Co-occurrence of two cases of progressive multifocal leukoencephalopathy in a natalizumab "infusion group".

We observed two cases of progressive multifocal leukoencephalopathy (PML) that occurred in the same "infusion group". The group consisted of four patients with relapsing-remitting multiple sclerosis (RRMS) who had been treated with natalizumab (NAT) in the same medical practice for more than four years at the same times and in the same room, raising concerns about viral transmission between members of the infusion group. DNA amplification and sequence comparison of the non-coding control region (NCCR) of JC virus (JCV) present in cerebrospinal fluid (CSF) samples from PML patients #1 and #2 revealed that the amplified JCV sequences differed from the JCV archetype. The NCRR of the viral DNA was unique to each patient, arguing against the possibility of viral transmission between patients. Statistical considerations predict that similar co-occurrences of PML are likely to happen in the future.

Management and outcome of CSF-JC virus PCR-negative PML in a natalizumab-treated patient with MS.

OBJECTIVE: To describe the diagnosis and management of a 49-year-old woman with multiple sclerosis (MS) developing a progressive hemiparesis and expanding MRI lesion suspicious of progressive multifocal leukoencephalopathy (PML) 19 months after starting natalizumab. RESULTS: Polyomavirus JC (JCV)-specific qPCR in CSF was repeatedly negative, but JCV-specific antibodies indicated intrathecal production. Brain biopsy tissue taken 17 weeks after natalizumab discontinuation and plasmapheresis was positive for JCV DNA with characteristic rearrangements of the noncoding control region, but histology and immunohistochemistry were not informative except for pathologic features compatible with immune reconstitution inflammatory syndrome. A total of 22 months later, the clinical status had returned close to baseline level paralleled by marked improvement of neuroradiologic abnormalities. CONCLUSIONS: This case illustrates diagnostic challenges in the context of incomplete suppression of immune surveillance and the potential of recovery of PML associated with efficient immune function restitution.

Analysis of cerebrospinal fluid and cerebrospinal fluid cells from patients with multiple sclerosis for detection of JC virus DNA.

OBJECTIVE: 1) To determine whether JC virus (JCV) DNA was present in the cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) in comparison with controls and 2) to find out if our clinical material, based on presence of JCV DNA, included any patient at risk for progressive multifocal leukoencephalopathy (PML). METHODS: The prevalence of JCV DNA was analyzed in CSF and plasma from 217 patients with MS, 86 patients with clinically isolated syndrome (CIS), and 212 patients with other neurological diseases (OND). In addition, we analyzed CSF cells, the first report of JCV DNA in CSF cells in a single sample, and peripheral blood cells in a subgroup of MS (n = 49), CIS (n = 14) and OND (n = 53). RESULTS: A low copy number of JCV DNA was detected in one MS cell free CSF sample and in one MS CSF cell samples. None of these had any signs of PML or developed this disease during follow-up. In addition, two OND plasma samples were JCV DNA positive, whereas all the other samples had no detectable virus. CONCLUSION: A low copy number of JCV DNA may occasionally be observed both in MS and other diseases and may occur as part of the normal biology of JC virus in humans. This study does not support the hypothesis that patients with MS would be at increased risk to develop PML, and consequently screening of CSF as a measurable risk for PML is not useful.

Molecular evaluation of BK polyomavirus nephropathy.

Understanding at a molecular level, the immunologic response of polyomavirus nephropathy (PVN), a critical cause of kidney graft loss, could lead to new targets for treatment and diagnosis. We undertook a transcriptional evaluation of kidney allograft biopsies from recipients with PVN or acute rejection (AR), as well as from recipients with stable allograft function (SF). In both the PVN and AR groups, Banff histologic scores and immunohistochemical analysis of inflammatory infiltrates were similar. Despite their different etiologies, the transcriptional profiles of PVN and AR were remarkably similar. However, transcription of genes previously linked to AR including CD8 (65.9 +/- 18.8) and related molecules IFN-gamma(55.1 +/- 17.0), CXCR3 (49.9 +/- 12.8) and perforin (153.8 +/- 50.4) were significantly higher in PVN compared to AR (30.9 +/- 2.0, 14.0 +/- 7.3, 12.1 +/- 7.3 and 15.6 +/- 3.8-fold, respectively; p < 0.01). Importantly, transcription of molecules associated with graft fibrosis including matrix collagens, TGFbeta, MMP2 and 9, as well as markers of epithelial-mesenchymal transformation (EMT) were significantly higher in PVN than AR. Thus, renal allografts with PVN transcribe proinflammatory genes equal in character and larger in magnitude to that seen during acute cellular rejection. BK infection creates a transcriptional microenvironment that promotes graft fibrosis. These findings provide new insights into the intrarenal inflammation of BK infection that promotes graft loss.

Predicted amino acid sequences for 100 JCV strains.

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.

Molecular genotyping of BK and JC viruses in human polyomavirus-associated interstitial nephritis after renal transplantation.

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.

Reconstructing population history using JC virus: Amerinds, Spanish, and Africans in the ancestry of modern Puerto Ricans.

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.

New JC virus (JCV) genotypes from papua new guinea and micronesia (type 8 and type 2E) and evolutionary analysis of 32 complete JCV genomes.

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.

Polyomavirus JC genotypes in an urban United States population reflect the history of African origin and genetic admixture in modern African Americans.

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.

Human polyomavirus JC variants in Papua New Guinea and Guam reflect ancient population settlement and viral evolution.

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.

Influence of JC virus coding region genotype on risk of multiple sclerosis and progressive multifocal leukoencephalopathy.

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.

JC virus as a marker of human migration to the Americas.

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.

Progressive multifocal leukoencephalopathy and JC virus genotypes in West African patients with acquired immunodeficiency syndrome: a pathologic and DNA sequence analysis of 4 cases.

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.

A novel JC virus variant found in the Highlands of Papua New Guinea has a 21-base pair deletion in the agnoprotein gene.

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.

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.

Reappraisal of progressive multifocal leukoencephalopathy due to simian virus 40.

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.

Phylogenetic analysis of 22 complete genomes of the human polyomavirus JC virus.

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.

Rearrangements of archetypal regulatory regions in JC virus genomes from urine.

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.

Tubulointerstitial nephritis due to a mutant polyomavirus BK virus strain, BKV(Cin), causing end-stage renal disease.

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.

JC virusType 2: definition of subtypes based on DNA sequence analysis of ten complete genomes.

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.