Vol. 29, No. 10JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1991, p. 2130-21380095-1137/91/102130-09$02.00/0Copyright C) 1991, American Society for Microbiology

Typing of Human Polyomavirus JC Virus on the Basis of RestrictionFragment Length Polymorphisms

YOSHIAKI YOGO,1* TAKAKO IIDA,2 FUMIAKI TAGUCHI,2 TADAICHI KITAMURA,3 AND YOSHIO ASO4Department of Viral Infection, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku,

Tokyo 108,1 Department of Microbiology, School of Hygienic Sciences, Kitasato University, Sagamihara,Kanagawa 228,2 Department of Urology, Branch Hospital, Faculty of Medicine, The University of Tokyo,

Mejirodai, Bunkyo-ku, Tokyo 112,3 and Department of Urology, Faculty of Medicine,The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113,4 Japan

Received 3 April 1991/Accepted 12 July 1991

JC virus DNA clones from the urine of nonimmunosuppressed Japanese individuals regularly contain anarchetypal regulatory sequence which may have generated various regulatory sequences of JC virus isolatesfrom patients with progressive multifocal leukoencephalopathy (PML). In this study, we established 15 newclones from the urine of Dutch, German, and Taiwanese healthy volunteers and patients. Most of these clonescontained regulatory sequences essentially identical to the archetypal regulatory sequence. These clones, alongwith two representative urine-derived clones in Japan and five clones from the brains of PML patients (fourestablished in the United States and one established in Japan), were analyzed with a number of restrictionenzymes. We found nine restriction fragment length polymorphisms by which all clones were classified intoeither of the two types, A and B. Type A contained only clones from the West, while type B contained somefrom the West and all from eastern Asia. Each type contained both urine-derived and PML-derived clones.Furthermore, there was a close relationship between some urine-derived clones and some PML-derived clonesin restriction site mapping analysis. These findings support the adaptation hypothesis which has beenpostulated to explain the genesis of PML-type JC viruses.

JC virus (JCV) is ubiquitous in the human population (17),infecting children asymptomatically and then persisting in alatent form, probably in the kidneys (2). Current data suggestthat the latent virus is reactivated and resumes replication inorgan transplant patients on immunosuppression therapy (1)or in older, nonimmunosuppressed individuals (11). SinceJCV progeny are excreted into the urine (1, 11), JCVcirculating in the human population can be readily recoveredto elucidate biological and biochemical properties.

Recently, we molecularly cloned JCV DNAs from theurine of nonimmunosuppressed or healthy Japanese individ-uals and analyzed their regulatory DNA sequences (26). Wefound that the regulatory sequences of all the cloned JCVDNAs were identical to each other, except for a few nucle-otide substitutions, but were remarkably different from thoseof PML-derived JCVs. We showed that this regulatorysequence, designated an archetypal sequence, may havegenerated various regulatory sequences of PML-derivedisolates (26).

In this study, we attempted to classify various JCV DNAclones by restriction fragment length polymorphisms(RFLPs). For this purpose, we newly established 15 clonesfrom the urine of Dutch, German, and Taiwanese individu-als. All contained regulatory sequences essentially identicalto the archetypal regulatory sequence. These clones, alongwith two representative urine-derived clones from Japan andfive clones isolated from the brains of PML patients (fourestablished in the United States and one established inJapan), were analyzed with a number of restriction enzymes.This analysis allowed the elucidation of the phylogeneticrelationship between urine-derived and PML-derived iso-lates.

* Corresponding author.

MATERIALS AND METHODS

Urine. Urine specimens were collected in Deventer, TheNetherlands, in Illertissen, Germany, and in Taipei, People'sRepublic of China. The donors were either healthy volun-teers (Illertissen) or outpatients (Deventer and Taipei), andnone of them were immunosuppressed. Urine was collectedfrom about 50 donors in each geographical region, frozen at-20°C immediately after collection, and sent in dry ice to theInstitute of Medical Science, University of Tokyo, wherethey were stored at -80°C until use.

Extraction of viral DNA from urine. Urine was centrifugedat 1,300 x g for 10 min at 4°C. The resultant supernatant wascentrifuged at 100,000 x g for 3 h at 4°C. The pellet obtained(virion fraction) was resuspended in 10 mM Tris-HCI-10 mMEDTA (pH 7.6), and the DNA was extracted as describedpreviously (26).

Cloning of viral DNA. DNA from the urine virion fractionwas mixed with BamHI-digested, alkaline phosphatase-treated pUC19 (this plasmid was used as a carrier and as avector), and the mixture was digested with BamHI, whichcleaves JCV DNA at a single site. The DNAs recoveredwere ligated with T4 DNA ligase in the presence of ATP.The ligated DNAs were used for the transformation ofEscherichia coli DH5a by high-voltage electroporation (5).Colonies containing recombinant plasmids were obtained bytwo-round colony hybridization (21) with JCV (MY)[32P]DNA as the probe. Plasmid DNAs were extracted fromsmall liquid cultures and analyzed with restriction enzymesto confirm that the recombinant plasmids contained JCVDNA.The origins of the recombinant JCV DNA clones that had

been previously established and used in this study are shownin Table 1. Five of these clones were kindly supplied by theJapanese Cancer Resources Bank [pJC(1-4)], R. J. Frisque(pHerl-Br, pMad8-Br, and pMadll-Br), and K. Yasui

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TABLE 1. Cloned JCV DNAs previously established and used in this study

Clone AbbreviationaOrigin of clone Method of Reference

Country Donor Tissue cloning'pJC (1-4) M1 United States PML Brain VC 10pHerl-Br Hi United States PML Brain DC 14pMad8-Br M8 United States PML Brain DC 8pMadll-Br Mul United States PML Brain DC 8pJCT-Br Ti Japan PML Brain DC 15pCY CY Japan Healthy Urine DC 26pMY MY Japan Healthy Urine DC 26

a Indicates the cloned viral DNA in an abbreviated form.b VC, viral culturing followed by molecular cloning; DC, direct molecular cloning.

(pJCT-Br). Since, in pHerl-Br, pMad8-Br, pMadll-Br, andpJCT-Br, JCV DNA was linked to vectors at EcoRI sites,JCV DNA was excised from the vectors and recloned at theBamHI site of pUC19.

Restriction analysis of viral DNA. Restriction enzymeswere obtained from Toyobo Co., Ltd. (BamHI, BglII, Cfrl3I[AsuI], HaeIII, HinclI, HindlIl, Hinfl, NcoI, PvuII, andSau3AI), Bethesda Research Laboratories, Inc. (SstI[Sacl]), and Takara Shuzo Co., Ltd. (EcoT14I [Styl]). Di-gestion with each enzyme was carried out as recommendedby the suppliers. Digested DNAs were separated on a 0.6 to1.8% horizontal agarose gel or on a 3.5% polyacrylamide gel,depending on the sizes of the fragments to be separated. Assize references, the HindlIl fragments of lambda phageDNA or the Hinfl fragments of plasmid pUC19 were elec-trophoresed in parallel.Mapping of RFLPs. An RFLP caused by the presence of

an extra restriction site in type A (a corresponding site wasabsent in type B) was mapped by use of the restriction sitemap deduced from the complete sequence data for a type AJCV, Madl-TC (7). An RFLP caused by the presence of anextra restriction site in type B (a corresponding site wasabsent in type A) was mapped as follows. For a restrictionsite present only in type B, two possible locations were firstdetermined by estimating the sizes of three fragmentspresent in only one type, one (fragment a) in type A and two(fragments b and c) in type B (the sum of the sizes offragments b and c should be equal to the size of fragment a).Type B DNA was then digested with a restriction enzymecleaving fragment a at a single site (this restriction enzymewas selected with the aid of the Madl-TC restriction map [71)and electrophoresed on an agarose gel to determine which offragments b and c was cleaved with the enzyme. In this way,it was determined which of the two possible sites was thecase.

Sequencing. Two HindIII-NcoI fragments, one containinga region from the origin of DNA replication to the start siteof the late leader protein (agnoprotein) and one containing aregion from the origin ofDNA replication to the start site ofthe T antigens, were inserted into M13mpl8 and M13mpl9between the SmaI and HindlIl sites. Furthermore, to deter-mine the joint sequence between the two NcoI-HindIIIfragments, an NcoI fragment containing both fragments wasinserted into M13mpl8 at the SmaI site. Single-strandedDNAs purified from recombinant phages were sequenced bychain termination (22). Sequencing was carried out withoverlapping clones representing both DNA strands.

Nucleotide sequence accession numbers. The nucleotidesequence accession numbers (EMBL/GenBank/DDBJ) areas follows: D00801, JCV MY; D00802, JCV Cl; D00803,

JCV C2; D00804, JCV N4; D00805, JCV N1; D00806, JCVN5; D00807, JCV Gi; and D00808, JCV G2.

RESULTS

Cloning ofJCV DNA from the urine of Dutch, German, andTaiwanese individuals. Urine specimens were collected inDeventer, The Netherlands, in Illertissen, Germany, and inTaipei, People's Republic of China. About 50 urine speci-mens were collected from different donors in each country,and DNA was extracted as described previously (26). DNAsamples were screened for the presence ofJCV DNA by blothybridization as described previously (24). JCV DNA wasdetected in 13, 8, and 9 specimens collected in The Nether-lands, Germany, and People's Republic of China, respec-tively. DNA samples containing JCV DNA were cut at theunique BamHI site, ligated with BamHI-cleaved, alkalinephosphatase-treated pUC19, and used to transform E. coliDH5a. To obtain recombinant JCV DNA efficiently from thelimited numbers of positive samples, we used the efficienttransformation procedure of high-voltage electroporationdescribed by Dower et al. (5) rather than that of Hanahan (9)used previously (26). We cloned JCV DNA from five donorsin each country. We designated clones from The Nether-lands as Ni to N5, those from Germany as Gl to G5, andthose from People's Republic of China as Ci to C5.

Regulatory sequences of urine-derived isolates from coun-tries other than Japan. We sequenced the whole noncodingregion of each JCV isolate obtained spanning from the startsite of the T antigens to that of the late leader protein(agnoprotein). The region examined consisted of the originofDNA replication and the early and late noncoding regions.The late noncoding region corresponds to the regulatoryregion whose structure differed remarkably among PML-derived isolates examined so far (4, 8, 14, 15, 18) but wasconserved among urine-derived isolates in Japan (26). (Thelatter was named an archetypal regulatory sequence, since itmay have generated various regulatory regions of PML-derived isolates by deletion and duplication [26]).

In Fig. 1, the sequence of a representative isolate (N1) isshown on the top line, and those of the others are shownbelow. Nucleotides identical to Ni are displayed as dashes,and deletions relative to Ni are displayed as gaps. We foundthat 10 of the 15 urine-derived isolates obtained from threegeographical regions other than Japan carried the archetypalregulatory sequence, with a few nucleotide mismatches (Fig.1). These isolates included four from The Netherlands (Ni toN4), five from Germany (Gi to G5), and one from Taiwan(C2). The rest of the urine-derived isolates (N5, Ci, C3, C4,and C5) had regulatory sequences that deviated slightly from

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TYPING OF JC VIRUS 2133

the archetypal regulatory sequence by deletion or duplica-tion. In N5, a 13-bp segment spanning from nucleotides (nt)166 to 178 (unless otherwise mentioned, nucleotide positionswill be expressed as those of an archetypal isolate, e.g., Ni)was duplicated. In Ci, C3, C4, and C5, a 5-bp segmentspanning from nt 218 to 222 was deleted. Recently, Flaegstadet al. (6) amplified and sequenced JCV regulatory regionsfrom four urine samples from Norway and Denmark andfound that all amplified regions were essentially identical tothe archetypal regulatory regions of Japanese JCV isolates(26). Along with this finding, our findings described aboveand previously (25, 26) indicate that JCV isolates carryingarchetypal regulatory sequences (or carrying sequences de-viating slightly from them) are spread worldwide.Typing of JCV isolates by RFLPs. We carried out a series

of restriction analyses to compare different JCV isolates: 15urine-derived isolates obtained in this study, 2 repre-

sentative urine-derived isolates from Japan, and 5 PML-derived isolates (4 from the United States and 1 from Japan).The origins of the isolates established previously are shownin Table 1. For some previously obtained clones in whichJCV DNAs were linked to vectors at EcoRI sites, full-lengthJCV DNAs were recloned at the BamHI site of pUC19. JCVDNAs were recovered from recombinant clones by BamHIdigestion and analyzed with restriction enzymes.

We first used two enzymes, BglII and HincII, which havebeen used to distinguish different PML-derived isolates (8,16, 26) (HincII fragment patterns are shown in Fig. 2A). Onthe basis of fragment patterns produced by each of theseenzymes, all isolates examined were classified into either oftwo types, one including only isolates from the West (Ni,N2, N5, G1 to G5, Mi, Hi, and Mll) and one including allisolates from eastern Asia (Ci to C5, CY, MY, and Ti) as

well as some from the West (N2, N3, and M8). The differ-ence in restriction patterns between the two types was

ascribed to the presence or absence of one (HincII) or two(BglII) restriction sites on the basis of the sizes of fragmentsproduced from isolates belonging to each type (data notshown).We assumed that the presence or absence of HincIl and

BglII cleavage sites arose from nucleotide substitutionswhich had occurred and been conserved after divergence ofthe two types from an ancestral JCV. If this assumption iscorrect, type A and type B viruses would have undergonemany other nucleotide substitutions, some producing new

RFLPs not recognized previously. We therefore attemptedto compare JCV isolates by using a number of restrictionenzymes known to cut JCV (Madl-TC) DNA at multiplesites (7). The enzymes used were Cfrl3I (AsuI), EcoT14I(StyI), HaeIII, Hinfl, PvuII, Sau3AI, and SstI (SacI). Wefound a number of RFLPs that could distinguish types A andB (Table 2). Some typical examples are shown in Fig. 2B toD (closed and open arrowheads denote fragments producedby only types A and B, respectively). Typing by use of eachof these RFLPs perfectly coincided with typing by use of theHincII and BglII RFLPs. In an analysis in which 10 restric-tion enzymes, including HincIl and BglII, were used, ninetype-specific RFLPs were identified. Eight of these RFLPswere located on the JCV genome (Fig. 3). All of them were

mapped in the regions encoding viral proteins, both earlyand late. In addition, an RFLP (Cfrl3I RFLP) whose exactlocation was not determined was mapped at the C-terminalregion of the VP1 gene.We used a new nomenclature for the two JCV types found

in the current study, although the names type I and type IIhave been used to classify PML-derived isolates (13-16, 23).

We did so because the latter classification was based on thestructures of the regulatory regions, while ours was based onRFLPs distributed throughout the JCV genome. Thus, thetype containing Ni, N2, N5, Gi to G5, Mi, Hi, and Mllwas named type A, while the type containing N3, N4, M8,Ci to C5, CY, MY, and Ti was named type B. It should benoted that both urine-derived and PML-derived isolates fellinto either of the two types.

Subtyping of JCV isolates by RFLPs. Analysis of JCVisolates with various restriction enzymes revealed that notonly type-specific RFLPs but also subtype-specific RFLPswere present on the JCV genome. The enzymes producingsubtype-specific RFLPs are shown in Table 2. For example,Hinfl produced an RFLP that could distinguish types A andB, as described above. On the other hand, the Hinfl restric-tion fragment patterns of N5 and Hi (type A isolates) weredifferent from those of the other members of type A (Fig.2D). Similarly, five distinctive Hinfl restriction fragmentpatterns were identified in type B (Fig. 2D). Another exam-ple was EcoT14I. The sizes of the shortest detectablefragments produced with this enzyme differed between someurine-derived isolates (Ni to N5, Gi to G5, and MY) and theother urine-derived isolates (Ci to C5 and CY) (Fig. 2B).Upon examining the sequence data in Fig. 1, we found thatthe nucleotide variation at position 217 (Fig. 1) caused theEcoTi4I RFLP described above.We wish to mention two subtype-specific RFLPs. First, in

addition to an RFLP distinguishing C2 from the others,PvuII generated another RFLP distinguishing three isolatesin type A (G2, Mi, and Mul) and one in type B (MY) fromthe others (Fig. 2E). The sequence data described above(Fig. 1) and previously (14, 26) revealed that, for types A andB, different nucleotide substitutions within the PvuII recog-nition sequence (nt 103 to 108) were responsible for thesecond PvuII RFLP. Thus, PvuII is not suitable for typing ofJCV, although it was previously used to type PML-derivedJCV isolates (14). Second, SstI is of interest, since it dividedtype B isolates into two further groups (Fig. 2F). Isolatesfrom eastern Asia (Ci to C5, CY, MY, and Ti) belonged tothe group carrying an extra cleavage site, while isolates fromthe West (N3, N4, and M8) belonged to the group notcarrying it. This SstI site was located in the region encodingthe N-terminal peptide of T antigens (data not shown).We found several cases in which sequence rearrangements

within the regulatory region gave rise to various RFLPs.Four cases will be described below. Two cases wereEcoT14I and SstI. With both, the patterns of shorter frag-ments differed between urine (e.g., MY)- and PML (e.g.,Ti)-derived isolates belonging to the same type (Fig. 2B);fragments unique to PML-derived isolates are indicated withwhite dots). This result is explained by the fact that sequencerearrangements in the regulatory regions involving duplica-tion of segments containing EcoT14I and SstI sites (nt 89 to94 and 80 to 85, respectively; Fig. 1) and deletion ofsegments not containing them occurred in most PML-de-rived isolates (7, 14, 15). Third, the Cfrl3I fragment patternof Mi differed from those of other isolates belonging to typeA. Since this result was incompatible with the Cfrl3I cleav-age map deduced from the sequence data of another Madlclone (Madl-TC) (both Mi and Madl-TC were molecularlycloned from the same JCV isolate, obtained by viral cultur-ing [7, 10]), we sequenced the regulatory region of Mi. Thestructure of the regulatory region of Mi was identical to thatof Madl-TC, except that Mi contained a tandem repeat of a102-bp sequence rather than the 98-bp sequence which isduplicated in Madl-TC (7). The 102-bp sequence was com-

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TYPING OF JC VIRUS 2135

TABLE 2. Various RFLPs that could distinguish types andsubtypes of JCV

Presence (+) or absence (-) of:Restrictionenzyme Type-specific Subtype-specific

RFLP (no.) RFLP

BgIII + (2)HincII + (1)Cfrl3I + (1)EcoT14I + (1) +Sau3AI + (1) +Hinfl + (2) +HaeIII + (1) +PvuII +SstI +

posed of the 98-bp sequence and two dinucleotides flankingthe 98-bp sequence. This extension of the repeated regioncreated a new Cfrl3I site not present in Madl-TC. Finally,the Cfrl3I fragment patterns of MY and Ti were different,although both were similar in most restriction enzyme anal-yses, except for that with SstI (Fig. 2). Upon examining theTi regulatory sequence reported previously (15), we foundthat the insertion of a 17-bp sequence produced a new Cfrl3Isite. We wish to emphasize that these RFLPs, caused bysequence rearrangements in the regulatory region, were

excluded from the subtyping of JCV isolates in this study.Subtypes identified in types A and B with nine restriction

enzymes are shown in Table 3; isolates on the same line wererelated to each other by the current restriction enzyme

analysis. Many subtypes were represented by single isolates,while several subtypes were represented by multiple iso-lates. Interestingly, each of three PML-derived isolates (Mi,M8, and Ti) and one urine-derived isolate fell into the same

subtype.Nucleotide differences in the noncoding regions of various

JCV isolates. To verify the classification of JCV isolates byRFLPs, we compared the sequences of the noncoding re-

gions of various isolates. For most of the previously ob-tained isolates (MY, Hi, M8, Mll, and Ti), the sequences ofthe noncoding regions on the early origin ofDNA replicationwere not described previously and therefore were sequencedin this study. The sequences of PML-derived isolates were

aligned relative to an archetypal regulatory sequence, show-ing duplication as parallel lines (26). Nucleotide numbers ofPML-derived isolates could thus be expressed in the sameway as those of urine-derived isolates with the archetypalregulatory sequence. The results are summarized in Table 4,in which only nucleotides variable among isolates are indi-cated.The nucleotides at three positions (-113, -104, and -97)

differed between types A and B, without exception. All typeA isolates carried A, C, and C and all type B isolates carriedT, T, and G at positions -113, -104, and -97, respectively.This finding is consistent with the typing of JCV isolates byRFLPs described above.

\ LP1 Hae III

~~~~T

K~~~~~~~~~

EcoT14 I

FIG. 3. Locations of type-specific RFLPs on the JCV genome.Various RFLPs by which types A and B are differentiated are

located outside of the circular JCV map. Arrows with closed andopen heads indicate restriction sites present only in types A and B,respectively. These restriction sites were mapped as described inMaterials and Methods. Inside the map are located the regionsencoding early (T and t antigens) and late (late leader protein [LP1],VP1, VP2, and VP3) proteins and the origin ofDNA replication (Ori)(7). In addition, the Cfri3I RFLP was mapped at the C-terminalregion of the VP1 gene, although its exact location was not deter-mined.

On the other hand, the noncoding regions containedseveral other positions (-97, -50, 107, 108, 109, 159, and217) which varied among isolates (Table 4). For example, thenucleotide (A) at position -97 was identical among mostisolates but was substituted for by a G in C2. The nucleotideat position 107 was usually T but was substituted for by an Ain two isolates (MY and T1). All of these changes could havebeen generated by point mutations from prototypic noncod-ing sequences sharing the same nucleotide at all positionsexcept for the three type-specific nucleotide positions. Theprototypes of type A and type B noncoding sequencesappeared to be identical to those of Ni (N2, G3, G4, or G5)and N3 (N4), respectively.

In general, the subtyping of JCV isolates by nucleotidesubstitutions within the noncoding regions (Table 4) ap-peared to have a lower resolution than did the subtyping ofJCV isolates by RFLPs distributed throughout the genome(Table 3). For example, in type A, Ni, N2, G3, G4, and G5were clustered together in Table 4, but according to RFLPs,Ni and G4 represented independent subtypes (Table 3).Likewise, in type B, N3 and N4 were clustered together inTable 4, but both isolates were distinguished by RFLPs(Table 3). On the other hand, one isolate (Gi) clustered with

FIG. 2. Restriction enzyme analysis of various JCV DNA clones. JCV DNAs were recovered from recombinants and digested with therestriction enzymes HinclI (A), EcoT14I (B), Cfrl3I (C), Hinfl (D), PvuII (E), and SstI (F). The fragments generated were electrophoresedon 1.5% (A, B, and D) and 1.8% (C, E, and F) agarose gels, stained with ethidium bromide, and photographed on a UV light transilluminator.The sizes of all Hinfl fragments of pUC19 DNA and some Hindlll fragments of lambda DNA are indicated in base pairs to the right of eachpanel. Closed and open arrowheads to the left of each panel denote the fragments unique to types A and B, respectively. White dots in panelsB, C, and F indicate fragments unique to PML-derived isolates (see the text).

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TABLE 3. Classification of 22 JCV isolates by RFLPsa

Type of Urine-derived PML-derivedJCV isolate(s) isolate

A NiN2, Gi, G3, G5bN5G2 MlG4

HiM11

B N3N4 M8Ci, C3, C4, CSC2CYMY Ti

a Isolates classified into the same subtype are on the same line (see thetext).-, a corresponding isolate was not identified.

b Gl was distinguished from the other three by a nucleotide difference in thenoncoding region (see Table 4).

others in RFLP analysis (Table 3) was separated from thatgroup in nucleotide substitution analysis (Table 4). Thus,classification by both methods was not contradictory butrather was complementary.

It should be noted again that except for the absence of onevariable nucleotide (nt 159) in PML-derived isolates, theother variable nucleotides were identical between someurine- and PML-derived isolates, e.g., G2 and Ml (or Mll),N3 (or N4) and M8, and MY and Ti (Table 4). Thus,classification by both methods pointed to the genetic rela-tionship between some PML- and urine-derived isolates.

DISCUSSION

Typing of JCV by RFLPs was first attempted by Grinnellet al. (8), who cloned JCV DNA directly from brain tissue of10 patients with PML. As criteria for classification, theyused the HincIl RFLP and the deletion of about 75 bp, bothlocated in the coding region, although they were aware thata region around the origin of DNA replication was variable

among PML-derived isolates. (It was later found that theregion containing the 75-bp deletion was not lacking anysequence but had nucleotide substitutions which affected thedegree of DNA bending [16].) They found that the PML-derived isolates could be classified into either of two types(they did not name them). Soon after the report by Grinnellet al. (8), Martin and Foster (13) distinguished two PML-derived isolates (Madl and Mad8) by both the Hincll RFLPand the structures of the regulatory regions and designatedisolates represented by Madl and Mad8 as types I and II,

respectively. Since then, JCV isolates have been classifiedonly on the basis of the structures of the regulatory regions(14-16, 23). Thus, Madl and isolates containing the sameregulatory sequence as Madl have been classified as type I,

and isolates containing regulatory sequences different fromthat of Madl have been classified as type 11 (23).

It is to be expected that different JCV isolates will containmultiple nucleotide substitutions which would have accumu-lated in the course of evolution. Most RFLPs located inregions encoding viral proteins were probably caused bysuch nucleotide substitutions. Therefore, the classificationof JCV isolates by RFLPs should reflect the phylogeneticrelationship between various isolates. On the other hand,differences in a hypervariable region, e.g., the regulatoryregion, do not necessarily represent a phylogenetic distancebetween the compared isolates. In fact, hypervariable re-

gions have not been used to study the molecular epidemiol-ogy of herpes simplex virus (19). Therefore, like Grinnell etal. (8), we used RFLPs distributed throughout the JCVgenome to classify urine- and PML-derived isolates.By using RFLPs, we unequivocally classified all isolates

into type A or B. The archetypal regulatory sequence, or itsslightly deviated version, was detected in all the urine-derived isolates of each type. Strains with the archetypalregulatory sequence seem to be widely distributed in theJCV population. It is likely that a common ancestor of typesA and B also had this sequence, which has been conservedin the course of JCV evolution.There was no discrimination between urine- and PML-

derived isolates in the classification of JCV isolates byRFLPs. Both types of isolates were grouped into either typeA or type B. Furthermore, there were at least three cases in

TABLE 4. Nucleotide substitutions between various JCV isolatesa

Type of Isolate(s) of Nucleotide substitution at position': OtherJCV JCV -113 -104 -97 -91 -50 107 108 109 159 217 changec

A Ni N2, G3, G4, G5 A C A C A T G A C AN5 A C A C A T G A C A dpGi A C A C A T G T C AG2 A C A C A T A A C AMi A C A C A T A A A dl, dpHi A C A C C T G A A dl, dpMul A C A C A T G A C A dl, dp

B N3, N4 T T A G A T G A C ACi, C3, C4, C5 T T A G A T G A C G dlC2 T T G G A T G A C GCY T T A G A T G A C GMY T T A G A A G A A AM8 T T A G A T G A A dl, dpTi T T A G A A G A A dl, dp

a Noncoding regions from the start site of the T antigens to that of the agnogene were compared.b See Fig. 1.-, absence of a corresponding nucleotide.' dp, duplication; dl, deletion.

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TYPING OF JC VIRUS 2137

which PML- and urine-derived isolates fell into the samesubtype. On the basis of these findings, the possibility thatPML- and urine-derived isolates represent different strainswhich have different pathogenicities and which divergedfrom an ancestral JCV in the course of evolution can beexcluded.

If a PML-derived isolate were generated from an arche-typal strain as an adaptation within a host, as suggestedpreviously (3, 4, 7, 12, 25, 26), the PML-derived isolatewould be genetically related to one of the archetypal strainscirculating in the human population to which the PMLpatient belonged. In type A, PML-derived isolate Mi wasclosely related to urine-derived isolate G2. In type B, twoPML-derived isolates, M8 and Ti, were closely related tourine-derived isolates N4 and Ti, respectively. However,two PML-derived isolates in type A (Hi and Mll) could notbe paired with any urine-derived isolates. It is possible thatwe missed urine counterparts of these PML-derived isolatesbecause we analyzed only a small number of urine-derivedisolates from the West. We are currently studying thephylogenetic relationship within pairs of related isolates, onefrom PML and one from urine, by using DNA sequence datafor the late genes.The present classification of JCV isolates by RFLPs

suggests that there is a correlation between the types of JCVand human populations. First, type A contained only isolatesfrom Europe and the United States. In addition, Flaegstad etal. (6) recently amplified and sequenced a JCV DNA frag-ment containing the noncoding region from urine obtained inNorway and Denmark. Upon examining their data, we foundthat the type-specific nucleotides at positions -113, -104,and -91 were those of type A for five of the six isolates thatthey analyzed. Thus, most of these isolates probably repre-sent type A isolates. These data, together with our findingsin the present study, suggest that type A isolates are the JCVisolates circulating in Caucasians.

Second, type B contained all the eastern Asia isolates andsome from Europe and the United States. Moreover, aGerman isolate (GS/K) which was previously obtained byDorries (4) from the kidneys of a PML patient is apparentlya type B isolate. GS/K carried a HincII cleavage site uniqueto type B (Fig. 2A) (4) and contained three type B-specificnucleotides within the noncoding region (12). Thus, type Bisolates are probably circulating in Caucasoid as well asMongoloid races. It is possible that type B isolates in bothraces represent subtypes of type B JCV, since we observedan SstI RFLP which distinguished type B isolates fromeastern Asia and from the West. The phylogenetic relation-ship between type B isolates from eastern Asia and Europeremains to be examined by a more detailed comparison; thatis, nucleotide sequences should be compared throughout thegenomes.

That only two types were identified in the isolates exam-ined here does not necessarily imply that there are only twotypes in the JCV population. This is because we did notexamine JCV isolates from Africa. Recently, Sakaoka et al.(20) reported a study in which herpes simplex virus type 1isolates from three geographical regions (Japan, Sweden,and Kenya) were compared by a number of RFLPs. Theyfound that herpes simplex virus type 1 isolates prevalent inone region (e.g., Kenya) differed from those prevalent ineach of the other regions. Similarly, it is likely that JCVisolates from Negroid races would differ from type A andtype B isolates and would thereby represent a third type.

In conclusion, analysis of JCV isolates by RFLPs ishelpful in identifying a possible archetypal JCV from which

a PML-derived isolate may have evolved. Phylogeneticcomparisons between both viruses by use of sequence datafor the coding regions would provide support for the adap-tation hypothesis which has been put forward to explain thevariations in the regulatory sequences of PML-derived iso-lates (3, 4, 7, 12, 25, 26). Furthermore, the present classifi-cation of JCV isolates by RFLPs has implications for newstudies of the relationship between JCV types and humanpopulations.

ACKNOWLEDGMENTS

We are grateful to H. Shibuta and M. Hayashi for helpfulsuggestions. We thank G. A. E. M. Buijs, A. Wildfeuer, S. K. Soh,and W. C. Hsieh for supplying urine specimens and R. J. Frisque,K. Yasui, and the Japanese Cancer Research Resources Bank forsupplying recombinant DNAs carrying JCV DNA.

This work was supported in part by grant 02670192 from theMinistry of Education, Science, and Culture of Japan.

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