A simple method for detecting human polyomavirus DNA in urine by the polymerase chain reaction.

In order to develop a simple and sensitive method for detecting human polyomavirus DNA in the urine of patients by the polymerase chain reaction (PCR), it was found that the viral DNA could be released from urine by proteinase K and then amplified by PCR directly, without additional treatment such as ultracentrifugation or DNA extraction. Direct PCR amplification of viral DNA from urine was volume limited and 5 microliters of urine appeared to be the optimum amount for direct PCR amplification. When the urine volume was greater than 10 microliters, the results of PCR were inconsistent. However, the urine volume could be increased after dialysis to remove possible inhibitor(s) which may interfere with PCR. Direct PCR amplification of patient urine is convenient and eliminates several steps which can cause loss of DNA from the sample.

Production of the antigen and the antibody of the JC virus major capsid protein VP1.

The DNA of the major capsid protein VP1 of the human polyomavirus JC virus (JCV), Taiwan-3 strain, was generated from the urine of an autoimmune disease patient by polymerase chain reaction (PRC). The VP1 DNA was cloned into a prokaryotic expression vector, pGEX-4T-1, for expression in E. coli. The nucleotide sequences and the deduced amino acid sequences were determined and compared with the JC virus prototype, Mad-1. Thirty nucleotides were different between these two strains. Six of the altered nucleotides affected amino acid coding and ten of them caused changes in endonuclease recognition sites. The recombinant VPI protein was purified and used to raise monospecific antiserum in rabbit. Recombinant JCV VP1 protein and its monospecific antiserum are important clinical reagents and could possibly be developed as a subunit vaccine and as a serological diagnostic antigen in the future. In addition, the region between amino acid residues 40 and 80 of JCV VP1 is predicted to be an antigenic epitope on the basis of its hydropathy plot and comparison with the VP1 sequences of SV40 and BK virus.

Human anti-JC virus serum reacts with native but not denatured JC virus major capsid protein VP1.

The immunoreactivity of human anti-JC virus (JCV) serum against the major capsid protein VP1 of JCV was analyzed by Western blot, dot blot, and hemagglutination inhibition (HAI) assays. JCV-positive human serum reacted with native but not denatured JCV major capsid protein VP1, as demonstrated by dot blot and Western blot. Rabbit antiserum raised against native JCV capsid had immunoreactivities similar to those of human anti-JCV serum. These results indicate that the antigenecity of native and denatured JCV VP1 is different. In addition, both JCV-positive human serum and rabbit antiserum raised against native JCV capsid protein inhibited the hemagglutination activity of JCV capsid particles. In contrast, rabbit antiserum raised against denatured JCV VP1 did not inhibit hemagglutination. These findings reveal that denaturation may alter the antigenic epitopes of JCV VP1. Therefore, keeping the JCV capsid protein native appears to be essential for serological or other immunological analyses of the virus.

Changes over time in survival of children after AIDS diagnosis in New York City.

We determined whether survival of children following AIDS diagnosis is improving over time through 1991. AIDS surveillance data from New York City Department of Health on 914 pediatric AIDS patients, diagnosed between 1979 and 1991 and presumed due to maternal-infant HIV transmission, were analyzed. Survival following AIDS diagnosis, established from hospital records and death certificates, was compared by calendar year of initial diagnosis using Kaplan-Meier lifetable analysis. Cox Proportional Hazards regression models were used to compare survival for patients diagnosed earlier or later in the decade, controlling for age at diagnosis, presenting opportunistic illness, and gender of the child. Patients diagnosed with AIDS from October 1987 to September 1989 survived longer, median survival 17 months after diagnosis, than patients diagnosed before September 1987, median survival 10 months (relative risk [RR] = 0.76; 95% confidence intervals [CI] = 0.62, 0.93). Patients diagnosed from October 1989 to December 1991 also survived a median of 17 months. Secular improvements in survival after AIDS diagnosis remained after controlling for age at diagnosis, presenting diagnosis, and gender, even if deaths within three months of diagnosis were excluded. These data suggest that for recent years, survival following AIDS diagnosis in those contracting the infection through maternal-infant transmission has been prolonged. Possible explanations for these findings include both methodological issues (changes in diagnostic criteria, incomplete ascertainment of deaths) and substantive issues (developments in therapeutic interventions and management of pediatric AIDS).

Circulating intercellular adhesion molecules-1 and autoantibodies including anti-endothelial cell, anti-cardiolipin, and anti-neutrophil cytoplasma antibodies in patients with vasculitis.

Circulating intercellular adhesion molecule-1 (ICAM-1) and 3 types of autoantibodies were measured in 30 patients with angiographical or pathological proved vasculitis. There were 18 patients with systemic vasculitis and 12 patients with cutaneous vasculitis. The measured antibodies included anti-endothelial cell antibodies (AECA), anti-cardiolipin (ACL) antibodies of 3 isotypes and anti-neutrophil cytoplasma antibodies (ANCA). The results showed that patients with systemic vasculitis had elevated levels of ICAM-1, AECA and IgG isotype antibody as compared with none or lower in patients with cutaneous vasculitis. Levels of ICAM-1 and IgG isotype ACL antibodies also decreased significantly after disease activity subsided in patients with systemic vasculitis. Measurement of ICAM-1 and autoantibodies may be useful in evaluating the extent of involvement and in following the disease activity of patients with vasculitis.

Genomic cloning and sequence analysis of Taiwan-3 human polyomavirus JC virus.

Four different-strains of human polyomavirus JC virus (JCV), CY, Taiwan-1, Taiwan-2, and Taiwan-3, have been found in pregnant women and autoimmune disease patients in Taiwan. In this study, we report the cloning and sequencing of the Taiwan-3 JCV, virus isolated from the urine of an immunosuppressed patient with rheumatoid arthritis. The viral genome was amplified by polymerase chain reaction and then cloned into a prokaryotic replicative plasmid, pGEM-7Zf(-). The genomic DNA was sequenced and found to comprise 5,111 base pairs. The enhancer-promoter region of the viral genome lacks a copy of pentanucleotide-A (GGGAA) and pentanucleotide-B (AAAGC) compared to the CY archetypal JCV. There are 108 nucleotides altered in the total genome, excluding the variable part of the enhancer-promoter region, between Mad-1 (the prototype JC virus) and Taiwan-3. The enhancer-promoter region has approximately 25% of the altered nucleotides, resulting in amino acid changes in the open reading frames for I.T. capsid proteins (VP1, VP2, and VP3), and the agno protein. The cloned Taiwan-3, genome will provide an source for physiologic and pathologic investigation of the JCV virus in the future.

Anticentromere antibodies in subjects with no apparent connective tissue disease.

OBJECTIVES: To study the association of anticentromere antibodies (ACA) in various diseases. METHODS: A total of 4800 consecutive serum samples were tested for ACA by indirect immunofluorescence using HEp-2 cells as substrates and by immunoblotting of Molt-4 cell mitotic chromosomal antigens and recombinant CENP-B protein. RESULTS: Anticentromere antibodies were identified in the serum samples of 24 subjects, including eight without apparent connective tissue diseases, six with primary biliary cirrhosis, two with diffuse scleroderma, one with pulmonary hypertension, one with primary Raynaud's phenomenon, one with CREST syndrome (calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly, telangiectasia), and five with other connective tissue diseases. By immunoblotting using Molt-4 cells mitotic chromosomal antigens three centromere antigens were recognised by these serum samples. These were: CENP-A (17 kilodalton recognised by 22 of 24 ACA positive serum samples); CENP-B (80 kilodalton recognised by 22 of 24 ACA positive serum samples); and CENP-C (140 kilodalton recognised by 19 of 24 ACA positive serum samples). There was no specific pattern for serum samples from patients with different groups of diseases on immunoblotting. Recombinant CENP-B proteins were all recognised by these samples. Patients without apparent connective tissue disease often had a lower ACA titre than patients with primary biliary cirrhosis. CONCLUSIONS: These data suggest that a positive result for ACA does not always indicate the presence of a connective tissue disease.

JC virus genotypes in a Taiwan aboriginal tribe (Bunun): implications for its population history.

The origin of Taiwanese aborigines remains obscure; it has been speculated that they may be from either mainland China or southeastern Asia. We used the JCV genotyping method to elucidate the origin of Bunun aborigines who now live in central mountain areas of Taiwan. We found that Bunun aborigines carried two major (B1-a2 and CY) and two minor JCV genotypes (B1-a1 and SC). This was contrasted with the JCV genotype profile in modern Taiwanese: one major (SC) and two minor genotypes (CY and B1-a1). It thus appears that B1-a2 and CY are indigenous to the Bunun tribe. B1-a2 was first identified in this study as a discrete cluster that contained only Bunun and Philippine JCV isolates and that was closely related to B1-a1, one of the three common JCV genotypes in China. CY predominates in North China, while SC predominates in South China and southeastern Asia. The present findings suggest that the Bunun tribe is an admixture of two ethnic groups, one carrying B1-a2 and the other carrying CY. In other words, it is likely that the Bunun tribe was established by two waves of immigrations from mainland Asia, predating those by southern Chinese which began in the 17th century.

Different genotypes of human polyomaviruses found in patients with autoimmune diseases in Taiwan.

We have assayed for the presence of human polyomaviruses in urine of autoimmune disease patients, such as systemic lupus erythematosus (SLE), Sjogren's syndrome (SS), rheumatoid arthritis (RA), or dermatomyositis/polymositis (DM/PM), by PCR. The results indicate that approximately 40% of patients were JCV positive and 15% of the JCV positive patients were also infected by BKV at the same time according to Southern blot and DNA sequencing of the PCR products. Interestingly, the JCV present in autoimmune diseases patients were Taiwan-1, Taiwan-2, and Taiwan-3 strains with pentanucleotide-A (GGGAA) and/or -B (AAAGC) deletions within the regulatory region. In addition, BKV found in the examined samples were Taichung-1 and Taichung-2 strains. Taichung-1 had two nucleotide alterations and Taichung-2 had six nucleotide differences within the regulatory region when compared to WW BKV archetype. Although the examined autoimmune diseases patients included RA, SLE, PM, DM, and SS patients, there appears to be no correlation between disease and virus strains. However, Taiwan-2 strain JCV with two copies of pentanucleotide-A deletion was present in the patient with the longest period of immunosuppressive medication.

Thyroid peroxidase autoantibodies and their effects on enzyme activity in patients with systemic lupus erythematosus.

Thyroid microsomal antibodies (Ms-Ab) are recently proved to be directed to thyroid peroxidase (TPO). The aim of this study was to investigate whether the sera of patients with systemic lupus erythematosus (SLE) contain anti-TPO antibodies (TPO-Ab) and whether these antibodies influence enzyme activity. Sera from patient with Hashimoto's thyroiditis was also studied. Serum samples were obtained from 37 patients with SLE, 20 patients with Hashimoto's thyroiditis and 20 healthy subjects. TPO-Ab were detected by immunoprecipitation using crude microsomal preparations or enzyme-linked immunoabsorbent assay (ELISA) with recombinant TPO. Positive TPO-Ab by ELISA were found in 11 (61%) of 18 patients with lupus whose serum contained Ms-Ab. Low levels of TPO-Ab also were found in three (16%) of 19 lupus sera that did not contain Ms-Ab. All patients with Hashimoto's thyroiditis had high levels of TPO-Ab in serum. When measured by ELISA, TPO-Ab were highly correlated with the results of a TPO immunoprecipitation assay and with the titers of Ms-Ab in patients with lupus (r = 0.83, n = 18, P < 0.01; r = 0.63, n = 18, P < 0.01) and in Hashimoto's thyroiditis (r = 0.89, n = 20, P < 0.01; r = 0.75, n = 20, P < 0.01). When evaluating the direct influence of TPO-Ab on the activity of TPO, we found no significant inhibition of enzymatic activity in both guaiacol and iodide assays by lupus sera in contrast with sera from Hashimoto's thyroiditis.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid disorders in Chinese patients with systemic lupus erythematosus.

To clarify the prevalence of thyroid disorders in Chinese patients with systemic lupus erythematosus (SLE), 45 SLE patients not receiving corticosteroid therapy for at least 6 months were selected over a period of 1 year. They were investigated by utilizing thyroid ultrasonography and by determinations of thyroid antibodies and thyroid function. Of these patients, 24 (53.3%) showed abnormal sonographic findings. Thyroid antibodies (microsomal and/or thyroglobulin) were detected in 21 patients (46.7%), but a low index of thyrotropin-binding inhibitory immunoglobulin (TBII) was found in only one euthyroid patients with a normal echogram. Ten patients (22.2%) had abnormal thyroid function. The mean disease duration was longer in patients with thyroid anomalies (p < 0.05). Hashimoto's thyroiditis was found in four patients (8.8%), two of whom had hypothyroidism. We concluded that thyroid anomalies are frequently found in patients with SLE in the area of this study. The possibility of the coexistence of thyroid disorders in systemic lupus erythematosus should be carefully considered throughout the course of patients' follow-up, especially in those with a long disease duration.

Genotypes of human polyomaviruses in urine samples of pregnant women in Taiwan.

The viral DNA of human polyomaviruses JC virus (JCV) and BK virus (BKV) was detected by the polymerase chain reaction (PCR) in urine samples from 31 pregnant women in Taiwan. A pair of appropriate primers amplified both JCV and BKV DNA of the regulatory region simultaneously in PCR. An oligonucleotide probe homologous to both JCV and BKV regulatory region was used subsequently to detect the viral DNA by Southern blotting after PCR amplification. Approximately 36% of the examined urine samples were human polyomavirus positive. The genotypes of JCV and BKV were determined by DNA sequencing of their regulatory regions. Besides CY archetype, a new strain (Taiwan-1) of JCV with a pentanucleotide (GGGAA) deletion and a new strain (Taichung-1) of BKV with two nucleotide alterations within the regulatory region were found in the urine samples. Eight of the examined samples were JCV infected, one was BKV infected, and two were JCV and BKV mix-infected. The JCV positive individuals were infected by CY archetype and Taiwan-1 strain equally. However, Taichung-1 strain was the only BKV strain found in the BKV positive individuals.

Treatment with cytotoxic immunosuppression agents increases urinary excretion of JCV in patients with autoimmune disease.

Human JC virus is ubiquitous in human populations and is reactivated frequently in immunosuppressed patients. Fifty-one patients with autoimmune disease receiving immunomodulating therapy were evaluated to study the possible relationship between immunosuppression and JCV viruria. Patients were divided into cytotoxic and noncytotoxic treatment groups based on their prescription. The incidence of JCV viruria in the cytotoxic treatment group was significantly higher than that in the noncytotoxic group (67% vs. 28%; P < 0.05). Most patients with JCV viruria were receiving corticosteroid (P = 0.03 for any dose and P < 0.001 for higher-dose treatments) and cytotoxic agents (P = 0.02). Age, disease duration, and medication duration appeared not to be the precipitating factors of JCV viruria in this study. The results of clinical evaluation indicate that cytotoxic immunosuppression may play an important role in JC virus reactivation.

Anticardiolipin antibodies in patients with acute myocardial infarction.

Fifty-six consecutive patients, 53 males and 3 females aged from 36 to 83 with a mean age of 61.0, all with acute myocardial infarction (AMI), were screened for anticardiolipin antibodies (ACA) by single sampling at time of admission to the medical intensive care unit; results were compared with those for age-matched, healthy controls. IgM and IgG-ACA were measured by an enzyme-linked immunosorbant assay technique. IgG-ACA were detected in 9 patients (16.1%); IgM-ACA were detected in 9 patients (16.1%). Only one of the patients had raised ACA of both isotypes. There was on difference in either ACA levels or frequency of ACA elevation between patients and controls. Risk factors of coronary artery disease showed no significant difference between patients with and without ACA. Low titer of IgG-ACA was found in one of ten patients with reinfarction and/or previous cerebral infarction. In conclusion, single measurements of anticardiolipin antibodies in general AMI patients are unlikely to yield diagnostically important information. The implication of occasional significant elevation of such antibodies in a general AMI population remains to be speculated.

Self-assembly of the JC virus major capsid protein, VP1, expressed in insect cells.

The major capsid protein of human polyomavirus JC virus, VP1, has been cloned into a baculovirus genome and expressed in insect cells. The VP1 protein was expressed in the cytoplasm and transported into the nucleus. It was then purified by a sucrose cushion and CsCI density gradient centrifugation to near homogeneity. Electron microscopy showed that isolated recombinant VP1 protein self-assembled into a capsid-like structure similar to the natural empty capsid. Both chelator (EDTA) and reducing agent (DTT) are required to disrupt the capsid structure into the pentameric capsomeres, as demonstrated by haemagglutination assay and electron microscopy. These results suggest that JC virus VP1 can be transported into the nucleus and self-assembled to form capsid-like particles without the involvement of the viral minor capsid proteins, VP2 and VP3. In addition, metal ions and disulphide bonds appear to be important in maintaining the integrity of the viral capsid structure.

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.

Incidence of JC viruria is higher than that of BK viruria in Taiwan.

To investigate the prevalence of human polyomaviruses in Taiwan, urine samples from immunocompetent (healthy), transient immunocompromised (pregnant), and prolonged immunosuppressed (autoimmune disease) individuals were collected throughout the island. The viral DNA in the urine was detected by the polymerase chain reaction (PCR) and Southern blot. The viral genotypes were determined by DNA sequencing within the regulatory region. The overall results, including cases reported previously, show that 13.3% (10/75) of immunocompetent individuals, 26.0% (20/77) of pregnant women, and 37.5% (18/48) of autoimmune disease patients are JCV positive. All of the immunocompetent individuals are BKV negative, but 3.9% (3/77) of the pregnant women and 6.2% (3/48) of autoimmune disease patients are BKV positive. Twenty-four percent (48/200) of the examined urine samples were JCV positive, but only 3% (6/200) were BKV positive. JCV positive individuals were mainly infected with CY (42%) and TW-1 (52%) subtypes. These results suggest that the incidence of urinary excretion of human polyomaviruses in immunosuppressed individuals is higher than that of immunocompetent individuals. The prevalence of JCV appears to be higher than that of BKV in Taiwan. In addition, CY and TW-1 are the predominant subtypes of JCV prevalent in the Taiwanese population.

Measurement of synovial tumor necrosis factor-alpha in diagnosing emergency patients with bacterial arthritis.

Because of the high morbidity and mortality in patients with bacterial arthritis, rapidly and correctly diagnosing this critical condition is a challenge to emergency clinicians. Synovial fluid samples were obtained from 75 patients with arthritis disorders who presented to an emergency service, and levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) were measured. Twenty patients with culture-proven bacterial arthritis had higher levels of synovial TNF-alpha than patients with osteoarthritis or with inflammatory arthritis, including gouty arthritis, rheumatoid arthritis, reactive arthritis, and lupus arthritis. There was a good sensitivity for synovial TNF-alpha level in diagnosing patients with bacterial arthritis. Nearly 100% of patients with bacterial arthritis had elevated synovial TNF-alpha levels. However, synovial IL-1 beta and IL-6 levels failed to discriminate bacterial arthritis from other inflammatory arthritis. Measurement of synovial TNF-alpha level may be useful as a diagnostic aid in emergency patients with bacterial arthritis disorders.