Review: JC virus infection of lymphocytes--revisited

JC virus (JCV), the causative agent of the fatal human demyelinating disease progressive multifocal leukoencephalopathy (PML), is an opportunistic papovavirus that infects and destroys oligodendrocytes, the myelin-producing cells of the central nervous system. Since its isolation from the brain of a PML patient, JCV has long been classed as a neurotropic virus. Many studies, however, have demonstrated that JCV can infect various other cell types, including immune system cells. Moreover, several recent studies have focused specifically on lymphocytes as a target of JCV. This review chronicles the association of JCV with lymphocytes, including cell type localization, molecular regulation, and viral sequences, and discusses clinical implications of these findings.     

Detection of reactivation and size variation in the regulatory region of JC virus in brain tissue

AIMS: To develop a sensitive and specific polymerase chain reaction (PCR) based system for detecting genomic variation in JC virus. To apply this system to formalin fixed, paraffin wax embedded brain tissue from patients with and without progressive multifocal leucoencephalopathy (PML). METHODS: A pair of primers (JC1 and JC2) were designed to be complementary to the early and late regions of JC and BK polyomaviruses, respectively. A third primer (JC3), internal to JC1 and JC2, was designed to be specific for JC virus. The specificity of JC3 was investigated by amplifying plasmids with BK or JC virus genomes. Sensitivity was estimated by titration of a plasmid containing JC virus genome. Seven brains from patients with PML (PMLB) and 30 from patients without PML (non-PMLB) were amplified using JC1 and JC2, followed by JC1 and JC3. Amplification of the beta globin gene was used as an amplification control. RESULTS: Amplification with JC1 and JC2 was common for JC and BK viruses, but with JC1 and JC3 it was specific for JC virus. The sensitivity of the system was 25 copies of JC plasmid per 10 microliters of digested tissue. Five out of seven PMLB and 28 of the 30 non-PMLB amplified for beta globin, but only the PMLB gave a signal with polyoma primers. Hypervariation of the length of the regulatory region of the JC isolates in the PML tissues was consistent with the presence of multiple strains of JC. CONCLUSIONS: Variation in the regulatory region of JC virus can be specifically and sensitively detected from routinely processed, paraffin wax embedded brain tissue. Variation in the regulatory region is common in PML derived JC strains, but JC virus was not detectable in non-PMLB tissue.     

DNA replication of chimeric JC virus-simian virus 40 genomes

The ubiquitous virus JCV is the etiologic agent of the human brain disease progressive multifocal leukoencephalopathy. Although infection usually occurs early in life and the virus can remain latent in human tissues, including brain, little information is available regarding its replication. It is known that DNA replication of primate polyomaviruses is dependent upon the synthesis of T antigen and the subsequent interactions of this protein with cellular factors and the viral origin of replication. We constructed chimeric genomes between JCV and SV40, two genetically similar viruses with distinct biologies, in which segments of the T antigen coding region and the replication origin were exchanged. Because the engineering of these genomes created a defect in the structural protein VP1, their DNA replicating activities could be compared without the complication of secondary infection of adjacent cells and amplification of the replication signal. The ability of the JCV-SV40 hybrid T antigens to initiate replication from the two viral origins in primate cells was investigated. A region of the JCV T antigen that includes the DNA binding and zinc finger domains was found to be responsible for the failure of JCV T antigen to interact productively with the SV40 origin. In addition, the ability to replicate in monkey cells was limited to constructs expressing T antigens which contained the carboxy-terminal host range domain of SV40.     

Lack of evidence for the transmission of JC polyomavirus between human populations

Human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy, is ubiquitous in humans, infecting children asymptomatically then persisting in renal tissue. Since JCV DNA can readily be detected from urine, it should be a useful tool with which to study the mode of virus transmission in humans. Based on this notion, we examined the extent to which JCV was transmitted from the American to Japanese populations in Okinawa Island, Japan. (A population of about 50 000 American soldiers and families have been stationed in Okinawa since 1945.) Four JCV types (A to D) were identified in American populations in U.S.A., whereas only type B was prevalent in elder Japanese in Okinawa who had reached adulthood by 1945. Thus, types A, C, and D served as indicators of the transmission of JCV from American to Japanese populations. We then examined whether types A, C, and D were detectable in Japanese in Okinawa aged 30-50 years who may have been in contact with Americans during childhood. However, all the 125 isolates from the younger Japanese population were type B without exception. From this finding, we concluded that JCV is rarely transmitted between human populations.     

Non-random deletions in human foamy virus long terminal repeat during viral infection

We have characterized a new form of human foamy virus (HFV) non-random deleted long terminal repeat (LTR) sizing 1078-bp, deleted in its U3 region, sensitive to the viral transactivator and functional in an infectious proviral clone. Besides two known HFV LTRs of 1260-bp and 1123-bp, this LTR represents the smallest, designed S. Analysis of the LTR sequence shows the presence of short direct repeats surrounding the deletions, suggesting a mechanism generating deletion by misalignment of the growing strand during replication. Our data suggest that the deleted LTRs, preferentially associated with chronic viral infection, could be related with viral persistence.     

Detection of viral proteins after infection of cultured hepatocytes with rabbit hemorrhagic disease virus

The calicivirus rabbit hemorrhagic disease virus (RHDV), which replicates predominantly in the livers of infected rabbits, cannot be propagated in tissue culture. To enable the performance of in vitro studies, rabbit hepatocytes were isolated by liver perfusion and gradient centrifugation. After inoculation with purified RHDV, more than 50% of the cells proved to be infected. Protein analyses led to the detection of 13 RHDV-specific polypeptides within the infected cells. These proteins were assigned to defined regions of the viral genome, resulting in a refined model of RHDV genome organization.     

Endogenous oncornaviral DNA sequences: evidence for two classes of viral DNA sequences in guinea pig cells

The nature of the endogenous viral DNA sequences in guinea pig cells was studied by hybridization. A segment of the viral RNA (r-VRNA) hybridizing to abundant (or reiterated) DNA sequences (R-VDNA) was isolated by recycling to a Cot of 300. The hybridization of the recycled VRNA, as well as the total VRNA, was followed by determining their kinetics and by Wetmur-Davidson analysis. The kinetics of hybridization of total VRNA were complex, did not follow a second-order kinetics, and revealed two slopes by Wetmur-Davidson analysis. The recycled RNA, on the other hand, had a second-order reaction rate expected of the hybridization between a single species of RNA and DNA sequences and yielded a single straight line in a Wetmur-Davidson plot. The Cot1/2 and slope of the recycled r-VRNA was almost identical to that of the abundant VDNA sequences obtained from the hybridization data of the total VRNA. Guinea pig 28S rRNA with or without recycling was used in monitoring hybridization rate. The kinetics of hybridization of 28S RNA followed a second-order reaction and produced a single straight line by Wetmur-Davidson plot, with a second-order reassociation rate constant of 9.6 x 10(-3) liters/mol-s, a Cot1/2 of 104 mol-s/liter, and reiteration frequency of 146. There was no difference in the kinetics of hybridization of 28S RNA before and after recycling. These experiments showed that guinea pig cells contain two classes of VDNA sequences. (i) R-VDNA sequences with a second-order reassociation rate constant of 8.2 x 10(-4) liters/mol-s, a Cot1/2 of 1,219 mol-s/liter, and a reiteration frequency of 12 represent 37.5% of the viral genome. (ii) Unique VDNA sequences with a second-order reassociation rate constant of 1.2 x 10(-4) liters/mol-s, a Cot1/2 of 7,692 mol-s/liter, and a reiteration frequency of 2 represent 62.5% of the viral genome.     

Detection of Epstein-Barr virus DNA in renal tissue from patients with interstitial nephritis

In order to demonstrate whether Epstein-Barr virus (EBV) infection might play a role in the pathogenesis of interstitial nephritis as suggested by many scholars, EBV DNA was detected in twelve specimens of frozen renal biopsy tissue from patients with interstitial nephritis by using nest polymerase chain reaction (nest PCR). For comparison, frozen renal biopsy tissue from ten patients with minimal change disease was used as control. Southern blot hybridization was used to check the specificity of PCR product. The results showed that eight of twelve frozen renal biopsy specimens from interstitial nephritis patients were EBV DNA positive (66.7%), as compared with negative in all the ten frozen renal specimens from minimal change disease patients. The differences was statistically significant (P < 0.01). The study strongly suggests that EBV infection may play an important role in the pathogenesis of interstitial nephritis. The location of EBV in renal tissue and the mechanism inducing interstitial nephritis by EBV are to be clarified.     

New mechanisms of viral persistence in primary human immunodeficiency virus (HIV) infection

Viruses, including the Human Immunodeficiency Virus (HIV), have evolved multiple strategies to overcome host immune defenses, allowing them to persist in the host. Molecular and cellular approaches were simultaneously used to provide sensitive and unbiased delineation of the diversity and dynamics of the immune response, and to study the relative compartimentalization of HIV-specific CTL clones in patients undergoing primary HIV infection. This approach revealed that some HIV-specific CTL clones can be deleted in presence of high levels of antigen, a phenomenon analogous to high-dose tolerance or clonal exhaustion described in murine models of persistent viral infections. Also, HIV-specific CTL clones were found to accumulate preferentially in peripheral blood as compared to lymph nodes, even though the large majority of viral replication during primary HIV infection takes place within lymph nodes. These two mechanisms may decrease the effectiveness of the host cell-mediated immune responses, and favor the establishment of virus persistence during primary HIV infection.     

Detection of human papillomavirus DNA and clinicopathological study of vulvar human papillomavirus infection

We studied 217 vulvar HPV infection patients by clinically, pathologically, and virologically. From 90.6% of the cauliflower-like vulvar lesions and 29.7% of the papillomatous and finger-like lesions, we detected HPV 6/11 DNA by dot blotting hybridization. The patients in 90.0% of the cauliflower-like group and 9.8% of the papillomatous and finger-like group had a high risk factor to intercourse with different sex partners (P < 0.0001). The pathological characteristics, nature history, and response to treatment were different. According to clinical, pathological, and virological findings divided three types: vulvar HPV infection type 1 (or condylomata acuminata), vulvar HPV infection type 2, and vulvar HPV infection type 3.     

Dynamics of viral replication in infants with vertically acquired human immunodeficiency virus type 1 infection

About one-third of vertically HIV-1 infected infants develop AIDS within the first months of life; the remainder show slower disease progression. We investigated the relationship between the pattern of HIV-1 replication early in life and disease outcome in eleven infected infants sequentially studied from birth. Viral load in cells and plasma was measured by highly sensitive competitive PCR-based methods. Although all infants showed an increase in the indices of viral replication within their first weeks of life, three distinct patterns emerged: (a) a rapid increase in plasma viral RNA and cell-associated proviral DNA during the first 4-6 wk, reaching high steady state levels (> 1,000 HIV-1 copies/10(5) PBMC and > 1,000,000 RNA copies/ml plasma) within 2-3 mo of age; (b) a similar initial rapid increase in viral load, followed by a 2.5-50-fold decline in viral levels; (c) a significantly lower (> 10-fold) viral increase during the first 4-6 wk of age. All infants displaying the first pattern developed early AIDS, while infants with slower clinical progression exhibited the second or third pattern. These findings demonstrate that the pattern of viral replication and clearance in the first 2-3 mo of life is strictly correlated with, and predictive of disease evolution in vertically infected infants.     

Progression of viral infection in twins born from a mother infected with human immunodeficiency virus

We studied the evolution of HIV-1 infection and immune response during six years in two twins born from an infected mother. The children had a continuous progression of the infection, proved by CD4+ cell count, serum anti-HIV antibodies, cultivable virus and proviral load. Now, both children are on antiviral treatment. The analysis of serum antibodies showed a different immune response in both children. One of them developed higher levels of antibodies directed against viral proteins and synthetic peptides derived from their aminoacid sequence. In this child, the amount of cultivable virus increased less than in his twin. Nucleotide sequencing of a part of viral genoma, showed that the virus belonged to the B subtype, prevalent in America and Europe. The observed differences in viral sequences suggest a different selective pressure in both twins. This phenomenon could be related to the observed differences in immune response.     

Molecular evidence for a viral etiology of human CNS tumors

The newer methods of molecular virology, including molecular hybridization and the "simultaneous detection test," were used to examine human brain tumors for evidence of RNA tumor viruses. It was found that they contained 70S RNA and RNA-directed DNA polymerase, both encapsulated in a particle possessing a density of 1.17 g/ml. These particles therefore satisfy the three diagnostic features that characterize the animal RNA tumor viruses. Of 26 of the most malignant (glioblastoma and medullo blastoma) brain tumors examined, 24 (92%) contained these virus-like entities. The possible usefulness of these particles as aids in diagnosis and monitoring therapy is briefly discussed.     

Dual NF1-requiring effect of human neurotropic JC virus composite pentanucleotide repeat elements on early and late viral gene expression

OBJECTIVE: The purpose of this study was to prepare a light-cured adhesive applicable for orthodontics by mixing monomers and a polymerized reactive organic composite filler (prepolymerized trimethylolpropane trimethacrylate-filler, TMPT-filler). METHODS: The monomer component was a mixture of 3.0 wt% 2-hydroxy-3-(2-naphthoxy)propyl methacrylate (HNPM) in triethylene glycol dimethacrylate. This was applied to extracted bovine tooth enamel after acid etching with 65 wt% phosphoric acid for 30 s. After 24 h in 37 degrees C water, the tensile bond strength was measured, and the data were analyzed with Duncan's new multiple range test (p < 0.01 or 0.05). RESULTS: The tensile bonding strength to enamel etched with 65 wt% phosphoric acid was 13.1 +/- 0.5 MPa, and the thermal stability of the bond was excellent. SEM examination of the cross-sectioned specimens modified with HCl demineralization showed that when the diffusion time prior to light irradiation was only 1 min, a well-developed resin honeycomb-like structure was created in the enamel surface in the formulation containing HNPM. SIGNIFICANCE: Monomer impregnation beyond the etched enamel surface was important for resin-enamel bonding, increasing bonding strength and thermal stability. HNPM was effective in enhancing monomer diffusion and impregnation of the etched enamel surface.     

Investigation of human urine for genomic sequences of the primate polyomaviruses simian virus 40, BK virus, and JC virus

Recent reports of the detection of simian virus 40 (SV40) nucleotide sequences in ependymomas, choroid plexus tumors, osteosarcomas, and mesotheliomas have raised the possibility that SV40, which naturally infects Asian macaques, is circulating among humans. This possibility was examined by performing polymerase chain reaction assays on urine samples of 166 homosexual men, 88 of them human immunodeficiency virus (HIV)-seropositive, for genomic sequences of SV40 as well as of human polyomaviruses BK virus (BKV) and JC virus (JCV). Tests with masked urine specimens spiked with SV40-transformed cells were included to monitor the SV40 assay. SV40, BKV, and JCV sequences were identified, respectively, in 0, 14%, and 34% of the urine specimens. JCV viruria was far more common (37%) than BKV viruria (5%) in HIV-seronegative persons. HIV infection and more severe immunosuppression were associated with a higher frequency of BKV viruria. In summary, SV40 viruria was not detected among homosexual men who shed human polyomaviruses at a high frequency.     

Varicella-zoster virus infection in children with underlying human immunodeficiency virus infection

This article describes a prospective longitudinal study of varicella-zoster virus (VZV) infections in human immunodeficiency virus (HIV)-infected children, designed to determine their natural history of VZV infection and possible effects of VZV on the progression of HIV infection. Varicella was usually not a serious acute problem, and it did not seem to precede clinical deterioration. The rate of zoster was high: 70% in children with low levels of CD4+ lymphocytes at the time of development of varicella. It is predicted that immunization with live attenuated varicella vaccine is unlikely to be deleterious to HIV-infected children. Moreover, if they are immunized when they still have relatively normal levels of CD4+ lymphocytes, they may have a lower rate of reactivation of VZV than if they were allowed to develop natural varicella when their CD4+ cell counts have fallen to low levels as a result of progressive HIV infection.     

Detection of human papillomaviruses in tissue specimens

During the past decade, molecular methods based on the detection of viral DNA have become a key tool for the detection of human papillomaviruses (HPVs) in tissue. The methods can be divided into two groups: those in which tissue destruction is unavoidable for the detection of HPV DNA, and those in which the detection of viral DNA is performed in a way that allows tissue morphology preservation. Polymerase chain reaction is currently the most sensitive method for HPV detection and an excellent research tool. However, because of frequent contamination problems and lack of standardization, it is not readily applicable to diagnostic laboratories. The recent improvements in in situ hybridization have made it possible for this method to become the most appropriate method for routine detection of HPVs in tissue. At present, however, the use of at least two independent HPV DNA detection methods is indispensable for accurate determination of HPVs.     

Characterization of JC virus DNA amplified from urine of chronic progressive multiple sclerosis patients

The present investigation was designed to study the biological responses in cultures of Madin-Darby canine kidney (MDCK) cells exposed to calcium oxalate monohydrate (COM) crystals, the most common type of urinary crystals. The addition of COM crystals significantly accelerated the multiplication of MDCK cells and significantly activated the cell viability. After exposure of MDCK cells to COM crystals, scanning electron microscopy revealed that some crystals adhered to the plasma membrane and others were endocytosed by the cell. This cellular uptake of crystals was time dependent from 1 to 8 h and showed a specificity according to crystal type. However, the endocytosis of aggregated COM crystals was less marked than that of non-aggregated crystals. Pre-treatment with each of the glycosaminoglycans (sodium pentosan polysulphate, heparin, and chondroitin sulphate C) produced a significant reduction of the cellular uptake of COM crystals, suggesting that these glycosaminoglycans may play some critical roles in preventing the cellular uptake of crystals. Although investigation in further detail is necessary, we speculate that these crystal-cell interactions, that is, the cellular uptake of crystals and cell proliferation, may be among the earliest processes in the formation of kidney stones.