Survey of urine from transplant recipients for polyomaviruses JC and BK using the polymerase chain reaction

Molecular and Cellular Probes (1991) 5, 125-128

Survey of urine from transplant recipients for polyomaviruses JC and BK using the polymerase chain reaction W . F. Marshall,' A. Telenti; J. Proper,' A. J . Aksamit' and T. F . Smith' 'Mayo Clinic and Mayo Foundation, Rochester, MN: 55905, USA and 2 lnstitut fur Med Mikrobiologie, Universitat Bern, Bern, Switzerland (Received 25 July 1990, Accepted 9 October 1990) Using polymerase chain reaction (PCR), we examined 108 urine specimens from 39 post transplant patients for polyomaviruses JC (JCV) and BK (BKV) . Urine sediments were collected and subjected to 30 cycles of amplification . PCR products were resolved by agarose gel electrophoresis, transferred to nylon membranes by Southern blot, and hybridized with radiolabelled probes . Polyomavirus DNA was found in urine specimens from 17 out of 39 patients (44%) . Both viruses were detected in specimens from nine patients, JCV alone in five, and BKV alone in three . In comparison, polyomavirus was detected in only five of 22 PCR positive specimens by shell vial cell culture assay . Our results show a high prevalence of polyomavirus shedding after transplantation and suggest a higher rate of JC viruria than previously reported.

KEYWORDS : JC virus, BK virus, polymerase chain reaction, transplantation, polyomavirus . INTRODUCTION The polyomaviruses JC (JCV) and BK (BKV) are small, non-enveloped, double-stranded DNA viruses . JCV is the etiological agent of progressive multifocal leukoencephalopathy while BKV has yet to be definitively linked to any human disease . Asymptomatic infection with both viruses is common' and latent virus persists in the kidneys after primary infection? Reactivation occurs with immunosuppression and is associated with viral excretion in the urine.' Previous studies have found varying rates of polyomavirus reactivation depending on the methods of detection used and the populations studied . The polymerase chain reaction (PCR) offers a sensitive method to identify viruses. In this study, we used PCR to examine urine from a well-characterized organ and bone marrow transplant population to identify JCV and BKV shedding.

transplant recipients and 12 specimens from 12 normal subjects were examined . Specimens were centrifuged at 700 x g for 10 min and the supernatants discarded. Sediments were resuspended in 1 ml of sterile water and subjected to three cycles of rapid freezing and thawing to liberate cell-associated viral DNA, and stored at -70 ° C until tested .

PCR A volume of 1 µl of resuspended urine sediment was added to 25 gl of PCR reaction mixture . The PCR reaction mixture contained a final concentration of 50 mm KCI, 10 mm Tris-HCI (pH 8.4), 15 MM MgCl 2 0 . 01 % (wt/vol) gelatin, 200 tM of each dNTP, 0. 1 µM of each primer, and 2. 5 units of Taq polymerase. The previously described primers were directed to the T antigen region of both BKV and JCV genome .' After denaturing at 94° C for 10 min, specimens were subjected to 30 cycles of amplification (1 . 5 min at 94° C, 1 . 5 min at 55° C, 2 min at 72 ° C) . DNA extracted from a hamster tumour cell line (HTBK) was used as the BKV

MATERIALS AND METHODS Specimens A total of 108 urine specimens from 39 recent 0890-8508/91/020125 + 04 $03 .00/0

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positive control . JCV cultured in SVG cells was used as the JCV positive control .

RESULTS Electrophoresis of PCR products yielded single bands of appropriate weights (173 by for JCV and 176 by for

Detection of amplified products

BKV) that hybridized with one or both virus specific probes (Fig . 1) . Bands were visible on gel in 21 (50%) of

PCR products were resolved by agarose gel electro-

the specimens that were positive by hybridization .

phoresis and visualized with u .v . light as a single band

Of 108 transplant recipient urine specimens tested, 42 (39%) were positive for one or both viruses . This corresponded to 17 (44%) of patients with positive results from at least one urine specimen . JCV was detected more often than BKV (14 vs 12 patients) ; however, nine patients shed both viruses (Table 1) . Polyomavirus amplification products were not found in any of the 12 specimens from normal subjects . Three patients (two liver recipients and one heart recipient) accounted for 20 of the 42 positive specimens, JCV was repeatedly found in the urine of the liver recipients while both viruses were detected in the heart recipient's urine . None of these patients had urinary symptoms or developed progressive multileukoencephalopathy . Twenty-two other focal patients were consistently negative despite testing of numerous urine specimens . The type of transplant did not seem to aff-ct our results (Table 1) . The liver transplant group was the largest and had the highest rate of viral shedding . Other transplant groups were relatively under-represented so that no statistically meaningful conclusions can be made .

by staining with ethidium bromide . Products were then transferred to a nylon membrane by Southern blotting . Membranes were prehybridized for 3 h at 43 ° C in a solution containing 5 x SSPE (1 x SSPE= 180 mm NaCl, 10 mm NaH,PO 4 , 1 mm EDTA [pH 7 . 4]), 0. 1% SDS, and 100 .tg ml - ' Escherichia coli RNA (Calbiochem, San Diego). The 32 P-end-labelled JCV specific internal probe was hybridized to the blots overnight . Blots were washed 30 min at room temperature and 30 min at 60 ° C with 2 x SSPE and 0-1 0/6 SDS . Membranes were then stripped in a solution of 0 . 1 X SSPE and 0-1% SDS at 80 ° C for 45 min prior to rehybridization with the BKV specific probe .

Shell vial assay A total of 49 urine specimens, 22 of which were positive by PCR, were inoculated into human embryonic kidney cell shell vials and stained by indirect immunofluorescence 36 h later as previously described .'

BK JC NEG

1 2 3 4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20

1 2 3 4

5

6

7 8

9 10 11 12 13 14 15 16 17 18 19 20

(a) 8K JC NEG

40

0

( b)

Fig . 1 . Example of resolution of PCR amplification products by gel electrophoresis (c), Southern blot hybridized with JCV probe (a), and with BKV probe (b) . Bands are at different levels because three separate gels were used . BKV probe cross-reactivity with the JCV positive control results from contamination of JCV stock culture with BKV DNA .



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Table 1. Polyomavirus urinary shedding after organ transplantation as detected by PCR . One to six specimens per patient were tested . All specimens were collected within the first 6 months of transplantation Organ Liver Heart Pancreas/kidney Kidney Bone marrow Total

Patients

JCV

BKV

Both BKV and JCV

Total

25 3 3 6 2 39

5 0 0 0 0 5

2 0 0 1 0 3

6 1 0 1 1 9

13 (52%) 1 (33%) 0 2 (33%) 1 (50%) 17 (44%)

Shell vial assay (SVA) was much less sensitive than PCR. Of 22 PCR positive specimens, only five were positive by SVA (two BKV, two JCV, one for both viruses) . The PCR negative specimens were all negative by SVA .

DISCUSSION DNA fragments from both JCV and BKV were ampli-

fied by the same set of primers .' The amplified sequences are sufficiently dissimilar that they can be differentiated by hybridization with virus-specific internal probes . This primer set has previously been used to detect JCV in brain tissue from patients with PML',' and both BKV and JCV in urine from renal and bone marrow transplant patients .' Previous studies have detected polyomavirus excretion in many groups of immunosuppressed patients . Reported rates of polyomaviruria after transplantation range from 13 . 5-% to 50% depending on methodologies used and populations sur.veyed .s,6,10-13 JC viruria is generally less common (420%) than is the detection of BKV (15-48%) .6. ' 0,13 Using PCR, we demonstrated an overall rate of excretion of 44% with 14 out of 39 transplant recipients excreting JCV and 12 out of 39 shedding BKV . Simultaneous shedding of both viruses was found in nine out of 39 patients. As expected, PCR was more sensitive than SVA in our study . PCR can produce positive results, at least theoretically, with as little as one copy of the viral genome present . SVA appears to require at least 1000 viral particles to consistently yield positive results . Both JCV and BKV grow very slowly and their replication is inefficient . Both of these factors affect the sensitivity of SVA and other culture techniques. PCR did not detect polyomavirus DNA in any of the 12 normal subjects tested . This suggests that urinary shedding of JCV and BKV is related to immunosuppression and that PCR can be used to discriminate between latent and reactivated infections .

CONCLUSIONS Urinary shedding of polyomaviruses is very common in organ transplant recipients as assessed by PCR . JCV DNA is more commonly detected than is BKV DNA . This technique can be used in studies monitoring for possible diseases associated with polyomavirus infection . The significance of polyomaviruria after organ transplantation has yet to be determined but warrants further study because of its frequency .

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of JC virus and BK virus in human urine by using immunofluorescence microscopy . Journal of Clinical Microbiology 11, 178-83 . '.1 . Hogan, T . F ., Borden, E. C ., McBain, J . A ., Padgett, B . L . & Walker, D . L . (1980). Human polyomavirus infections with JC virus and BK virus in renal transplant patients . Annals of Internal Medicine 92, 373-8 . 12 . Arthur, R . R ., Beckmann, A . M ., Li, C . C ., Saral, R . &

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