ORIGINAL ARTICLE
Detection of Polyomavirus JC Genotype from Transplant Patients by Capillary Electrophoresis: Comparison to Fragment Length Polymorphism Analysis Ning-Sheng Lai,1 Ming-Chi Lu,1 Ming-Che Lee,1 Teng-Yi Lin,2 Wen-Yao Yin3* Background/Purpose: Among the genotypes of human polyomavirus JC (JCV) reported in Taiwan, CY, TW1, TW2 and TW3 are the most commonly correlated with human diseases. JCV is usually detected using nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis. The aim of this study was to detect the rate of positivity and genotype of the JCV genome in urine by RFLP or capillary electrophoresis (CE) in renal transplant patients and healthy volunteers. Methods: We compared CE analysis to the methods of nucleotide sequencing and RFLP analysis for detection of JCV viruria among 60 renal transplant patients and 50 unrelated healthy controls. Genotyping of the positive PCR products was performed using CE and RFLP analysis simultaneously. Results: The urine JCV-positive rate was significantly higher in renal transplant patients than in healthy volunteers (40% [24/60] vs. 20% [10/50]; p = 0.0238). In addition, multiple genotypes of JCV could be detected by CE, but only one genotype could be detected by RFLP. In our study, 20% (2/10) of urine JCV-positive samples from healthy volunteers had two different genotypes. In renal transplant patients 66% (16/24) of JCV-positive samples had two different genotypes and 12% (3/24) had three different genotypes. Conclusion: In comparison with RFLP, CE can detect multiple genotypes in urine JCV-positive samples and requires only 1/200 of the volume of specimen required for RFLP analysis. The CE method has sensitivity and specificity suitable for use in the clinical laboratory, and identifies more genotypes than RFLP analysis. [J Formos Med Assoc 2008;107(3):239–244] Key Words: capillary zone electrophoresis, human polyomavirus JC, immunosuppression, kidney transplantation, restriction fragment length polymorphism, RFLP
Virions of human polyomavirus JC (JCV) are 40– 55 nm in diameter, non-enveloped icosahedrals with 72 capsomers containing double-stranded circular genome DNA.1 In the clinical setting, JCV has been isolated from a patient with progressive multifocal leukoencephalopathy (PML)2 and it
has also been isolated from the urine of a renal transplant patient by Horgan et al.3 JCV DNA can be isolated from urine, and scientists have used this technique for human polyomavirus research including research on strains, regionalism, and evolutionism.
©2008 Elsevier & Formosan Medical Association .
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Departments of 1Rheumatology and 3General Surgery, Buddhist Dalin Tzu Chi General Hospital, Chia-Yi, and 2 Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. Received: August 15, 2007 Revised: September 28, 2007 Accepted: December 11, 2007
J Formos Med Assoc | 2008 • Vol 107 • No 3
*Correspondence to: Dr Wen-Yao Yin, Department of General Surgery, Buddhist Dalin Tzu Chi General Hospital, 2 Min Sheng Road, Dalin, Chiayi, Taiwan. E-mail:
[email protected]
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Previous studies revealed that JCV infection often occurs in childhood, and most of the first time infections do not cause clinical signs or they are not obvious. When JCV infects the urethra or other organs (including the brain), uremia occurs in some patients.4,5 JCV is an opportunistic infection in humans and causes demyelination diseases such as PML.6,7 As we can isolate JCV from the urine of an infected person, this could be because the virus is in the kidneys or is secreted into the urine from other organs.8,9 When T cell function declines, the virus becomes active, as evidenced by rising virus copy numbers in the serum, and increasing numbers of virions secreted into the urine. Virus activation by immunosuppression or the relationship between JCV activation and immunity can be seen in renal transplant patients,3,10 bone marrow transplant patients, pregnant mothers,9,11 cancer patients,3,12 and AIDS patients.13 PML occurs in the brain when patients remain in a long-term immunosuppressive situation in which the virus becomes active and new virus strains come up in the promoter-enhancer regulatory region and deletion or recombination or replication of JCV take place.14–18 The original JCV strain, CY, has three copies of GGGAA pentanucleotide-A (PA) and two copies of AAAGC pentanucleotideB (PB). The PA is located in the regulatory region of nucleotides 30 to 34 (PA-1), 198 to 202 (PA2), and 218 to 222 (PA-3); the PB is located in nucleotides 188 to 192 (PB-1) and 207 to 211 (PB-2). We have demonstrated that in the three strains of JCV, there is a PA-3 deletion in TW1 and PA-2 and PA-3 deletions in TW2. There are also PB-1 and PA-3 deletions in TW3. To better understand the relationships between human JCV genetics and disease, many epidemiologic studies are focusing on infection ability and virus genotypes in different regions and populations.9,19–21 We used polymerase chain reaction (PCR) to detect the existence of JCV in renal transplant patients and healthy volunteers and then analyzed the genome type of the infectious JCV strains. The traditional way to detect different JCV genomes is by RFLP. However, capillary electrophoresis 240
(CE) separates DNA by electrophoresis migration in a capillary tube. The amount of sample is much less and it is possible to detect several DNA sequences at one time. Therefore, we compared CE and RFLP in detection of genotypes in JCVpositive samples from renal transplant patients and healthy volunteers.
Methods Patients and specimens Urine specimens from different patient groups (50 subjects who did not receive any immunosuppressive agent; mean age, 40 years) and 60 transplant patients (mean age, 39 years; receiving long-term, high-dose immunosuppressive therapy with tacrolimus [FK 506; Fujisawa, Japan] 2–18 mg/day) were included in JCV screening by PCR. Single urine specimens were collected in a sterile container without transport medium and immediately frozen at −20°C until analyzed.
DNA extraction Genomic DNA was extracted from urine samples using the QIAamp DNA blood mini kit (Qiagen, cat no. 51106, France) according to the manufacturer’s instructions.
Amplification of JCV DNA by PCR PCR primers flanking the region were JCVF-5⬘GACATGCCATGGTTTTGCTTTTTGTAGCA-3⬘ (NCBI U61771 nucleotides 4995–5012) with JCVR-5⬘-GAAGAACCATGGCCAGCTGGTGACAAG-3⬘ (NCBIU61771 nucleotides 241–267). Genomic JCV DNA (100–200 ng) was subjected to PCR. The amplification reaction mixture contained 10 X buffer (ABgene, UK) and 0.1 mM of each primer. Two units of DNA polymerase (ABgene, UK) were added to each 50-µL PCR reaction mixture. PCR amplification was performed using the GeneAmp PCR system (Perkin-Elmer Cetus, USA). The reaction mixture was subjected to denaturation at 94°C for 2 minutes, followed immediately by 40 cycles of denaturation at 94°C for1 minute, annealing at 54°C for 1 minute, and extension at J Formos Med Assoc | 2008 • Vol 107 • No 3
Detection of polyomavirus JC genotype
72°C for 2 minutes. Sequencing primers were the same as those used for PCR amplification.
50
p = 0.0238
Each sequencing reaction mixture consisted of 50 ng of PCR product. After purification, the PCR product was sequenced using an ABI Prism 310 Automated Genetic Analyzer (Perkin Elmer Applied Biosystems, USA) and the Big Dye Terminator Ready Reaction Cycle Sequencing Kit (Perkin Elmer Applied Biosystems) according to the manufacturer’s instructions.
JCV positive rate (%)
40
Direct autosequencing
Renal transplants (n = 60) Healthy volunteers (n = 50)
30
20
10
0 Transplant
Amplification of JCV genotype by capillary electrophoresis The detailed procedure for JCV DNA amplification and genotyping by CE was as described in the literature.22 To determine the number of triplet repeats in the flanking region of the gene of the PCR products, the amplified products were denatured for 5 minutes at 95°C, mixed with formamide containing a stop buffer, and electrophoresed on POP4 gels in an automated DNA sequencer (model 310; Applied Biosystems). The number of microsatellite repeats was estimated automatically using the GeneScan software 3.1 (Applied Biosystems) employing a local Southern blot method, with a size standard marker of 500 TAMRA (Applied Biosystems). Five distinct alleles consisting of the base pair size were designated.
PCR–RFLP To subtype VP1, PCR products were subjected to RFLP assay. Ten µL of product was digested with 1 ± 2 U of the appropriate endonuclease (BstN I ) for 2 hours at 60°C, in a total volume of 10 µL of the supplied buffer. The reaction mixtures were then subjected to electrophoresis on 5% agarose gels and the products were detected by ethidium bromide staining.
Data analysis Statistical analyses were performed with the aid of the Epi Info Database and Statistics Software Program, version 6 (CDC/WHO, Atlanta, GA, USA). J Formos Med Assoc | 2008 • Vol 107 • No 3
Control
Figure 1. Urine samples collected from patients for polymerase chain reaction screening for shedding of polyomavirus JC (JCV).
Results JCV may be activated and released into the urine of patients who receive long-term immunosuppressant therapy. We collected renal transplant patients’ urine specimens and compared the differences in JCV DNA and genotypes between patients and healthy subjects (no immunosuppression). We collected 60 specimens from renal transplant patients and another 50 specimens from unrelated healthy controls. The results showed that 10 out of 50 (20%) controls and 24 out of 60 (40%) renal transplant patients were positive for JCV DNA by PCR (Figure 1). Using the CE technique, 34 specimens were positive for JCV in renal transplant patients. Five out of 24 (21%) renal transplant patients had single genotypes (either CY, TW1 or TW3); 16 out of 24 (66%) patients had CY and TW1 genotypes, or genotypes CY and TW3; and three out of 24 (12%) had three viral genotypes of JCV. In addition to CY, TW1, or TW2, we also identified the mutated genotypes of the VP1 region. Among the healthy people who were positive for JCV, eight out of 10 (80%) had genotypes CY or TW1; and only two out of 10 (20%) had multiple genotypes (Figure 2). Using RFLP for detecting the 34 urine JCVpositive specimens from renal transplant patients, 241
N.S. Lai, et al
RFLP
CE
100 Transplant Control
JCV-positive rate (%)
80 60 40 20 0
Single-genotype Single-genotype Two-genotype Multi-genotype Figure 2. Restriction fragment length polymorphism (RFLP) analysis of polyomavirus JC (JCV) samples positive by polymerase chain reaction (PCR) and capillary electrophoresis (CE) analysis of JCV-positive samples by PCR.
Table 1. Characteristics of genotypes in urinary specimens in renal transplant patients and healthy controls by capillary electrophoresis
Mean age (yr) JCV-DNA (−) JCV-DNA (+) Single-genotype Two-genotype Multi-genotype
Normal (n = 50)
Transplant patients (n = 60)
40 40 10 8 2 0
39 36 24 5 16 3
four single genotypes were found among the patients and controls: CY, TW1, TW2 , TW3 or others (Table 1). We compared the DNA replication templates identified by CE and RFLP showing obvious variation in DNA replication numbers. The RFLP technique required DNA templates from 1 ng to 0.1 ng, but CE required only 0.01 ng templates to perform the analysis (Table 2). We found mutations occurring in the regulatory regions of JCV DNA that differed from the original JCV strain.
Discussion In this study, we found renal transplant patients have not only an increased rate of JCV infection, but different genotypes as well. Renal transplant patients often receive long term immunosuppressive agents such as steroids, cyclosporine or 242
p
0.0238 0.0015
Table 2. DNA replication templates by restriction fragment length polymorphism (RFLP) and capillary electrophoresis (CE) show obvious variations in DNA replication numbers (+ = positive; − = negative) Sample
RFLP
CE
1 ng 0.1 ng 0.01 ng
+ +/− −
+ + +
FK-506 to suppress refection. However, at the same time, these immunosuppressants impair the patients’ immune response and render them susceptible to infection. Among these infections, virus infection is frequently overlooked. Actually, cytomegalovirus and BK virus infection could cause severe morbidity and even death in renal transplant patients. The development of more sensitive J Formos Med Assoc | 2008 • Vol 107 • No 3
Detection of polyomavirus JC genotype
and specific methods to detect virus infection is crucial. Immunosuppressant therapy such as corticosteroid therapy suppresses immune system activity and alleviates autoimmune disorders. However, the risk of infection and cancer among these patients is increased.23,24 There are many reports on the relationship between virus activation and immunosuppression.3,4,10,25 The pathogenic mechanisms of JCV strains and their related consequences are still under investigation. Tada and Khalil discovered that one brain-derived DNA binding protein, lytic control element-binding protein 1 (LCP-1) binds to the lytic control element (LCE; AGGGAAGGGA) to regulate the early gene expression of JCV in neural glial cells.26 Kumar et al also found that, in vivo, TGGAAAGCAGCCA in embryonic carcinoma cells is among the nuclear factor 1 (NF-1) motifs,27 and is important for glial cell-specific expression of JCV. Kumar et al showed that the GGG region in the LCE is necessary for glial cell-specific transcription. Others demonstrated that NF-1 in neural glial cells can bind to nucleotides 207 to 231 in the JCV regulatory region,4,28 and this part includes PB-2 (AAAGC) and PA-3 (GGGAA), which may involve gene regulation of JCV in neural glial cells. Based on this information, TW1, TW2, and TW3 will not be activated in neural glial cells because TW1 has one GGGAA deletion, TW2 has two GGGAA deletions, and TW3 has one GGGAA and one AAAGC deletion. These genotypes of JCV may infect and remain in the adrenal cells. According to previous reports, the renal transplant patients with JCV infection who take immunosuppressive drugs are at higher risk of developing PML than healthy individuals. We hypothesize there is a relationship between patient immunity and virus genotype. We wish to have a deeper understanding of reactivation, infection, and specious differentiation with the disease. CE separates DNA by electrophoretic migration and electro-osmotic flow using a high-voltage electrical field in a capillary tube, with a diameter of 20–200 µm. CE requires no gel making, no running of polyacrylamide gel electrophoresis, and J Formos Med Assoc | 2008 • Vol 107 • No 3
can automatically separate PCR products rapidly, especially for identifying different lengths of PCR products. This powerful technique enables differentiation of small changes in DNA sequence up to 1 bp, which makes it suitable for DNA polymorphism analysis. In conclusion, we have established a simple, delicate, and accurate method for detecting DNA viruses requiring very little urine sample volume. We can use CE to detect 0.01 ng JCV DNA with genotyping. The further advantage of CE is analysis of the data by computer to reduce manpower or to avoid the artificial blind spot. Providing a highly accurate and delicate platform is beneficial for future clinical diagnosis of JCV. We can also use CE for monitoring the reduction of viral load among renal transplant patients and tracing the virus in transplant patients. We also believe that CE can be used to detect other viruses.
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