Dissertations / Theses on the topic 'Human Polyomavirus JC (JCV)'

Os poliomavírus compõem uma grande família de vírus que causam infecções primárias geralmente na infância, e se mantem em condições subclínicas. Em situações de imunossupressão podem causar algumas doenças. Os indivíduos com HIV/AIDS frequentemente apresentam deficiência imunológica e por isso podem exibir maior risco de doenças causadas pelos poliomavírus. A utilização da saliva no diagnóstico e acompanhamento de doenças infecciosas tem sido explorado na literatura. As vantagens de usar a saliva para rastreio se pautam especialmente na coleta não invasiva e segurança no manuseio. O presente estudo teve como objetivo, detectar e quantificar o DNA dos poliomavírus BKV, JCV, de células Merkel e TSV, em fluídos orais (saliva, lavado bucal e fluído gengival crevicular) e comparar com a detecção em soro e urina, meios usualmente utilizados para detecção. Foram coletadas 299 amostras de 42 indivíduos, sendo 22 HIV positivos (GE) e 20 pacientes controle (GC). No GE, 63,6% dos pacientes apresentaram positividade para JCV em pelo menos uma amostra analisada, 54,5% foram positivos para BKV, 18,2% para células Merkel e não houve amostra positiva para TSV. No GC, 45% exibiu positividade para o JCV em pelo menos uma amostra analisada, 80% para BKV e nenhuma participante controle exibiu positividade para células Merkel e TSV. Não houve diferença de frequência de detecção viral entre os grupos estudados em relação às amostras coletadas, ou ainda em relação à idade ou sexo. Entretanto, nas amostras de fluídos orais houve maior prevalência de detecção para o BKV e para células Merkel. Concluímos que fluídos orais, especialmente saliva e lavado bucal, podem ser usados para o rastreamento do BK e JC; e que os indivíduos HIV positivos, sob tratamento antirretroviral não exibem frequências maior de poliomavírus, comparativamente a indivíduos controle.
Polyomavirus is one of the large family of viruses that cause primary infections usually in childhood, and can remain subclinical. In immunosuppression may cause some diseases. Individuals with HIV/AIDS often have immune deficiencies and may be at increased risk for diseases caused by polyomaviruses. The use of saliva in the diagnosis and follow-up of infectious diseases has been explored in the literature. The advantages of using saliva for screening are based on non-invasive collection and handling safety. The aim of present study was to detect and quantify the DNA from BKV, JCV, Merkel cell and TSV polyomaviruses in oral fluids (saliva, mouthwash and gingival crevicular fluid) and to compare it with serum and urine detection, the means usually used for detection. A total of 299 samples were collected from 42 individuals, 22 HIV positive (GE) and 20 control patients (GC). In GE, 63,6% of the patients presented positive for JCV in at least one sample analyzed, 54,5% were positive for BKV, 18,2% for Merkel cell and there was no positive sample for TSV. In GC, 45% showed JCV positivity in at least one analyzed sample, 80% in BKV, and no control participant exhibited positivity for Merkel cell and TSV. There was no difference in the frequency of viral detection among the groups studied in relation to the samples collected, or in relation to age or gender. However, in oral fluid samples there was a higher prevalence of detection for BKV and Merkel cell. We conclude that oral fluids, especially saliva and mouthwash, can be used for the screening of BK e JC; and that HIV positive individuals under antiretroviral treatment do not exhibit higher frequencies of polyomavirus compared to healthy control subjects.

Novas abordagens clínicas para o diagnóstico e monitoramento de pessoas com doenças sistêmicas têm sido empregadas, através da utilização de fluidos biológicos orais, como a saliva e o fluido gengival crevicular (FGC). Alguns autores têm avaliado o potencial desses fluidos no diagnóstico e acompanhamento de doenças, por apresentarem vantagens tais como coleta não invasiva e segurança no manuseio. Até o presente momento, poucos trabalhos detectaram os poliomavírus humano BK (BKV) e JC (JCV) em saliva e nenhum trabalho procurou sua presença no FGC. Esses poliomavírus infectam assintomaticamente cerca de 80% da população geral, mantendo-se latente no trato urinário. No caso de imunossupressão mediada por células, pode ocorrer o aumento da replicação e indução de reação inflamatória. Uma das doenças causadas pela replicação do BKV é a nefropatia associada ao poliomavírus (NAP), caracterizada pela disfunção e perda do próprio rim ou do rim transplantado, enquanto a Leucoencefalopatia Multifocal Progressiva (LMP), causada pela replicação do JCV, infecta os oligodendrócitos, causando desmielinização. Métodos não invasivos para o screening dos poliomavírus podem facilitar a detecção de novos casos e a monitoração de casos previamente conhecidos. O objetivo deste estudo foi verificar a possibilidade de detecção e quantificação do BKV e JCV em fluidos orais (saliva, lavado bucal e FGC) de indivíduos com insuficiência renal crônica (IRC), transplantados renais (TR), e controles em relação ao sangue e urina, fluidos frequentemente usados para esse teste. Para tanto, foram incluídos no estudo 38 sujeitos, divididos em 3 grupos, sendo 14 indivíduos no grupo com IRC (GIR), 12 TR no grupo transplantado renal (GTR) e 12 indivíduos saudáveis no grupo controle (GC). No total, coletamos 283 amostras dos participantes, sendo 151 de FGC, 38 amostras de saliva, 38 de lavado bucal, 35 de soro e 21 amostras de urina. No GIR, 100% (14) dos indivíduos apresentaram positividade para BKV em pelo menos uma amostra analisada e 14% (2) foram positivos para JCV. No GTR, 91,7% (11) dos indivíduos foram positivos para BKV e 51,7% (5) foram positivos para JCV. Dentre os sujeitos do GC, 91,7% (11) foram positivos para BKV e 50% (6) para JCV, em pelo menos uma amostra testada. Não houve diferença de frequência de detecção viral entre os 3 grupos de participantes, com relação às amostras coletadas. As amostras de fluidos orais (saliva, lavado e FGC) exibiram alta prevalência de detecção, principalmente do BKV, com muitas amostras com níveis quantificáveis de carga viral. Concluímos que fluidos orais, especialmente saliva e lavado bucal, podem ser usados para o rastreamento do BKV e JCV.
New clinical approaches for diagnosis and monitoring of individuals with systemic diseases have been employed through the use of oral biological fluids such as saliva and gingival crevicular fluid (GCF). Some authors have evaluated the potential of these fluids in the diagnosis and monitoring of diseases, because they have advantages such as noninvasive collection and safe handling. To date, few studies have demonstrated the detection of human polyomavirus BK (BKV) and JC (JCV) in saliva and no study reached for its presence in GCF. These polyomavirus infect asymptomatically around 80% of general population, remaining latent in the urinary tract. In case of immunosuppression mediated by cells, there is increased inflammation and induction of replication. One of the diseases caused by BKV replication is polyomavirus associated to the nephropathy (PVAN), characterized by the dysfunction or loss of the kidney or transplanted kidney, while the progressive multifocal leukoencephalopathy (PML) is caused by replication of JCV, infects oligodendrocytes causing demyelination. Noninvasive screening could facilitate the detection of new cases and monitoring of cases previously known. The objective of this study was to investigate the possibility of BKV and JCV detection and quantification in oral fluids (saliva, mouthwash and GCF) of individuals with chronic kidney failure (CKF), kidney transplantation (KT), and controls compared with blood and urine, often used for this test. Therefore, we included 38 subjects, divided into 3 groups, being 14 individuals with CKF (KFG), 12 individuals with KT (KTG) and 12 healthy control individuals (CG). In a total, we collected 283 samples, being 151 of GCF, 38 of saliva, 38 of mouthwash, 35 of serum and 21 samples of urine. In the KFG, 100% (14) of the individuals were positive for BKV in at least one of the collected sample and 14% (2) were positive for JCV. In the KTG, 91.7% (11) were positive for BKV and 51.7% for JCV. Among the subjects of the CG, 91.7% (11) were positive for BKV and 50% (6) to JCV, in at least one tested sample. There was no difference in viral detection frequency between the 3 studied groups with respect to the collected samples. Oral fluids samples (saliva, mouthwash and GCF) exhibited high prevalence of detection, especially of BKV, and several samples showed detectable viral load. We conclude that oral fluids, especially saliva and mouthwash, can be used for the screening of BKV and JCV.

E' noto che il sistema immunitario rappresenta la base per la protezione dell'organismo dalle infezioni e quindi ogni suo deficit facilita l'insorgenza di malattie infettive e le rende più gravi. Lo stato di immunodepressione, in cui possono trovarsi alcuni soggetti a causa di diversi eventi patologici, diventa il presupposto per la riattivazione di agenti patogeni virali già presenti in forma latente nell organismo. Il JCV è un polyomavirus ubiquitario che infetta l uomo in età pediatrica e permane latente, dopo la prima infezione, nell organismo ospite, alternandosi talvolta ad episodi di attiva replicazione e stati di quiescenza a seconda della capacità reattiva del soggetto infetto. Nonostante la comparsa degli anticorpi, il virus non viene eliminato dall organismo ma rimane latente nel rene, nel midollo osseo, nelle cellule del tessuto nervoso, nei linfonodi e nell epitelio intestinale, rendendo l ospite portatore sano fino ad un eventuale riattivazione. In condizioni di severa immunosoppressione il virus potrebbe riattivare e indurre una fatale malattia demielinizzante conosciuta come Leucoencefalopatia Multifocale Progressiva (PML). Il meccanismo della riattivazione sembra essere strettamente legato ai processi di replicazione e all espressione di particolari sequenze geniche. E stato, quindi, oggetto di questo studio, la valutazione della presenza del DNA di JCV in termini di espressione genica di due differenti regioni del virus: la regione precoce Large-T (early) o l introne late mRNA di mVP1/mVP2 (late) di JCV in cinque distinti gruppi di soggetti immunodepressi. Sono stati analizzati 200 campioni di plasma di pazienti ricoverati presso le U.O. di ematologia, trapianti, gastroenterologia appartenenti a diversi nosocomi catanesi. Inoltre venivano inclusi 55 campioni bioptici a fresco e paraffinati provenienti da un numero corrispondente di pazienti affetti da RCU (mucosa intestinale), appartenenti a soggetti con forme precancerose o cancro del colon (formazione neoplastica) e trapiantati di rene (rene trapiantato). Per lo studio retrospettivo sono state applicate metodiche di Nested-PCR e Real-Time PCR sia per confermare la presenza del DNA virale di JCV sia per la valutazione dell espressione delle due sequenze geniche ricercate. Di tutti i plasma analizzati solo il 26% (52/200) risultava negativo, per gli altri si poteva apprezzare una positiva solo alla regione tardiva del 38,5% (77/200) e una positività per la sola regione precoce del 25% (51/200). La copresenza delle due regioni ricercate si notava nel 10% dei casi (20/200). Data la prevalenza di positività alla regione tardiva, sembra che, nonostante la regione early sia una sequenza di riferimento diagnostico, la sequenza late rivesta un ruolo fondamentale nella diagnosi di tale tipologia di soggetti confermato altresì da un associazione statisticamente significativa tra le due regioni (p<0,05). Per quanto riguarda i campioni bioptici, si aveva positività solo alle sequenza VP1/VP2; ciò potrebbe dipendere dai meccanismi di replicazione che si instaurano in seguito allo stato di latenza o riattivazione del virus nelle cellule per esso non permissive come nel caso delle cellule dell epitelio intestinale. Poiché l espressione di Large-T e VP1/VP2 è strettamente correlata al completamento del ciclo virale, il loro reperimento dipende dalle diverse fasi della replicazione. Il nuovo bersaglio diagnostico, quindi, confrontato ed affiancato a quello tradizionale, potrebbe chiarire l evoluzione delle patologie connesse a questo virus. La ricerca di due regioni differenti del virus potrebbe essere di aiuto nel chiarire la diagnosi e, laddove fosse in corso una terapia, permettere un corretto monitoraggio e l ottimizzazione dell intervento terapeutico.

Biomedical Neuroscience
Ph.D.
The human neurotropic virus, JC virus (JCV), is the etiologic agent of the fatal demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML) that is seen primarily in immunodeficient individuals. Productive infection of JCV occurs only in glial cells and this restriction is to a great extent due to the activation of the viral promoter that has cell type-specific characteristics. The cell types that support the JCV infection cycle in culture are limited to primary human fetal glial cells and several transformed cell lines of glial origin. We developed a new hybrid cell system permissive for JC virus infection in order to gain insight into the mechanisms responsible for cell type specificity of JCV. The new cell system was created through the use of polyethylene glycol (PEG)-mediated cell fusion of primary human fetal astrocytes (PHFA) with an HPRT-deficient glioblastoma cell line, U-87MG. The new hybrid system was then used to analyze the ability of JCV replication and gene expression by infection studies. Results demonstrated that the new hybrid lines efficiently support JCV propagation during the early passages but lost that property in later passages. Earlier studies led to the assumption that glial-specific activation of the JCV promoter is mediated through the involvement of positive and negative transcription factors that control reactivation of the JCV genome under normal physiological conditions and suppress its activation in non-glial cells. Here we demonstrate that the alternative splicing factor, SF2/ASF, has the capacity to exert a negative effect on transcription of the JCV promoter in glial cells through direct association with a specific DNA sequence within the viral enhancer/promoter region. Our results show that down-regulation of SF2/ASF in fetal and adult glial cells increases the level of JCV gene expression and replication indicating that negative regulation of the JCV promoter by SF2/ASF may control reactivation of JCV replication in brain. JCV induces a broad range of neural-origin tumors in experimental animals has been repeatedly detected in several human cancer most notably neural-crest origin tumors, including medulloblastomas and glioblastomas. The oncogenic activity of JCV is attributed to the viral early gene products, large T and small t antigen, as evidenced by the results from in vitro cell culture and in vivo transgenic animal studies. We demonstrate that SF2/ASF suppresses the expression of large T antigen and small t antigen in JCV-transformed tumor cell lines. Expression of SF2/ASF in such tumor cells ultimately hinders the transforming capacity of the viral tumor antigens. Moreover, downregulation of SF2/ASF in viral-transformed tumor cell lines induces the growth and proliferation rate of the tumor cells. Altogether, we have created a new hybrid cell system which may serve as a good model system to study the biology of JCV aimed at identifying cellular determinants of the virus replication and gene expression as well as developing novel therapeutic intervention strategies against JCV-induced disease, PML. We have also demonstrated a novel role of the cellular alternative splicing factor, SF2/ASF, in the regulation of JCV gene expression and transformation. These observations provide a new avenue of research to understand pathogenesis of JCV-induced diseases through interplay between JCV regulatory proteins and host factors, such as SF2/ASF.
Temple University--Theses

Le polyomavirus humain JC (JCV) est un virus ubiquitaire qui infecte de façon persistante et asymptomatique la majorité de la population, entrainant occasionnellement une excrétion urinaire. Dans le contexte d'immunodépression, le JCV peut infecter les cellules gliales du système nerveux central (SNC), provoquant une maladie démyélinisante fatale, la Leucoencéphalopathie Multifocale Progressive (LEMP). Le génome du JCV est un ADN double brin circulaire de 5 kb composé de 2 régions codantes opposées -précoce et tardive- séparées par une région non codante de contrôle (NCCR). En comparaison à la séquence NCCR urinaire archétypale les séquences obtenues du SNC de patients atteints de LEMP sont constituées de réarrangements dont le rôle n'est pas entièrement connu. Pour étudier l'effet de ces réarrangements en culture cellulaire, la technologie des minicercles d'ADN a été adaptée afin de produire 4 vecteurs bidirectionnels exprimant deux gènes rapporteurs fluorescents sour le contrôle d'une NCCR archétypale (NCCR at) ou réarrangée (NCCR rr) comportant une délétion de 66 pb. Après transfection de cellules humaines gliales U-87MG et rénales HEK293, l'expression des rapporteurs à partir des NCCR at et rr a été mesurée par cytométrie en flux. Dans les cellules HEK293, l'expression des régions codantes précoce et tardive à partir de la NCCR at est similaire tandis que dans les cellules U-87MG, l'expression précoce est 2.1 fois supérieure à l'expression tardive (p <0.001). Par ailleurs, l'expression tardive à partir de la NCCR rr est similaire à l'expression précoce dans les cellules HEK293 et U-87MG. Ces résultats suggèrent que la délétion de 66 pb restaure l'expression tardive dans la lignée de glioblastome. L'utilisation d'un modèle in vitro a permis de mettre en évidence un lien particulier entre la séquence NCCR et l'expression dépendant du type cellulaire. En plus de la variabilité inter compartiment déjà décrite pour un même patient atteint de LEMP, la variabilité intra compartiment a été évaluée au moyen d'une technique de « single molecule real-time (SMRT) sequencing » à partir de 23 liquides céphalorachidiens (LCR), 1 biopsie cérébrale et 19 prélèvements urinaires de patients atteints de LEMP ainsi que 5 prélèvement urinaires de patients non atteints de LEMP. L'ensemble du génome du JCV a été amplifié en 2 fragments chevauchants aux deux extrémités, chacun constitué de la NCCR et de l'une ou l'autre des régions codantes. Les amplicons ont été séquencés par la technique SMRT PacBio. L'analyse phylogénétique montre une répartition des souches cérébrales parmi 6 génotypes différents, révélant l'absence de pathogénicité spécifique de type. Les séquences NCCR cérébrales comportent diverses délétions touchant principalement les sections b, d, et f ainsi que des insertions des sections c et e dupliquées. Chez la majorité des patients atteints de LEMP (18/23), la population virale cérébrale est composée d'au moins 2 formes de NCCR distinctes dont la structure suggère un lien d'apparition chronologique entre ces deux variants. Par ailleurs, des substitutions d'acide aminé au niveau de 7 emplacements déjà décrits dans la protéine VP1 ont été identifiées exclusivement dans les souches cérébrales. Hormis plusieurs mutations en rapport avec des polymorphismes de souches, 2 nouvelles substitutions ont été observées à partir du LCR de deux patients différents situées respectivement dans le domaine hélicase de la séquence AgLT (Tyr407Asn) et dans le domaine N-terminal du gène VP2 (Pro65Ala). Ces mutations pourraient jouer un rôle dans la pathogénicité en modifiant les capacités réplicatives virales, en créant un changement de structure de la particule virale ou en favorisant l'échappement à la réponse immune. Ce travail fournit un argument supplémentaire en faveur de l'implication des réarrangements de la NCCR dans la neuropathogénicité du JCV et apporte un éclairage nouveau sur les populations virales associées à la LEMP
JC Polyomavirus (JCV) is a ubiquitous human virus which causes asymptomatic persistent infections, and occasional urine shedding. In immune depression conditions, JCV causes a fatal disease, progressive multifocal leukoencephalopathy (PML), by infecting oligodendroglial cells of the central nervous system (CNS). The JCV double-stranded circular 5 kb genome is composed of two opposite coding regions - early and late - transcribed from opposite strands of DNA, and separated by the regulator non-coding control region (NCCR). The hallmark of NCCR prototype sequences recovered from PML brain lesions is the presence of rearrangements (rr) of unknown function, compared with urine archetype (at) NCCR sequences. To analyse the effects of such mutations on early and late expression in tissue-specific cultured cells, we produced bidirectional reporter vectors expressing two distinct fluorescent reporters under control of either rr or at JCV NCCR. We adapted the technology involving DNA circles devoid of bacterial plasmid backbone and generated four expression vector maxicircles, to investigate the effects of a single 66 bp deletion differentiating rr and at NCCR. After transfection of U-87MG (human glioblastoma cell line) and HEK293 (human kidney cell line), fluorescent reporter expressions from at and rr NCCR were analysed by cytometry analysis. In HEK293 cells, early and late expressions from at NCCR were similar, whereas in U-87 MG cells, early expression was 2.1-fold higher than late expression (p <0.001, Welch's t-test). This suggests that late expression from at NCCR is impaired in this glioblastoma cell line. Interestingly, late expression from mutated rr NCCR was similar to early expression in both HEK293 and U-87 MG cells, indicating that the 66 bp deletion restored late expression in the glioblastoma cell line. By using this in vitro model, we evidenced a relevant link between JCV NCCR sequence and cell-type dependent expression. In addition to the inter-compartment variability within patients, we further investigated the previously reported intra-compartment variability. By using a single-molecule real-time (SMRT) sequencing technology (PacBio, Pacific Biosciences) in order to obtain 3 kb amplicon sequences in a single read, we analysed precisely the JCV genomic populations in 23 cerebrospinal fluid (CSF), 1 cerebral biopsy (CB) and 19 urine samples of PML patients and 5 urine samples from non PML patients. JCV full-length genome was amplified in 2 overlapping opposite fragments, each encompassing the NCCR and either the early or the late coding sequence. Phylogenetic analysis revealed distribution of PML strains among 6 distinct genotypes, suggesting absence of specific pathogenic JCV genotype. PML JCV NCCR from cerebral samples displayed various deletions affecting mainly b, d and f sections and insertions of duplicated c and e sections. In 18/23 cerebral samples, intra compartment variability consisted in detection of at least two JCV variants and suggested a chronological emergence relationship between the two rearranged forms. In VP1, previously reported aminoacid substitutions at 7 distinct positions of sialic acid binding regions and antigenic epitopes were observed exclusively in cerebral strains. Apart single nucleotide polymorphisms evidenced over the whole viral genome, we observed, in two distinct PML CSF strains, two novel missense mutations, located in the helicase domain of LTAg sequence (Tyr407Asn) and in the N terminal domain of VP2 coding gene (Pro65Ala) respectively. These mutations could play a role in PML pathogenesis by modifying viral and/or cellular replication and transcription, by changing viral particle conformational structure and by immune response escape. This work supports the role of JCV NCCR rearrangements in PML neuropathogenesis and provides further insights in the genesis of neurotropic strains in PML lesions

Doenças neurológicas focais em pacientes com aids podem ser causadas por vários patógenos oportunistas. Dentre estas se inclui a encefalite por Toxoplasma gondii, os linfomas primários do sistema nervoso central causados pelo vírus Epstein-Barr, as encefalites virais (CMV, HSV, VZV) e a leucoencefalopatia multifocal progressiva (LEMP), causada pelo vírus JC (VJC). O presente estudo teve por objetivos detectar o DNA do vírus JC em amostras de líquido cefalorraquidiano de pacientes com aids e lesões não expansivas de substância branca do SNC, bem como caracterizar esses pacientes com relação ao número de células TCD4+, sexo, idade e ocorrência de outros diagnósticos etiológicos. A detecção do DNA do VJC foi realizada através da técnica de reação em cadeia por polimerase. O protocolo de PCR empregado, anteriormente descrito, utiliza um par de primers complementar à região precoce do vírus JC (antígeno T), resultando em um fragmento de 173 pb. Todas as amostras positivas foram submetidas a etapa posterior de tipagem com enzima de restrição Bam H1, resultando em dois fragmentos menores (120 e 53 pb), característicos do vírus JC. Com o intuito de estimar a sensibilidade da técnica empregada, um controle positivo qüantificável foi padronizado. O fragmento de 173 pb amplificado de uma das amostras de líquor estudadas foi inserido em plasmídio, e o recombinante obtido foi quantificado através de espectrofotometria, titulado e submetido a PCR. Através desta metodologia foi possível estimar que o teste é capaz de detectar a partir de 200 cópias/ µl. A especificidade do teste foi avaliada através da análise de amostras de líquor de pacientes com e sem aids e outros diagnósticos neurológicos, não compatíveis com LEMP. A pesquisa do DNA do vírus JC foi negativa em 119 de 120 amostras testadas, demonstrando uma especificidade de 99,17%. Foram incluídas no estudo 56 amostras de líquor de pacientes com lesão focal não expansiva de substância branca, compatível com LEMP, sendo positiva em 27/56 (48,2%) e negativa em 29/56 (51,8%). Em 23 dos 29 (79,3%) pacientes negativos para o vírus JC foi possível estabelecer um diagnóstico diferencial para os quadros encefalíticos: Toxoplasma gondii (nove casos), complexo cognitivo motor do HIV (CCMHIV) (cinco casos), tuberculose (três casos) e outros diagnósticos (oito casos). Em seis pacientes DNA-VJC negativos não houve um diagnóstico final. A caracterização da população avaliada, dividida em dois grupos, de acordo com o resultado da PCR (DNA-VJC positivo ou DNA-VJC negativo), não demonstrou diferença estatisticamente significante no que diz respeito ao sexo ou idade. No grupo de pacientes DNA-VJC positivos, o número de células TCD4+ foi significativamente mais baixo. Os resultados do presente estudo demonstraram uma alta prevalência do DNA do VJC (48,2%) nesse grupo de pacientes. Foi possível concluir também que, em pacientes com aids e encefalite focal com lesões não expansivas de substância branca do sistema nervoso central, com PCR negativa para o VJC, é necessária uma investigação diagnóstica mais aprofundada já que a maioria desses casos apresenta outros agentes etiológicos, na maioria das vezes passíveis de tratamento.
Focal neurological diseases in aids patients can be caused by a range of opportunistic pathogens such as Toxoplasma gondii, EBV-associated primary CNS lymphomas, viral encephalitis (CMV, HSV, VZV) and JC virus causing the progressive multifocal leukoencephalopathy (PML). In the present study, we evaluated the detection of JC virus DNA in CSF samples from aids patients with white matter non-expansive lesions of CNS by polymerase chain reaction (PCR) and characterize this finding in relation to the number of TCD4+, age, gender, and other etiological diagnosis. The primers used to amplify the T antigen region of JC virus resulted in a fragment of 173 base pairs. Since JC virus harbor a BAM H1 restriction site in this region, digestion of the PCR product with the enzyme resulted in two fragments of 120 and 53 base pairs, characteristic of JC virus. To estimate the sensitivity of the assay, the 173 bp fragment obtained from one of the samples was inserted into a plasmid and the recombinant quantified by spectrophotometry. The sensitivity of the PCR was 200 copies / µL. The specificity of the assay was evaluated in CSF samples from patients with and without aids and other neurological conditions, not suggestive of PML. The PCR resulted negative in 119 of the 120 CSF samples tested showing a specificity of 99,17%. In 56 CSF samples from patients with neurological symptoms and radiological signs of PML, JC virus was detected in 27 (48.2%) by PCR. In 23 of the remaining 29 patients (79.3%) other neurological conditions were diagnosed: T. gondii encephalitis (9 cases), HIV encephalitis (5 cases), tuberculosis (3 cases) and other diagnosis (8 cases). In six patients no neurological disease diagnosis could be established. In the group of patients characterized as JC virus-DNA positive the mean number of TCD4+ was significantly lower as compared to the JC virus-DNA negative patients. No statistical difference was seen in relation to gender or age distribution between the two groups. The results of the present study demonstrated a high prevalence of JC virus DNA (48,2%) in patients with clinical and radiological signs of PML. We concluded that the polymerase chain reaction for JC-virus DNA detection can represent an advance in the diagnosis of PML. aids patients with non-expansive focal lesions of CNS white matter and JC virus-DNA negative by PCR probably have other treatable neurological conditions that must be extensively investigated.

碩士
中山醫學院
免疫學研究所
89
Abstract Human JC virus (JCV) belongs to the family of polyomaviridae. The viral capsid is composed of 72 capsomeres. Five VP1 molecules make up a capsomere structure. To investigate the minimal sequences on JCV VP1 polypeptide required for capsid assembly, the first twelve (△N12) and nineteen (△N19) amino acids at the N-terminus and the last sixteen (△C16), seventeen (△C17) and thirty-one (△C31) amino acids at the C-terminus of VP1 were truncated and expressed in E. coli. The VP1 proteins of △N12 and △C16 were able to self-assemble into a virus-like particle similar to that of wild type (WT) VP1. However, the mutant proteins of △N19, △C17 and △C31 formed a pentameric capsomere structure as demonstrated by a 10-30% sucrose gradient centrifugation and 5-20% sucrose gradient centrifugation and electron microscopy. These results suggest that the first nineteen and the last seventeen residues are the minimal sequences on VP1 polypeptide for JCV capsid assembly. Although DNA sequences of JCV,SV40 and Py are highly homologous,their host ranges are different. The results of immunological analysis showed that the surface domains,BC,DE and HI loops of the viruses may play an important role in host determination.

Primary contact with human polyomaviruses is followed by lifelong asymptomatic persistence of viral DNA. Under severe immunosuppression JCV activation may lead to unrestricted virus growth in the CNS followed by development of progressive multifocal leukoencephalopathy (PML). Besides the kidney and the brain, target cells of persistent infection were also found in the hematopoietic system. This included the presence of JCV genomes in peripheral blood cells (PBCs). In the attempt to understand the role of PBCs for the JCV infection in humans, we asked for the type of cells affected as well as for virus interaction with PBCs. Analysis of separated subpopulations by highly sensitive and specific polymerase chain reaction and Southern blot hybridization revealed the presence of JCV DNA mostly in circulating granulocytes. These cells have important functions in innate immunity and are professional phagocytes. This suggested that PCR amplified DNA might be the result of an extranuclear association of the virus due to membrane attachment or phagocytosis rather than JCV infection with presence of viral DNA in the nucleus. In the attempt to answer this question JCV DNA was subcellularly localized in the blood of 22 healthy donors by JCV specific fluorescence in situ hybridization (FISH). Granulocytes and peripheral blood mononuclear cells (PBMCs) were separated by Percoll gradient centrifugation. Intracellular JCV DNA was hybridized with Digoxigenin-labeled JCV specific DNA probes covering half of the viral genome. As the sensitivity of the anti-digoxigenin antibody system was lower than the PCR detection level, a chemical amplification step was included consisting of peroxidase labeled secondary antibody precipitating biotinylated tyramide followed by detection with streptavidin-Texas-Red and fluorescence microscopy. Comparison of the number of cells affected in healthy individuals with 15 HIV-1 infected patients with and without PML revealed that the rate of affected PBMCs was comparable in both groups (2.5±0.4 and 14.5±0.9 per 1000). In contrast, the rate of JCV positive granulocytes in the immunosuppressed group was 92.6±1.7% compared to 4±1.4% in healthy donors thus confirming that granulocytes are the major group of circulating cells affected by JCV and that HIV-1 associated immune impairment has an important effect on the virus-cell association. Localization revealed that JCV DNA was predominantly located within the cytoplasm, although hybridizing signals occasionally covered the nuclear compartment. The fluorescent glow of chemical amplification combined with classical fluorescence microscopy did not allow an unequivocal localization of viral DNA. However, confocal microscopy of 24 sections through single cells combined with FISH without chemical amplification confirmed cytoplasmic localization of JCV DNA in a large number of cells. Additionally, it clearly demonstrated that JCV DNA was also located in the nucleus and nuclear localization directly correlated with the number of cells affected. Calculation of the virus load in subcellular compartments revealed that up to 50% of the JCV genomes were located in the nucleus thus pointing to viral infection at least in the granulocytes of HIV-1 infected patients. This may contribute to the distribution of the virus from sites of peripheral infection to the CNS and may promote the development of active PML in the severely immune impaired patients
Primärer Kontakt mit dem humanen Polyomavirus JC führt zu lebenslanger asymptomatischer Persistenz der viralen DNA in den Zielorganen der Infektion insbesondere der Niere und dem ZNS. Unter schwerer Immunsuppression kann die Aktivierung des JCV zu uneingeschränkter Vermehrung des Virus im ZNS und zur Entwicklung einer zentralnervösen Erkrankung, der progressiven multifokalen Leukoenzephalopathie (PML) führen Neuerdings wurde JCV DNA auch in Zellen des blutbildenden Systems insbesondere in peripheren Blutzellen (PBCs) beschrieben. Um die Rolle der PBCs für die JCV-Infektion beim Menschen besser zu verstehen, sollte der virus-assoziierte Zelltyp bestimmt und die Virus-Zell Interaktion näher untersucht werden. Die Analyse von isolierten Blutzellsubpopulationen durch eine sensitive und spezifische Polymerase-Kettenreaktion mit folgender Southern Blot-Hybridisierung ergab die Präsenz von JCV-DNA zumeist in zirkulierenden Granulozyten. Diese Zellen haben eine wichtige Funktion in der angeborenen Immunität und sind professionelle Phagozyten. Dies legte nahe, dass die PCR-amplifizierte DNA eher das Ergebnis einer extranukleären Assoziation des Virus durch Membranassoziation oder Phagozytose als einer JCV-Infektion ist, die durch Virus-DNA im Kern charakterisiert ist. Bei dem Versuch, diese Frage zu klären, wurde JCV-DNA in Blutzellen von gesunden Spendern mittels JCV-spezifischer Fluoreszenz in situ Hybridisierung (FISH) subzellulär lokalisiert. Granulozyten und periphere mononukleäre Blutzellen (PBMCs) wurden isoliert und intrazelluläre JCV-DNA mit Digoxigenin-markierten JCV DNA-Sonden, die die Hälfte des viralen Genoms representierten, hybridisiert. Da die Empfindlichkeit des Anti-Digoxigenin-Antikörper-Systems niedriger war als die PCR-Nachweisgrenze, wurde ein chemischer Amplifikationsschritt benutzt, das sogenannte Tyramidsystem, um die Sensitivität der FISH in Kombination mit der klassischen Fluoreszenzmikroskopie zu erhöhen. Der Vergleich der Anzahl von JCV betroffenen Zellen in gesunden Individuen mit Zellen von HIV-1-infizierten Patienten mit und ohne PML zeigte, dass die Rate der betroffenen PBMCs in beiden Gruppen (2,5 ± 0,4 und 14,5 ± 0,9 pro 1000) vergleichbar war. Im Gegensatz dazu war die Rate der JCV positiven Granulozyten in der immunsupprimierten Gruppe, 92,6 ± 1,7%, im Vergleich zu denen bei gesunden Spendern 4 ± 1,4% deutlich höher. Dies bestätigte, dass mit den Granulozyten die größte Gruppe von zirkulierenden Zellen von JCV betroffen sind und dass die schwere Beeinträchtigung der immunologischen Kompetenz durch die HIV-1 Infektion einen bedeutenden Einfluss auf auf die Virus-Zell Interaktion hat. Die intrazelluläre Lokalisation der viralen DNA ergab, dass die Signale überwiegend im Zytoplasma lokalisiert waren, wenngleich gelegentlich auch nukleäre Kompartimente betroffen waren. Durch die chemischen Verstärkung der Fluoreszenzsignale in Kombination mit klassischer Fluoreszenzmikroskopie war es jedoch nicht möglich eine eindeutige Lokalisierung der viraler DNA zu erreichen. Erst die Anwendung der konfokalen Mikroskopie bestätigte die predominant zytoplasmatische Lokalisierung von JCV-DNA in einer großen Anzahl von Zellen und hat eindeutig gezeigt, dass JCV-DNA zusätzlich im Kern lokalisiert ist. Die Kern Lokalisation korreliert direkt mit der Anzahl der betroffenen Zellen. Berechnung der Viruslast in subzellulären Kompartimenten hat gezeigt, dass bis zu 50% der JCV Genome im Kern von Granulozyten von HIV-1 Patienten lokalisiert waren. Dies deutet auf eine virale Infektion der Granulozyten hin und lässt vermuten, dass sie unter der HIV-1 Infektion an der Disseminierung des JC Virus aus den Organen der peripheren Infektion in das ZNS beteiligt sind und in der Konsequenz auch bei der Entwicklung der PML eine wesentliche Rolle spielen könnten

博士
中山醫學大學
醫學研究所
93
JC virus (JCV), a human polyomavirus, belongs to the polyomaviridae. The JC virion conatins three capsid proteins（VP1, VP2 and VP3）and a viral minichromosome. VP1 is the major capsid protein constituting approximately 75 ％ of the total proteins. We generated JCV virus-like particles（VLPs）when the major capsid protein VP1 was expressed in E. coli. The recombinant VLPs were demonstrated to be able to package and deliver exogenous DNA into mammalian cell. These findings indicate that the recombinant JCV VLP potentially could be used as a human gene transfer vector for gene therapy. A reporter plasmid, pEGFP, was introduced into the E. coli carrying JCV VP1 expressing plasmid. The VLPs purified from the E. coli with dual plasmids were found to package both plasmids as demonstrated by PCR and E. coli transformation. Furthermore, the VLPs which purified from in vivo packaging system were able to deliver the reporter plasmid, pEGFP, into 13 different mammalian cell lines and transduce the gene for expression. In this study, we also tried to determint the limitation of DNA size that could be packaged and protected in the VLP from in vivo packaging system. The maximum size of DNA molecule could be packaged in the VLP was approximately 10 Kbp. The findings demonstrate that the JCV VLP may provide more flexible gene delivery vector in terms of accommodation of large size of DNA molecule. Furthermore, it is easy to purify the VLPs just by using sucrose or CsCl gradient centrifugation. The findings in the thesis indicate that the human JCV VLPs generated in E. coli are potentially to be developed as a gene delivery vector for human gene therapy in the future.

博士
國立清華大學
生命科學系
89
JC virus (JCV), a human polyomavirus, is an important pathogen of human central nervous system (CNS) demyelinating disease. The purpose of this thesis is to investigate the molecular characteristics of the major structure protein, VP1, of JC virus and assembly mechanism of the virus particles. First of all, we amplified a new JCV genome (Taiwan-3, TW-3) from an autoimmune diseases patient by PCR and then cloned into a prokaryotic replicative plasmid, pGEM-7Zf(-). This clone is an important material to study the biological functions of TW-3. The TW-3 genome was sequenced and found to comprise of 5,111 base pairs. The major capsid protein VP1 gene of JCV was amplified from the TW-3 JCV genome and cloned into a prokaryotic expression plasmid. The VP1 protein expressed in E. coli was self-assembled into capsid-like particles and caused hemagglutination of human O-type red blood cells. Cesium chloride density gradient centrifugation analysis found that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The pseudovirion packaged DNA and RNA molecules but the pseudocapsid did not contain any nucleic acid. The DNA binding activity of VP1 was also demonstrated by the Southwestern probing method in vitro. The pseudocapsid was further demonstrated to be able to deliver exogenous DNA into human fetal kidney epithelial cells. In order to study the mechanism of DNA packaging, we deleted the first 12 amino acids of JCV VP1. The expressed truncated VP1 (△N12VP1) failed to encapsidate the host DNA although the integrity of the capsid-like structure was maintained. In addition, capsid-like particles of △N12VP1 did not package exogenous DNA in vitro. The results indicate that the N-terminal region of the human polyomavirus major capsid protein VP1 may be involved in viral genome encapsidation during progeny maturation. The calcium ions play an important role in the assembly process of JCV progeny. In this thesis, we tried to find out the calcium binding sites in the VP1 molecule for capsid assembly. Site-directed mutagenesis was performed to replace Glu149 and Glu152 within EF loop, Asp239 and Glu240 within GH loop and Asp338 within C-arm by alanine respectively. Sucrose cushion and CsCl density centrifugation demonstrated that Asp239 and Asp338 mutated VP1 proteins could not form a capsid-like particle. These results indicate that Asp239 and Asp338 may be involved in calcium binding and then cause associations of inter-pentameric molecules for capsid assembly.

碩士
中山醫學院
生物化學研究所
87
The major capsid protein VP1 of human polyomavirus, JC virus, has been cloned and expressed in yeast cells. VP1 protein expressed in yeast was able to self-assemble into a capsid-like structure and cause hemagglutination.The capsid-like particles were comprised of virion-like and empty capsid-like particles with 1.34 and 1.29 g/cm3 of density respectively. Morphology of the particles has been observed by electron microscopy.The virion-like particles contained host DNA and RNA molecules.The empty capsid-like particles were able to package and deliver exogeneous DNA into human kidney 293 cell. JCV capsid could be disrupted into pentameric capsomeres in the presence of both EGTA and DTT but the roles of metal ion and disulfide in capsid are still not known. In this study, disulfide linkage was found in the VP1 capsid as demonstrated by non-reducing gel. Capsid treated with DTT remained structural integrity but the disulfide has been abolished. Subsequently, the DTT treated capsid could be dissociated into capsomeres by EGTA alone. In addition, capsomeres were able to re-assemble into capsid-like particle in the presence of calicium ions. The re-assembled capsid without disulfide could be disrupted into capsomeres by EGTA alone. These results indicate that calcium ion is essential for capsid formation and disulfide is crucial for keeping capsid integrity and presumably protects calcium ion from chelation.

碩士
國立中正大學
分子生物研究所
95
JC virus (JCV), a human polyomavirus, is a small DNA virus. JCV is usually latent but can be reactivated under immunosuppressing conditions. For latent infection, JCV was able to develop neural-origin tumors in the inoculated hamsters. These transformed hamster cells showed an integrated JCV genome and expressed the viral large tumor antigen (T-Ag). T-Ag could bind p53 and retinoblastoma proteins resulting in uncontrolled cellular proliferation. In order to investigate the possible correlation between JCV infection and human cancers, formalin-fixed paraffin-embedded tissues from colon, lung, and breast cancers were examined. T-Ag, viral late capsid protein (VP1), and p53 were determined by immunohistochemistry (IHC). Quantitative real-time PCR and nested PCR were used to detect JCV genomic DNA. The product of nested PCR flanking JCV regulatory region was sequenced. Results from IHC revealed that expression of viral oncogenic T-Ag was detected in 63.6％ (14/22), 61.1％ (11/18), 20％ (2/10), and 55.6% (5/9) of colon, lung, breast, and bladder cancer samples, respectively. Expression of viral structural protein VP1 was only detected in 3 of bladder cancer samples. In addition, p53 was also detected in colon (50.00％), lung (66.67％), breast (10％), and bladder cancers (44.4%). JCV genomic DNA was detected in 95.5％, 77.8％, and 90％ of colon, lung and breast cancer tissues by real-time PCR. JCV regulatory region was found in 72.7％ colon cancer, 83.3％ lung cancer, 90％ breast cancer, and 55.6% bladder cancer tissues by nested PCR. The viral genotypes included Mad-1, TW3, and CY strains in colon cancers, CY, Mad-1, Mad-1R, TW3, TW9, and UT strains in lung cancers, CY and TW4 strains in breast cancers, and CY and TW3 strains in bladder cancers. Therefore these findings suggest a possible correlation between JCV and human colon, lung, breast, and bladder cancers

碩士
中山醫學院
生物化學研究所
87
The major capsid protein VP1 of human plyomavirus, JC virus, has been expressed in E. coli and found to self-assembled into a capsid-like structure. The capsid-like particles have been purified by sucrose cushion, CsCl density gradient centrifugation and sucrose gradient centrifugation from E. coli lysate. The purified capsid-like particles were capable of packaging exogeneous DNA by osmotic shock and delivering the DNA into human kidney cells(293 cell line). Osmotic shock for 90 min was the optimum period for DNA packaging. The capsid-like particles could protect exogeneous DNA from DNase I digestion at the biological osmotic condition. One kilobase-pair DNA in length was the maximun for accommodation in the particles. Capsid-like particles could be dissociated into pentameric capsomeres by treating EDTA and DTT. Capsid-like particles could be re-assembled from capsomeres in the presence of calcium ions. The exogeneous DNA was packaged in the re-assembled capsid and delivered into 293 cells. In addition, VP1 protein expressed in E. coli was able to form capsid-like particles and package host DNA and RNA molecules. Based on this finding, a reporter plasmid, pEGFP, was introduced into the E. coli carrying JCV VP1 expressing plasmid, ΔpFJCV1. The capsid-like particles purified from E. coli with dual plasmids were found to package both plasmids as demonstrated by PCR and E. coli transformation. Furthermore, the capsid-like particles packaging reporter plasmid, pEGFP, in vivo were able to deliver the plasmid DNA into human kidney 293 cells for expression. These findings indicate that human JC virus capsid-like particles self-assembled in E. coli were potentially to be developed as a gene transfer vecter for human gene therapy in the future.

碩士
國立中正大學
分子生物研究所
93
Gene therapy refers sending deoxyribonucleic acids to the host cell by using genetic engineering, to substitute the abnormal genes and restore physiological functions. Gene therapy may divide into three steps that are supply, delivery and expression. Supply includes “ex vivo”,“in situ”and “in vivo”three ways. Delivery vectors usually include viral carrier and non-viral carrier. This study focuses on the gene transfer ability and transduction efficiency using the JCV VLP as a gene delivery vector. The major capsid protein, VP1, of the human polyomavirus, JCV, that can self-assemble into virus-like particle (VLP). We attempted to understand whether this vector could carry foreign gene (green fluorescence protein gene) and express the gene in various kinds of human tumors. The results showed that JCV VP1 VLP could carry and transduce green fluorescence protein (GFP) gene into lung adenocarcinoma and colon adenocarcinoma. Not only did JCV VP1 VLP transduce the green fluorescence protein gene in metastatic colorectal adenocarcinoma (SW620), but also transduce the gene into the liver of nude mice. However, JCV VP1 VLP may potentially be developed as a gene delivery vector for human gene therapy in the future.

博士
國立中正大學
化學所
98
The JC virus (JCV) may infect human oligodendrocytes and, consequently, cause progressive multifocal leukoencephalopathy (PML) in patients with immune¬ deficiency. In addition, the virus has also been detected in other human tissues, including kidney, B lymphocytes and gastro-intestinal tissue. The recombinant major structural protein, VP1, of JCV is able to self-assemble to form a virus-like particle (VLP). It has been shown that the VLP is capable of packaging and delivering exogenous DNA into human cells for gene expression. However, gene transfer is not efficient when using in vitro DNA packaging methods with VLPs. In the current study, a novel in vivo DNA packaging method using the JCV VLP was employed to obtain more highly efficient gene transfer. A reporter gene, the green fluorescence protein (gfp), and a suicide gene, the herpes simplex virus thymidine kinase (tk), were encapsidated into VLPs in E. coli. The VLP was used to specifically target human colon carcinoma (COLO-320 HSR) cells in a nude mouse model. Intra-peritoneal administration of ganciclovir (GCV) in the tk-VLP treated mice greatly reduced tumor volume. These findings suggest that it will be possible to develop the JCV VLP as a gene delivery vector for human colon cancer therapy in the future.