JC virus regulatory region tandem repeats in plasma and central nervous system isolates correlate with poor clinical outcome in patients with progressive multifocal leukoencephalopathy

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), has a hypervariable regulatory region (JCV RR). A conserved archetype form is found in the urines of healthy and immunocompromised individuals, whereas forms with tandem repeats and deletions are found in the brains of PML patients. Type I JCV RR, seen in MAD-1, the first sequenced strain of JCV, contains two 98-bp tandem repeats each containing a TATA box. Type II JCV RR has additional 23-bp and 66-bp inserts or fragments thereof and only one TATA box. We cloned and sequenced JCV RR from different anatomic compartments of PML patients and controls and correlated our findings with the patients' clinical outcome. Twenty-three different sequences were defined in 198 clones obtained from 16 patients. All 104 clones with tandem repeats were type II JCV RR. Patients with poor clinical outcome had high proportions of JCV RR clones with both tandem repeats in plasma (54%) and brain or cerebrospinal fluid (85%). In those who became survivors of PML, archetype sequences predominated in these anatomic compartments (75 and 100%, respectively). In patients with advanced human immunodeficiency virus infection without PML, only 8% of JCV RR clones obtained in the plasma contained tandem repeats. These data suggest that the presence of tandem repeats in plasma and CNS JCV RR clones is associated with poor clinical outcome in patients with PML.     

Rearrangement of simian virus 40 regulatory region is not required for induction of progressive multifocal leukoencephalopathy in immunosuppressed rhesus monkeys

Rearrangements of the JC virus (JCV) regulatory region (RR) are consistently found in the brains of patients with progressive multifocal leukoencephalopathy (PML), whereas the archetype RR is present in their kidneys. In addition, the C terminus of the large T antigen (T-Ag) shows greater variability in PML than does the rest of the coding region. To determine whether similar changes in simian virus 40 (SV40) are necessary for disease induction in monkeys, we sequenced the SV40 RR and the C terminus of the T-Ag from the brain of simian/human immunodeficiency virus (SHIV)-infected monkey 18429, which presented spontaneously with an SV40-associated PML-like disease, as well as from the peripheral blood mononuclear cells (PBMC), kidneys, and brains of SV40-seronegative, SHIV-infected monkeys 21289 and 21306, which were inoculated with the 18429 brain SV40 isolate. These animals developed both SV40-associated PML and meningoencephalitis. Thirteen types of SV40 RR were characterized. Compared to the SV40 archetype, we identified RRs with variable deletions in either the origin of replication, the 21-bp repeat elements, or the late promoter, as well as deletions or duplications of the 72-bp enhancer. The archetype was the most prominent RR in the brain of monkey 18429. Shortly after inoculation, a wide range of RRs could be found in the PBMC of monkeys 21289 and 21306. However, the archetype RR became the predominant type in their blood, kidneys, and brains at the time of sacrifice. On the contrary, the T-Ag C termini remained identical in all compartments of the three animals. These results indicate that unlike JCV in humans, rearrangements of SV40 RR are not required for brain disease induction in immunosuppressed monkeys.     

Archetype JC Virus Efficiently Replicates in COS-7 Cells, Simian Cells Constitutively Expressing Simian Virus 40 T Antigen

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is ubiquitous in humans, infecting children asymptomatically and then persisting in the kidney. Renal JCV is not latent but replicates to excrete progeny in the urine. The renal-urinary JCV DNAs carry the archetype regulatory region that generates various rearranged regulatory regions occurring in JCVs derived from the brains of PML patients. Tissue cultures that support the efficient growth of archetype JCV have not been reported. We studied whether archetype JCV could replicate in COS-7 cells, simian cells transformed with an origin-defective mutant of simian virus 40 (SV40). Efficient JCV replication, as detected by a hemagglutination assay, was observed in cultures transfected with five of the six archetype DNAs. The progeny JCVs could be passaged to fresh COS-7 cells. However, when the parental cells of COS-7 not expressing T antigen were transfected with archetype JCV DNAs, no viral replication was detected, indicating that SV40 T antigen is essential for the growth of JCV in COS-7 cells. The archetype regulatory region was conserved during viral growth in COS-7 cells, although a small proportion of JCV DNAs underwent rearrangements outside the regulatory region. We then attempted to recover archetype JCV from urine by viral culture in COS-7 cells. Efficient JCV production was observed in COS-7 cells infected with five of the six JCV-positive urine samples examined. Thus, COS-7 cells should be of use not only for the production of archetype JCV on a large scale but also for the isolation of archetype JCV from urine.     

Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat

Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter.
In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.     

JC Virus Latency in the Brain and Extraneural Organs of Patients with and without Progressive Multifocal Leukoencephalopathy

JC virus (JCV) is latent in the kidneys and lymphoid organs of healthy individuals, and its reactivation in the context of immunosuppression may lead to progressive multifocal leukoencephalopathy (PML). Whether JCV is present in the brains or other organs of healthy people and in immunosuppressed patients without PML has been a matter of debate. We detected JCV large T DNA by quantitative PCR of archival brain samples of 9/24 (38%) HIV-positive PML patients, 5/18 (28%) HIV-positive individuals, and 5/19 (26%) HIV-negative individuals. In the same samples, we detected JCV regulatory region DNA by nested PCR in 6/19 (32%) HIV-positive PML patients, 2/11 (18%) HIV-positive individuals, and 3/17 (18%) HIV-negative individuals. In addition, JCV DNA was detected in some spleen, lymph node, bone, and kidney samples from the same groups. In situ hybridization data confirmed the presence of JCV DNA in the brains of patients without PML. However, JCV proteins (VP1 or T antigen) were detected mainly in the brains of 23/24 HIV-positive PML patients, in only a few kidney samples of HIV-positive patients, with or without PML, and rarely in the bones of HIV-positive patients with PML. JCV proteins were not detected in the spleen or lymph nodes in any study group. Furthermore, analysis of the JCV regulatory region sequences showed both rearranged and archetype forms in brain and extraneural organs in all three study groups. Regulatory regions contained increased variations of rearrangements correlating with immunosuppression. These results provide evidence of JCV latency in the brain prior to severe immunosuppression and suggest new paradigms in JCV latency, compartmentalization, and reactivation.JC virus (JCV) is the etiologic agent of the often fatal brain-demyelinating disease progressive multifocal leukoencephalopathy (PML) (23a). JCV remains latent in the kidneys, lymph nodes, and bone marrow of healthy and immunosuppressed individuals without PML (2, 21, 24) and, upon reactivation, can cause a lytic infection of oligodendrocytes in the brain, leading to PML (14). Although JCV is often found in the urine of healthy individuals (12, 18), it is not usually detected in the blood of patients without PML (15). The pathway leading to viral reactivation and replication in the brains of immunosuppressed individuals is not well defined. Molecular analysis of JCV has prompted hypotheses on how the virus emerges from latency and becomes pathogenic. JCV has a double-stranded, circular DNA of 5,130 bp. While the coding region is well conserved, the noncoding regulatory region (RR) of JCV is hypervariable. The kidneys and urine usually contain JCV with a well-conserved, nonpathogenic RR which is called the “archetype” (30). The JCV RR detected in the brains and the cerebrospinal fluid (CSF) of PML patients usually has duplications, tandem repeats, and deletions and has been called “rearranged” compared to the archetype. Although it is not clear which form of JCV RR is propagated at the time of primary infection, it has been hypothesized that JCV with the archetype RR remains confined in the kidneys of most healthy individuals and that rearrangements which confer neurotropism need to occur prior to viral migration to the brain to destroy the myelin-producing glial cells. Whether JCV can reach the brain and establish latency in the central nervous systems (CNS) of otherwise-healthy individuals are matters of debate. While some investigators detected JCV DNA in 28 to 68% of frozen (8, 27) and 18 to 71% of formalin-fixed, paraffin-embedded (FFPE) (4, 7, 20) brain samples of patients without PML, others reported negative results (3, 6, 10, 23). Clearly, characterizing JCV sites of latency is imperative in the prevention of viral reactivation and PML. Recently, a group of PML patients has emerged among those treated with monoclonal antibodies, including natalizumab (13, 17, 26), efalizumab (16, 19a), and rituximab (5), for multiple sclerosis, psoriasis, hematological malignancies, and rheumatologic diseases. Mechanisms of JCV reactivation in these patients has yet to be defined. To better understand JCV organ tropism and characterize the types of JCV RRs in different compartments, we used archival pathology samples to detect JCV DNA and proteins and to analyze JCV RRs in various organ systems in HIV-positive individuals with and without PML and in HIV-negative subjects.     

JC virus encephalopathy is associated with a novel agnoprotein-deletion JCV variant

JC virus encephalopathy (JCVE) is a newly described gray matter disease of the brain caused by productive infection of cortical pyramidal neurons. We characterized the full length sequence of JCV isolated from the brain of a JCVE patient, analyzed its distribution in various compartments by PCR, and determined viral gene expression in the brain by immunohistochemistry(IHC). We identified a novel JCV variant, JCV(CPN1), with a unique 143 bp deletion in the Agno gene encoding a truncated 10 amino acid peptide, and harboring an archetype-like regulatory region. This variant lacked one of three nuclear protein binding regions in the Agno gene. It was predominant in the brain, where it coexisted with an Agno-intact wild-type strain. Double immunostaining with anti-Agno and anti- VP1 antibodies demonstrated that the truncated JCV(CPN1) Agno peptide was present in the majority of cortical cells productively infected with JCV. We then screened 68 DNA samples from 8 brain, 30 CSF and 30 PBMC samples of PML patients, HIV+ and HIV- control subjects. Another JCV(CPN) strain with a different pattern of Agno-deletion was found in the CSF of an HIV+/PML patient, where it also coexisted with wild-type, Agno-intact JCV. These findings suggest that the novel tropism for cortical pyramidal neurons of JCV(CPN1), may be associated with the Agno deletion. Productive and lytic infection of these cells, resulting in fulminant JCV encephalopathy and death may have been facilitated by the co-infection with a wild-type strain of JCV.     

Agnogene Deletion in a Novel Pathogenic JC Virus Isolate Impairs VP1 Expression and Virion Production

Infection of glial cells by the human polyomavirus JC (JCV) causes progressive multifocal leukoencephalopathy (PML). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. The virus JCV_CPN associated with JCVE contains a unique 143 base pair deletion in the agnogene. Contrary to most JCV brain isolates, JCV_CPN has an archetype-like regulatory region (RR) usually found in kidney strains. This provided us with the unique opportunity to determine for the first time how each of these regions contributed to the phenotype of JCV_CPN. We characterized the replication of JCV_CPN compared to the prototype virus JCV_Mad-1 in kidney, glial and neuronal cell lines. We found that JCV_CPN is capable of replicating viral DNA in all cell lines tested, but is unable to establish persistent infection seen with JCV_Mad-1. JCV_CPN does not have an increased ability to replicate in the neuronal cell line tested. To determine whether this phenotype results from the archetype-like RR or the agnogene deletion, we generated chimeric viruses between JCV_CPN of JCV_Mad-1. We found that the deletion in the agnogene is the predominant cause of the inability of the virus to maintain a persistent infection, with the introduction of a full length agnogene, either with or without agnoprotein expression, rescues the replication of JCV_CPN. Studying this naturally occurring pathogenic variant of JCV provides a valuable tool for understanding the functions of the agnogene and RR form in JCV replication.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

Is progressive multifocal leukoencephalopathy a chronic disease because of defective interfering particles or temperature-sensitive mutants of JC virus?

JC virus was examined for temperature sensitivity and for evidence of defective interfering particles as a means of explaining the slow chronic nature of progressive multifocal leukoencephalopathy (PML). JC virus direct from the brain tissue of seven persons with PML was not temperature sensitive as indicated by in vitro assay at 37 and 39 degrees C. In fact, more cells contained viral antigen at 39 than at 37 degrees C. The amount of infectious virus also was increased at 39 degrees C. Virions isolated directly from diseased brain tissue had a higher buoyant density than did virus from the same PML patient passaged in culture and containing genomic deletions. In contrast to DNA from culture-passed virus, DNA extracted from virions direct from brain tissue was homogeneous in length. In 13 separate cases examined, the viral DNA direct from the brain was homogeneous although variations in length were noted among DNAs from different cases. Restriction enzyme cleavage patterns identified all as JC virus DNA. It was concluded that neither temperature sensitivity nor DI particles can be used to explain the slow, progressive nature of PML.     

Potential transmission of human polyomaviruses through the gastrointestinal tract after exposure to virions or viral DNA

The mechanism of human-to-human transmission of the polyomaviruses JC virus (JCV) and BK virus (BKV) has not been firmly established with regard to possible human exposure. JCV and BKV have been found in sewage samples from different geographical areas in Europe, Africa, and the United States, with average concentrations of 10(2) to 10(3) JCV particles/ml and 10(1) to 10(2) BKV particles/ml. Selected polyomavirus-positive sewage samples were further characterized. The JCV and BKV present in these samples were identified by sequencing of the intergenic region (the region found between the T antigen and VP coding regions) of JCV and the VP1 region of BKV. The regulatory region of the JCV and BKV strains found in sewage samples presented archetypal or archetype-like genetic structures, as described for urine samples. The stability (the time required for a 90% reduction in the virus concentration) of the viral particles in sewage at 20 degrees C was estimated to be 26.7 days for JCV and 53.6 days for BKV. The presence of JCV in 50% of the shellfish samples analyzed confirmed the stability of these viral particles in the environment. BKV and JCV particles were also found to be stable at pH 5; however, treatment at a pH lower than 3 resulted in the detection of free viral DNA. Since most humans are infected with JCV and BKV, these data indicate that the ingestion of contaminated water or food could represent a possible portal of entrance of these viruses or polyomavirus DNA into the human population.     

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.     

JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency.

The human polyomavirus JC virus (JCV) infects myelin-producing cells in the central nervous system, resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV-induced PML occurs most frequently in immunosuppressed individuals, with the highest incidence in human immunodeficiency type 1-infected patients, ranging between 4 and 6% of all AIDS cases. Although JCV targets a highly specialized cell in the central nervous system, infection is widespread, with more than 80% of the human population worldwide demonstrating serum antibodies. A number of clinical and laboratory studies have now linked the pathogenesis of PML with JCV infection in lymphoid cells. For example, JCV-infected lymphocytes have been suggested as possible carriers of virus to the brain following reactivation of a latent infection in lymphoid tissues. To further define the cellular tropism associated with JCV, we have attempted to infect immune system cells, including CD34+ hematopoietic progenitor cells derived from human fetal liver, primary human B lymphocytes, and human tonsillar stromal cells. Our results demonstrate that these cell types as well as a CD34+ human cell line, KG-1a, are susceptible to JCV infection. JCV cannot, however, infect KG-1, a CD34+ cell line which differentiates into a macrophage-like cell when treated with phorbol esters. In addition, peripheral blood B lymphocytes isolated by flow cytometry from a PML patient demonstrate JCV infection. These results provide direct evidence that JCV is not strictly neurotropic but can infect CD34+ hematopoietic progenitor cells and those cells which have differentiated into a lymphocytic, but not monocytic, lineage.     

Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate

Polyomavirus JC (JCV) infects ∼60% of the general population, followed by asymptomatic urinary shedding in ∼20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs). Although JCV NCCR rearrangements are deemed pathognomonic for PML, their role as a viral determinant is unclear. We sequenced JCV NCCRs found in CSF of eight HIV/AIDS patients newly diagnosed with PML and analyzed their effect on early and late gene expression using a bidirectional reporter vector recapitulating the circular polyomavirus early and late gene organization. The rr-NCCR sequences were highly diverse, but all increased viral early reporter gene expression in progenitor-derived astrocytes, glia-derived cells, and human kidney compared to the expression levels with the at-NCCR. The expression of simian virus 40 (SV40) large T antigen or HIV Tat expression in trans was associated with a strong increase of at-NCCR-controlled early gene expression, while rr-NCCRs were less responsive. The insertion of rr-NCCRs into the JCV genome backbone revealed higher viral replication rates for rr-NCCR compared to those of the at-NCCR JCV in human progenitor-derived astrocytes or glia cells, which was abrogated in SV40 large T-expressing COS-7 cells. We conclude that naturally occurring JCV rr-NCCR variants from PML patients confer increased early gene expression and higher replication rates compared to those of at-NCCR JCV and thereby increase cytopathology.Polyomavirus JC (JCV) infects approximately 60% of the general population, followed by asymptomatic urinary shedding in 20% of healthy individuals (20). Although JCV-associated nephropathy may occur in kidney transplant (14, 33) and HIV/AIDS patients (6, 27), the most prominent JCV disease is progressive multifocal leukoencephalopathy (PML) (44, 60). The pathology of PML was first described in 1958 as a rare complication of patients with chronic lymphocytic leukemia or Hodgkin''s lymphoma (3). Today, PML is recognized as a rare, virus-mediated demyelinating disease of the white brain matter in highly immunocompromised patients, including HIV/AIDS, transplantation, and chemotherapy patients and those exposed to immunomodulatory or depleting biologicals for the treatment of autoimmune diseases (29, 40). During the human immunodeficiency virus type 1 (HIV-1) pandemic, the incidence of PML rose significantly to rates of 1 to 8% prior to the use of highly active antiretroviral therapy (2, 5, 34). The definitive diagnosis requires brain tissue, but the detection of JCV by PCR in cerebrospinal fluid (CSF) is generally accepted for a laboratory-confirmed diagnosis in immunocompromised patients with (multi-)focal neurological deficits and corresponding radiological findings (8, 26). Due to the lack of effective antiviral therapy (13), the treatment of PML is based on improving overall immune functions. While this is difficult to achieve in cancer, chemotherapy, and transplantation, prompt antiretroviral therapy in HIV/AIDS patients has significantly improved PML survival, with increasing JCV-specific immune responses and declining intracerebral JCV replication (7, 15, 23, 35, 37). In patients diagnosed with PML after treatment with natalizumab for multiple sclerosis or inflammatory bowel disease, the removal of the monoclonal antibody by plasmapheresis has been tried to restore lymphocyte homing to, and the immune surveillance of, JCV replication sites in the central nervous system (38, 40, 52). However, the success of immune reconstitution in HIV/AIDS- and natalizumab-associated PML cases is limited by the fact that PML is typically diagnosed clinically by neurological deficits resulting from significant brain damage, where mounting antiviral immunity often may be too slow to modify the outcome. On the other hand, rapid recovery may cause immune reconstitution inflammatory syndrome with paradoxical clinical worsening and fatal outcomes (9, 16, 25, 38, 46). Although the etiologic role of JCV in PML is well documented, the pathogenesis and, in particular, the role of viral determinants is less clear. Virtually all JCV strains isolated from the brain or CSF of PML patients are characterized by highly variable genomic rearrangements of the noncoding control region (NCCR), which governs viral early and late genes in opposite directions of the circular polyomavirus DNA genome (1, 4, 31, 39, 41, 43, 49, 54, 59). In contrast, JCV detected in the urine of immunocompetent individuals show a consistent linear architecture called the archetype NCCR (at-NCCR). Thus, detecting rearranged NCCRs (rr-NCCRs) JCV in the central nervous system has been viewed as being derived from the archetype and closely linked to PML (4), but the functional consequences of rearrangements are unclear. To address the consequences of the rr-NCCR for JCV gene expression and replication, we characterized the sequences of JCV rr-NCCR from patients with PML and analyzed their effect on viral gene expression and replication with JCV at-NCCR in a bidirectional reporter assay and in recombinant JCV.     

Genetic analysis of simian virus 40 from brains and kidneys of macaque monkeys.

Simian virus 40 (SV40) was isolated from the brains of three rhesus monkeys and the kidneys of two other rhesus monkeys with simian immunodeficiency virus-induced immunodeficiency. A striking feature of these five cases was the tissue specificity of the SV40 replication. SV40 was also isolated from the kidney of a Taiwanese rock macaque with immunodeficiency probably caused by type D retrovirus infection. Multiple full-length clones were derived from all six fresh SV40 isolates, and two separate regions of their genomes were sequenced: the origin (ori)-enhancer region and the coding region for the carboxy terminus of T antigen (T-ag). None of the 23 clones analyzed had two 72-bp enhancer elements as are present in the commonly used laboratory strain 776 of SV40; 22 of these 23 clones were identical in their ori-enhancer sequences, and these had only a single 72-bp enhancer element. We found no evidence for differences in ori-enhancer sequences associated with tissue-specific SV40 replication. The T-ag coding sequence that was analyzed was identical in all clones from kidney. However, significant variation was observed in the carboxy-terminal region of T-ag in SV40 isolated from brain tissues. This sequence variation was located in a region previously reported to be responsible for SV40 host range in cultured cell lines. Thus, SV40 appears to be an opportunistic pathogen in the setting of simian immunodeficiency virus-induced immunodeficiency, similarly to JC virus in human immunodeficiency virus-infected humans, the enhancer sequence organization generally attributed to SV40 is not representative of natural SV40 isolates, and sequence variation near the carboxy terminus of T-ag may play a role in tissue-specific replication of SV40.