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JC virus (JCV) is a human polyomavirus that infects the central nervous system (CNS) of immunocompromised patients. JCV granule cell neuronopathy (JCV-GCN) is caused by infection of cerebellar granule cells, causing ataxia. A 77-year-old man with iatrogenic lymphopenia presented with severe ataxia and was diagnosed with JCV-GCN. His ataxia and cerebrospinal fluid (CSF) improved with intravenous immunoglobulin, high-dose intravenous methylprednisolone, mirtazapine, and mefloquine. Interleukin-7 (IL-7) therapy reconstituted his lymphocytes and reduced his CSF JCV load. One month after IL-7 therapy, he developed worsening ataxia and CSF inflammation, which raised suspicion for immune reconstitution inflammatory syndrome. Steroids were restarted and his ataxia stabilized.

Progressive multifocal leukoencephalopathy (PML)-derived noncoding control region (NCCR) sequences permitted greater early viral gene expression than kidney-associated NCCR sequences. This was driven in part by binding of the transcription factor Spi-B to unique PML-associated Spi-B binding sites. Spi-B is upregulated in developing B cells in response to natalizumab therapy, a known risk factor for PML. Naturally occurring JCV sequence variation, together with drug treatment-induced cellular changes, may synergize to create an environment leading to an increased risk of PML.

Background: JC virus (JCV) is the etiologic agent for progressive multifocal leukoencephalopathy (PML), a demyelinating disease occurring in the brain of patients with underlying immune compromised states. All viable JCV genomes contain a conserved region in the T protein coding nucleotide sequence that when detected by PCR in CSF is a confirmatory diagnostic marker for PML along with clinical and neuroradiological evidence. The non-coding regulatory region (NCRR) is hypervariable, as evidenced by nucleotide sequence of the non-virulent variant, which is predominantly excreted in urine, versus that of virulent variants found in brain and CSF of PML patients. All variants can be found in blood., Objective: A single assay that quantifies and identifies JCV DNA in clinical samples and discriminates between variants has significant value to physicians and patients at risk for PML., Study Design: Separate primer pairs were tested together to quantitatively detect conserved viral DNA nucleotide sequence in patient samples, while simultaneously detecting the NCRR specific for the non-virulent variant., Results: In testing using control plasmids and patients' CSF, blood, and urine, PML patients predictably demonstrated the non-virulent, archetype NCRR in urine, but virulent NCRR variants in CSF and blood., Conclusion: The JCV qPCR multiplex assay targets two regions in JCV genomes to simultaneously identify and measure viral DNA, as well as distinguish between variants associated with PML and those that are not. The multiplex results could signal risk for PML if patients are viremic with JCV variants closely associated with PML pathogenesis., (Published by Elsevier B.V.)

JC virus (JCV) DNA in the cerebrospinal fluid (CSF) provides the laboratory confirmatory diagnosis of progressive multifocal leukoencephalopathy (PML) in patients whose clinical symptoms and magnetic resonance imaging findings are consistent with PML.The Laboratory of Molecular Medicine and Neuroscience (LMMN), National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), made the confirmatory laboratory diagnosis in 35 multiple sclerosis (MS) patients treated with natalizumab. Thirteen patients had 3 or more CSF samples taken from weeks to months following PML diagnosis. Seven of the 13 patients demonstrated persistence of JCV DNA in the CSF even though all patients experienced immune reconstitution inflammatory syndrome (IRIS), 11 patients had plasma exchange, and 2 had immunoabsorption. Specific anti-JCV antibody was measured in plasma/sera samples from 25 of the 35 patients. Most of the samples showed moderate to high or rising antibody levels from the time of PML diagnosis. However, plasma from 1 patient at or near the time of PML diagnosis had a titer considered seronegative and 2 other plasma samples from patients had titers considered at baseline for seropositivity. In several PML cases, viral persistence and neurological deficits have continued for several years, indicating that once initiated, JCV infection may not entirely clear, even with IRIS.

We have further characterized the Asian genotypes (Types 2 and 7) and subtypes of JC virus (JCV). Urine samples from 224 individuals with Han and Mongolian populations were collected in five regions in eastern China: Kunming, Chengdu, Shenyang, Chifeng, and Manzhouli. Also, 99 urine samples were collected from coastal and hill groups in Kerala, southern India, and 23 urine samples from Seoul, Korea. PCR products of four typing fragments were sequenced, including two in the VP1 gene, as well as one each in the VT intergenic region and regulatory region. It was possible to clone and sequence a total of 42 JCV whole genomes (approximately 5120 bp). Five genotypes of JCV (Types 7A, 7B, 7C, 2D, and 4) were found in China, four genotypes (Types 2D, 7C, 4, and 1B) in southern India, and three genotypes (Types 7B, 2A, and 1A) in Korea. Type 7A was most prevalent in South China (59-64%) and Type 7B was predominant in northeast China and Inner Mongolia (67-77%). Type 7C strains were spread throughout North and South China (3-14%), while Type 2D strains were found only in the two Mongolian groups (9-10%). In southern India, Type 2D was predominant in the coastal group (95%), and two major types, Type 7C (50%) and Type 2D (35%), were prevalent in the tribal hill groups. In Korea two major genotypes were found: Type 7B (50%) and Type 2A (43%). Phylogenetic reconstruction places the Chinese genotypes in the Afro-Asiatic supercluster, but distinct from the Mongolian and Indian strains (Type 2D), as well as the Korean and Japanese genotype (Type 2A) that predominates in the Americas.

Distinct genotypes of human polyomavirus JC (JCV) have remained population associated possibly from the time of dispersal of modern humans from Africa. Seven major genotypes with additional subtypes serve as plausible markers for following early and more recent human migrations in all parts of the world. Phylogenetic trees of JCV sequences from the major continental population groups show a trifurcation at the base indicating early division into European, African, and Asian branches. Here, we have explored JCV relationships in the island populations of the western Pacific. Since these islands were settled from the Asian mainland and islands of Southeast Asia, we expected that their virus genotypes might show an Asian connection. We found that Type 2E (Austronesian) and Type 8 (non-Austronesian) are widely distributed in western Pacific populations. A few south China strains were found (Type 7A). A subtype of Type 8, Type 8A, was confined to Papua New Guinea. In keeping with these assignments we find that phylogenetic analysis by neighbor-joining and maximum parsimony methods places Type 2E in a closer relationship to east Asian mainland strains such as Type 2A and Type 7. Our findings support the Asian origins of the western Pacific JCV strains, and suggest three broad movements: an ancient one characterized by Type 8A, and then Type 8B, followed much later by migrations carrying Type 2E, which may correlate with the arrival of Austronesian-language speakers, the bearers of the "Lapita" cultural complex (approximately 3,500 to 5,000 years ago), and relatively recent movements carrying largely Type 7A (south China) strains directly from the West.

Previously we showed that strains of human polyoma virus JC among the Navajo in New Mexico, speakers of an Athapaskan language in the Na-Dene language phylum, and among the Salish people in Montana, speakers of a language of the Salishan group in the Amerind family, were mainly of a northeast Asian genotype found in Japan (type 2A). We now report partial VP1-gene, regulatory region, and complete genome sequences of JC virus (JCV) from the Guaraní Indians of Argentina. The Tupí-Guaraní language represents the Equatorial branch of the Amerind language family proposed by Greenberg ([1987] Language in the Americas, Stanford: Stanford University Press). The partial VP1 gene sequences of the Guaraní revealed several variants of strains found in northeast Asia (Japan), as did the Salish. In contrast, the strains in the Navajo largely conformed to the prototype type 2A sequence (MY). Phylogenetic reconstruction with both the neighbor-joining and maximum parsimony methods utilized three complete Guaraní JCV genome sequences, three genomes from the Salish people, and 27 other complete JCV genomes, including three from the Navajo and three from Japan. Both trees showed that all type 2A JCV strains from the North and South Americans are closely related phylogenetically to strains in present-day Japan. However, variant sites in the coding regions, the T-antigen intron, and the regulatory region link the type 2A strains in Amerind groups (Guaraní and Salish), but differentiate them from those in a Na-Dene-speaking (Navajo) population. The data suggest separation from a population ancestral to modern Japanese, followed by a second division within the ancestral group that led to Amerind- and Na-Dene-speaking groups. The data cannot, however, localize the latter split to the Asian mainland (two migrations) or to North America (one migration).

BK virus (BKV) was recovered by polymerase chain reaction (PCR) from brain, kidney, lung, urine, and cerebrospinal fluid (CSF) of a fatal case of BKV tubulointerstitial nephritis with dissemination to lung and brain. Viral regulatory regions in PCR-amplified urine and the lung samples were identical to the archetypal structure, WWT. In the brain and CSF, a rearranged sequence predominated, however. A 94-bp deletion preceded a 71-bp tandem duplication because the same 94-bp segment was deleted from both copies. PCR-amplified regulatory region products were cloned and sequenced to define further the extent of the rearranged structures. Two kidney clones were archetypal, whereas two others were rearranged differently from the brain and from each other. In contrast to the brain clones, the kidney rearrangements seemed to involve deletion after duplication. Three of four brain clones sequenced were identical to the rearrangement found to dominate in the PCR product. A fourth clone showed two short deletions without any duplication. The four CSF clones all showed rearrangements identical to that which was amplified by PCR from CSF and brain. This represents the first molecular analysis of a BKV strain obtained from a central nervous system infection, and it reveals regulatory region rearrangements reminiscent of those described in JC virus from brains with progressive multifocal leukoencephalopathy. We suggest that the presence in the CSF of BKV with a dominant rearranged regulatory region may be useful in the diagnosis of BKV meningoencephalitis secondary to BKV nephritis., (Copyright 2002 by the National Kidney Foundation, Inc.)

The human polyomaviruses BK virus (BKV) and JC virus (JCV) have been linked to ureteric stenosis and allograft interstitial nephritis, but molecular characterization of the species involved has not been performed. We studied paraffin-embedded renal tissue from 19 cases of allograft viral interstitial nephritis. Histological sections were subjected to polymerase chain reaction amplification using consensus, BKV-, and JCV-specific primers, with subsequent DNA sequencing for strain determination. BKV was present in all (100%) interstitial nephritis kidneys and placed in genotypes corresponding to serological groups I (n = 11), II (n = 1), and IV (n = 5). Fourteen of 17 isolates (82%) showed sequence variations in the viral capsid protein-1 (VP1) capsid region, with predicted changes in the encoded amino acids and sometimes with potential implications for the secondary and tertiary structure of the corresponding protein molecules. An additional case showed a previously reported glutamine-->leucine T-antigen region mutation. JCV was seen in seven interstitial nephritis kidneys (37%), with types 4 (n = 3), 3A (n = 2), and 2A (n = 1) identified. Most white individuals with asymptomatic infection are reported to shed type 1 JCV in the urine. Simian 40 polyomavirus was not identified in any case. These observations may have pathogenic relevance to the development of an extremely refractory form of polyomavirus interstitial nephritis seen after kidney transplantation.

DNA sequence variation between JCV genotypes is confined largely to noncoding intergenic regions and introns. Nevertheless, evidence suggests that the amino acid sequence variations among the 8 genotypes of JCV can influence the potential for neurovirulence of the virus. In the current study, the amino acid sequences for 100 JCV genomes were translated and grouped into genotype families. Subtype consensus sequences were determined and the type-specific amino acid sequence variants were identified.

The roots of the Hispanic populations of the Caribbean Islands and Central and South America go back to three continents of the Old World. In Puerto Rico major genetic contributions have come from (1) Asians in the form of the aboriginal Taino population, an Arawak tribe, present when Columbus arrived on the Island, (2) Europeans, largely Spanish explorers, settlers, government administrators, and soldiers, and (3) Africans who came as part of the slave trade. Since JC virus (JCV) genotypes characteristic of Asia, Europe, and Africa have been identified, and excretion of JCV in urine has been proposed as a marker for human migrations, we sought to characterize the JCV strains present in a Caribbean Hispanic population. We found that the strains of JCV present today in Puerto Rico are those derived from the Old World populations represented there: Types 1B and 4 from Spain, Types 3A, 3B, and 6 from Africa, and Type 2A from Asia. The Type 2A genotype represents the indigenous Taino people. This JCV genotype was represented much more frequently (61%) than would be predicted by the trihybrid model of genetic admixture. This might be attributable to characteristics of JCV Type 2A itself, as well as to the nature of the early relationships between Spanish men and native women. These findings indicate that the JCV strains carried by the Taino Indians can be found in today's Puerto Rican population despite the apparent demise of these people more than two centuries ago. Therefore, molecular characterization of JCV provides a tool to supplement genetic techniques for reconstructing population histories including admixed populations.

Distinctive genotypes of JC virus have been described for the major continental landmasses. Studies on European-Americans and small cohorts in Europe showed predominantly Type 1. Types 2 and 7 are found in Asia, and Types 3 and 6 in Africa. These genotypes differ in sequence by about 1--3%. Each genotype may have several subtypes which differ from each other by about 0.5--1%. The genotypes can be defined by a distinctive pattern of nucleotides in a typing region of the VP1 gene. This genotyping approach has been confirmed by phylogenetic reconstruction using the entire genome exclusive of the rearranging regulatory region. In this first large European study, we report on the urinary excretion of JCV DNA of 350 individuals from Poland, Hungary, Germany and Spain. We included Gypsy cohorts in Hungary (Roma), Germany (Sinti), and Spain (Gitano), as well as Basques in Spain. We show that while Type 1 predominates in Europe, the proportions of Type 1A and 1B may differ from East to Southwest Europe. Type 4, closely related to the Type 1 sequence (only approximately 1% difference) was a minor genotype in Germany, Poland and Spain, but represented the majority in Basques. The Gitanos in Spain showed a variant Type 4 sequence termed 'Rom-1'. Interestingly, neither the Gitanos in Spain, nor Sinti or Roma in Germany or Hungary showed the Type 2 or Type 7 genotype that might be expected if their origins were in an Asian population.

The JC virus (JCV) is a ubiquitous human polyomavirus that frequently resides in the kidneys of healthy individuals and is excreted in the urine of a large percentage of the population. Geographic-specific JCV variants, isolated from urine and from brain of progressive multifocal leukoencephalopathy (PML) patients, have been grouped into seven distinct genotypes based on whole genome analysis and by individual polymorphic nucleotides (typing sites) in the VP1 coding region. Mutations in the archetypal regulatory region, sometimes consisting of deletions and/or duplications, are also useful taxonomic characters for further characterizing and subdividing genotypes. Investigation of JCV variation in Papua New Guinea (PNG) revealed three distinct variants called PNG- 1, PNG-2, and PNG-3. These variants exhibited consistent coding region and regulatory region mutations. Evolutionary analysis of 32 complete JCV genomes including six new viral genomes from the western Pacific suggests that the new PNG JCV variants are closely associated with the broad group of Type 2 strains of JCV found throughout Asia, forming a monophyletic group with the Northeast Asian strains (Type 2A). Within the Type 2 clade, however, the PNG JCV variants cluster as two distinct groups and are therefore described here as new JCV genotypes designated Type 2E and Type 8.

JC virus is a ubiquitous human polyomavirus present in populations worldwide. Seven genotypes differing in DNA sequence by approximately 1-3% characterize three Old World population groups (African, European and Asian) as well as Oceania. It is possible to follow Old World populations into the New World by the JC virus genotypes they carried. The first population to settle in the Americas, the Native Americans, brought with them type 2A from northeast Asia. European settlers arriving after Columbus carried primarily type 1 and type 4. Africans brought by the slave trade carried type 3 and type 6.

The human polyomavirus JC (JC virus), a small, circular, double-stranded DNA virus, has a worldwide distribution and is excreted harmlessly in urine by 20% to 70% of adults. DNA sequence analysis has identified seven distinct genotypes that likely coevolved with modern humans, although the mode of virus transmission is unknown. Type 1 is European in its distribution. Types 2 and 7 are Asian, while Types 3 and 6 are African. Type 4, closely related to Type 1, is of uncertain origin, having been found in population groups in parts of Europe and in the United States, but not in Africa. Here we have studied the JCV partial genomic DNA sequences amplified by polymerase chain reaction techniques from urines of an urban, mainly African American population cohort from Washington, D.C. The predominant genotype identified was Type 4 (32/78 JCV strains, 41%). Type 1 strain was found in 32% of African Americans, while JCV Type 3 strain was found in 18% of African Americans. These African strains have persisted in modern African Americans after 200 to 400 years of minority existence and genetic admixture in the New World. An ancient West African genotype, Type 6, was absent in this African American cohort. However, one Type 6 strain was found in a patient from Sierra Leone (West Africa), domiciled in the United States for 20 years. Type 2A, the most common subtype in Native Americans, was seen in only two African-Americans (3%). A Type 7 strain, previously reported only in Taiwan and South China, was identified in a Vietnamese immigrant. These data support the history of African origin, migration, and genetic admixture of modern African Americans. Analysis of JCV strains in the present American populations provides a novel tool for reconstructing human migrations and genetic admixture in the New World.

The peopling of the Pacific was a complex sequence of events that is best reconstructed by reconciling insights from various disciplines. Here we analyze the human polyomavirus JC (JCV) in Highlanders of Papua New Guinea (PNG), in Austronesian-speaking Tolai people on the island of New Britain, and in nearby non-Austronesian-speaking Baining people. We also characterize JCV from the Chamorro of Guam, a Micronesian population. All JCV strains from PNG and Guam fall within the broad Asian group previously defined in the VP1 gene as Type 2 or Type 7, but the PNG strains were distinct from both genotypes. Among the Chamorro JCV samples, 8 strains (Guam-1) were like the Type 7 strains found in Southeast Asia, while nine strains (Guam-2) were distinct from both the mainland strains and most PNG strains. We identified three JCV variants within Papua New Guinea (PNG-1, PNG-2 and PNG-3), but none of the Southeast Asian (Type 7) strains. PNG-1 strains were present in all three populations (Highlanders and the Baining and Tolai of New Britain), but PNG-2 strains were restricted to the Highlanders. Their relative lack of DNA sequence variation suggests that they arose comparatively recently. The single PNG-3 strain, identified in an Austronesian-speaking Tolai individual, was closely related to the Chamorro variants (Guam-2), consistent with a common Austronesian ancestor. In PNG-2 variants a complex regulatory region mutation inserts a duplication into a nearby deletion, a change reminiscent of those seen in the brains of progressive multifocal leukoencephalopathy patients. This is the first instance of a complex JCV rearrangement circulating in a human population.

Two features of the biology of JC virus make it a particularly suitable candidate for an agent in MS-like disease: its neurotropic capability targeting glial cells as evidenced in progressive multifocal leukoencephalopathy lesions, and its capacity for latency and persistence as illustrated by its behaviour in the kidney. JC virus is chronically or intermittently excreted in the urine by some 40% of the population. The existence of JC virus in multiple coding-region genotypes provides a unique approach to the study of JC virus-induced neurological disease. We have previously shown that a genotype originating in Asia but also present in Europe and the US, called Type 2B, is more frequently found in PML brain than expected based on its prevalence in urine samples from a control population. In contrast, we find that the excretion of JCV in MS patients is similar in both genotype and frequency to that of control individuals, and appears to be regulated by factors unrelated to those that control CNS disease activity.

Objectives: This paper describes a unique JC virus (JCV) variant recovered from the Highlands of Papua New Guinea that contains an inframe 21-bp deletion in the agnoprotein gene. We characterize the mutation and suggest possible roles for the deletion in JCV evolution., Study Design/methods: JCV DNA was extracted from urine and polymerase chain reaction (PCR) amplified using whole genome primers. PCR products were cloned, and multiple clones were sequenced. The JCV agnogene was PCR amplified to verify the presence of the agnogene deletion., Results: This mutation creates a 21-bp deletion near the 3' end, which alters the predicted secondary structure of the messenger RNA and changes local codon usage at the 3' end of the agnogene. Protein secondary structure predictions suggest the deleted portion of the agnoprotein may be a flexible surface feature., Conclusions: We describe the first stable coding region deletion in JCV that presumably signifies a single evolutionary event that led to the split from other Highlands viral groups and occurred well after the human expansions that led to the peopling of the Southwest Pacific.

Objective: Progressive multifocal leukoencephalopathy is caused by polyomavirus JC in immunosuppressed patients. JC virus genotypes are identified by sequence analysis of the viral genome. Despite the prevalence of acquired immunodeficiency syndrome in sub-Saharan Africa, few cases of progressive multifocal leukoencephalopathy have been reported from this region. Here we describe 4 African cases and provide an analysis of viral genotypes., Methods: Immunohistochemical staining by labeled streptavidin-biotin for capsid protein antigen was performed on all cases. Polymerase chain reaction amplification of viral genomic DNA was followed by direct cycle sequencing., Results: JC virus type 3 was identified in 2 cases, and type 6 was isolated in 1 case. The viral regulatory region from 1 case showed an uncommon rearrangement pattern., Conclusions: Progressive multifocal leukoencephalopathy in West African patients with acquired immunodeficiency syndrome is caused by African genotypes of JC virus (types 3 and 6). The prevalence of disease in this autopsy series from sub-Saharan Africa (1.5%) was less than has been reported from Europe and the United States (4% to 10%) and may be partly due to biological differences in JC virus genotypes. Further studies will be needed to confirm this observation.

The polyomavirus JC (JCV) establishes a persistent infection in the kidneys, and is the virus agent that causes the demyelinating disease progressive multifocal leukoencephalopathy. PCR and DNA sequence analyses of partial JCV genomes have shown that there are at least four main JCV types, each associated with a specific geographical region. Type 1 is of European origin, Type 2 is Asian, Type 3 is found in individuals of African decent and Type 4 is a potential recombinant of Types 1 and 3, and is widely distributed throughout the population of the United States. A comprehensive phylogenetic analysis of 22 complete JCV genomes excluding part of the regulatory region was accomplished using neighbour-joining, UPGMA and maximum parsimony methods. The resulting UPGMA and parsimony phylogenies suggest that the European Type 1 strains diverged from the other types during the evolution of JCV and that each of the other genotypes (and subtypes) falls into well-supported clades. This is the first whole genome approach to phylogeny reconstruction for JCV and represents a significant improvement over earlier studies that were limited to partial JCV sequences and the neighbour-joining method.

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.

Several cases of progressive multifocal leukoencephalopathy (PML) have been associated with simian virus 40 (SV40), rather than with JC virus (JCV), the polyomavirus originally isolated from PML tissue. PML has, therefore, been defined as a demyelinating syndrome with possible multiple viral etiologies. Tissues from three of the cases thought to be associated with SV40 were available for reexamination. Monoclonal antibodies specific for SV40 capsid antigen VPI, virus-specific biotinylated DNA probes for in situ hybridization, and virus-specific primers in the polymerase chain reaction (PCR) were used. Macaque PML brain served as a positive control tissue for SV40 brain infection. Monoclonal antibodies to SV40 VPI failed to recognize viral antigen in lesions from all three human PML cases. The biotinylated DNA probe, which reacted with SV40 in macaque PML, failed to detect SV40 in human PML. However, JCV could be detected by in situ hybridization with a JCV-specific DNA probe. Moreover, JCV DNA sequences were amplified by PCR from the human PML tissues, whereas SV40 DNA sequences were amplified only from the macaque brain. Thus, we could not confirm the original reports that the demyelinating agent in these three cases of PML was SV40, rather than JCV. We conclude that SV40 infection of the central nervous system need not be ruled out in the differential diagnosis of PML.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating central nervous system (CNS) infection, affecting mainly oligodendrocytes, but also occasional astrocytes. In the USA, Europe and Asia, PML is caused by the human polyomavirus JC virus (JCV) and in autopsy series occurs in about 4-7% of AIDS patients. In Africa, the prevalence of PML in AIDS patients is uncertain and the causative agent is unknown. This study reports immunocytochemical and PCR confirmation of PML in the CNS of an AIDS patient dying in Uganda, East Africa (case 1). In a Gambian patient infected with HIV-2 who died 3 months after onset of AIDS/PML in Germany (case 2), it was possible to confirm the identity of the virus by DNA sequencing of the PCR amplified JCV product. This African genotype of the virus (type 3) showed an unusual re-arrangement of the regulatory region, and could be distinguished at several sites from East African and African-American JCV strains described previously. This study has confirmed that PML is a complication of African AIDS as it is in Europe and the USA, and that JCV type 3 is pathogenic in African AIDS patients. Furthermore, the finding of an African genotype of JCV in a patient dying in Germany suggests that in this individual JCV represented a latent infection acquired in Africa.

A renal biopsy from a 36-year-old man with AIDS showed a severe tubulointerstitial nephritis with intranuclear inclusions in epithelial cells. Electron microscopy revealed the characteristic findings of a polyomavirus (PyV) infection, and immunofluorescence indicated the presence of BK virus (BKV) antigen. Inoculation of rhesus monkey kidney cell cultures both with urine and with buffy coat blood cells resulted in a cytopathic response which was subsequently confirmed to be due to BKV. Further characterization of the viral DNA from the kidney by PCR amplification and Southern blot analysis with PyV and strain-specific primers and probes indicated that the virus was closely related to the BK(Dun) strain but different in its apparent sequence arrangement. Subsequent cycle sequencing showed a dinucleotide mutation of TG-->AA which substitutes hydrophilic Gln for hydrophobic Leu in a sequence homologous to an origin DNA-binding domain of simian virus 40 T antigen. It is suggested that the mutation and a coding region rearrangement of this strain of BKV designated BKV(Cin) has the potential to alter viral DNA replication and enhance pathogenicity.

Five major genotypes of JC virus (JCV) have been defined based on nucleotide differences in the VP1 gene of the DNA sequence. These types are probably a result of virus evolution in geographically isolated population groups. One of the first genotypes identified, Type 2, was found to represent strains of Asian origin. In order to further define the spectrum within Type 2 strains, the entire 5.1 kb genome of nine urinary strains of JCV was amplified by PCR with one pair of primers. These urine samples were obtained in the USA (California and New Mexico) from three European Americans, three Native Americans, two African Americans and one Hispanic American. The complete genome of an Asian JCV strain (Tokyo-1) isolated from progressive multifocal leukoencephalopathy (PML) brain in Japan was also sequenced. Here, we report the analysis of these ten DNA sequences and their deduced protein translations. Two phylogenetically distinct subtypes of Type 2 were found, 2A and 2B, which differ from each other by 0.8-1.1% of the coding region sequence. A 215 bp product amplified with primers in the VP1 gene contains enough sequence information to distinguish the major types and subtypes of JCV and is suitable for application in viral epidemiological studies. The investigation of these genomic variations is of special interest because JCV Type 2 strains are found at a significantly higher frequency in brain tissue of patients with PML than would be predicted from their excretion in a control population.

The regulatory region of progressive multifocal leukoencephalopathy-type JC virus (JCV) is rearranged in each host by a process of deletion and duplication. Of the more than 40 that have been examined, no two regulatory regions have been rearranged identically in the brain. The substrate for this rearrangement appears to be a highly stable archetypal regulatory region excreted in the urine. Its role as the transmissible form of the virus, although inferred, has never been proven. We have now amplified by PCR and cycle-sequenced the regulatory regions from 48 urinary strains of the virus. We find that the urinary form of the regulatory region is not entirely stable. Short deletions and duplications in the range of 2-16 bp were observed in seven of these strains. One of these, an inverted repeat, is a pattern of rearrangement not yet found in the brain. Two others (#208 and 230) showed a 2-bp deletion at position nos. 221 and 222, and an unusual mutation at position no. 219. These two urines were collected in different states of the USA at different times and analysed months apart. It is very unlikely that these unusual changes represent sample contamination or that they arose independently. This finding indicates that archetypal forms of the JCV regulatory region are infectious, despite their relative inactivity in tissue culture. While changes in the archetypal structure can be found, it is clear that rearrangements in the kidney are rare or rarely infectious.

Objectives: The major genotypes of the human polyomavirus JC (JCV) include type 1 (European), type 2 (Asian), type 3 (African), and type 4 (United States). Here we report characterization of the complete genome of a genotype obtained from the brain of an African American with systemic lupus erythematosus (SLE) and progressive multifocal leukoencephalopathy (PML)., Study Design/methods: DNA extracted from JCV-infected brain tissue was subjected to whole-genome polymerase chain reaction (PCR) amplification and direct cycle sequencing. Relations to other JCV genotypes and the predicted amino acid sequence were analyzed., Results: This African-American type 6 strain (#601) differs from strains of all other genotypes in about 2% of its DNA sequence. The length of the total coding region of strain #601 is increased to 4855 bp by the insertion of a single nucleotide in the large T-antigen intron. This strain, originally placed with the type 2 group on the basis of its sequence in the VT-intergenic region, is very closely related to strains recently identified in the urine of individuals from Ghana, West Africa., Conclusions: This is the first example of an African JCV genotype identified in the brain of an African-American PML patient. The extent of sequence divergence of JCV type 6 suggests a split of type 6 strains before the separation of types 2 and 3. These findings confirm that distinctive African genotypes of JCV have been maintained in the African-American population and that they are capable of causing PML.

JC virus (JCV), a human polyomavirus, is the agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV exists in four main genotypes in the USA. Type 1, including subtypes Type 1A and Type 1B, makes up about 64% of strains in the USA and is thought to be of European origin. Type 2 is found in Asia, and Type 3 in Africa. A fourth type is found only in the USA. In general, these genotypes differ in 1-2.5% of their DNA sequence. Thirty MS patients and 30 paired controls from Budapest were studied. The clinical course of MS was mainly secondary progressive, and patients were stable at the time of testing. Most of the controls were relatives of the probands: a spouse, parent, or child. Overall, 25 of 60 (42%) of the urines tested positive for JCV by PCR. These included 13 of 30 MS patients, and 12 of 30 controls. Genotyping in the VPI gene showed all 25 JCV strains to be Type 1. Among the MS patients, seven were Type 1A and six were Type 1B. Among the controls, nine were Type 1A and three were Type 1B. In five pairs of MS patients and controls, both were positive for JCV by PCR. Two of these were husband/wife pairs of which one pair was matched for subtype (both Type 1A), and the other was not. Two of them were mother/daughter pairs, and both were matched for subtype (both Type 1B). These findings demonstrate that JCV Type 1 predominates among Hungarians, and suggest that parent/child pairs can be used to trace JCV transmission within the MS family.

The complete genomes of three new Type 1 strains of JC virus (JCV) from urine have been analysed. These were subtype 1A, subtype 1B and Type 4 as assigned from a short typing fragment in the VP1 gene. They differ from Mad1 (subtype 1A) by less than 1.0% of the DNA sequence. Based on its complete genome, the JCV Type 4 strain falls into a Type 1 subgroup. Type 4, with several Type 3-like sites in the short typing fragment, is a possible recombinant strain. The consensus of Type 1 DNA sequences is distinguished within the coding region from both Type 2 (strain GS/B) and five Type 3 (African and African American) strains at 64 sites. Most mutations are silent, but at 21 positions amino acid changes occur. Our findings define the subtypes of JCV Type 1 and support the validity of genotyping within the short VP1 fragment.

Objectives: Previous studies have shown that strains of human polyomavirus JC (JCV) of Asian origin (type 2) are much more highly represented in progressive multifocal leukoencephalopathy (PML) brain than would be expected from their frequency of excretion in urine samples of a comparable control group. The present studies were designed to test whether one subtype of type 2 was preferentially elevated., Study Design/methods: The statistical relation between JCV subtypes represented in PML brain tissue from 51 probands and those in urine samples from 115 control individuals was examined., Results: The proportion of the JCV subtype 2B in PML brain (36%) was highly significantly increased relative to its occurrence in control urine samples (5.9%; P < .001). Type 1 and its subtypes were not different in the PML brain and control urine cohorts. The number of type 4 strains in PML brains was reduced, although the difference did not reach statistical significance (P = .08)., Conclusions: The results predict that the human immunodeficiency virus (HIV)-positive individuals at highest risk of PML infection are those carrying the JCV genotype known as type 2B. Prospective studies will be required to confirm this finding.

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.

Progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus JC (JCV), and there are at least 4 different genotypes of JCV in the United States. Type 1 strains are of European origin, whereas type 2 and 3 strains are of Asian and African origin, respectively. JCV type 4 strains are derived from a type 1/3 recombinant. In this study, the genotype distribution of JCV strains found in brain tissue or cerebrospinal fluid of 50 PML patients was compared with JCV genotypes excreted in the urine of 103 control subjects. Type determination was based on the polymerase chain reaction-amplified partial sequence of the VP1 coding gene and the noncoding region left of ori. Brain tissues from patients with PML were infected with a significantly higher proportion of JCV type 2 strains than were urine samples from the control group (P = .004). This evidence indicates a biologic difference between JCV genotypes and suggests a difference in their potential to cause PML.

JC virus (JCV) causes the central demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV strains excreted in the urine are distinguishable from those in PML tissue by the configuration of their regulatory region to the right of ori: the archetypal regulatory region, 267 nucleotides long, is rearranged in PML tissue by deletion and duplication. Within the coding region JCV shows variations as a result of virus evolution. Four major genotypes are distinguishable of which Type 1 is based in Europe and Type 2 in Asia. Here, the regulatory region rearrangements and the viral genotypes of 29 JCV strains from PML brain were determined. Rearrangement patterns and genotypes were not associated. In general, deletions occurred before duplications, but exceptions to this rule exist. Each configuration of the 29 rearranged regulatory regions was unique and could be derived directly from the non-rearranged, archetypal form.

The central demyelinating disease progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus JC virus (JCV). JCV evolved as geographically based genotypes of which Type 3 is an African variant first characterized in HIV-1 positive patients from Tanzania. This study reports the complete sequence of five JCV Type 3 strains. The entire JCV genome was PCR amplified from urine specimens of three African and two African-American individuals. The African consensus sequence was compared to the Type 1 and Type 2 prototype strains, JCV (Mad-1) and JCV(GS/B), respectively. Type 3 differed in 2.2% of its coding region genome from JCV (Mad-1) and in 1.3% from JCV(GS/B). Within the coding region the sequence variation among the three types was higher in the capsid protein VP1 and in the regulatory protein large T antigen than in the agnoprotein or in VP2/3. Notable Type 3-specific changes were located at sites adjacent to the zinc finger motif and near the major donor and acceptor splice junctions of large T antigen. Four of the five urinary Type 3 strains had an unrearranged, archetypal regulatory region. African strain #309 showed a 10-bp deletion at a location similar to that previously described for #307 from Tanzania. The African-American Type 3 strain #312 was closely related to the African consensus sequence. The complete genome of a urinary JCV strain from another African-American male, previously reported as a possible Type 5, showed a sequence difference of only 0.52% from the Tanzanian consensus and has been reclassified as a subtype of Type 3.

The human polyomavirus JC (JCV), which exists in different geographically based genotypes, causes the central demyelinating disease known as progressive multifocal leukoencephalopathy (PML). A coding region recombinant JCV Type 1/Type 3 (Type 4) is excreted in the urine of some 16% of individuals in the USA. In addition, occasional 'crossovers' in viral DNA sequence at type-specific sites in the coding region occur between JCV genotypes amplified from PML brain. For recombination to occur requires the existence of two different genotypes in the same host. Here we provide evidence from direct cycle sequencing of PCR products that different genotypes of JCV can be found in a single tissue sample. After non-type-specific PCR amplification, cycle sequencing produced 'split bands' at type determining sites which were resolved into type or subtype-specific sequences by subcloning of the PCR products. PCR products with split bands at typing sites were found in two brain samples and in one cerebrospinal fluid (CSF) from AIDS patients with PML and in the urine of four immunocompetent individuals. This indicates that co-infection with two viral types does not depend on severe immunocompromise. Combinations of genotypes found were Types 1A & 1B, 1A & 2, 1B & 2 and 2 & 3. In one doubly infected patient the major JCV type excreted in the urine changed within 1 week.

Thirty-seven chronic progressive multiple sclerosis (MS) patients, 20 of whom were taking cyclosporine, were examined for excretion of JC virus (JCV) in the urine. Polymerase chain reaction (PCR) amplification of DNA in urinary cell extracts detected JCV in 30% of the MS urines. In the cyclosporine treated group four of 20 (20%) excreted JCV, whereas in the untreated group seven of 17 (41%) excreted JCV. Thus, cyclosporine treatment did not enhance urinary excretion of the virus. A control group consisting of an unselected series of 89 patients donating urine in a general medical clinic and 16 healthy volunteers showed 41% with detectable urinary JCV. Thirty-three percent of the control females excreted JCV (18/54), as did 49% of the control males (25/51). Although the percentage of MS patients excreting detectable virus was not increased compared to the control group, the presence of JCV in the urine provides a convenient source of the virus for further characterization. Genotyping of DNA fragments amplified from the VP1 region indicates mainly the presence of JCV Type 1 in these chronic progressive MS patients. This is also the type that predominates in the control group. An apparent recombinant between Type 1 and Type 3 (African) within the VP1 region, tentatively designated Type 1/3 (or Type 4), was found in both the MS group and the controls. A larger series of MS patients that includes relapsing/remitting disease will be required to determine whether the genotype profile of JCV excreted in the urine of MS patients differs significantly from controls.

The human polyomavirus JC (JCV) causes the central demyelinating disease progressive multifocal leukoencephalopathy in about 5% of AIDS patients. To characterize the type profile of JCV in a control population in the United States, 54 females (10 to 79 years of age; average age, 43.4 years) and 51 males (18 to 94 years of age; average age, 47.9 years) were examined for the excretion of different genotypes of JCV in their urine by PCR followed by direct cycle sequencing. The group consisted of 89 patients of a general medical clinic in addition to 16 healthy volunteers. The overall incidence of JC viruria was 43 of 105 (40.9%) subjects, with a marked increase for those subjects above the age of 30 years. Two men were found to excrete two different types of JCV at the same time, indicating double infections. Of the three different genotypes of JCV identified to date, type 1 strains (European) were the most common in this cohort (64% of total strains) followed by type 2 (East Asian) (18%). No type 3 (East African) strains were detected. Indirect evidence for the existence of JCV type 3 was found in seven individuals (16%) in the form of a type 1/3 recombinant (also called type 4). In addition, a single example of JCV which differs from types 1, 2, and 3 and may represent a phylogenetically older type (type 5) was found in a 59-year-old African-American. Delineation of sequence variations between JCV types is essential for the design of primers for sensitive PCR with clinical samples.

PCR on 52 cerebrospinal fluid (CSF) specimens and 33 brain biopsies obtained from HIV-1 positive patients utilized pairs of primers from both the early region (JTP) and late region (JLP). In these patients, in whom progressive multifocal leukoencephalopathy (PML) was suspected on the basis of clinical symptoms and magnetic resonance imaging (MRI) studies, eight CSFs (15%) and 14 brain biopsy specimens (42%) contained JCV DNA sequences. In two patients' samples, the CSFs were positive for JCV DNA in the VP1 region using the primer pair for the VP1 region (JLP), but the fragment amplified migrated more rapidly than the 129-bp product obtained from prototype JCV(Mad-1) or the fragment amplified from the antigenic variant of JCV known as Mad-11. These patients died 3-4 months after onset of progressive neurological symptoms. Cycle sequencing of the fragments revealed overlapping deletions of 24 and 27 nucleotides. These strains were of different genotypes, designated strain 107 and strain 206. Computer analysis of the VP1 amino acid sequence predicts that the eight or nine amino acid residue deletions represent a surface loop with a high antigenic index. These naturally occurring deletion mutants are the first examples of a phenomenon observed experimentally in the mouse polyoma virus capsid protein VP2.

HIV-1 positive patients from Tanzanian villages near Shirati were examined for urinary excretion of the human polyomaviruses JC and BK using the polymerase chain reaction (PCR). BK virus (BKV) was detected in 11 of 23 individuals tested. The BKV DNA sequences were all closely related to prototype Gardner strain and BKV (DUN). In contrast, a new type of JCV, termed Type 3 [or JCV (Shi)], was identified in seven of these same 23 individuals by comparison with Type 1 and Type 2 sequences of the VP1/intergenic/T antigen region of U.S., European and Asian strains. This suggests that JCV and BKV, although closely related, have different evolutionary histories within the African population. The six BKV regulatory regions amplified all showed the archetypal configuration. However, two of the seven JCV regulatory regions showed rearrangements: a small deletion and an inverted repeat. JCV causes a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), in about 5% of AIDS patients in Europe and the U.S.A., but only one case has been reported in Africa. Our results suggest that this rarity of PML is not due to the absence of JCV in the African population.

Two types of JC virus (JCV) are found in infected brain and kidney tissues. A highly reliable PCR assay to determine viral type in tissue is presented. This type-specific system is analogous to allele-specific PCR used to detect point mutations in cellular genes. Specific amplification of two fragments, using four pairs of type-specific primers, is based on a single nucleotide difference at the 3'-ends of the primers. A combination of three conditions in the PCR reaction was required for specificity: 'hot start', a ramped ('touchdown') cycle profile, and a slightly lowered molar concentration of the specific primers and dNTPs. Efficient yield of PCR product is not lost under these conditions, and even the least selective mismatches (C:A and T:G) provided specific amplification. Type-specific restriction enzyme sites within the amplified fragments confirm type designation.

The large T-antigens of papovaviruses JC (JCV) and BK share a C-terminal subsequence with myelin basic protein (MBP). Since this sequence functions as a phosphate acceptor site in MBP, expression of a competing T-antigen sequence in oligodendroglia might adversely affect their ability to post-translationally process MBP and thus to maintain myelin. We have used techniques which demonstrate JCV T-antigen in small oligodendroglial cells from progressive multifocal leukoencephalopathy tissue to search for a possible latent JCV infection expressing T-antigen in nine cases of multiple sclerosis (MS) and three normal brains. No cells expressing T-antigen were detected in plaque or periplaque regions of the MS brains or in control CNS tissue.

Progressive multifocal leukoencephalopathy (PML) is a JC papovavirus infection of the central nervous system in immunocompromised patients. It is well established that demyelination in PML is caused by JC virus infection of oligodendroglia, but whether the nonstructural regulatory protein, large tumor (T) antigen, is detectable in infected human tissue was not known. Using a modification of the peroxidase-antiperoxidase technique, we found T antigen expressed in the nuclei of cells in virus-infected sites in five cases of PML studied, including two with acquired immune deficiency syndrome (AIDS). PML occurs in AIDS at a much higher frequency than in other immunosuppressive disorders, and PML in AIDS may represent a more severe form of JC virus infection of the central nervous system.

Antisera raised against porcine myelin basic protein (MBP) in Syrian hamsters were assayed by an ELISA method. The specificity of a high-titered antiserum was probed with synthetic peptides representing a hexapeptide and a decapeptide of the JC virus (JCV) large T-antigen C-terminus which is homologous to the MBP triproline region, a decapeptide from MBP which is encephalitogenic in guinea pigs, and peptides unrelated to MBP, i.e., substance P and poly-L-lysine. In an ELISA inhibition assay, preincubation of the hamster antiserum to MBP with either the JCV T-antigen C-terminal decapeptide or the encephalitogenic determinant inhibited binding activity in a dose-dependent manner. In contrast, the T-antigen C-terminal hexapeptide, substance P, and poly-L-lysine were not inhibitory. These results suggest that the triproline region of MBP can be immunogenic in hamsters, and support the concept that a conformation of the MBP triproline region is shared with certain of its viral homologues. In an effort to detect similar cross-reactive specificities in hamster antisera to JCV T-antigen, sera of 50 hamsters bearing subcutaneous tumors induced by JCV-transformed glial cells were tested for ability to bind to MBP in the ELISA assay. While significant increases in response compared to prebleed levels were observed in about one-fourth of the sera, some of them showed similar increases in binding to other basic proteins such as histones, and the binding to MBP was not inhibited by the triproline-containing decapeptide.

A new double-label immunocytochemical method detects JC virus (JCV) early (T-antigen) and late (capsid) proteins simultaneously in cryostat sections of progressive multifocal leukoencephalopathy (PML) brain tissue from both acquired immunodeficiency syndrome (AIDS) and non-AIDS patients. T-antigen is detected with a monoclonal antibody (PAb 416) followed by goat anti-mouse IgG and mouse Clono-PAP, while capsid proteins are detected by a rabbit polyclonal antiserum to capsid proteins followed by biotinylated goat anti-rabbit IgG and streptavidin-alkaline phosphatase conjugate. The substrates are 3,3'-diaminobenzidine and Vector Red I, respectively. With this method some infected glial cells stain for late (capsid) antigens in the nucleus, while others show early protein (large T-antigen) immunoreactivity. The latter are likely to be astrocytes infected abortively or oligodendrocytes in the early stages of a productive JCV infection.

Thirty monoclonal antibodies to SV40 large T-antigen were tested for reactivity on the JC virus-transformed hamster glial cell line known as HJC-15. Two of them (PAb 416 and PAb 108) detected a nuclear antigen in both SV40-transformed CCL 75.1 cells and in HJC-15 cells, but not in control cells lacking T-antigen. These same antibodies also labeled a nuclear antigen in hamster tumor tissue derived from HJC-15 cells. In addition, the monoclonal antibody PAb 416 detected a nuclear antigen in progressive multifocal leukoencephalopathy (PML) tissue infected with JC virus, but not in normal brain tissue or tissue from other neurological diseases. Staining by PAb 416 was reduced by prior incubation with hamster anti-JCV tumor serum, suggesting that the polyclonal antiserum to JCV T-antigen may compete for an epitope at or near the PAb 416 binding site.