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Polyomavirus JC (JCPyV) is a ubiquitous human neurotropic virus that can cause progressive multifocal leukoencephalopathy (PML), sometimes as a consequence of drug treatment for disabling diseases, including Multiple Sclerosis. JCPyV expresses microRNAs (miRNAs), and in particular miR-J1-5p, but at now we have limited knowledge regarding this aspect. In the present study the expression of JCPyV miR-J1-5p was measured in infected COS-7, to verify if and when this miRNA is expressed in a cell model of JCPyV-MAD-4 strain infection. Results showed that miR-J1-5p expression was relatively constant inside the cells from 11 days to 35 days after infection (mean: 4.13 × 105 copies/μg), and became measurable in supernatants 18 days after infection (mean: 7.20 × 104 copies/μl). miR-J1-5p expression in supernatants peaked (3.76 × 105 copies/μl) 25 days after infection and started to decrease 32 days after infection (7.20 × 104 copies/μl). These data show that COS-7 cells, already used as model for JCPyV replication cycle, can be also utilized to study JCPyV miRNAs expression, potentially opening new research avenues for diseases in which current therapeutic approaches could result in severe adverse effects (e.g. Natalizumab-associated JCPyV reactivation in Multiple Sclerosis patients). In these situations monitoring of miR-J1-5p may shed light on the mechanisms of virus reactivation and may help the clarification of the mechanisms responsible for such severe side effects.

Polyomavirus JC (JCPyV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). JCPyV infection is very common in childhood and, under conditions of severe immunosuppression, JCPyV may reactivate to cause PML. JC viral proteins expression is regulated by the JCPyV non-coding control region (NCCR), which contains binding sites for cellular transcriptional factors which regulate JCPyV transcription. Our earlier studies suggest that JCPyV reactivation occurs within glial cells due to cytokines such as TNF-α which stimulate viral gene expression. In this study, we examined interferon-α (IFNα) or β (IFNβ) which have a negative effect on JCPyV transcriptional regulation. We also showed that these interferons induce the endogenous liver inhibitory protein (LIP), an isoform of CAAT/enhancer binding protein beta (C/EBPβ). Treatment of glial cell line with interferons increases the endogenous level of C/EBPβ-LIP. Furthermore, we showed that the negative regulatory role of the interferons in JCPyV early and late transcription and viral replication is more pronounced in the presence of C/EBPβ-LIP. Knockdown of C/EBPβ-LIP by shRNA reverse the inhibitory effect on JCPyV viral replication. Therefore, IFNα and IFNβ negatively regulate JCPyV through induction of C/EBPβ-LIP, which together with other cellular transcriptional factors may control the balance between JCPyV latency and activation.

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.

Human polyomavirus JC (JCPyV) is a widely distributed viral agent and because it high resistance against environmental conditions it is frequently recovered from diverse sources of water and is considered a good marker for human pollution. Phylogenetic analysis of JCPyV isolated in different part of the world has revealed 7 genotypes, which have been associated with specific populations or ethnics groups. This feature has been used to trace pre-historic and historic human migration patterns across the world. Although there are many reports describing genotypes distribution around the world, data on JCPyV genotypes in the southernmost areas of South America are scarce. The goal of this study is to detect and characterize the JCPyV that circulates in Santiago, Chile using sewage samples from wastewater treatment plants (WWTP). Sewage samples were obtained monthly during 1 year from three WWTPs which together process about 80% of wastewater generated in the city of Santiago, Chile. Our results show that JCPyV profusely circulates in Santiago, Chile, because it was detected in 80.56% of the samples, reinforcing the use of JCPyV as a feasible marker to assess human environmental pollution. JCPyV was detected in high frequency in influents and effluents samples, with the largest WWTPs showing the highest percentage of detection and viral loads. In the phylogenetic analysis the Chilean sequences clustered mainly with genotype 2A (Asian genotype). This is similar to that previously reported from Buenos Aires, Argentina and divergent to data from Brazil, where the circulation of European subtypes 1 and 4 and African subtypes 3 and 6 has been described.

The objective of this study was to evaluate the prevalence, genotypic characterization, and determination of the patterns of shedding of human polyomavirus JC (JCPyV) and BK (BKPyV) in consecutive urine samples collected from healthy adults. Urine samples collected monthly over a 6 month period were screened by polymerase chain reaction (PCR) with two sets of primers complementary to the VP1 protein region specific for the JCPyV or BKPyV genome. The viral load of JCPyV and BKPyV in positive samples was determined by quantitative real time PCR. Seventy-one healthy individuals (ages between 18 and 65) were included in the study. Polyomavirus DNA urinary shedding was identified in 44 (62%) of the 71 individuals evaluated: BKPyV only in 16 (22.5%); JCPyV only in 19 (26.7%); and both in 9 (12.7%). Among the 28 individuals shedding JCPyV, the shedding was nearly continuous in 13 (46.4%) and sporadic in 15 (53.6%), whereas all BKPyV shedding was sporadic. A total of 45 (19 BKPyV and 26 JCPyV) strains were identified. Of the BKPyV strains, individuals were observed that excreted all genotypes except genotype 3 and the JCPyV strains, excretion of 5 different genotypes. Evaluating the age of individuals who excrete JCPyV and BKPyV, mostly are young adults, with a slight increase with increasing age and observing the viral load can not draw any parallel between the increase or decrease of age or excreted genotype as there was a wide variation both in the excretion of BKPyV and JCPyV. The high occurrence of isolated or simultaneous urinary shedding of JCPyV and BKPyV in healthy individuals merits further study. J. Med. Virol. 88:153–158, 2016. © 2015 Wiley Periodicals, Inc.

Background Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or “inside-out” NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of post-translational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation. Methods We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots. Results We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. Conclusions We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression.

The human polyomavirus JC (JCV) is the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is extremely common and after primary infection, JCV persists in a latent state. However, PML is a very rare disease suggesting that the virus is tightly regulated. Previously, we showed that NF-κB and C/EBPβ regulate the JCV early and late promoters via a DNA control element, KB, which also mediates the stimulatory effects of proinflammatory cytokines such as TNF-α on JCV gene expression. Other studies have implicated NFAT4 in JCV regulation. We now report that NFAT4 and NF-κB interact at the KB element to co-operatively activate both JCV early and late transcription and viral DNA replication. This interplay is inhibited by C/EBPβ and by agents that block the calcineurin/NFAT signaling pathway. The importance of these events in the regulation of JCV latency and reactivation is discussed.

Polyomavirus JC (JCPyV) is largely excreted by the human population through the urinary route and has been recognized as a potential viral marker for human waste contamination. This study aims to investigate the dissemination of JCPyV in waste water from a sewage treatment plant (STP) located in Rio de Janeiro, Brazil, and to describe the prevalence of JCPyV subtypes currently present in this population. Raw and treated sewage samples were collected bimonthly during one year, and examined for the presence of JCPyV using nested polymerase chain reaction (nPCR) and quantitative real time PCR (qPCR). JCPyV was detected by nPCR in 96% and 43% of raw and treated sewage samples, respectively. The concentration of JCPyV present in the samples ranged from 1.2 × 103 to 3.2 × 105 and 2.6 × 102 to 6.2 × 103 genome copies per 2 ml of concentrated raw and treated sewage sample, respectively. The strains were characterized and the obtained nucleotide sequences indicated that the detected JCPyV strains clustered with subtypes of East African, West African and European origin. To our knowledge, this is the first study describing the incidence and diversity of JCPyV strains in raw and treated sewage in Brazil.

Background Quantitative PCR (qPCR) is the standard molecular method for detection of polyomavirus JC (JCPyV) DNA reactivation in serum and cerebrospinal fluid (CSF) in patients at risk of progressive multifocal leukoencephalopathy (PML). Recently, digital PCR has shown potential benefits over qPCR in viral diagnostics. Objective To evaluate the performance of droplet digital PCR (ddPCR) assay in assessing JCPyV-DNA status in clinical samples of patients at risk for PML. Study design JCPyV specific ddPCR was developed with primers/probes targeting Large T and the noncoding control region used in qPCR. The ddPCR accuracy of JCPyV-DNA quantification was investigated using serial dilutions of genomic JCPyV-DNA. The ddPCR JCPyV-DNA quantification and qPCR confirmation were performed on 150 CSF and 100 serum clinical samples. Results Using genomic JCPyV-DNA, ddPCR was highly sensitive, repeatable and reproducible for both molecular targets. Using clinical samples, JCPyV-DNA was detected in 13% of CSF and in 50% of serum samples with limit of detection of 30 copies/ml. Among the 19 JCPyV-DNA-positive CSF detected using the ddPCR, 15 also tested positive with the qPCR. Among the 50 JCPyV-DNA-positive serum identified with ddPCR, 41 tested positive with qPCR. All the ddPCR-negative samples were negative when assessed using qPCR. Additionally, the mean JCPyV-DNA viral load obtained with ddPCR in all samples was not significantly different from that of qPCR. Conclusion The results demonstrate that ddPCR is a highly sensitive alternative for measuring JCPyV-DNA that should be considered in clinical diagnostic testing of JCPyV-DNA in patients at risk of PML and other associated diseases.

The ubiquitous human polyomavirus JC (JCV) is a small double-stranded DNA virus that establishes a persistent infection, and it is often transmitted from parents to children. There are at least 14 subtypes of the virus associated with different human populations. Because of its presumed codivergence with humans, JCV has been used as a genetic marker for human evolution and migration. Codivergence has also been used as a basis for estimating the rate of nucleotide substitution in JCV. We tested the hypothesis of host-virus codivergence by (i) performing a reconciliation analysis of phylogenetic trees of human and JCV populations and (ii) providing the first estimate of the evolutionary rate of JCV that is independent from the assumption of codivergence. Strikingly, our comparisons of JCV and human phylogenies provided no evidence for codivergence, suggesting that this virus should not be used as a marker for human population history. Further, while the estimated nucleotide substitution rate of JCV has large confidence intervals due to limited sampling, our analysis suggests that this virus may evolve nearly two orders of magnitude faster than predicted under the codivergence hypothesis.

Many genetic studies using human mtDNA or the Y chromosome have been conducted to elucidate the relationships among the three Native American groups speaking Amerind, Na-Dene, and Eskimo-Aleut. Human polyomavirus JC (JCV) may also help to gain insights into this issue. JCV isolates are classified into more than 10 geographically distinct genotypes (designated subtypes here), which were generated by splits in the three superclusters, Types A, B, and C. A particular subtype of JCV (named MY) belonging to Type B is spread in both Japanese/Koreans and Native Americans speaking Amerind or Na-Dene. In this study, we evaluated the phylogenetic relationships among MY isolates worldwide, using the whole-genome approach, with which a highly reliable phylogeny of JCV isolates can be reconstructed. Thirty-six complete sequences belonging to MY (10 from Japanese/Koreans, 24 from Native Americans, and 2 from others), together with 54 belonging to other subtypes around the world, were aligned and subjected to phylogenetic analysis using the neighbor-joining and maximum-likelihood methods. In the resultant phylogenetic trees, the MY sequences diverged into two Japanese/Korean and five Native American clades with high bootstrap probabilities. Two of the Native American clades contained isolates mainly from Na-Denes and the others contained isolates mainly from Amerinds. The Na-Dene clades were not clustered together, nor were the Amerind clades. In contrast, the two Japanese/Korean clades were clustered at a high bootstrap probability. We concluded that there is no distinction between Amerinds and Na-Denes in terms of indigenous JCVs, although they are linguistically distinguished from each other.

Volume 88, no. 12, p. [6556–6575][1], 2014. Page 6556, abstract, line 5: “Glu55” should read “Gln52.” Page 6556, importance, line 4: “Q54” should read “Q52.” Page 6557, column 2, second full paragraph, line 3: “Glu54” should read “Gln52.” The authors regret these errors

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.

For almost 40 years, polyomavirus JC and BK were the only known human polyomaviruses but in the last 7 years, increased interest and innovative molecular screening techniques have led to the identification of 10 previously unknown polyomaviruses in humans. Two of these, Merkel cell polyomavirus and Trichodysplasia spinulosa polyomavirus, have also been found to cause disease in immunocompromised patients. Seroprevalence studies indicate that human polyomaviruses are transmitted independently of one another in humans and carry different risks of exposure and reexposure throughout life. The potential coexistence of 12 or more different polyomavirus species in the same host and possibly even in the same organ raises the question of potential interactions. Careful review of polyomavirus biology may facilitate new discoveries concerning these formerly underestimated viral agents and their influence on human health.

The present study evaluated the contamination of a surface water lagoon (Peri Lagoon) in Florianópolis, Santa Catarina, Brazil, by human adenovirus (HAdV), polyomavirus JC (JCPyV), hepatitis A virus (HAV) and rotavirus species A (RVA). Efforts were driven to determine the correlation between viral presence and the physicochemical parameters of the lagoon and measure the distribution of these viruses throughout the year (June 2010 to May 2011). A total of 48 samples were collected, concentrated and analyzed by qPCR (quantitative polymerase chain reaction). Approximately 96% of the samples were positive for HAdV (46/48), 65% were positive for RVA (31/48), 21% were positive for JCPyV (10/48) and 12% were positive for HAV (6/48). The presence of JCPyV was positively correlated with that of NO2−N, and also there was a positive correlation between the presence of each one of the viruses (HAdV, HAV and RVA) in winter. Samples from water dedicated for human consumption and recreation tested positive for HAdV by qPCR. These samples were also subjected to viral integrity and viability assays: 83% (10/12) contained intact viral particles and 66% (8/12) contained infectious particles. Our results demonstrate the release of human waste into water sources, justifying the urgent need to add viral parameters to water quality surveillance.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the neurotropic human polyomavirus JC (JCV) lytic infection of oligodendrocytes. PML was first described as a complication of lymphoproliferative disorders more than 50 years ago and emerged as a major complication of human immunodeficiency virus (HIV) infection in the 1980s. Despite the ubiquity of this virus, PML is rare and always seen in association with underlying immunosuppressive condition, such as HIV infection, autoimmune diseases, cancer, and organ transplantation. JCV remains quiescent in the kidneys, where it displays a stable archetypal non-coding control region (NCCR). Conversely, rearranged JCV NCCR, including tandem repeat patterns found in the brain of PML patients, have been associated with neurovirulence. The specific site and mechanism of JCV NCCR transformation is unknown. According to one model, during the course of immunosuppression, JCV departs from its latent state and after entering the brain, productively infects and destroys oligodendrocytes. Although the majority of PML cases occur in severely immunesuppressed individuals, PML has been increasingly diagnosed in patients treated with biological therapies such as monoclonal antibodies (mAbs) that modulate immune system functions: in fact, CD4+ and CD8+ T lymphopenia, resulting from this immunomodulatory therapy, are the primary risk factor. Furthermore, JCV reactivation in nonpermissive cells after treatment with mAbs, such as intestinal epithelial cells in Crohn's disease patients, in association with other host tumor-inducing factors, could provide valid information on the role of JCV in several malignancies, such as colorectal cancer.

Polyomavirus JC (JCV) is ubiquitous in humans and causes a chronic demyelinating disease of the central nervous system , progressive multifocal leukoencephalopathy which is common in AIDS. JCV is excreted in urine of 30-70% of adults worldwide. Based on sequence analysis of JCV complete genomes or fragments thereof, JCV can be classified into geographically derived genotypes. Types 1 and 2 are of European and Asian origin respectively while Types 3 and 6 are African in origin. Type 4, a possible recombinant of European and African genotypes (1 and 3) is common in the USA. To delineate the JCV genotypes in an aboriginal African population, random urine samples were collected from the Biaka Pygmies and Bantu from the Central African Republic. There were 43 males and 25 females aged 4-55 years, with an average age of 26 years. After PCR amplification of JCV in urine, products were directly cycle sequenced. Five of 23 Pygmy adults (22%) and four of 20 Bantu adults (20%) were positive for JC viruria. DNA sequence analysis revealed JCV Type 3 (two), Type 6 (two) and one Type 1 variant in Biaka Pygmies. All the Bantu strains were Type 6. Type 3 and 6 strains of JCV are the predominant strains in central Africa. The presence of multiple subtypes of JCV in Biaka Pygmies may be a result of extensive interactions of Pygmies with their African tribal neighbors during their itinerant movements in the equatorial forest.

The JC polyomavirus (JCV) is ubiquitous in humans, infecting children asymptomatically, then persisting in renal tissue. Since JCV DNA can be readily isolated from urine, it should be a useful tool with which to study the evolution of DNA viruses in humans. We showed that JCV DNA from the urine of Japanese, Taiwanese, Dutch and German patients can be classified into A and B types, based upon restriction fragment length polymorphisms (RFLPs). This work was extended in the present study. We established multiple JCV DNA clones from the UK, Spain, Italy, Sweden, South Korea, People's Republic of China, Malaysia, Indonesia, Mongolia, India, Sri Lanka, Saudi Arabia, Ethiopia, Kenya, Zambia, South Africa and Ghana. Using type-specific RFLPs, most clones except the four clones from Ghana were classified as either type A or B. We constructed a molecular phylogenetic tree for the Ghanaian clones and several representative type A and B clones. According to the phylogenetic tree, the Ghanaian clones constituted a major new group, tentatively named type C. From the findings presented here and elsewhere, the following conclusions were drawn: (i) type A is prevalent only in Europe; (ii) type B is found mainly in Asia and Africa; and (iii) type C is localized to part of Africa. Our findings should help to clarify how JCV evolved in humans.

In the last years, the treatment of multiple sclerosis (MS) patients with natalizumab has been associated with the occurrence of progressive multifocal leukoencephalopathy (PML) caused by human polyomavirus JC (JCV). Here, we have shown a significant correlation between patients with JC viruria and positive JC-specific antibody response and patients without JCV-specific antibodies after 1 year of natalizumab (p = 0.0006). Furthermore, JCV-specific quantitative PCR on urine and plasma samples, collected at the enrollment (t0) and every 4 months (t1, t2, t3) in the first year and at two time points (t4 and t5) in the second year of natalizumab treatment, indicated the prevalence of JC viremia rather than JC viruria only in the second year of treatment (p = 0.04). Moreover, the analysis of JCV non-coding control region (NCCR) sequences in peripheral blood mononuclear cells of patients with JC-specific antibodies after 12 natalizumab infusions (t3) revealed the presence of rearranged sequences, whereas the prevalence of genotypes 1A, 1B, and 4 was detected in these patients by VP1 sequence analysis. In summary, JC viruria evaluation seems to be useful to identify early those patients who do not already develop a humoral immune response against JCV. It may also be interesting to study the JCV NCCR rearrangements since they could give us new insights on the onset of neuro-invasive viral variants.

Agnoprotein is required for the successful completion of the JC virus (JCV) life cycle and was previously shown to interact with JCV large T-antigen (LT-Ag). Here, we further characterized agnoprotein's involvement in viral DNA replication. Agnoprotein enhances the DNA binding activity of LT-Ag to the viral origin (Ori) without directly interacting with DNA. The predicted amphipathic α-helix of agnoprotein plays a major role in this enhancement. All three phenylalanine (Phe) residues of agnoprotein localize to this α-helix and Phe residues in general are known to play critical roles in protein–protein interaction, protein folding and stability. The functional relevance of all Phe residues was investigated by mutagenesis. When all were mutated to alanine (Ala), the mutant virus (F31AF35AF39A) replicated significantly less efficiently than each individual Phe mutant virus alone, indicating the importance of Phe residues for agnoprotein function. Collectively, these studies indicate a close involvement of agnoprotein in viral DNA replication.