Auswahl der wissenschaftlichen Literatur zum Thema „Human Polyomavirus JC (JCV)“

More than 20 near full-length genome sequences have been reported for human polyomavirus JC (JCV). These have previously been classified into seven genotypes, and additional subtypes, which exhibit geographical associations. One of these genotypes, Type 4, has been suggested to be a recombinant of Types 1 and 3. We have investigated the pattern of diversity, and evolutionary relationships, among these sequences. In direct contradiction of a recent report, we found that different phylogenetic methods gave consistent results for the phylogenetic relationships among strains. The single known strain representing Type 5 was shown to be a mosaic of sequences from Types 2 and 6, although whether this recombination occurred in vivo or in vitro is not clear. In contrast, there was no substantial evidence that Type 4 strains are recombinant; rather they seem to be simply divergent examples of Type 1. On the assumption that the major genotypes of JCV diverged with human populations, the rate of synonymous nucleotide substitution was estimated to be around 4×10−7 per site per year, about 10 times higher than a previous estimate for primate polyomaviruses.

Polyomaviruses are a family of small, nonenveloped viruses with a circular double-stranded DNA genome of ∼5,000 base pairs protected by an icosahedral protein structure. So far, members of this family have been identified in birds and mammals. Until 2006, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) were the only polyomaviruses known to circulate in the human population. Their occurrence in individuals was mainly confirmed by PCR and the presence of virus-specific antibodies. Using the same methods, lymphotropic polyomavirus, originally isolated in monkeys, was recently shown to be present in healthy individuals although with much lower incidence than BKV, JCV, and SV40. The use of advanced high-throughput sequencing and improved rolling circle amplification techniques have identified the novel human polyomaviruses KI, WU, Merkel cell polyomavirus, HPyV6, HPyV7, trichodysplasia spinulosa-associated polyomavirus, and HPyV9. The skin tropism of human polyomaviruses and their dermatopathologic potentials are the focus of this paper.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCVE promoter and to a lesser extent the JCVL promoter. Mutations in the NF-1 sites in the JCVE promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.

The polyomavirus (PyV), JC virus (JCV), is a small nonenveloped DNA virus that asymptomatically infects about 80% of healthy adults and establishes latency in the kidney tissue. In case of immunodeficient hosts, JCV can lytically infect the oligodendrocytes, causing a fatal demyelinating disease, known as progressive multifocal leukoencephalopathy (PML). Although the reactivation of another human PyV, BK virus (BKV), is relatively common and its association with the polyomavirus associated nephropathy (PyVAN) following renal transplantation is proven, JCV replication and its impact on graft function and survival are less well studied. Here we describe the biology of JCV and its pathological features and we review the literature regarding the JCV infection analyzed in the setting of transplantations.

JC virus (JCV) is ubiquitous in the human population. Primary infection normally occurs during childhood and is followed by a lifelong persistent infection. The main mode of transmission remains unknown. Several authors have hypothesized that JCV transmission occurs through the respiratory route, and that respiratory secretions could represent a possible source of viral particles. The present study intended to evaluate oropharyngeal fluids from patients infected with JCV, in order to ascertain if respiratory secretions could indeed constitute a source of exposure to this polyomavirus. Oropharyngeal washing samples from 25 patients co-infected with JCV and human immunodeficiency virus type 1 were evaluated for the presence of JCV DNA. Regardless of the titre of antibodies or the presence of viral urinary excretion, JCV genome was not detected in oropharyngeal samples collected from any of the patients infected with JCV included in this study, which may suggest that oropharyngeal fluids are an unlikely source for JCV infection.

ABSTRACT The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal α(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.

Many human neurological diseases involve demyelination of the central and/or peripheral nervous systems. These include the hereditary leukodystrophies -which have a genetic basis; multiple sclerosis (MS) -where the underlying cause of demyelination remains unknown; and progressive multifocal leukoencephalopathy (PML) -where the etiology is well-established as being viral. The human neurotropic polyomavirus -JC virus (JCV) -is the etiologic agent of PML, a fatal demyelinating disease of the central nervous system that occurs mainly in immunosuppressed patients, especially those with HIV/AIDS. JCV belongs to the polyomavirus family of tumor viruses that are characterized by non-enveloped icosahedral capsids containing small, circular, double-stranded DNA genomes. Serological studies have shown that JCV is widespread throughout the human population, but infections are usually restricted by the immune system, particularly cell-mediated immunity, causing the virus to enter a latent phase. An important corollary of this is that situations of severe immunosuppression may permit JCV to replicate and are thus a risk factor for PML.

The BK (BKV) and JC viruses (JCV) are human polyomaviruses. After primary infection, they persist as latent infection in the kidneys. Immunosuppression leads to their reactivation, which is associated with life-threatening diseases such as polyomavirus-induced nephropathy and progressive multifocal leukoencephalopathy. However, the behavior of these viruses in immunocompetent individuals is still an open question with no right answer. The aim of this study was to determine the prevalence of BKV and JCV shedding in the urine of immunocompetent individuals from the Serbian population. Sixty-five urine samples were collected and tested for the presence of BKV and JCV DNA by PCR. JCV DNA was detected in 19/65 (29.2%) and BKV DNA in 3/65 (4.6%) of the urine samples. Forty-three (66.2%) urine samples of the immunocompetent donors were negative for both viruses. The present study provides the first results of urinary excretion of human polyomaviruses in the Serbian population.

JC virus (JCV) is a human polyomavirus known as the causative agent of the fatal demyelinating disease, Progressive Multifocal Leukoencephalopathy (PML). Further, in experimental animals this virus causes a broad range of tumors of central nervous system origin. Recent studies have suggested the association of JCV with several human tumors most notably malignant brain tumors of childhood, medulloblastoma. The development of tumors by JCV is most likely through mechanisms involving inactivation of tumor suppressors and de-regulation of signaling pathways such as Wnt by the viral early protein, T-antigen. The neurotropic nature of JCV along with the overwhelming evidence for its oncogenic potential in laboratory animals and its detection in significant numbers of human medulloblastomas invite the re-evaluation of the role for JCV in the development of human brain tumors.

Cancer is the second leading cause of mortality worldwide. The study of DNA tumor-inducing viruses and their oncoproteins as a causative agent in cancer initiation and tumor progression has greatly enhanced our understanding of cancer cell biology. The initiation of oncogenesis is a complex process. Specific gene mutations cause functional changes in the cell that ultimately result in the inability to regulate cell differentiation and proliferation effectively. The human neurotropic Polyomavirus JC (JCV) belongs to the family Polyomaviridae and it is the causative agent of progressive multifocal leukoencephalopathy (PML), which is a fatal neurodegenerative disease in an immunosuppressed state. Sero-epidemiological studies have indicated JCV infection is prevalent in the population (85%) and that initial infection usually occurs during childhood. The JC virus has small circular, double-stranded DNA that includes coding sequences for viral early and late proteins. Persistence of the virus in the brain and other tissues, as well as its potential to transform cells, has made it a subject of study for its role in brain tumor development. Earlier observation of malignant astrocytes and oligodendrocytes in PML, as well as glioblastoma formation in non-human primates inoculated with JCV, led to the hypothesis that JCV plays a role in central nervous system (CNS) tumorigenesis. Some studies have reported the presence of both JC viral DNA and its proteins in several primary brain tumor specimens. The discovery of new Polyomaviruses such as the Merkel cell Polyomavirus, which is associated with Merkel cell carcinomas in humans, ignited our interest in the role of the JC virus in CNS tumors. The current evidence known about JCV and its effects, which are sufficient to produce tumors in animal models, suggest it can be a causative factor in central nervous system tumorigenesis. However, there is no clear association between JCV presence in CNS and its ability to initiate CNS cancer and tumor formation in humans. In this review, we will discuss the correlation between JCV and tumorigenesis of CNS in animal models, and we will give an overview of the current evidence for the JC virus’s role in brain tumor formation.

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.

Abstract Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) resulting from the lytic infection of oligodendrocytes by a human polyomavirus, JC virus (JCV). This disease develops mostly in patients with underlying immunosuppressive conditions including AIDS, Hodgkin’s lymphoma, lymphoproliferative diseases and those undergoing antineoplastic therapy (Berger and Concha 1995; Berger and Major 1999; Berger 2000; Berger et al. 2001; Major et al. 1992). In addition, in a small number of cases, PML was also found to affect individuals with no underlying disease (Berger and Concha 1995; Major et al. 1992).

This chapter is about the prevention of HIV and the prevention of opportunistic infections in people living with HIV/AIDs (PLWH). It covers treatment as prevention (TasP), HIV pre-exposure prophylaxis (PrEP) (including indications, initial assessment, and contraindications to PrEP), HIV post-exposure prophylaxis (PEP) (including estimating risk of transmission, timing of PEP initiation, counseling at time of PEP evaluation, and overlap of PEP and PrEP), Mycobacterium tuberculosis (MTB), infections due to bacteria, Treponema pallidum (syphilis), infections due to viruses, human papilloma virus (HPV), influenza A and B, JC polyomavirus (JCV), cryptosporidiosis, mycobacterium avium complex infection, and Pneumocystis jiroveci (formerly Pneumocystis carinii) pneumonia.

Progressive multifocal leucoencephalopathy (PML) is an opportunistic infection of the central nervous system caused by the JC polyomavirus (JCV) It is seen almost exclusively in individuals with severe immunosuppression and has become much more widely recognized since the AIDS/HIV epidemic. Classical imaging appearances can be highly suggestive of the diagnosis but definitive evidence would include detection of JCV in CSF or a brain biopsy. Treatment is supportive and focused on immune reconstitution.