Dynamics of pregnancy-associated polyomavirus urinary excretion: a prospective longitudinal study

Asymptomatic polyomaviruria of pregnancy has been documented in point prevalence studies, but little attention has been given to the dynamics of polyomavirus excretion during pregnancy because of its benign course. We tested the hypothesis that the frequency and/or magnitude of polyomavirus excretion would increase as pregnancy progresses. Urine specimens were obtained prospectively from 179 healthy women during uncomplicated pregnancies and 37 healthy non‐pregnant women. Real‐time polymerase chain reaction was used to determine BK virus (BKV) and JC virus (JCV) viral loads in urine, blood, and rectal and vaginal swabs collected during routine obstetric and gynecologic clinic visits. Asymptomatic urinary shedding of BKV and/or JCV was observed in 384 (48.0%) of 800 specimens from 100 (55.8%) pregnant women. BKV excretion was more common in pregnant than non‐pregnant women (41.3% vs. 13.5%, P = 0.0026). The frequency of JCV excretion was no different in pregnant compared to non‐pregnant women. The frequency and magnitude of polyomavirus shedding did not vary with gestational age. Post‐partum shedding of BKV, but not JCV, rapidly decreased to undetectable levels. Pregnancy‐associated BKV excretion begins early in pregnancy and terminates rapidly post‐partum. Neither the frequency nor magnitude of BKV or JCV shedding increased with pregnancy progression. Further study into the host factors that regulate pregnancy‐associated BKV excretion may allow identification of the host factors that predict susceptibility to BKV‐associated diseases in immune compromised patients. J. Med. Virol. 84: 1312–1322, 2012. © 2012 Wiley Periodicals, Inc.     

Dynamics of urinary polyomavirus shedding in healthy adult women

The hypothesis was examined that physiologic variation of estrogen concentrations during the menstrual cycle can provoke BK virus (BKV) excretion. BKV and JCV viral loads were determined in urine specimens obtained almost daily from 20 healthy, non-pregnant women over 2 months. Asymptomatic urinary shedding of BKV was observed in 123 (12.0%) of 1,021 specimens from 11 (55%) study subjects. Two subjects excreted JCV in their urine, with one subject excreting detectable JCV in all urine specimens. Analysis of 36 complete menstrual cycles revealed no difference in the prevalence of BKV excretion between pre-ovulatory and post-ovulatory phases of the menstrual cycle. The unexpected day-to-day variability in BKV excretion suggests that as yet unidentified factors may contribute to the periodic shedding of BKV by healthy women.     

The VP1 subunit of JC polyomavirus recapitulates early events in viral trafficking and is a novel tool to study polyomavirus entry

JC polyomavirus (JCV) is an important human pathogen that causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In this study we further delineate the early events of JCV entry in human glial cells and demonstrate that a pentameric subunit of the viral capsid is able to recapitulate early events in viral trafficking. We show that JCV traffics to the endoplasmic reticulum (ER) by 6 h post infection, and that VP1 pentamers arrive at the ER with similar kinetics. Further, this JCV localization to the ER is critical for infection, as treatment of cells with agents that prevent ER trafficking, ER function, or ER quality control reduce JCV infectivity. These pentamers represent a new tool to study polyomavirus entry, and will be particularly useful in studying recently identified polyomaviruses that are difficult to propagate.     

Use of a molecular probe for detecting JCV DNA directly in human brain material

A hybridot assay using a radiolabelled JC virus probe has been used to detect the presence of JCV DNA in brain biopsy and postmortem brain samples from patients with neurological disease and possible progressive multifocal leucoencephalopathy. Sixty-nine brain samples from 45 patients were examined. Eleven samples from eight patients had detectable JCV DNA sequences. In seven of the eight patients this result was confirmed by electron microscopy and/or virus isolation or immunofluorescence.     

Transplacental transmission of human polyomavirus BK

The presence of BK virus (BKV) and JC virus (JCV) in autopsy materials (placenta, brain, and kidney) of aborted fetuses was investigated by PCR using two sets of primers, specific for the regulatory region (RR) and for the capsid protein VP1, respectively. The RR of BKV was detected in 12 samples of placenta and brain and in nine samples of kidney obtained from 15 fetuses. Out of the 12 positive cases, four placentas, one brain, and three kidney samples also showed the presence of BKV DNA in the VP1 region. Of 12 placentas from a control group with a normal pregnancy outcome, the RR of BKV was detected in six samples, four of which were also positive for the VP1 region. None of the samples from either group was positive for the RR of JCV. In two cases, the nucleotide sequence of the BK RR demonstrated that the viruses isolated from maternal and fetal tissues showed a high homology with one another and had a characteristic deletion of the R₆₃ box compared to the archetype strain. The results indicate that BKV may be transmitted vertically. J. Med. Virol. 56:372–376, 1998 . © 1998 Wiley-Liss, Inc.     

Pseudovirus mimics cell entry and trafficking of the human polyomavirus JCPyV

The normally asymptomatic human polyomavirus, JCPyV, is the causative agent of a rare but fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Individuals at risk for developing PML include those with AIDS, with other underlying immunosuppressive diseases, and in patients treated with immunomodulatory regimens. Drugs to prevent viral reactivation in the setting of immunosuppression or immunomodulation could be used to sustain lives. Development of such drugs has been impeded by the difficulty of growing and studying the virus. We sought to develop a more efficient method for screening drugs that inhibit viral infection. Pseudovirus models have been developed which may be of use in pharmaceutical research. The use of pseudoviruses as models for viral infection is dependent on them using similar pathways for infection as virus. We screened known inhibitors of viral entry for their ability to block pseudovirus infection. Here we show that the pseudovirus based on the human polyomavirus JCPyV recapitulates virus binding, entry and trafficking. This system can be used for high-throughput screening of antiviral drugs.     

Characterization of JC human polyomavirus infection in a Portuguese population

JC virus (JCV) is ubiquitous in the human population, infecting children asymptomatically. After primary infection, JCV persists in the host throughout life and is often excreted in the urine. Two hundred thirty‐four urine samples and 78 serum samples, collected from 171 healthy individuals and 63 patients infected with HIV, were used to characterize JCV infection in a Portuguese population. Using PCR, JCV DNA was detected in 38% of the urine samples. A significant difference in the excretion rate was observed between patients infected with HIV (51%) and healthy individuals (33%). The frequency of JCV viruria increased with age in healthy individuals, but not in patients infected with HIV. JCV urinary load was determined by real‐time quantitative PCR and was independent of gender, age, HIV infection, and CD4+ cell count. Overall, the JCV genotype detected most commonly was 1B, followed by genotypes 2B and 4. The detection and quantitation of JCV‐specific antibodies were performed in serum samples by an established enzyme immunoassay (EIA). Antibodies to JCV were observed in 91% of the patients tested, irrespective of HIV infection. A positive correlation between JCV urinary load and antibody titers was demonstrated. The present study provides the first characterization of seroprevalence and urinary excretion of JCV in a Portuguese population and revealed similar results to those observed in other European countries. A comparison between healthy individuals and patients infected with HIV, despite identical values of seroprevalence, showed some differences in the pattern of urinary excretion. J. Med. Virol. 82:494–504, 2010. © 2010 Wiley‐Liss, Inc.     

Quantification of human polyomavirus JC virus load in urine and blood samples of healthy tribal populations of North-Eastern part of West Bengal,India

Background: Human polyomavirus JC (JCV) is a widespread human virus with profound pathogenic potential. A study was undertaken to quantify JCV load in urine and peripheral blood samples of immunocompetent, apparently healthy tribal individuals of North-Eastern part of West Bengal, India for the first time. Materials and Methods: One hundred and thirteen samples of urine or blood were collected from different tribal groups of this region. For the quantitative estimation of the viral load in each sample, real-time polymerase chain reaction method using the SYBR Green dye was employed. Results: The viral load estimated was found in the range between 3.5 × 10² and 2.12 × 10⁶ copies/ml of samples having a mean and median viral copy numbers of 8.67 × 10⁵ and 9.19 × 10⁵ copies/ml of sample respectively. Conclusion: The mean viral DNA load in urine samples of the studied immunocompetent population was found to be higher than that found in a study conducted in the USA, but lower than similar groups of Italy and healthy adult women in the USA. However when compared with median values of viral DNA loads in urine samples of immunocompetent human subjects of Kuwait, Portugal, and Switzerland the observed viral DNA load was found to be substantially higher.     

A prospective study of renal transplant recipients reveals an absence of primary JC polyomavirus infections

BackgroundBoth JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) are acquired at an early age. JCPyV causes progressive multifocal leukoencephalopathy and has been described in association with nephropathy.ObjectivesUrine and plasma samples from renal transplant recipients (RTRs) were examined for JCPyV to determine its involvement in causing infection and disease.Study designJCPyV testing was performed on 112 RTRs included in a randomised controlled study of steroid-sparing immunosuppressive regimens [1]. Urine and EDTA blood samples were collected pre- and post-transplantation and analysed for JCPyV using real-time PCR and sequencing to determine genotype and viral variation. Donor and recipient IgG antibody status to JCPyV was also determined.ResultsOverall, 13.3% of RTRs were positive for JCPyV of which one patient developed viraemia without viruria. JCPyV DNA was detected early following transplantation (defined as five days post transplantation) from recipients with donors that were positive for JCPyV IgG antibodies. No dual cases of JCPyV and BKPyV were observed. One patient sample had sequence duplication in the non-coding control region.ConclusionsLike BKPyV, JCPyV tends to occur early post transplantation but did not result in sustained viraemia. There was no deterioration of renal function in patients positive for JCPyV. As with other viruses, JCPyV donor serostatus was a risk factor for detection of JCPyV DNA. JCPyV appears to protect individuals from BKPyV infection, as recipients were twice as likely to develop BKPyV with a negative JCPyV donor.     

VP-1 quasispecies in human infection with polyomavirus BK

Polyomavirus BK is a recognized cause of nephropathy and hemorrhagic cystitis in kidney or allogeneic hematopoietic stem cell transplant recipients. This study explored a role of genetic variations in capsid protein VP-1 gene as a factor in viral pathogenesis. VP-1 was amplified from 7 healthy subjects with viruria, 7 transplant patients with viruria, and 11 patients with viremia or nephropathy. PCR products were cloned and a total of 558 clonal sequences were subjected to phylogenetic analysis using standard methods. VP-1 quasispecies were found in 25/25 and coinfection with different genotypes in 12/25 subjects. Genotype II was found as an unexpected minority species in 5/25 individuals. Recombinant strains of uncertain biologic significance, which frequently contained genotype II and IV sequences were identified in 9/25 subjects. Viremia/nephropathy group was characterized by (a) greater sequence complexity in whole VP-1 versus BC loop and BC loop compared to the HI loop, (b) greater intra-strain genetic diversity in the BC loop compared to whole VP-1 protein and HI loop, (c) more non-synonymous substitutions (dN) in the BC loop compared to whole VP-1 and HI loop, (e) fewer synonymous substitutions (dS) compared to healthy-viruria group, and (f) selection pressure (dN/dS >1.0) exerted on VP-1. In conclusion, this study documents frequent occurrence of quasispecies in a host DNA polymerase dependent virus, which is theoretically expected to show high replication fidelity. Quasispecies occur even in healthy subjects with viruria, but evolutionary selection pressure directed at the viral capsid protein (VP-1) is seen only in patients with viremia or nephropathy.     

Detection of Merkel cell polyomavirus and human papillomavirus DNA in porocarcinoma

BackgroundIncreasing evidences support the role of Merkel cell polyomavirus (MCPyV) and human papillomavirus (HPV) in non-cutaneous and cutaneous tumours. Porocarcinoma is a rare malignant neoplasm that arises from the intraepidermal ductal portion of the eccrine sweat glands. The aetiology of porocarcinoma is largely unknown and no systematic studies have been done to investigate the implication of infectious agents in the pathogenesis of this tumour.ObjectivesTo investigate the possible association between MCPyV and/or HPV infection and porocarcinoma.Study designForty-four formalin-fixed paraffin-embedded (FFPE) porocarcinomas (40 primary and 4 metastatic) and 10 healthy skin specimens (controls), were analysed for the presence of MCPyV and HPV DNA using molecular detection methods.ResultsMCPyV DNA was found in 27/40 (68%) primary porocarcinomas and in 3/10 (30%) controls (Fisher exact test: p < 0.04). No significant difference in viral load was observed between tumours and healthy skin. Moreover, 2/40 primary porocarcinomas tested positive for high-risk HPV16. Cutaneous beta-HPV infection was detected in 16/40 (40%) porocarcinomas and in 6/10 (60%) controls. No particular beta-HPV types were significantly associated with tumour or with healthy skin. Two out of 4 metastatic biopsies were MCPyV DNA positive. All metastatic samples had mixed infections with cutaneous HPV types.ConclusionsThis study demonstrated a significantly high prevalence of MCPyV and the presence of a broad spectrum of HPV types in porocarcinoma and provided the first available data about viral infections in this tumour. To understand the role, if any, of viral infections in the pathogenesis of porocarcinoma further studies are needed.     

Frequency and large T (LT) sequence of JC polyomavirus DNA in oligodendrocytes, astrocytes and granular cells in non-PML brain

Progressive multifocal leukoencephalopathy (PML) and JCV granular cell neuronopathy occur secondary to JCV polyomavirus (JCV) infection of oligodendrocytes and cerebellar granular cell neurons (CGNs) during immunosuppression. Pure populations of astrocytes, oligodendrocytes, CGNs and microglia from frontal cortex and cerebellum of 17 non-PML patients (9 immunocompetent; 8 immunosuppressed) were isolated by laser capture microdissection (LCM). JCV large T (LT) antigen DNA was detected by triple nested polymerase chain reaction (PCR). Sequence analysis was performed to assess LT gene variation. JCV DNA was detected in oligodendrocytes, astrocytes and CGNs of non-PML brains. The most common site for viral latency was cortical oligodendrocytes (65% of samples analyzed). Immunosuppressed patients were significantly more likely to harbor JCV DNA in CGN populations than immunocompetent patients (P = 0.01). Sequence analysis of the LT region revealed eight novel single nucleotide polymorphisms (SNPs) in four immunosuppressed patients. Of the eight novel SNPs detected, six were silent and two resulted in amino acid changes. JCV DNA is present within cells of the non-PML brain, known to be infected during PML and granular cell neuronopathy. This supports the argument for a brain only reservoir of JCV and supports the hypothesis that reactivation of latent brain JCV may be central to disease pathogenesis.     

High prevalence of BK polyomavirus sequences in human papillomavirus-16-positive precancerous cervical lesions

High- and low-grade cervical lesions were analyzed for the presence of polyomavirus (PYV) and human papillomavirus (HPV) sequences. In precancerous cervical lesions, the overall prevalence of PYV sequences was 44% (41/93). Specifically, among the PYV-positive samples, 83% (34/41) tested positive for BK polyomavirus (BKV) sequences, whereas 17% (7/41) were positive for JC-virus. None of the samples were positive for simian virus 40. The presence of BKV DNA in high-grade squamous intraepithelial lesions was confirmed by in situ PCR. BKV sequences were detected more frequently in high-grade squamous intraepithelial lesions, together with the genotype HPV-16. The association of BKV with precancerous cervical lesions suggests that this polyomavirus participates with HPV-16 in the cell transformation process. Alternatively, BKV might multiply better in HPV-16-positive cells from precancerous cervical lesions than in HPV-16-negative cells.     

Biologic diversity of polyomavirus BK genomic sequences: Implications for molecular diagnostic laboratories

Data on polyomavirus genomic diversity has greatly expanded in the past few years. The implications of viral DNA sequence variation on the performance of molecular diagnostic assays have not been systematically examined. 716 BK, 1626 JC, and 73 SV40 virus sequences available in GenBank were aligned using Clustal-X. Five different published BKV PCR assays currently in use at major medical centers were evaluated for primer and probe mismatches with available GenBank sequences. Coverage of naturally occurring BKV strains varied amongst different assay methods. Targeted viral sequences showed major mismatch with primer or probe sequence in up to 30.7% of known BKV strains. BKV subtypes IVa, IVb, and IVc were more prone to this problem, reflecting common use of Type I Dun sequence for assay design. Despite the known polymorphism of this gene, 484 VP-1 sequences with conserved areas potentially suitable for PCR assay design are available. Assay targets in the Large T-antigen and agnogene are less subject to genetic variation, but sequence information corresponding to the latter two genes is available only for 164 and 174 published strains, respectively. Cross reactivity of appropriately selected BKV primers with JCV and SV40 sequences available in current databases was not a significant problem.     

Investigation of pre‐diagnostic virological markers for progressive multifocal leukoencephalopathy in human immunodeficiency virus‐infected patients

Progressive multifocal leukoencephalopathy (PML) is a severe neurological disorder due to JC virus (JCV) infection. Pre‐diagnostic biological markers and risk factors for PML are not well understood. We conducted a case–control study nested within the Multicenter AIDS Cohort Study to examine the association between JCV viruria and viremia and serum antibody to JCV capsids, in relation to subsequent PML diagnoses, 5 months to 12 years later. Other demographic and immunologic factors were also examined. The study population included 28 incident cases of PML, 26 matched HIV‐positive controls, and 50 HIV‐negative controls. Prevalence of JCV viruria was 37% in cases, 42% in HIV‐positive controls, and 28% in HIV‐negative controls (P = 0.43). Among persons with JCV viruria, persistent viruria was more common in cases (89%) than in HIV‐positive controls (33%) (P = 0.02). Presence of JCV viruria was not related to the time to PML diagnosis (OR: 1.03, 95% CI: 0.8–1.4); however, the urinary concentration of JCV DNA increased with proximity to the date of PML diagnosis in cases. JCV seropositivity did not differ between cases or controls (P = 0.42). Four cases tested JCV seronegative, including one case only 5 months prior to diagnosis with PML. JCV DNA was detected in the serum of one HIV‐positive control. Smoking was the only demographic variable analyzed associated with an increased risk for PML (MOR: 9.0, 95% CI: 1.2–394.5). The results suggest that persistent JCV viruria and increasing urinary concentration of JCV DNA may be predictive of PML for some patients. J. Med. Virol. 81:1140–1150, 2009. © 2009 Wiley‐Liss, Inc.     

Genotyping of the JC virus in urine samples of healthy Korean individuals

A human polyomavirus, JC virus (JCV) is ubiquitous in humans and infects children asymptomatically. It persists in renal tissue and is excreted progeny in urine. DNAs from urine samples of 100 healthy Korean individuals were screened for the presence of JCV by polymerase chain reaction (PCR). Twenty of the samples were positive for JCV. JCV DNA was found in one individual (4%) in the 1-19-year group, two individuals (9%) in the 20-39-year group, ten individuals (38%) in the 40-59-year group, seven individuals (28%) in the over 60-year group. The prevalence of JC viral DNA was the highest in the 40-59-year-old Korean population. To investigate genotypes of JCV in Korea, the genotypes were determined by DNA sequence analysis of the regulatory region (333 bp) and the VT-intergenic region (656 bp) of DNA from the 20 JCV isolates. We have identified three distinctive JCV strains in the regulatory region and ten distinctive JCV strains in the VT-intergenic region of DNA from the 20 isolates. Based on restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis of the VT-intergenic region of JCV, two distinct subtypes, CY and type 2A (MY), were found to be prevalent in this Korean population. CY and type 2A of JCV were identified in 13 individuals (65%) and four individuals (20%), respectively. Interestingly, type 1, which was distributed mostly in Europe, was found in 3 (15%) isolates from healthy Korean individuals.