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<p>Vi&scaron;e od 100 vrsta virusa ljudi i životinja se izlučuje u spolja&scaron;nju sredinu. Prisustvo ovih virusa u povr&scaron;inskim vodama reflektuje fekalnu kontaminaciju i ukazuje na<br />opasnost za zdravlje ljudi i životinja. Na području Srbije se ne prati prisustvo patogenih virusa u povr&scaron;inskim vodama, pa&nbsp; čak ni u vodama za piće, a nije uspostavljena&nbsp; ni&nbsp; metodologija ovih ispitivanja. Shodno tome, cilj disertacije je da se utvrdi i analizira prisustvo animalnih i humanih virusa u povr&scaron;inskim vodama primenom najsavremenijih metoda koncentrovanja i detekcije virusa. U okviru disertacije ispitano je prisustvo sledećih virusa u povr&scaron;inskim vodama na teritoriji Vojvodine: humanih adenovirusa (HAdV); norovirusa (NoV) i hepatitis A virusa (HAV), adenovirusa svinja (PAdV),&nbsp; poliomavirusa goveda (BPyV) i hepatitis E virus (HEV).</p><p>Ispitano je ukupno 108 uzoraka povr&scaron;inskih i otpadnih voda koji su prikupljani od oktobra 2012. godine do juna 2014. godine. U radu su primenjene najsavremenije metode koncentrovanja i detekcije virusa u vodi, koje se u Srbiji nisu koristile za ovu namenu. Sprovedenim ispitivanjima dokazano je da su animalni i humani virusi prisutni u povr&scaron;inskim vodama na području Vojvodine. Najče&scaron;će detektovan&nbsp; virus u povr&scaron;inskim vodama je humani adenovirus (42,4%), a potom norovirusi GII i GI (40,4% i 15,2%), adenovirus svinja (11,1%), poliomavirus goveda (7,1%) i hepatitis E virus (3,0%). U ukupno 9 testiranih uzoraka gradske kanalizacione vode najče&scaron;će je detektovan HAdV (44,4%), NoV GII i GI&nbsp; (66,7% i 22,2%), BPyV je detektovan u samo jednom od 9 uzoraka, a niti u jednom nisu detektovani PAdV i HEV. Hepatitis A virus nije detektovan u uzorcima, a eksperimentalno je potvrđeno da su metode primenljive i za detekciju ovog virusa. Na osnovu rezultata prinosa procesne kontrole i utvrđenog prisustva virusa u uzorcima,&nbsp; zaključeno&nbsp; je da se ove metode mogu veoma uspe&scaron;no koristiti za detekciju virusne kontaminacije&nbsp; povr&scaron;inskih voda. Izvr&scaron;ena je igenotipizacija virusa iz odabranih uzoraka metodom sekvenciranja dela virusnog genoma. Indirektno je potvrđeno da su infekcije&nbsp;&nbsp; detektovanim virusima prisutne u populaciji životinja i ljudi. Prisustvo virusa u&nbsp;&nbsp; povr&scaron;inskim vodama i uzorcima gradske kanalizacije odražava infektivni status stanovni&scaron;tva, ali predstavlja i značajan rizik za zdravlje životinja i ljudi na području koje gravitira ispitanim vodama.&nbsp;</p> / <p>Over 100 types of pathogenic viruses are excreted in human and animal wastes. The presence of human and animal pathogenic enteric viruses in water environments reflects fecal contamination and indicates a risk to public health.&nbsp; Republic of Serbia does not implement surveillance for the presence of pathogenic human and animal viruses in surface waters and even in drinking water, neither is the established methodology of these studies in any institution in Serbia.&nbsp; Accordingly, the aim of the study was to determine and analyze the presence of human and animal viruses in surface water,&nbsp; using the latest methods&nbsp; of&nbsp; concentration and detection of the viruses.&nbsp; Within the dissertation examined the presence of the following viruses in surface waters in Vojvodina:&nbsp; Human adenoviruses&nbsp; (HAdV), noroviruses (NoV)&nbsp; and hepatitis A virus), Porcine adenovirus (PAdV) and Bovine polyomavirus (BPyV)&nbsp; and&nbsp; Hepatitis E virus (HEV).<br />A total of 108 samples of surface water and waste water were collected from October 2012 to June 2014. The paper are applied the most advanced methods and the concentration of virus detection in water, which in Serbia are not used for this purpose. The conducted tests have proven that the animal and human viruses present in surface waters in Vojvodina. The most commonly detected virus in surface water was human adenovirus (42.4%), followed by Norovirus GI&nbsp; and GII (40.4% and 15.2%),&nbsp; Porcine adenovirus&nbsp; (11,1%),&nbsp; Bovine polyomavirus&nbsp; (7.07%) and hepatitis E virus (3,0%).<br />In total of&nbsp; nine analysed sewage samples human adenovirus was detected in 44,4%&nbsp; of&nbsp; samples. The prevalence of norovirus GII and GI in sewage&nbsp; samples was&nbsp; 66,7%&nbsp; and 22,2%. Bovine&nbsp; polyomavirus was detected in one of nine samples while porcine adenovirus and hepatitis E virus were not detected in any of analyzed samples.&nbsp; Hepatitis A virus was not detected in samples, but&nbsp; it has been experimentally confirmed that the methods applicable for detection of the virus. Based on the results of process control and yield determined the presence of virus insamples, it was found that these methods can be successfully used to detect viral contamination of surface waters. Also, in these study was performed genotyping of viruses from selected samples by sequencing a part of the viral genome. Indirectly it is confirmed that the infection detected viruses present in a population of animals and humans. The presence of virus in samples of surface water and urban sewage reflects the infectious status of the population, but also constitutes a significant risk to the health of animals and people in the area that gravitates with tested waters.</p><p>&nbsp;</p>

To understand molecular mechanisms of individual steps of virus infection is a prerequisite for successful design of specific and effective antiviral drugs. Polyomaviruses, replicating in the cell nucleus, travel from plasma membrane to the endoplasmic reticulum (ER) in endosomes. However, it is not clear how they deliver their DNA genomes from ER to the nucleus. In this thesis, we found that partially disassembled virions of the Murine polyomavirus (MPyV) interact with importin β1 at around 6 hours post infection. Mutational disruption of the nuclear localization signal (NLS) of the major capsid protein, VP1, and/or common NLS sequence of the minor capsid proteins VP2 and VP3 did not affect the structure and composition of virions, but it resulted in decreased viral infectivity (up to 80%). Virions are thus released from ER to cytosol and translocate to the nucleus via nucleopores. Mutation analyses of NLSs of individual capsid proteins showed that MPyV virions can utilize VP1 and VP2/VP3 NLSs in concert. However, one functional NLS, either that of VP1 or VP2/3 seems to be sufficient for the delivery of VP1-VP2/3 complexes into the nucleus, although none of these proteins is delivered into the nucleus separately. Thus, the conformation of NLS regions given by the presence of all three capsid...

In this dissertation the investigation of potential applications of hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) harboring CTL epitopes for anticancer vaccine development is presented. The objective of this study was to investigate the potential of recombinant HaPyV VP1 based VLPs for anticancer vaccine generation in model systems, including investigation of VP1 applicability for heterologous CTL epitopes insertions, VLPs assembly and ability to induce insert specific immune response in vivo. HaPyV VP1 VLPs carrying CLT epitopes derived from different proteins were generated, most suitable positions for insertion into VP1 protein were selected, the ways to improve assembly and yield of the chimeric VLPs were determined and new VLPs purification procedure was created allowing to purify VLPs cheaper, faster and more efficiently. HaPyV VP1 based VLPs ability to induce CTL immune response in vivo was evaluated for the first time. It was demonstrated that model chimeric VLPs were able to stimulate antigen specific CTL cells in vitro and in vivo, induced insert specific humoral and CTL immune response in vivo and protected mice from insert specific virus infection and antigen-specific tumor growth. Presented data confirmed that HaPyV protein VP1 is universal carrier for CTL epitopes, capable to tolerate insertions, to form VLPs and to induce effective, long lasting immune response against inserted antigens in vivo. / Disertacijoje yra aprašomas perspektyvų panaudoti žiurkėno poliomos viruso (HaPyV) pagrindinio struktūrinio baltymo VP1 formuojamas į virusus panašias daleles priešvėžinių vakcinų kūrimui tyrimas. Pagrindinis disertacijos darbo tikslas buvo modelinėse sistemose parodyti rekombinantinių HaPyV VP1 baltymų formuojamų į virusus panašių dalelių panaudojimo priešvėžinių vakcinų kūrimui galimybes, įvertinant svetimų CTL epitopų įterpimo į VP1 baltymą toleravimą, VPD formavimosi efektyvumą bei sukeltą įterptam antigenui specifinį imuninį atsaką. Disertacijoje atlikta tyrimo srities literatūros apžvalga, smulkiai aprašomi darbe naudoti metodai, atlikti eksperimentai, pateikiami bei analizuojami gauti rezultatai. Darbe pirmą kartą buvo nuodugniai ištirtos HaPyV viruso VP1 baltymo formuojamų VPD savybės, parodytas jų tinkamumas būti CTL epitopų nešikliais, ištirtos įterpimui palankiausios VP1 baltymo vietos, išbandyti nauji VPD gavimo ir gryninimo būdai, pagerinantys chimerinių VPD formavimąsi bei išeigas. Panaudojant modelines chimerines VPD in vivo buvo ištirtas chimerinių HaPyV VP1 pagrindu sukonstruotų VPD sukeliamas humoralinis ir ląstelinis imuninis atsakas. Gauti rezultatai parodė, kad HaPyV VP1 baltymas yra vienas iš nedaugelio virusų struktūrinių baltymų, kurie ne tik formuoja VPD, bet pasižymi ir universaliomis baltymo – nešiklio savybėmis, o in vivo sukelia efektyvų, ilgalaikį, įterptam epitopui specifinį imuninį atsaką.

Mouse polyomavirus is a type species of Polyomaviridae family and serves as model for studying viral infection of human pathogenic polyomaviruses. Minor proteins of viral capsid have been found to be necessary for effective initiation of infection. In order to study their role in the early steps of infection we utilized the novel Cre-LoxP system for production of the viral mutant lacking both minor proteins. Virus produced this way was compared with virus produced by standard method and we found that both systems facilitate production of mutant virus with the comparable quality and quantity. The mutant virus contained reduced amount of viral DNA and formed virions with impaired stability. For further studies of intracellular virion trafficking we prepared virions with genomes modified by thymidine analogues 5- bromo-2'-deoxyuridine (BrdU) and 5-Ethynyl-2'-deoxyuridine (EdU) and optimized the methods for analogue detection. The viral genome become accessible for detection 4 hours post infection. For ultramicroscopic analysis of translocation of virus to the nucleus we used freeze substitution. All this methods will be utilized for detailed study of distinct steps in viral infection. Key words: Mouse polyomavirus, minor proteins,...

Os Poliomavírus JC e BK são os mais importantes integrantes da família Polyomaviridae, gênero Orthopolyomavirus, devido ao seu grau de patogenicidade no homem. O poliomavírus humano JC (JCV) foi isolado a partir de fragmento do cérebro de um paciente com linfoma de Hodgkin e leucoencefalopatia multifocal progressiva por Padgett e colaboradores. Já o poliomavírus humano BK foi isolado em 1971 por Gardner e colaboradores a partir da semeadura da urina de um paciente, submetido a transplante renal, em cultura de células da linhagem VERO Poucos são os dados sobre os poliomavírus humanos JC e BK em indivíduos sadios no mundo e no Brasil. Além disso, as formas de excreção e transmissão destes vírus ainda não estão completamente elucidadas. Este estudo teve como objetivos principais determinar a incidência, a dinâmica de excreção e a caracterização genotípica dos poliomavírus JC e BK em amostras sequenciais de urina de indivíduos sadios. Como objetivo secundário, foram analisados filogeneticamente os subtipos dos vírus encontrados. Foram incluídos 71 indivíduos de ambos os sexos, com idades variando entre 21 e 65 anos e destes foram coletadas amostras mensais de urina durante 6 meses. Todas as amostras do estudo foram submetidas a um teste de PCR em tempo real, que amplifica um fragmento do gene que codifica o antígeno T, e a um PCR convencional que amplifica um fragmento do gene da proteína VP1 do vírus. Ao final do período de 6 meses de acompanhamento a incidência de excreção urinária dos poliomavírus JCV e BKV na população estudada foi de 53,52% e 64,79% respectivamente. O perfil de excreção do JCV mostrou-se continuo em 42% dos casos , intermitente em 8% e esporádico em 50% dos casos. Analisando o perfil de excreção do BKV, em 80% dos casos mostrou-se esporádico, em 17% intermitente e em 3% dos casos contínuo. Posteriormente, as amostras positivas foram sequenciadas e analisadas filogeneticamente observando-se que os genótipos mais prevalentes do JCV foram o 1, subtipo 1B e 3, seguidos dos genótipos 1, subtipo 1A e 4. Com relação ao BKV, o genótipo 1, subtipo 1A foi o mais prevalente, seguido do 4 e do 1, subtipo 1B. / The Polyomavirus JC and BK are the most important members of Polyomaviridae family, genus Orthopolyomavirus, due to their degree of pathogenicity in humans. The human polyomavirus JC (JCV) was isolated from a fragment of the brain of a patient with Hodgkin\'s lymphoma and progressive multifocal leukoencephalopathy by Padgett et al. On the other hand the human polyomavirus BK was also isolated in 1971 by Gardner et al from the urine of a patient who underwent renal transplantation in VERO cell line. There are few data on human polyomavirus JC and BK in healthy individuals on the world and in Brazil. Moreover the forms of excretion and transmission of these viruses are not yet fully elucidated. This study aimed to determine the incidence, the dynamics of excretion, and the molecular characterization of polyomavirus JC and BK in serial samples of urine of healthy individuals. A secondary objective was to analyze phylogenetically the subtypes of the viruses found during the study. Were included 71 patients of both genders, aged from 21 to 65 yearsold. Urine samples were collected every month for six months. All samples in the study were screened by a real time PCR which amplifies a fragment of the T antigen gene, and to a conventional PCR that amplifies a fragment of the gene of VP1 protein of the virus. At the end of 6 months of follow-up, the incidence of urinary excretion of polyomaviruses BKV and JCV in the study population was 53.52% and 64.79% respectively The profile of excretion of JCV was continuous in 42% of cases, intermittent in 8% and sporadic in 50% of cases. The profile of excretion of BKV was shown to be sporadic in 80% of cases, intermittent in 17% and continuous in only 3% of cases. Subsequently, the positive samples were sequenced and analyzed phylogenetically showing that the more prevalent genotypes were JCV 1, subtype 1b and 3, followed by genotype 1, subtypes 1a and 4. Regarding the BKV genotype 1 subtype 1a was the most prevalent, followed by 4 and 1, subtype 1b.

A Esclerose Múltipla (EM) é uma doença autoimune caracterizada por um processo neuroinflamatório com degeneração axonal progressiva. O medicamento Natalizumab (Biogen Idec, NC, USA) representa hoje um dos tratamentos mais promissores para EM. Entretanto, pacientes sob esse tratamento possuem maiores chances de desenvolver Leucoencefalopatia Multifocal Progressiva (LEMP), em decorrência de uma possível reativação do poliomavírus JC (VJC). Além do VJC, o poliomavírus BK (VBK) pode representar uma preocupação adicional para tais pacientes, uma vez que também apresenta capacidade de causar encefalopatias. Apesar do Natalizumab ser uma ótima ferramenta contra a EM, o fato de interagir de alguma maneira com os poliomavírus, em especial o VJC, impede que seja utilizado em larga escala. Dessa forma,o objetivo desse trabalho foi investigar os padrões de excreção e reativação dos VJC e VBK em pacientes com EM durante o tratamento com Natalizumab e comparar aos padrões observados em pacientes que se encontram sob outros tratamentos. Amostras seriadas de sangue e urina foram coletadas e submetidas a testes de biologia molecular para detecção do vírus e caracterização molecular. Foram analisados 97 pacientes em diferentes tempos de acompanhamento. Não foi observada presença de poliomavírus no sangue de nenhum dos indivíduos analisados. Entretanto, 36% excretavam poliomavírus na urina em pelo menos uma das coletas, sendo que 21,7% excretavam VJC, 9,3% excretavam VBK e 5,1% excretavam ambos os poliomavírus. Não foi observada diferença entre as taxas de excreção urinária de poliomavírus entre pacientes que tratavam com Natalizumab (38,9%) e pacientes que sob outros tratamentos (34,5%), sendo que para o Grupo Controle (GC); 21,3%, 8,2% e 4,9% excretavam VJC, VBK e ambos os vírus, respectivamente e para o grupo Grupo Natalizumab (GN) 22,2%, 11,1% e 5,6% excretavam VJC, VBK e ambos os vírus, respectivamente. As análises moleculares da Região Regulatória do VJC revelaram sequências de característica arquetípica. A reconstrução filogenética de sequências do gene VP1 do VJC revelou predominância do genótipo 3 e do genótipo 1 para o VBK. Não foi observada diferença estatística da carga viral do VJC e do VBK entre os dois grupos. Foi detectada uma mutação (E29G) na VP1 de uma paciente que apresentou alta carga viral do VJC. Do grupo GN, 14 apresentaram anticorpos para VJC, sendo que desses 58% apresentou excreção de VJC, 42% não apresentou excreção urinária, interessantemente uma paciente não apresentou anticorpos contra o VJC, mas apresentou excreção de VJC. Pode-se concluir principalmente que a detecção de anticorpos, concomitantemente com a investigação molecular do VJC poderá contribuir para uma melhor determinação da estratificação do risco de desenvolvimento de LEMP em indivíduos com EM sob tratamento com Natalizumab. / Multiple sclerosis (MS) is an autoimmune disease characterized by neuronal inflamatory process with progressive axonal degeneration. The drug Natalizumab (Biogen Idec, NC, USA) is today one of the most promising treatments for MS. However, patients undergoing this treatment have higher chances of developing progressive multifocal leukoencephalopathy (PML), due to a possible reactivation of the polyomavirus JC (VJC). Besides VJC, the BK polyomavirus (VBK) may represent an additional concern for such patients, since it also has ability to cause encephalopathies. Despite Natalizumab be a great tool against MS, the fact that drug some way may interact with polyomavirus, especially VJC, prevents it from being used on a large scale. Thus, aim of this study was to investigate the patterns of excretion and reactivation of VJC and VBK in MS patients during treatment with Natalizumab and compare the patterns observed in patients who are under other treatments. Serial blood samples and urine were collected and submitted to molecular biology tests for virus detection and molecular characterization. Ninety seven patients were analyzed at different follow-up times. There was no polyomavirus DNA in the blood of none subjects analyzed. However, 36% of patients excreted polyomavirus in the urine in at least one of the samples, of those 21.7%, 9.3% and 5.1% excreted VJC, VBK and both polyomavirus, respectively. No difference was observed between the rates of urinary excretion of polyomavirus patients treated with Natalizumab (38.9%) and patients treated with other drugs (34.5%), for the Control Group (GC); 21,3%, 8,2% and 4,9 shed VJC, VBK and both viruses, respectevely and for the Natalizumab Group (GN) 22,2%, 11,1% and 5,6% shed VJC, VBK and both viruses, respectvely. Molecular analysis of the Regulatory Region of VJC revealed sequences similar to the archetype form of VJC. A phylogenetic reconstruction of the VP1 gene sequences revealed VJC predominance of genotype 3 and genotype 1 for VBK. There was no statistical difference in the viral load VJC and VBK between the two groups. It was detected a mutation (E29G) in VP1 of a patient who had a high viral load VJC, however the mutation disappeared after a few months of monitoring. Fourteen patients of GN had antibodies to VJC, and of these 58% had excretion VJC, 42% showed no urinary excretion, interestingly one patient had no antibodies against VJC but showed excretion of VJC. It can be concluded that mostly anti-VJC antibodies detection, concurrently with the VJC molecular research may contribute to a better determination of risk stratification for development of PML in patients with MS undergoing treatment with Natalizumab.

En 2008, le génome d'un nouveau virus a été caractérisé au sein d'un cancer cutané rare survenant préférentiellement chez l'immunodéprimé, le carcinome de Merkel. Ce nouveau virus appartenait à la famille des Polyomaviridae qui comprend des virus dont le caractère cancérigène chez l'animal est avéré depuis plus de 50 ans. Dénommé Polyomavirus de Merkel puisqu'il semblait lié à la survenue du cancer du même nom, il constituait le premier Polyomavirus impliqué de manière consistante dans un cancer humain. Cette implication reposant sur une étude unique limitée à 10 cas, l'objectif de notre travail de thèse était de confirmer le rôle étiologique du Polyomavirus de Merkel dans le carcinome de Merkel.Nous avons montré que le génome du Polyomavirus de Merkel était présent dans les trois quarts des cas de carcinome de Merkel, mais également que le virus infecte la population générale de manière quasi-ubiquitaire et de nombreux tissus en dehors de la peau. Les faits que chez les sujets atteints de carcinome de Merkel, l'ADN viral soit présent à des taux décelables de manière chronique dans différents tissus et que les titres d'anticorps sériques spécifiques du virus soient élevés suggèrent que ces sujets développent une infection chronique active. Celle-ci pourrait faciliter la survenue de mutations et d'intégrations de l'ADN viral qui sont spécifiquement associées aux carcinomes de Merkel. Ces modifications secondaires du génome viral aboutissent à la production d'oncoprotéines virales par les cellules tumorales, mais à l'abolition des capacités réplicatives donc lytiques du virus et constitueraient ainsi le support de la transformation tumorale.

Os Poliomavírus JC e BK são os mais importantes integrantes da família Polyomaviridae, gênero Orthopolyomavirus, devido ao seu grau de patogenicidade no homem. O poliomavírus humano JC (JCV) foi isolado a partir de fragmento do cérebro de um paciente com linfoma de Hodgkin e leucoencefalopatia multifocal progressiva por Padgett e colaboradores. Já o poliomavírus humano BK foi isolado em 1971 por Gardner e colaboradores a partir da semeadura da urina de um paciente, submetido a transplante renal, em cultura de células da linhagem VERO Poucos são os dados sobre os poliomavírus humanos JC e BK em indivíduos sadios no mundo e no Brasil. Além disso, as formas de excreção e transmissão destes vírus ainda não estão completamente elucidadas. Este estudo teve como objetivos principais determinar a incidência, a dinâmica de excreção e a caracterização genotípica dos poliomavírus JC e BK em amostras sequenciais de urina de indivíduos sadios. Como objetivo secundário, foram analisados filogeneticamente os subtipos dos vírus encontrados. Foram incluídos 71 indivíduos de ambos os sexos, com idades variando entre 21 e 65 anos e destes foram coletadas amostras mensais de urina durante 6 meses. Todas as amostras do estudo foram submetidas a um teste de PCR em tempo real, que amplifica um fragmento do gene que codifica o antígeno T, e a um PCR convencional que amplifica um fragmento do gene da proteína VP1 do vírus. Ao final do período de 6 meses de acompanhamento a incidência de excreção urinária dos poliomavírus JCV e BKV na população estudada foi de 53,52% e 64,79% respectivamente. O perfil de excreção do JCV mostrou-se continuo em 42% dos casos , intermitente em 8% e esporádico em 50% dos casos. Analisando o perfil de excreção do BKV, em 80% dos casos mostrou-se esporádico, em 17% intermitente e em 3% dos casos contínuo. Posteriormente, as amostras positivas foram sequenciadas e analisadas filogeneticamente observando-se que os genótipos mais prevalentes do JCV foram o 1, subtipo 1B e 3, seguidos dos genótipos 1, subtipo 1A e 4. Com relação ao BKV, o genótipo 1, subtipo 1A foi o mais prevalente, seguido do 4 e do 1, subtipo 1B. / The Polyomavirus JC and BK are the most important members of Polyomaviridae family, genus Orthopolyomavirus, due to their degree of pathogenicity in humans. The human polyomavirus JC (JCV) was isolated from a fragment of the brain of a patient with Hodgkin\'s lymphoma and progressive multifocal leukoencephalopathy by Padgett et al. On the other hand the human polyomavirus BK was also isolated in 1971 by Gardner et al from the urine of a patient who underwent renal transplantation in VERO cell line. There are few data on human polyomavirus JC and BK in healthy individuals on the world and in Brazil. Moreover the forms of excretion and transmission of these viruses are not yet fully elucidated. This study aimed to determine the incidence, the dynamics of excretion, and the molecular characterization of polyomavirus JC and BK in serial samples of urine of healthy individuals. A secondary objective was to analyze phylogenetically the subtypes of the viruses found during the study. Were included 71 patients of both genders, aged from 21 to 65 yearsold. Urine samples were collected every month for six months. All samples in the study were screened by a real time PCR which amplifies a fragment of the T antigen gene, and to a conventional PCR that amplifies a fragment of the gene of VP1 protein of the virus. At the end of 6 months of follow-up, the incidence of urinary excretion of polyomaviruses BKV and JCV in the study population was 53.52% and 64.79% respectively The profile of excretion of JCV was continuous in 42% of cases, intermittent in 8% and sporadic in 50% of cases. The profile of excretion of BKV was shown to be sporadic in 80% of cases, intermittent in 17% and continuous in only 3% of cases. Subsequently, the positive samples were sequenced and analyzed phylogenetically showing that the more prevalent genotypes were JCV 1, subtype 1b and 3, followed by genotype 1, subtypes 1a and 4. Regarding the BKV genotype 1 subtype 1a was the most prevalent, followed by 4 and 1, subtype 1b.

A Esclerose Múltipla (EM) é uma doença autoimune caracterizada por um processo neuroinflamatório com degeneração axonal progressiva. O medicamento Natalizumab (Biogen Idec, NC, USA) representa hoje um dos tratamentos mais promissores para EM. Entretanto, pacientes sob esse tratamento possuem maiores chances de desenvolver Leucoencefalopatia Multifocal Progressiva (LEMP), em decorrência de uma possível reativação do poliomavírus JC (VJC). Além do VJC, o poliomavírus BK (VBK) pode representar uma preocupação adicional para tais pacientes, uma vez que também apresenta capacidade de causar encefalopatias. Apesar do Natalizumab ser uma ótima ferramenta contra a EM, o fato de interagir de alguma maneira com os poliomavírus, em especial o VJC, impede que seja utilizado em larga escala. Dessa forma,o objetivo desse trabalho foi investigar os padrões de excreção e reativação dos VJC e VBK em pacientes com EM durante o tratamento com Natalizumab e comparar aos padrões observados em pacientes que se encontram sob outros tratamentos. Amostras seriadas de sangue e urina foram coletadas e submetidas a testes de biologia molecular para detecção do vírus e caracterização molecular. Foram analisados 97 pacientes em diferentes tempos de acompanhamento. Não foi observada presença de poliomavírus no sangue de nenhum dos indivíduos analisados. Entretanto, 36% excretavam poliomavírus na urina em pelo menos uma das coletas, sendo que 21,7% excretavam VJC, 9,3% excretavam VBK e 5,1% excretavam ambos os poliomavírus. Não foi observada diferença entre as taxas de excreção urinária de poliomavírus entre pacientes que tratavam com Natalizumab (38,9%) e pacientes que sob outros tratamentos (34,5%), sendo que para o Grupo Controle (GC); 21,3%, 8,2% e 4,9% excretavam VJC, VBK e ambos os vírus, respectivamente e para o grupo Grupo Natalizumab (GN) 22,2%, 11,1% e 5,6% excretavam VJC, VBK e ambos os vírus, respectivamente. As análises moleculares da Região Regulatória do VJC revelaram sequências de característica arquetípica. A reconstrução filogenética de sequências do gene VP1 do VJC revelou predominância do genótipo 3 e do genótipo 1 para o VBK. Não foi observada diferença estatística da carga viral do VJC e do VBK entre os dois grupos. Foi detectada uma mutação (E29G) na VP1 de uma paciente que apresentou alta carga viral do VJC. Do grupo GN, 14 apresentaram anticorpos para VJC, sendo que desses 58% apresentou excreção de VJC, 42% não apresentou excreção urinária, interessantemente uma paciente não apresentou anticorpos contra o VJC, mas apresentou excreção de VJC. Pode-se concluir principalmente que a detecção de anticorpos, concomitantemente com a investigação molecular do VJC poderá contribuir para uma melhor determinação da estratificação do risco de desenvolvimento de LEMP em indivíduos com EM sob tratamento com Natalizumab. / Multiple sclerosis (MS) is an autoimmune disease characterized by neuronal inflamatory process with progressive axonal degeneration. The drug Natalizumab (Biogen Idec, NC, USA) is today one of the most promising treatments for MS. However, patients undergoing this treatment have higher chances of developing progressive multifocal leukoencephalopathy (PML), due to a possible reactivation of the polyomavirus JC (VJC). Besides VJC, the BK polyomavirus (VBK) may represent an additional concern for such patients, since it also has ability to cause encephalopathies. Despite Natalizumab be a great tool against MS, the fact that drug some way may interact with polyomavirus, especially VJC, prevents it from being used on a large scale. Thus, aim of this study was to investigate the patterns of excretion and reactivation of VJC and VBK in MS patients during treatment with Natalizumab and compare the patterns observed in patients who are under other treatments. Serial blood samples and urine were collected and submitted to molecular biology tests for virus detection and molecular characterization. Ninety seven patients were analyzed at different follow-up times. There was no polyomavirus DNA in the blood of none subjects analyzed. However, 36% of patients excreted polyomavirus in the urine in at least one of the samples, of those 21.7%, 9.3% and 5.1% excreted VJC, VBK and both polyomavirus, respectively. No difference was observed between the rates of urinary excretion of polyomavirus patients treated with Natalizumab (38.9%) and patients treated with other drugs (34.5%), for the Control Group (GC); 21,3%, 8,2% and 4,9 shed VJC, VBK and both viruses, respectevely and for the Natalizumab Group (GN) 22,2%, 11,1% and 5,6% shed VJC, VBK and both viruses, respectvely. Molecular analysis of the Regulatory Region of VJC revealed sequences similar to the archetype form of VJC. A phylogenetic reconstruction of the VP1 gene sequences revealed VJC predominance of genotype 3 and genotype 1 for VBK. There was no statistical difference in the viral load VJC and VBK between the two groups. It was detected a mutation (E29G) in VP1 of a patient who had a high viral load VJC, however the mutation disappeared after a few months of monitoring. Fourteen patients of GN had antibodies to VJC, and of these 58% had excretion VJC, 42% showed no urinary excretion, interestingly one patient had no antibodies against VJC but showed excretion of VJC. It can be concluded that mostly anti-VJC antibodies detection, concurrently with the VJC molecular research may contribute to a better determination of risk stratification for development of PML in patients with MS undergoing treatment with Natalizumab.