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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AB181539AB181608.
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INTRODUCTION |
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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, IIV, 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.
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METHODS |
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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 16301649) and 327-2HIN (5'-GCAAGCTTGCATGAAGGTTAAGCATGC-3'; nt 19561937). Those used to amplify the TCR were RR-1PST (5'-GCCTGCAGGCCTCAGAAAAAGCCTCCACAC-3'; nt 4972) and RR-2HIN (5'-CGAAGCTTGTCGTGACAGCTGGCGCAGAAC-3'; nt 412391). 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 µl1 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 IIV (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 1020 U each enzyme. The digest was resolved by electrophoresis on a 3 % NuSieve agarose gel (Takara Bio) stained with ethidium bromide.
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RESULTS |
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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 7080, 23 and 1020 %, 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
).
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 15 June 2004;
accepted 9 July 2004.