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BKV and JCV belong to the Polyomaviridae family and are opportunistic agents associated with complications in immunocompromised individuals. Although a single screening assay for both viruses would be convenient, the diversity of BKV and JCV serotypes and genotypes is a methodological challenge. In this paper, we developed a PCR method able to detect and segregate BKV and JCV, despite these genetic discrepancies. A duplex semi-nested PCR (duplex snPCR) was designed to target a conserved region (639nt-1516nt) within the VP2 gene. In the first PCR, a primer set common to all BKV and JCV serotypes/ genotypes was used, followed by a semi-nested PCR with internal primers for BKV and JCV segregation. The limit of detection of the duplex snPCR was as low as 10 copies of BKV or JCV plasmids/μL. Specific products were observed when JCV and BKV plasmids were mixed in the same reaction. In field sample testing, the duplex snPCR detected and distinguished both viruses in different biological samples. Results were confirmed by Sanger's sequencing. The geographical complexity of BKV and JCV serotypes and genotypes imposes limits to a simple and universal method that could detect each virus. However, we describe here a sensitive and reliable PCR technique for BKV and JCV diagnosis that overcomes these limitations and could be universally applied.

JC virus (JCV) causes progressive multifocal leukoencephalopathy under conditions of immunosuppression, especially associated with HIV. Despite the high prevalence of HIV-1 infection, few cases of progressive multifocal leukoencephalopathy have been reported and only a small number of JCV strains have been characterized in AIDS patients in Southern Africa. Diagnosis of progressive multifocal leukoencephalopathy through PCR detection of JCV DNA in CSF may result in false negative results if variable regions of the genome are targeted. Characterization of circulating JCV genotypes in Southern Africa may assist with designing appropriate diagnostic assays and characterizing the molecular epidemiology of JCV in African AIDS patients. In this study, a new real-time PCR using fluorescence resonance energy transfer hybridization probes targeting the conserved large T-antigen was developed and compared to a conventional nested PCR targeting the variable VP1 region. Validation against known JCV positive specimens confirmed its specificity while amplification of a serial dilution of JCV control DNA suggests that the new real-time PCR can detect lower concentrations of JCV than the VP1 nested PCR. Investigation of CSF specimens from 44 suspected progressive multifocal leukoencephalopathy patients suggest that the new assay is more sensitive being able to detect JCV in 12 specimens versus 5 specimens with the VP1 nested PCR. Sequence analysis confirmed that the T-antigen region is completely conserved while phylogenetic analysis of the five VP1 products identified two genotype 3, one genotype 1 and two genotype 4 strains, the latter two identified for the first time in South African AIDS patients.

Mixtures of polyomaviruses can be present in the central nervous system, the gastrointestinal tract, the genitourinary tract, blood, and urban sewage. We have developed 12 primer/probe sets (four per virus) for real-time, quantitative PCR assays (TaqMan) that can specifically detect BKV, JCV, and SV40 genomes present in mixtures of these viruses. The specificities of these primer/probe sets were determined by evaluating their level of interaction with the DNA from other polyomaviruses and their ability to estimate the number of copies of homologous viral DNA in blinded samples of defined mixtures of three polyomaviral DNAs. Three early region and three late region primer/probe sets determined, within a two-fold range, the number of copies of their respective DNAs. Four sets of SV40 primer/probes also detected 1.1-2.4 copies of SV40 DNA per COS-1 cell, cells estimated to contain a single copy of SV40 DNA. Three JCV primer/probe sets detected 3.7-4.2 copies per cell of JCV DNA in the JCV-transformed cell line M1-HR, cells estimated to contain between 0.5 and 1 copy of the JCV genome. We suggest that the virus-specific primer/probe sets in this study be considered sufficiently characterized to initiate the quantification of polyomavirus DNA in biological samples.

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.

Human polyomaviruses JCV and BKV can cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy (PML) and haemorrhagic cystitis. Molecular detection by polymerase chain reaction (PCR) is recognised as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, we developed a PCR assay using a single primer pair to amplify a segment of the VP1 gene of JCV and BKV. An enzyme linked amplicon hybridisation assay (ELAHA) using species-specific biotinylated oligonucleotide probes was used to differentiate between JCV and BKV. This assay (VP1-PCR-ELAHA) was evaluated and compared to a PCR assay targeting the human polyomavirus T antigen gene (pol-PCR). DNA sequencing was used to confirm the polyomavirus species identified by the VP1-PCR-ELAHA and to determine the subtype of each JCV isolate. A total of 297 urine specimens were tested and human polyomavirus was detected in 105 specimens (35.4%) by both PCR assays. The differentiation of JCV and BKV by the VP1-PCR-ELAHA showed good agreement with the results of DNA sequencing. Further, DNA sequencing of the JCV positive specimens showed the most prevalent JCV subtype in our cohort was 2a (27%) followed by 1b (20%), 1a (15%), 2c (14%), 4 (14%) and 2b (10%). The results of this study show that the VP1-PCR-ELAHA is a sensitive, specific and rapid method for detecting and differentiating human polyomaviruses JC and BK and is highly suitable for routine use in the clinical laboratory., (Copyright 2004 Wiley-Liss, Inc.)

Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an "in-house" PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.

A combined PCR assay was developed for the detection and typing of human polyomavirus (huPoV) in clinical samples, consisting of (i) a qualitative seminested PCR assay (snPCR) to discriminate between huPoV BK and JC and (ii) a high-throughput, quantitative TaqMan PCR assay (TM-PCR) for the general detection of huPoV. The TM-PCR detects huPoV DNA in a linear range from 10(7) to 10(1) copies per assay. In reproducibility runs, the inter- and intra-assay variabilities were < or =60 and < or =50%, respectively. The snPCR assay uses a set of four primers for the same region of the BK and JC viral genomes. In the first round of amplification, two general primers were used; in the second round, one of these general primers and two additional, BK- or JC-specific primers were used simultaneously to produce amplicons of different sizes specific for BK virus (246 bp) and JC virus (199 bp), respectively. We tested different urine dilutions in order to determine the inhibitory effects of urine on PCR amplification. Furthermore, we compared the use of native urine with DNA purified by different preparation procedures. Our results show, that a 1:10 dilution of the urine led to complete reduction of the amplification inhibition found with 6% of undiluted urine samples. In a clinical study including 600 urine specimens, our assay turned out to be fast, cheap, and reliable in both qualitative and quantitative aspects.