Molecular Epidemiology of Human Polyomavirus JC in the Biaka Pygmies and Bantu of Central Africa

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.

Molecular characterization of human polyomavirus JC in Brazilian AIDS patients with and without progressive multifocal leukoencephalopathy

Background: JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is classified in 8 different genotypes. Previous reports have suggested a positive association between specific genotypes and PML. Objective: To compare genotypes and adaptive mutations of JCV strains from Brazilian AIDS patients with and without PML. Study design: The VP1 region of JCV was amplified by polymerase chain reaction from cerebrospinal fluid samples from 51 patients with PML and from urine samples of 47 patients with AIDS without central nervous system disease. Genotyping was done by phylogenetic analysis. Amino acid replacement and selection pressures were also investigated. Results: JCV genotype frequency distributions showed that genotypes 2 (32.7%), 1 (26.5%) and 3 (23.5%) were the most prevalent. Genotype 1 had a positive association (p < 0.0001) and genotype 3 showed an inverse association (p < 0.001) with PML. A previously undescribed point mutation at residue 91 (L/I or L/V) and (L/P), non-genotype-associated, was found in 5/49 (10.2%) and 2/47 (4.3%) JCV sequences from PML and non-PML patients, respectively. This mutation was under positive selection only in PML patients. A previously described substitution of T-A in position 128 showed a significant difference between PML and non-PML cases (70% versus 16%, respectively, p < 0.0005). Conclusion: In Brazilian patients with AIDS, JCV genotype 1 showed a strong association with PML (p < 0.0001) and JCV genotype 3 showed an inverse association with PML. The possible association of aminoacids substitution in residues 91 and 128 with PML in patients with AIDS must be further investigated. (C) 2010 Elsevier B.V. All rights reserved.

Prevalência da infecção pelos Polyomavirus JC e BK em pacientes com Doença Renal Crônica e transplantados

O presente estudo teve como objetivo realizar a investigação molecular da infecção pelos Poliomavírus JC e BK em pacientes com Doença Renal Crônica (DRC) terminal, transplantados e em indivíduos sem DRC. Foram testadas 295 amostras de urina, que após a extração de DNA, foram submetidas à amplificação de um fragmento de 173 pb do gene do antígeno-T de Polyomavirus por meio da PCR seguida pela análise de RFLP, utilizando a endonuclease de restrição BamHI, na qual foi detectado 17,6% (52/295) de infecção por Polyomavirus, sendo 3,9% (4/102) nos pacientes com DRC, 30,5% (18/59) nos pacientes transplantados e 22,4% (30/134) nos assintomáticos. A prevalência da infecção pelo BKV foi de 88,9% (16/18) nos transplantados e de 10,0% (3/30) nos assintomáticos, não sendo detectada a infecção pelo BKV em pacientes com DRC. A prevalência de infecção pelo JCV foi de 3,9% (4/102) nos pacientes com DRC, de 11,1% (2/16) no transplantados e de 90,0% (27/30) nos assintomáticos. O risco de infecção por BKV foi determinada ser 72 vezes maior em pacientes transplantados do que em assintomáticos. A baixa frequência de infecção encontrada entre os pacientes com DRC pode estar relacionada ao fato de que esses pacientes apresentam uma elevada taxa de excreção de uréia na urina, assim como, baixo volume e densidade urinária, podem ser outros dois fatores contribuintes para a ausência de amplificação por estarem associados à baixa carga viral presente. De acordo com estes resultados, sugere-se que a investigação da infecção por Polyomavirus deve ser realizada, rotineiramente, nos pacientes pré e póstransplante, assim como nos doadores de órgãos, uma vez que a infecção por BKV tem sido associada com rejeição de enxerto em transplante de rins.

Presence of JC virus-specific CTL in the cerebrospinal fluid of PML patients: rationale for immune-based therapeutic strategies.

OBJECTIVES: There is urgent need of a treatment for progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). To evaluate the rationale for immunotherapy of PML, we explored whether JCV-specific cytotoxic T lymphocytes (CTL) can penetrate the central nervous system (CNS). In addition, we studied the breadth of their T-cell receptor (TCR) repertoire, and sought to establish a reliable method to expand these cells in vitro. DESIGN AND METHODS: We enrolled 18 patients in this study, including 16 with proven or possible PML (15 HIV-positive and one HIV-negative), and two HIV-positive patients with other neurological diseases. Detection of JCV-specific CTL in the blood and the cerebrospinal fluid was performed by Cr release and tetramer staining assays in 15 patients. RESULTS: Of 11 PML patients with analyzable cerebrospinal fluid (CSF), two had no detectable JCV-specific CTL in the blood and CSF and died 3.7 and 7.2 months later. The nine remaining patients had an inactive course of PML and detectable JCV-specific CTL in the blood. In addition, four of them (44%) also had detectable JCV-specific CTL in the CSF. Both HIV-positive patients with OND had detectable JCV-specific CTL in the blood and one in the CSF. Using tetramer technology, we obtained highly enriched JCV-specific CTL lines that were able to kill target cells presenting JCV peptides. The breadth of the TCR repertoire was CTL epitope dependent. CONCLUSIONS: These results indicate that JCV-specific CTL are present in the CNS of PML patients and pave the way for an immune-based therapeutic approach.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

Typing of urinary JC virus DNA offers a novel means of tracing human migrations

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

Asian genotypes of JC virus in Native Americans and in a Pacific Island population: Markers of viral evolution and human migration

The human polyomavirus JC (JCV) causes the central nervous system demyelinating disease progressive multifocal leukoencephalopathy. Previously, we showed that 40% of Caucasians in the United States excrete JCV in the urine as detected by PCR. We have now studied 68 Navaho from New Mexico, 25 Flathead from Montana, and 29 Chamorro from Guam. By using PCR amplification of a fragment of the VP1 gene, JCV DNA was detected in the urine of 45 (66%) Navaho, 14 (56%) Flathead, and 20 (69%) Chamorro. Genotyping of viral DNAs in these cohorts by cycle sequencing showed predominantly type 2 (Asian), rather than type 1 (European). Type 1 is the major type in the United States and Hungary. Type 2 can be further subdivided into 2A, 2B, and 2C. Type 2A is found in China and Japan. Type 2B is a subtype related to the East Asian type, and is now found in Europe and the United States. The large majority (56–89%) of strains excreted by Native Americans and Pacific Islanders were the type 2A subtype, consistent with the origin of these strains in Asia. These findings indicate that JCV infection of Native Americans predates contact with Europeans, and likely predates migration of Amerind ancestors across the Bering land bridge around 12,000–30,000 years ago. If JCV had already differentiated into stable modern genotypes and subtypes prior to first settlement, the origin of JCV in humans may date from 50,000 to 100,000 years ago or more. We conclude that JCV may have coevolved with the human species, and that it provides a convenient marker for human migrations in both prehistoric and modern times.

Viral- and myelin-specific cellular immune response in patients treated with natalizumab: a cross-sectional and a longitudinal prospective study

Introduction: Natalizumab, a monoclonal antibody binding to the alpha4 integrins, is efficient in preventing relapses and progression of disability in multiple sclerosis (MS) patients. However, a total of seven MS patients treated with natalizumab suffered from progressive multifocal leukoencephalopathy (PML), on a total of 53?000 patients (data of March 6, 2009) treated with this drug. PML is a disease affecting immunosuppressed people, which is caused by the polyomavirus JC (JCV). This virus produces a lytic infection of the oligodendrocytes. Yet, natalizumab cannot be considered as a classical immunosuppressant, such as suggested by the fact that no increased incidence of other opportunistic infections was reported with this drug. It has been postulated that, by closing the blood-brain, natalizumab might prevent JCV-specific CD8_ T cells to reach the CNS and perform immune surveillance. Alternatively, it has been suggested that this drug acts by releasing JCV from the bone marrow, one of its site of latency. In this study, we address the question whether there is an increased activity of JCV in the blood of natalizumab-treated MS patients. Material and Methods: In this prospective longitudinal study, we are following a cohort of 24 MS patients receiving monthly injections of natalizumab. Blood and urine are drawn every one to three months, up to 12 months. As a control group, we follow 16 MS patients treated with IFN-beta. For this control group, there are two time-points: before and 1094 months after treatment onset. We are analysing the viral (JCV-, EBV- and CMV-) as well as the myelin- (MOG-, MOBP-) specific cellular immune responses using proliferation and ELISPOT (IFNgamma) assays. For JCV, we study the response against VP1, the major capsid protein. For JCV VP1, MOG and MOBP, we use 15-mer peptides overlapping by 10 amino acids, thus eliciting CD4_ as well as CD8_ T cell response. These peptides encompasse the whole sequence of the proteins. For EBV and CMV, we use pools of immunodominant 8- to 10-mer peptides eliciting CD8_ T cells. At the same time-points, using RTPCR, we determine the presence of JCV DNA coding for the VP1 protein in the PBMC, plasma, and urine. Results: At the time of writing this abstract, 16 patients have reached the 9-month (T9), and 11 the T12 time-point. We expect that by the ISNV meeting in June 2009, 18 and 14 patients will be at T9 and T12, respectively. Virological and immunological results will be presented. 9th International Symposium on NeuroVirology 2_6 June 2009 39 J Neurovirol Downloaded from informahealthcare.com by Cantonale et Universitaire on 06/25/10 For personal use only. Conclusions: This ongoing longitudinal prospective study should tell us whether there is an enhanced JCV activity in the peripheral blood of patients on natalizumab. This work is supported by the FNS (PP00B-106716), the Swiss MS Society and a research grant from Biogen Dompe.

Towards the preparative and large-scale precision manafacture of virus-like particles

Virus-like particles (VLPs) are of interest in vaccination, gene therapy and drug delivery, but their potential has yet to be fully realized. This is because existing laboratory processes, when scaled, do not easily give a compositionally and architecturally consistent product. Research suggests that new process routes might ultimately be based on chemical processing by self-assembly, involving the precision manufacture of precursor capsomeres followed by in vitro VLP self-assembly and scale-up to required levels. A synergistic interaction of biomolecular design and bioprocess engineering (i.e. biomolecular engineering) is required if these alternative process routes and, thus, the promise of new VLP products, are to be realized.

JC polyomavirus infection in candidates for kidney transplantation living in the Brazilian Amazon Region

This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.

JC polyomavirus infection in candidates for kidney transplantation living in the Brazilian Amazon Region

This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.

Detection and differentiation of human polyomaviruses JC and BK by LightCycler PCR

Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an in-house PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.

High prevalence of the simultaneous excretion of polyomaviruses JC and BK in the urine of HIV-infected patients without neurological symptoms in São Paulo, Brazil

OBJECTIVE: To evaluate the prevalence of the urinary excretion of BKV and JCV in HIV-infected patients without neurological symptoms. METHODS: Urine samples from HIV-infected patients without neurological symptoms were tested for JC virus and BK virus by PCR. Samples were screened for the presence of polyomavirus with sets of primers complementary to the early region of JCV and BKV genome (AgT). The presence of JC virus or BK virus were confirmed by two other PCR assays using sets of primers complementary to the VP1 gene of each virus. Analysis of the data was performed by the Kruskal-Wallis test for numerical data and Pearson or Yates for categorical variables. RESULTS: A total of 75 patients were included in the study. The overall prevalence of polyomavirus DNA urinary shedding was 67/75 (89.3%). Only BKV DNA was detected in 14/75 (18.7%) urine samples, and only JCV DNA was detected in 11/75 (14.7%) samples. Both BKV and JCV DNA were present in 42/75 (56.0%) samples. CONCLUSION: In this study we found high rates of excretion of JCV, BKV, and simultaneous excretion in HIV+ patients. Also these results differ from the others available on the literature.

Human polyomaviruses JC and BK in the urine of Brazilian children and adolescents vertically infected by HIV

The aim of this study was to characterize the urinary excretion of the BK (BKV) and JC (JCV) human polyomaviruses in a cohort of human immunodeficiency virus (HIV)-infected children and adolescents. One hundred and fifty-six patients were enrolled: Group I included 116 HIV-infected children and adolescents [median age = 11.4 years (y); range 1-22 y]; Group II included 40 non-HIV-infected healthy controls (median age = 11.37 y; range 7-16 y). Single urine samples from both groups were screened for the presence of JCV and BKV DNA by polymerase chain reaction at enrolment. The overall rate of JCV and BKV urinary excretion was found to be 24.4% and 40.4%, respectively (n = 156). Group I had urinary excretion of JCV and BKV in 27.6% and 54.3% of subjects, respectively. In contrast, Group II showed positive results for JCV in 17.5% of subjects and for BKV in 12.5% of subjects (p Pearson JCV = 0.20; p Pearson BKV < 0.0001). In Group I, there was no association between JCV/BKV shedding and age, gender or CD4 values. Patients with an HIV viral load < 50 copies/mL had a lower excretion of BKV (p < 0.001) and a trend of lower JCV excretion (p = 0.07). One patient in Group I (1/116, 0.9%) showed clinical and radiological features consistent with progressive multifocal leukoencephalopathy, suggesting that children with HIV/polyomavirus coinfection should be kept under surveillance.