Subtypes of BK virus prevalent in Japan and variation in their transcriptional control region

Tomokazu Takasaka1, Nobuyuki Goya2, Tadahiko Tokumoto2, Kazunari Tanabe2, Hiroshi Toma2, Yoshihide Ogawa3, Sanehiro Hokama3, Akishi Momose4, Tomihisa Funyu4, Tomoaki Fujioka5, So Omori5, Hideki Akiyama6, Qin Chen1, Huai-Ying Zheng1, Nobutaka Ohta1, Tadaichi Kitamura1 and Yoshiaki Yogo1

1 Department of Urology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2 Department of Urology, Tokyo Women's Medical University, Tokyo, Japan
3 Department of Urology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
4 Department of Medicine, Oyokyo Kidney Research Institute, Hirosaki Hospital, Hirosaki, Japan
5 Department of Urology, Iwate Medical University School of Medicine, Morioka, Japan
6 Department of Medicine, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan

Correspondence
Yoshiaki Yogo
yogo-tky{at}umin.ac.jp


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
BK polyomavirus (BKV) is ubiquitous in the human population, infecting children without obvious symptoms, and persisting in the kidney in a latent state. In immunosuppressed patients, BKV is reactivated and excreted in urine. BKV isolates have been classified into four subtypes (I–IV) using either serological or genotyping methods. To elucidate the subtypes of BKV prevalent in Japan, the 287 bp typing region in the viral genome was PCR-amplified from urine samples of 45 renal transplant (RT) and 31 bone-marrow transplant (BMT) recipients. The amplified fragments were subjected to a phylogenetic or RFLP analysis to determine the subtypes of BKV isolates in urine samples. Subtypes I, II, III and IV were detected, respectively, in 70–80, 0, 2–3 and 10–20 % of the BKV-positive patients in both patient groups. This pattern of distribution was virtually identical to patterns previously demonstrated in England, Tanzania and the United States, suggesting that BKV subtypes are distributed similarly in various human populations. Furthermore, transcriptional control regions (TCRs) were PCR-amplified from the urine samples of 25 RT and 20 BMT recipients, and their nucleotide sequences were determined. The basic TCR structure (the so-called archetype configuration) was observed in most isolates belonging to subtypes I, III and IV (subtype II isolates were not available), albeit with several nucleotide substitutions and a few single-nucleotide deletions (or insertions). Only three TCRs carried extensive sequence rearrangements. Thus, it was concluded that the archetypal configuration of the BKV TCR has been conserved during the evolution of BKV.

The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AB181539AB181608.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human polyomavirus BK virus (BKV) was first isolated in the urine of a renal transplant (RT) patient (Gardner et al., 1971). Seroepidemiological surveys conducted in various countries have since demonstrated that this virus is ubiquitous in humans (Knowles, 2001). Infection most frequently occurs during childhood, with adult levels of seroprevalence (65–90 %) reached between the ages of 5 and 10 years (Knowles, 2001). It is thought that BKV persists in renal tissue (Heritage et al., 1981; Chesters et al., 1983). The urinary excretion (viruria) of BKV is rather rare in immunocompetent individuals, but is frequent in immunocompromised individuals, including organ transplant recipients, HIV-infected patients and pregnant women (Knowles, 2001). In immuocompromised patients, the reactivation of BKV sometimes results in renal dysfunction, such as BKV-associated nephropathy (Moens & Rekvig, 2001).

BKV is the only primate polyomavirus that has subtypes distinguishable by immunological reactivity (Knowles, 2001). Knowles et al. (1989) introduced a typing scheme using a set of rabbit antisera against various isolates shown to differ from the prototype BKV (Gardner strain) either antigenically or by restriction enzyme cleavage patterns. However, this typing method requires BKV isolates previously obtained by viral culture. Jin et al. (Jin et al., 1993b; Jin, 1993) developed a direct and convenient method based on the polymerase chain reaction. A partial VP1 gene sequence probably containing nucleotide substitutions responsible for antigenic diversity (Jin et al., 1993a) was PCR-amplified from clinical samples (usually urine specimens), and the resultant amplified fragments were subjected to either DNA sequencing or restriction enzyme analysis. Based on these analyses, Jin et al. (Jin et al., 1993b; Jin, 1993) classified various laboratory BKV strains as well as clinical isolates into four subtypes, I–IV, which corresponded well to groups based on the serological assay.

It is of interest to examine whether a correlation exists between BKV subtypes and human populations, as JC virus (JCV), a related human polyomavirus, shows a close correlation between subtypes and human populations (Agostini et al., 2001; Yogo et al., 2004). The distribution of BKV subtypes has been studied in several patient groups in England (Jin et al., 1993b, 1995), Italy (Di Taranto et al., 1997), Tanzania (Agostini et al., 1995) and the United States (Baksh et al., 2001), and it was found that subtype I was predominant in all of these studies. Nevertheless, no information is available about the distribution pattern of BKV subtypes in Asia. To gain an overall picture of the distribution of BKV subtypes in the world, here, we genotyped BKV isolates detected in the urine of RT and bone-marrow transplant (BMT) recipients in Japan.

The BKV genome has a transcriptional control region (TCR) between the origin of replication and the start site of the late leader protein (agnoprotein) (Seif et al., 1979). The BKV TCR readily undergoes DNA sequence rearrangement during passage of the virus in cell culture (Yoshiike & Takemoto, 1986; Hara et al., 1986; Rubinstein et al., 1987). Therefore, the TCRs of BKV isolates obtained by viral culture could contain alterations introduced in vitro. In contrast, those obtained by molecular cloning or PCR should represent naturally occurring BKV TCRs. Analysis of BKV TCRs isolated using the latter method thus revealed that naturally occurring BKV TCRs have a common structure named the archetype (Moens & Rekvig, 2001).

Nevertheless, information about BKV TCRs has been obtained mainly for subtype I, as this subtype is most prevalent (Knowles, 2001). A complete DNA sequence was reported for a strain (AS) belonging to subtype III, but this strain was isolated by viral culture (Coleman et al., 1980). Indeed, AS carries a 32 bp deletion encompassing an origin-distal region of the TCR and the start site of the agnogene (Tavis et al., 1989). Furthermore, Negrini et al. (1991) detected AS-like TCRs in 2 of 13 isolates from the urine of BMT recipients, but they did not sequence the origin-distal portion of these TCRs. Thus, the relationship between BKV subtype and TCR structure remains to be clarified. In this study, we examined TCR structures for many isolates belonging to various subtypes of BKV.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Urine samples.
Urine samples were collected with informed consent from 186 RT recipients, who underwent a renal transplantation at Tokyo Women's Medical University, University of the Ryukyus Faculty of Medicine, Oyokyo Kidney Research Institute and Iwate Medical University School of Medicine. No patient had active graft rejection episodes or developed nephropathy during the study. About 40 ml urine was collected in a 50 ml plastic tube that contained 0·5 ml 0·5 M EDTA, pH 8·0, and the samples were sent to the Department of Urology, Faculty of Medicine, University of Tokyo, where DNA was extracted as described previously (Kitamura et al., 1990). Urine samples previously obtained from BMT recipients and shown to contain BKV DNA (Akiyama et al., 2001) were also used.

PCR.
The 287 bp typing region and the TCR were amplified from urinary DNA by PCR using ProofStart DNA polymerase (Qiagen). The 287 bp region spanned from 1650 to 1936 nt in the BKV (Dunlop) genome (GenBank accession no. V01108; NCBI no. NC_001538), and contained the whole effective sequence within the 327 bp typing region (Jin et al., 1993b). Primers used to amplify the typing region were 327-1PST (5'-GCCTGCAGCAAGTGCCAAAACTACTAAT-3'; nt 1630–1649) and 327-2HIN (5'-GCAAGCTTGCATGAAGGTTAAGCATGC-3'; nt 1956–1937). Those used to amplify the TCR were RR-1PST (5'-GCCTGCAGGCCTCAGAAAAAGCCTCCACAC-3'; nt 49–72) and RR-2HIN (5'-CGAAGCTTGTCGTGACAGCTGGCGCAGAAC-3'; nt 412–391). Underlined nucleotides were added to create a PstI or HindIII cleavage site. Primers 327-1PST and 327-2HIN were similar to 327-1 and 327-2 reported by Jin et al. (1993b) but carried restriction sites for PstI and HindIII, respectively. The total reaction volume of 50 µ1 contained 2·5 µl crude viral DNA, 1·25 U ProofStart DNA polymerase (Qiagen), 200 µM each dNTP, 0·5 µM primers and PCR buffer, supplied by the manufacturer. After activation at 95 °C for 5 min, the amplification reaction was performed for 50 cycles. The cycle profile was 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min. Both activation and amplification were carried out in a Thermal Sequencer (Asahi Techno Glass Corporation).

Molecular cloning.
The amplified fragments were cleaved with a combination of HindIII and PstI (Takara Bio), and ligated to pBluescript II SK (+) (Stratagene), which was previously digested with HindIII and PstI and dephosphorylated with bacterial alkaline phosphatase (Takara Bio). The ligation products were used to transform competent cells (Escherichia coli HB101; Takara Bio). Recombinant plasmids were prepared using a plasmid mini kit (Qiagen).

Sequencing.
Purified plasmids were used for a cycle sequencing reaction set up using the DYEnamic ET Terminator cycle sequencing kit (Amersham Biosciences). Primers used were the T3 and T7 promoters (Toyobo). The primers were added to a final concentration of 0·25 pmol µl–1 in a final reaction volume of 20 µl. The cycling conditions were 25 cycles of 30 s at 96 °C, 15 s at 50 °C and 60 s at 60 °C. The reaction was terminated at 4 °C. Cycle sequencing products were purified on Centri-Sep columns (Princeton Separations). DNA sequencing was performed using an automated sequencer (ABI Prism 373S DNA sequencer; Applied Biosystems).

Phylogenetic analysis.
A neighbour-joining (NJ) phylogenetic tree (Saitou & Nei, 1987) was constructed using the CLUSTAL W program (Thompson et al., 1994). Divergences were estimated with the two-parameter method (Kimura, 1980). The phylogenetic tree was visualized using DendroMaker for Macintosh ver. 4.1 (Imanishi, 1998). The confidence of branching patterns of the NJ trees was assessed based on 1000 bootstrap replicates (Felsenstein, 1985).

RFLP analysis.
Sequences of 287 bp typing fragments were available for several BKV isolates belonging to I–IV (Seif et al., 1979; Yang & Wu, 1979; Tavis et al., 1989; Sugimoto et al., 1990; Jin, 1993). Using a computer program, we examined these 287 bp sequences, together with those determined in this study (see below), for the presence or absence of restriction sites that would identify the four BKV subtypes. It was found that RFLPs generated by three restriction enzymes, AluI, Cfr13I and RsaI, can be used to identify BKV subtypes (Table 1). The RFLP analysis involving these enzymes was carried out as follows. PCR mixtures were extracted with phenol and filtered through spin-columns containing Sephadex G-25 superfine (Amersham Biosciences). Typically, 2·5 µl aliquot of a purified PCR mixture was digested at 37 °C for 1 h with 10–20 U each enzyme. The digest was resolved by electrophoresis on a 3 % NuSieve agarose gel (Takara Bio) stained with ethidium bromide.


View this table:
[in this window]
[in a new window]
 
Table 1. BKV subtype discrimination according to the RFLP analysis

A 342 bp fragment containing the 287 bp typing region was PCR-amplified from each BKV subtype as described in Methods. The sizes of subfragments detected after digestion of the amplified fragment with indicated restriction enzymes are shown.

 

   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
BKV subtypes prevalent in Japan
Using PCR to amplify the 287 bp typing region, we screened urine samples of 186 RT recipients for the presence of BKV DNA. We detected BKV DNA in 45 urine samples in total. We determined the subtypes of these BKV DNAs by phylogenetic analysis (n=27) and RFLP analysis (n=18). (We will describe the phylogenetic analysis in the following section.) We also determined the subtypes of BKV DNA previously detected in 31 urine samples from BMT recipients (Akiyama et al., 2001) by the phylogenetic method.

BKV subtype frequencies in the RT and BMT recipients analysed in this study are shown in Table 2. The RT recipients were classified into three groups according to their geographical origins, and BKV subtype frequencies are shown for each of these groups (Table 2). Subtypes I, III and IV were detected in 70–80, 2–3 and 10–20 %, respectively, of the BKV-positive patients in both patient groups. Nevertheless, subtype II was not detected in either RT or BMT recipients. Subtype distribution patterns were apparently similar among the two patient groups and geographical origins of patients (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. BKV subtype in RT and BMT recipients in Japan

 
Phylogenetic analysis of BKV isolates based on the 287 bp sequences
We cloned 287 bp typing regions amplified from urine samples, and sequenced representative clones for each urine sample (Table 3). We obtained single sequences from all urine samples examined. From these sequences, together with reference sequences reported previously (Table 4), a phylogenetic tree was constructed using the NJ method (Saitou & Nei, 1987). According to the resultant phylogenetic tree (Fig. 1), all isolates detected in this study diverged into three clusters corresponding to subtype I, III and IV. Nevertheless, isolate SB (reference for subtype II) did not cluster with any of the present isolates. It should be noted that the grouping of isolates belonging to subtype I, III or IV was supported by higher bootstrap probabilities (95–100 %) (Fig. 1). In addition, subtype I apparently subdivided into at least three subclusters, Ia, Ib and Ic. Ia contained one isolate from Sudanese and two isolates from Americans; Ib contained one isolate from a South African, one isolate from English, two isolates from Dutch and five isolates from Japanese; and Ic contained 41 isolates from Japanese. Bootstrap probabilities ranged from 63 to 86 %.


View this table:
[in this window]
[in a new window]
 
Table 3. BKV isolates analysed in this study

Isolates analysed by RFLP are not included.

 

View this table:
[in this window]
[in a new window]
 
Table 4. BKV isolates used as references in the phylogenetic analysis (Fig. 1)

 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. Phylogenetic tree used to classify the BKV isolates into subtypes. The 287 bp typing sequences detected in the present and previous studies were used to construct an NJ phylogenetic tree using CLUSTAL W (Thompson et al., 1994). The phylogenetic tree was visualized using DendroMaker for Macintosh ver. 4.1. The tree was rooted at the midpoint, assuming various BKV strains evolved at roughly the same rate. Subtypes and possible subgroups within subtype I are indicated to the right of the tree. Asterisks identify isolates reported previously and used as references. Origins of isolates are shown in Tables 3 and 4. The numbers at nodes give the bootstrap confidence level (%) obtained for 1000 replicates (only values >=50 % are shown for major nodes).

 
TCR sequences detected from urine-derived isolates
We cloned TCRs amplified from 45 urine samples (Table 3), and sequenced a few representative clones for each urine sample. We obtained single sequences from all urine samples examined. We also sequenced the TCRs of 2 complete BKV DNA clones (WW and MT-1) obtained directly from urine previously (Chauhan et al., 1984; Sugimoto et al., 1989). Alignment of the resultant 47 sequences gave rise to 10 unique sequences, designated Seq-1 to Seq-10 (Fig. 2). In Fig. 2, Seq-1 (the TCR commonly found in subtype IV isolates, see below) is shown at the top, and the other sequences are shown below in relation to Seq-1. BKV isolates carrying each TCR sequence are shown in Table 5.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2. Ten TCR sequences detected from the urine of RT and BMT patients. Sequences between the midpoint of the origin of replication and the start site of the agnogene are shown (for convenience, the nucleotide numbering starts at the midpoint of the origin of replication). Seq-1 (the TCR commonly found in subtype IV isolates, see text) is shown at the top. The other sequences (Seq-2 to Seq-10) are shown below in relation to Seq-1, with the same nucleotides indicated by dashes and deletions by rectangles. Parallel sequences connected with an oblique line in Seq-6 indicate a duplication. The subtype in which each sequence was detected is indicated within parentheses.

 

View this table:
[in this window]
[in a new window]
 
Table 5. TCR sequences of various BKV isolates directly obtained from urine

Isolates cloned previously (Rubinstein et al., 1987; Sugimoto et al., 1989) are underlined.

 
Interestingly, we found a correlation between BKV subtypes and TCR structures. Thus, Seq-1, -4, -5 and -7 TCRs were commonly detected in subtypes IV, III, Ib and Ic, respectively (the TCRs of naturally occurring BKV strains belonging to subtypes II and Ia were not available). Seq-5 was detected in strain WW frequently referred to as the representative archetypal BKV strain. The subtype-specific TCR sequences (Seq-1, -4, -5 and -7) were distinguished from each other by single-nucleotide substitutions at 11 positions and by single-nucleotide deletions at 2 positions (Table 6).


View this table:
[in this window]
[in a new window]
 
Table 6. Differences in TCR sequences among BKV subtypes

 
The other TCR sequences (Seq-2, -3, -6, -8, -9 and -10) were probably derived from the consensus sequences in individual subtypes by nucleotide substitutions or sequence rearrangements (deletions or duplications). Thus, it was inferred that Seq-2 and -3 were generated from Seq-1; Seq-6 from Seq-5; and Seq-8 to -10 from Seq-7. Three sequences (Seq-3, -6 and -10) carried rather extensive rearrangements, involving deletions or duplications. However, these were detected in only three isolates.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Distribution of BKV subtypes in human populations
BKV is the only primate polyomavirus that can be classified into antigenically distinct subgroups. In this study, we attempted to clarify the correlation between BKV subtypes and human populations. The distribution of BKV subtypes in human populations was previously studied in England, Tanzania and the United States (Jin et al., 1993b, 1995; Agostini et al., 1995; Baksh et al., 2001). In this study, we clarified the distribution of BKV subtypes in Japan. Although the populations studied varied in terms of the clinical state of the subjects, the results of the studies conducted so far suggest that subtype I is predominant in all human populations around the world.

However, relative proportions of the minor subtypes differed among populations studied. In renal transplant recipients (Jin et al., 1993b, 1995; Baksh et al., 2001; this study), subtype IV was detected at lower rates and subtypes II and III were not or rarely detected. Jin et al. (1995) reported that dual BKV infection frequently occurred in HIV-infected patients and that subtype III was more often detected than in the other subject groups. The reactivation of BKV in HIV-positive patients remains to be investigated further.

Molecular epidemiological studies of BKV conducted thus far (see above) suggest that there is no significant correlation between BKV subtypes and geographical regions. This is in striking contrast to the established correlation between JCV subtypes and geographical regions (Sugimoto et al., 1997; Agostini et al., 2001; Yogo et al., 2004). However, it should be noted that the subtypes of BKV are antigenically distinguished, while those of JCV are only discernable in terms of nucleotide sequences. If it can be assumed that BKV and JCV have essentially the same evolutionary rate, it may be speculated that it took longer for the BKV subtypes than JCV subtypes to be generated. Thus, it is conceivable that BKV originated before the formation of modern humans (i.e. the four BKV subtypes would have already existed in ancestral populations of modern humans). In contrast, JCV subtypes would have been generated, after the emergence of modern humans, in association with the division of human populations (Yogo et al., 2004).

Nevertheless, we consider that subgroups within each subtype may have a correlation with human populations. In this study, we analysed many isolates from the Japanese population, and found that a majority of Japanese isolates occurred in Ic, suggesting a correlation between Japanese isolates and subgroup Ic. If a larger number of isolates derived from Europeans, Africans and Asians (other than Japanese) are sequenced and a phylogenetic tree is constructed using the resultant sequence data, a correlation between BKV subgroups and human populations will be evident.

Relationships between TCR structures and BKV subtypes
We compared TCR structures, and found that there were several nucleotide substitutions and a few single-nucleotide deletions (or insertions) among BKV subtypes. We sometimes detected BKV TCRs with rather extensive rearrangements, but the BKVs with these rearranged TCRs rarely predominated in a population. Thus, we concluded that the basic TCR structure (the so-called archetype configuration) were conserved among subtypes of BKV.

The TCR detected in strain WW has been considered to represent the archetypal TCR of BKV (Knowles, 2001). Nevertheless, the present findings suggested that like other parts of the genome, the BKV TCR underwent evolutionary changes, involving nucleotide substitutions and single-nucleotide deletions/insertions. Thus, each subtype of BKV has a unique set of nucleotide substitutions and deletions/insertions. We therefore suggest that ‘archetype’ be used as a conceptual word that denotes the prototypical structure that can generate various rearranged TCRs typically observed in BKV strains passaged in cell culture (Yoshiike & Takemoto, 1986; Hara et al., 1986; Rubinstein et al., 1987). In this sense, TCR structures (e.g. Seq-1, -4, -5 and -7) shared by most isolates belonging to the same subtypes are all archetypal.


   ACKNOWLEDGEMENTS
 
We are grateful to all urine donors. This study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan and from the Ministry of Health, Labour and Welfare, Japan.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Agostini, H. T., Brubaker, G. R., Shao, J., Levin, A., Ryschkewitsch, C. F., Blattner, W. A. & Stoner, G. L. (1995). BK virus and a new type of JC virus excreted by HIV-1 positive patients in rural Tanzania. Arch Virol 140, 1919–1934.[Medline]

Agostini, H. T., Jobes, D. V. & Stoner, G. L. (2001). Molecular evolution and epidemiology of JC virus. In Human Polyomaviruses: Molecular and Clinical Perspectives, pp. 491–526. Edited by K. Khalili & G. L. Stoner. New York: Wiley.

Akiyama, H., Kurosu, T., Sakashita, C. & 9 other authors (2001). Adenovirus is a key pathogen in hemorrhagic cystitis associated with bone marrow transplantation. Clin Infect Dis 32, 1325–1330.[CrossRef][Medline]

Baksh, F. K., Finkelstein, S. D., Swalsky, P. A., Stoner, G. L., Ryschkewitsch, C. F. & Randhawa, P. (2001). Molecular genotyping of BK and JC viruses in human polyomavirus-associated interstitial nephritis after renal transplantation. Am J Kidney Dis 38, 354–365.[Medline]

Chauhan, S., Lecatsas, G. & Harley, E. H. (1984). Genome analysis of BK (WW) viral DNA cloned directly from human urine. Intervirology 22, 170–176.[Medline]

Chesters, P. M., Heritage, J. & McCance, D. J. (1983). Persistence of DNA sequences of BK virus and JC virus in normal human tissues and in diseased tissues. J Infect Dis 147, 676–684.[Medline]

Coleman, D. V., Wolfendale, M. R., Daniel, R. A., Dhanjal, N. K., Gardner, S. D., Gibson, P. E. & Field, A. M. (1980). A prospective study of human polyomavirus infection in pregnancy. J Infect Dis 142, 1–8.[Medline]

Di Taranto, C., Pietropaolo, V., Orsi, G. B., Jin, L., Sinibaldi, L. & Degener, A. M. (1997). Detection of BK polyomavirus genotypes in healthy and HIV-positive children. Eur J Epidemiol 13, 653–657.[CrossRef][Medline]

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.

Gardner, S. D., Field, A. M., Coleman, D. V. & Hulme, B. (1971). New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1, 1253–1257.[Medline]

Hara, K., Oya, Y., Kinoshita, H., Taguchi, F. & Yogo, Y. (1986). Sequence reiteration required for the efficient growth of BK virus. J Gen Virol 67, 2555–2559.[Abstract]

Heritage, J., Chesters, P. M. & McCance, D. J. (1981). The persistence of papovavirus BK DNA sequences in normal human renal tissue. J Med Virol 8, 143–150.[Medline]

Imanishi, T. (1998). DendroMaker for Macintosh ver. 4.1. http://www.cib.nig.ac.jp/dda/timanish/dendromaker/home.html.

Jin, L. (1993). Rapid genomic typing of BK virus directly from clinical specimens. Mol Cell Probes 7, 331–334.[CrossRef][Medline]

Jin, L., Gibson, P. E., Knowles, W. A. & Clewley, J. P. (1993a). BK virus antigenic variants: sequence analysis within the capsid VP1 epitope. J Med Virol 39, 50–56.[Medline]

Jin, L., Gibson, P. E., Booth, J. C. & Clewley, J. P. (1993b). Genomic typing of BK virus in clinical specimens by direct sequencing of polymerase chain reaction products. J Med Virol 41, 11–17.[Medline]

Jin, L., Pietropaolo, V., Booth, J. C., Ward, K. H. & Brown, D. W. (1995). Prevalence and distribution of BK virus subtypes in healthy people and immunocompromised patients detected by PCR-restriction enzyme analysis. Clin Diag Virol 3, 285–295.

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[Medline]

Kitamura, T., Aso, Y., Kuniyoshi, N., Hara, K. & Yogo, Y. (1990). High incidence of urinary JC virus excretion in nonimmunosuppressed older patients. J Infect Dis 161, 1128–1133.[Medline]

Knowles, W. A. (2001). The epidemiology of BK virus and the occurrence of antigenic and genomic subtypes. In Human Polyomaviruses: Molecular and Clinical Perspectives, pp. 527–559. Edited by K. Khalili & G. L. Stoner. New York: Wiley.

Knowles, W. A., Gibson, P. E. & Gardner, S. D. (1989). Serological typing scheme for BK-like isolates of human polyomavirus. J Med Virol 28, 118–123.[Medline]

Moens, U. & Rekvig, O. P. (2001). Molecular biology of BK virus and clinical and basic aspects of BK virus renal infection. In Human Polyomaviruses: Molecular and Clinical Perspectives, pp. 359–408. Edited by K. Khalili & G. L. Stoner. New York: Wiley.

Negrini, M., Sabbioni, S., Arthur, R. R., Castagnoli, A. & Barbanti-Brodano, G. (1991). Prevalence of the archetypal regulatory region and sequence polymorphisms in nonpassaged BK virus variants. J Virol 65, 5092–5095.[Medline]

Rubinstein, R., Pare, N. & Harley, E. H. (1987). Structure and function of the transcriptional control region of nonpassaged BK virus. J Virol 61, 1747–1750.[Medline]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Seif, I., Khoury, G. & Dhar, R. (1979). The genome of human papovavirus BKV. Cell 18, 963–977.[Medline]

Sugimoto, C., Hara, K., Taguchi, F. & Yogo, Y. (1989). Growth efficiency of naturally occurring BK virus variants in vivo and in vitro. J Virol 63, 3195–3199.[Medline]

Sugimoto, C., Hara, K., Taguchi, F. & Yogo, Y. (1990). Regulatory DNA sequence conserved in the course of BK virus evolution. J Mol Evol 31, 485–492.[Medline]

Sugimoto, C., Kitamura, T., Guo, J. & 16 other authors (1997). Typing of urinary JC virus DNA offers a novel means of tracing human migrations. Proc Natl Acad Sci U S A 94, 9191–9196.[Abstract/Free Full Text]

Tavis, J. E., Walker, D. L., Gardner, S. D. & Frisque, R. J. (1989). Nucleotide sequence of the human polyomavirus AS virus, an antigenic variant of BK virus. J Virol 63, 901–911.[Medline]

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[Abstract]

Yang, R. C. & Wu, R. (1979). BK virus DNA: complete nucleotide sequence of a human tumor virus. Science 206, 456–462.[Medline]

Yogo, Y., Sugimoto, C., Zheng, H.-Y., Ikegaya, H., Takasaka, T. & Kitamura, T. (2004). JC virus genotyping offers a new paradigm in the study of human populations. Rev Med Virol 14, 179–191.[CrossRef][Medline]

Yoshiike, K. & Takemoto, K. K. (1986). Studies with BK virus and monkey lymphotropic papovavirus. In The Papovaviridae, vol. 1, The Polyomaviruses, pp. 295–326. Edited by N. P. Salzman. New York: Plenum.

Received 15 June 2004; accepted 9 July 2004.