e recomendações que conduziram à interrupção da atividade nos consultórios e clínicas de medicina dentária em março daquele ano, e à retoma daquela atividade dois meses depois. Em que, para além das precauções universais, recomendou-se a adoção de medidas que assegurem a proteção pelo contágio através de gotículas e aerossóis contaminados. Esta doença é de fácil e rápido contágio, visto que a sua principal via de transmissão é por contacto direto com gotículas salivares portadoras do vírus. Os resultados desta revisão revelaram que as principais alterações orais associadas á COVID-19 são os distúrbios do paladar, xerostomia e ulcerações. Diversas manifestações orais foram observadas, contudo, não há evidências suficientes que comprovem uma relação de causalidade com a infeção por COVID-19. As implicações da COVID-19 no estado de saúde oral assentam na privação no acesso aos cuidados de saúde oral na fase de mitigação da COVID-19, o subdiagnóstico das manifestações orais na fase inicial da pandemia, e atualmente, desconhece-se uma associação cientificamente consistente entre a COVID-19 e as manifestações orais da doença.
In the context of the SARS-CoV-2 pandemic, in 2020 measures were implemented for a controlled management of the spread of contagion, minimizing the risk of virus transmission as much as possible. The General Directorate of Health legislated norms and recommendations that led to the interruption of activity in dental offices and clinics in March of that year, and the resumption of that activity two months later. In which, in addition to universal precautions, the adoption of measures to ensure protection from contagion through contaminated droplets and aerosols was recommended. This disease is easy and fast to spread, as its main transmission route is through direct contact with salivary droplets carrying the virus. The results of this review revealed that the main oral alterations associated with COVID-19 are taste disturbances, xerostomia and ulcerations. Several oral manifestations were observed, however, there is insufficient evidence to prove a causal relationship with COVID-19 infection. The implications of COVID-19 in the oral health status are based on the deprivation of access to oral health care in the mitigation phase of COVID-19, the underdiagnosis of oral manifestations in the initial phase of the pandemic, and currently, a scientific association is unknown. consistent between COVID-19 and the oral manifestations of the disease.