J Mol Evol (2003) 56:18±27 DOI: 10.1007/s00239-002-2376-3
Phylogenetic Relationships Among JC Virus Strains in Japanese/Koreans and Native Americans Speaking Amerind or Na-Dene Huai-Ying Zheng,1,2 Chie Sugimoto,1 Masami Hasegawa,3 Nobuyoshi Kobayashi,1 Akihiro Kanayama,4 Antonieta Rodas,5 Mildred Mejia,5 Jesus Nakamichi,6 Jing Guo,7 Tadaichi Kitamura,2 Yoshiaki Yogo1 1
Laboratory of Viral Infection, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan 2 Department of Urology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan 3 Department of Prediction and Control, The Institute of Statistical Mathematics, 4-6-7 Minami-Azabu, Minato-ku, Tokyo 106-8569, Japan 4 Yokohama City Institute of Health, 1-2-17 Takigashira, Isogo-ku, Yokohama 235-0012, Japan 5 Faculty of Chemistry and Pharmacy, University of San Carlos of Guatemala, Guatemala, Guatemala 6 Calle Jose Cueto y Cerapio Santiago, Jurisdiccion Sanitaria No. 6, Torreon Coahuila, Mexico 7 Departments of Cell Biology and Biochemistry, University of Alberta, 449 Medical Science Building, Edmonton, Canada T6G 2H7 Received: 20 March 2002 / Accepted: 12 July 2002
Abstract. Many genetic studies using human mtDNA or the Y chromosome have been conducted to elucidate the relationships among the three Native American groups speaking Amerind, Na-Dene, and Eskimo-Aleut. Human polyomavirus JC (JCV) may also help to gain insights into this issue. JCV isolates are classi®ed into more than 10 geographically distinct genotypes (designated subtypes here), which were generated by splits in the three superclusters, Types A, B, and C. A particular subtype of JCV (named MY) belonging to Type B is spread in both Japanese/Koreans and Native Americans speaking Amerind or Na-Dene. In this study, we evaluated the phylogenetic relationships among MY isolates worldwide, using the whole-genome approach, with which a highly reliable phylogeny of JCV isolates can be reconstructed. Thirty-six complete sequences belonging to MY (10 from Japanese/Koreans, 24 from Native Americans, and 2 from others), together with 54 belonging to other subtypes around the world, were aligned and subjected to phylogenetic analysis
Correspondence to: Yoshiaki Yogo; email:
[email protected]. ac.jp
using the neighbor-joining and maximum-likelihood methods. In the resultant phylogenetic trees, the MY sequences diverged into two Japanese/Korean and ®ve Native American clades with high bootstrap probabilities. Two of the Native American clades contained isolates mainly from Na-Denes and the others contained isolates mainly from Amerinds. The Na-Dene clades were not clustered together, nor were the Amerind clades. In contrast, the two Japanese/ Korean clades were clustered at a high bootstrap probability. We concluded that there is no distinction between Amerinds and Na-Denes in terms of indigenous JCVs, although they are linguistically distinguished from each other. Key words: JC virus Ð Subtype MY Ð Phylogenetic analysis Ð Native Americans Ð Japanese/ Koreans Ð Peopling of the Americas Introduction JCV is a member of the Polyomaviridae family. Its genome is a single molecule of covalently closed, circular double-stranded DNA about 5100 bp in length (Cole and Conzen 2001). JCV is ubiquitous in
19
the human population, although it causes a fatal demyelinating disease in the central nervous system, known as progressive multifocal leukoencephalopathy (PML), in immunocompromised patients (Padgett et al. 1971). Primary infection with this virus usually occurs asymptomatically during childhood (Padgett and Walker 1973; Walker and Padgett 1983). JCV persists in the renal tissue of most adults, who excrete progeny viruses in the urine (Chesters et al. 1983; Tominaga et al. 1992; Kitamura et al. 1990, 1997). Although JCV transmission is categorized as horizontal transmission (i.e., transmission among individuals after birth) (Daniel et al. 1981), JCV is frequently transmitted from parents to children during long-term cohabitation (Kunitake et al. 1995; Suzuki et al. 2002) and is rarely transmitted between human populations (Kato et al. 1997). By phylogenetic analysis of JCV DNA sequences, more than 10 JCV genotypes (designated subtypes here) that occupy unique domains in Europe, Africa, and Asia have been identi®ed (Sugimoto et al. 1997, 2002a; Guo et al. 1998). Europe is occupied mainly by two subtypes (EU-a and -b), but a part of Europe (e.g., The Netherlands) is also occupied by another subtype (B1-c). Almost all of Africa is occupied by one subtype (Af2), but Central and West Africa is also occupied by another subtype (Af1 and Af3, respectively). Finally, partially overlapping domains in Asia are occupied by seven subtypes (B1-a, -b, and -d, B2, MY, CY, and SC). Overall, the distribution patterns of JCV subtypes are compatible with the migration and expansion of modern humans that occurred after their departure from Africa (Cann et al. 1987; Underhill et al. 2001; Hammer et al. 2001). However, it remains to be investigated whether JCV evolved as a consistent molecular clock. Using the whole-genome approach, with which a highly reliable phylogeny of JCV isolates can be reconstructed (Jobes et al. 1998; Hatwell and Sharp 2000), it was recently shown that subtypes of JCV were generated by splits in the three superclusters, Types A, B, and C (Sugimoto et al. 2002a). Two subtypes of JCV (MY and CY), both generated by splits in Type B (Sugimoto et al. 2002a), are distributed mainly in northeastern Asia (Sugimoto et al. 1997) (MY and CY were also designated Types 2A and 7b, respectively, by Agostini et al. [1997b, 2001]). These subtypes have unique patterns of geographic distribution. Thus, MY is restricted to Japan and South Korea, whereas CY is widespread in northeastern Asia, including Japan, South Korea, and China. Interestingly, MY, and not CY, is prevalent in various Native American populations throughout the Americas, excluding those in the Arctic (Sugimoto et al. 1998, 2000, 2002b; Stoner et al. 2000; Yogo et al. 2001; Fernandez-Cobo et al. 2002) and in Hispanic populations who are thought to have been formed by the intermixture of three
ethnic groups, Native Americans, Spanish Americans, and African Americans (Agostini et al. 1998c; Fernandez-Cobo et al. 2001). A recent phylogenetic study using the whole-genome approach con®rmed that Japanese/Korean and Native American MY isolates constitute a single clade with a high bootstrap probability (Sugimoto et al. 2002a). In this study, we evaluated the phylogenetic relationships among JCV isolates identi®ed in Japanese/ Koreans and Native Americans speaking Amerind or Na-Dene. To reconstruct a reliable phylogeny of MY, we sequenced complete MY genomes cloned from native Japanese, Koreans, Mexicans, Guatemalans, Peruvians, and Canadians. Twenty-six complete sequences thus obtained and 10 complete MY sequences reported previously (Agostini et al. 1998c; Kato et al. 2000; Sugimoto et al. 2002a) were subjected to phylogenetic analysis using two methods, the neighbor-joining (NJ) (Saitou and Nei 1987) and maximum-likelihood (ML) methods. The ®ndings obtained in the present study, along with our recent ®nding that Eskimo-Aleuts carry a unique genotype of JCV (EU-a/Arc) belonging to Type A (Sugimoto et al. 2002b), suggested anity between Amerinds and Na-Denes and distinction between Amerinds/ Na-Denes and Eskimo-Aleuts. Materials and Methods Urine Samples We collected urine samples from Native Americans living in Guatemala, Peru, Mexico, and Canada. The Guatemalan urine donors were native patients in hospitals located at Totonicapan, Santa Cruz del Quiche, and Solola, Guatemala. The Peruvian donors were native inhabitants of villages located in Ollantay Tambo and Chuquito. The Mexican and Canadian urine donors were volunteers living in the state of Chihuahua, Mexico, and the northern part of Alberta, Canada, respectively. All urine donors were over 40 years old. The urine donors in Mexico, Guatemala, and Peru belonged to various subgroups of the Amerind language group, and those in Canada belonged to the Na-Dene language group (Ruhlen 1991) (Table 1).
DNA Analysis From the viral DNA that was extracted from urine as described previously (Kitamura et al. 1990), the 610-bp IG region (Ault and Stoner 1992), which encompasses the 30 -terminal regions of both the large T-antigen (LTag) and the VP1 genes, was ampli®ed by PCR as described previously (Kunitake et al. 1995) using Pwo DNA polymerase with proofreading activity (Roche Diagnostics GmbH, Mannheim, Germany). Ampli®ed IG fragments were cloned into pBluescript SK(+) as described previously (Kunitake et al. 1995), and puri®ed recombinant plasmids were sequenced with an autosequencer (ABI PRISM 3700 DNA Analyzer, Applied Biosystems, Foster City, CA). Entire JCV DNAs were cloned into pUC19 at the unique BamHI site as described previously (Yogo et al. 1991). The resultant complete JCV DNA clones were pre-
20 Table 1.
Detection of the JCV DNA from urine samples collected in various Native American populationsa
Population
Ethnic group
Language familyb
No. of JCV DNA-positive samples/ No. of samples examined (%)
Native Native Native Native
Tarahumara Mayan Aymara/Quechua Beaver/Dene Tha0
Amerind Amerind Amerind Na-Dene
13/38 (34) 42/125 (34) 23/96 (24) 30/58 (52)
a b
Mexicans Guatemalans Peruvians Canadians
From the DNA extracted from urine, the IG region was detected by PCR as described under Materials and Methods. According to Ruhlen (1991).
pared using a QIAGEN Plasmid Maxi kit (QIAGEN GmbH, Hilden, Germany). Puri®ed plasmids were sequenced as described previously (Sugimoto et al. 2002a).
Phylogenetic Analysis The noncoding regulatory region of the JCV genome was excluded from phylogenetic analysis, as this region is hypervariable, especially in JCV isolates derived from the brains of PML patients (Yogo and Sugimoto 2001). Rates of synonymous substitution were estimated using the Diverge program in the GCG Wisconsin package. DNA sequences were aligned using CLUSTAL W (Thompson et al. 1994), with a gap opening penalty of 15.00 and a gap extension penalty of 6.66. To evaluate the phylogenetic relationships among DNA sequences, we used the NJ (Saitou and Nei 1987) and ML methods. For analysis with the NJ method, we used the CLUSTAL W program. Divergences were estimated by Kimura's (1980) two-parameter method. To assess the con®dence of branching patterns of the NJ tree, bootstrap probabilities were estimated with 1000 bootstrap replicates (Felsenstein 1985) using CLUSTAL W. ML analysis was performed by the quartet-puzzling method implemented in the TREE-PUZZLE program (version 4.0.2) (Strimmer and von Haeseler 1996). The optimum transition/ transversion parameter of the HKY model was 2.15, and the a parameter of the G distribution was 0.13. For nucleotide substitution, the HKY model (Hasegawa et al. 1985) with the discrete G distribution for site heterogeneity was used. All phylogenetic trees were visualized using the TREEVIEW program (Page 1996).
Results The JCV Subtype Indigenous to Native Americans Using PCR to amplify the IG region, JCV DNA was detected in various Native American populations at rates ranging from 24% (native Peruvians) to 52% (native Canadians) (Table 1). All IG regions ampli®ed from native Mexicans, Peruvians, and Canadians were sequenced, but only those ampli®ed from 28 representative Guatemalans were sequenced. A NJ phylogenetic tree was constructed from the 94 determined sequences together with reference sequences from the Old World (Sugimoto et al. 1997). According to the phylogenetic tree (the tree is not shown, to save space, but a similar tree is presented by Sugimoto et al. [1997]), all of the JCV sequences from native Mexicans, Peruvians, and Canadians were classi®ed as belonging to MY. Twenty-®ve of the 28 Guatemalan JCV sequences were of the MY
type, two were found to be of the major European type (EU-a), and one was of the major African type (Af2) (Sugimoto et al. 1997, 2002a). From these results, we concluded that MY is the subtype of JCV indigenous to native Mexicans, Guatemalans Peruvians, and Canadians. The rare JCVs (i.e., those belonging to EU-a or Af2) in native Guatemalans, were probably introduced by Europeans and Africans who migrated to the New World in the last several hundred years. Complete JCV (MY) DNA Sequences We attempted to establish complete viral DNA clones from JCV (MY)-positive urine samples from native Mexicans, Guatemalans, Peruvians, and Canadians. We established ®ve or six clones per geographical region (Table 2). These clones and ®ve complete MY clones obtained in this and previous studies from Japanese and Koreans (Yogo et al. 1990; Kato et al. 1994; Guo et al. 1996) (Table 2) were sequenced. We con®rmed that these complete sequences were not recombinant using the method described previously (Sugimoto et al. 2002a) (data not shown). In addition, ®ve Japanese and ®ve American complete MY sequences have been reported (Agostini et al. 1998c; Kato et al. 2000; Sugimoto et al. 2002a) (the latter included three sequences, #225, #226, and #228 identi®ed in the Navaho tribe). Thus, we had 36 complete MY sequences, in total, derived from Japanese and Koreans as well as various Native American populations (Table 2). Using CLUSTAL W, we aligned the 36 complete MY sequences plus the 54 complete sequences of the other subtypes, excluding the rare African subtype Af3 (Sugimoto et al. 1997). This alignment required seven gaps shown in Table 3. Four of these gaps involved Native American MY isolates. Phylogenetic Analysis Using the Whole-Genome Approach We constructed a NJ phylogenetic tree from the 90 complete sequences aligned as described above. The
21 Table 2. Thirty-six JCV (MY) isolates analyzed using the whole genome Geographic origin (ethnic origin) Japan (Japanese)
South Korea (Korean) Mexico (Tarahumalan)
Guatemala (Mayan)
Canada (Beaver/Dene Tha0 )
Peru (Andean)
USA (Navaho) USA (Hispanic) USA (European American)
Referencea Isolate
For isolates
For sequences
Accession No.
Tokyo-1 Tky-1 MY AT-8 HR-7 JP-7 Aic-1 YI SK-1 SK-4 ME-4 ME-5 ME-8 ME-12 ME-14 ME-16 GU-4 GU-8 GU-15 GU-21 GU-25 CN-1 CN-13 CN-15 CN-25 CN-28 PE-1 PE-11 PE-12 PE-16 PE-21 #225 #226 #228 #224 #229
1 3 2 5 5 5 3 2 4 4 Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð 6 6 6 6 6
6 7 7 8 8 Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð Ð 6 6 6 6 6
AF030085 AB038254 AB038250 AB048577 AB048578 AB081016 AB081005 AB081030 AB081028 AB081029 AB081020 AB081021 AB081022 AB081017 AB081018 AB081019 AB081014 AB081015 AB081011 AB081012 AB081013 AB081007 AB081006 AB081008 AB081009 AB081010 AB081023 AB081024 AB081025 AB081026 AB081027 AF015530 AF015531 AF015534 AF015529 AF015535
a Ðthis study; 1, Matsuda et al. (1987); 2, Yogo et al. (1990); 3, Kato et al. (1994); 4, Guo et al. (1996); 5, Kitamura et al. (1998); 6, Agostini et al. (1998c); 7, Kato et al. (2000); 8, Sugimoto et al. (2002a).
Table 3. Gaps required to align 90 complete JCV DNA sequences (excluding regulatory sequences)a Locationb
Change causing each gap
nt 236 nt 4163
Insertion of a single nucleotide (T) Insertion of a single nucleotide (A)
nt 4177 nt 4185
Deletion of a single nucleotide (T) Deletion of a single nucleotide (T)
nt 4211 nt 128±139 nt 128±136
Insertion of a single nucleotide (A) Deletion of a 12-nucleotide stretch Deletion of a 9-nucleotide stretch
a b
Subtypes (isolates) in which changes were found MY (GU-8, ME-5, -14, and -16, #224) Af1 (GH-1, -3), EU-b (GR-3, MR-7, N-25, SP-1, UK-1) CY (all isolates) MY (most Native American isolates excluding GU-8, ME-5, -14, and -16, and #224) Af1 (GH-1 to -4, #601) MY (CN-15) MY (CN-25)
Aligned using the CLUSTAL W program with a gap opening penalty of 15.00 and a gap extension penalty of 6.66. nt, nucleotide number of the consensus sequence starting at the ®rst nucleotide of the initiation codon of the agnogene.
22
resultant tree is shown in Fig. 1, and the branching pattern of JCV isolates is summarized below. 1. The major divisions generating the three superclusters (Types A, B, and C) were con®rmed (Sugimoto et al. 2002a). 2. The splits in Types A and B were also con®rmed (Sugimoto et al. 2002a). Thus, Type A generated two subtypes, EU-a and -b. Type B generated various subtypes, the major African subtype (Af2), a minor European subtype (B1-c), and many Asian subtypes (B1-a, -b, and -d, B2, CY, MY, and SC). 3. MY generated seven clades (MY-a through MYg), which were tightly grouped with high bootstrap probabilities, ranging from 96 to 100%. 4. MY-a and -b were clustered together with a signi®cantly high bootstrap probability (91%). 5. The ®ve Native American clades separated into one clade (MY-c) and a group of clades comprising MY-d to MY-g with a lower bootstrap probability (61%). We also analyzed the same set of complete MY sequences using an independent phylogenetic method, i.e., the ML method. With the large number of sequences in our sequence data set, it was impossible to investigate the topologies of all possible trees by the ML method because of the explosive increase in the number of possible tree topologies. Therefore, we adopted the quartet-puzzling method implemented in the TREE-PUZZLE program (Strimmer and von Haeseler 1996), and the results are shown in Fig. 2. All aspects of the evolution of MY observed in the NJ analysis (see above) were reproduced in the ML analysis. Time Scale of the Divergence of MY JCVs Assuming that JCV coevolved with human populations, Hatwell and Sharp (2000) and Sugimoto et al. (2002a) estimated the rate of synonymous substitutions to be 4 ´ 10)7 substitution per synonymous substitution site per year. Using this substitution rate, we attempted to elucidate the time scale of the divergence of MY JCVs. We calculated the average Ks values (synonymous substitutions per synonymous site) between isolates belonging to three representative clades (i.e., MY-b, -f, and -g) for each of three genes, VP1, VP2, and L Tag (Table 4). As the mean of these Ks values, calculated with weight for the length of each gene, we obtained 0.015, 0.015, and 0.013 synonymous substitution per synonymous site for MY-b vs MY-f, MY-b vs MY-g, and MY-f vs MY-g, respectively. According to the rate of synonymous substitutions (4 ´ 10)7/synonymous site/year), we estimated that splits between MY-b and MY-f,
between MY-b and MY-g, and between MY-f and MY-g occurred about 19,000, 19,000, and 15,000 years ago, respectively. Taking account of statistical errors, we estimated that splits into various withinMY clades occurred between 10,000 and 30,000 years ago. This estimate is in rough agreement with the timing (12,000 or more years ago) of the arrival of the ``First Americans'' estimated based on archeological evidence (Fiedel 2000). Discussion Correlation Between Within-MY Clades and Human Populations As it appeared that there is a correlation between within-MY clades and human populations, we assigned within-MY clades to ethnic groups, as shown in Table 5. Some isolates (in bold face in Table 5), however, were not consistent with these assignments. For example, a Guatemalan isolate (GU-8) was found in MY-c that was assigned to Tarahumalans (a tribe within Mexico), while a Mexican isolate (ME-8) was found in MY-e that was assigned to Mayans (an ethnic group within Guatemala). These discrepancies may have been caused by the partial intermixture of neighboring populations. In this connection, it is of interest to note that one of the Navaho strains (#228) fell into MY-g that contained isolates mainly from the Canadian Na-Dene groups (Beaver and Dene Tha0 ). Both the Canadian groups and the Navahos speak the Athabaskan language of the Na-Dene family (Ruhlen 1991). Although they are now far removed from each other, they may have lived in close proximity sometime in the past. In addition, two Mexican isolates (ME-4 and -12) remained unassigned in this study. However, if a phylogenetic tree is constructed from a more comprehensive set of complete sequences, ME-4 or -12, together with other isolates, may form a new clade, which may be assigned to a Native American population not studied here. After we submitted a manuscript describing the present study, Fernandez-Cobo et al. (2002) reported six complete MY sequences identi®ed in two Amerind-speaking groups, Guarani and Salish, living in Argentina and the United States, respectively. We included these sequences in our data set and constructed a NJ phylogenetic tree. MY sequences identi®ed in Guarani and Salish peoples were rather dispersed on the phylogenetic tree, with only two sequences in Salish grouped weakly (data not shown). As the numbers of available sequences in these ethnic groups were small, we consider that more sequences are needed to draw a de®nite conclusion regarding the sequence diversity of JCV strains in these groups.
23
Fig. 1. NJ phylogenetic tree relating 90 complete JCV DNA sequences. A NJ phylogenetic tree was constructed from the complete sequences excluding regulatory regions using CLUSTAL W. The phylogenetic tree was visualized using the TREEVIEW 1.4 program. The numbers at nodes in the tree indicate the bootstrap probabilities (percentage) obtained by 1000 replicates (only those ³50% are shown). The MY isolates analyzed are listed in Table 2.
The isolates belonging to the other subtypes were described previously (Frisque et al. 1984; Loeber and DoÈrries 1988; Agostini et al. 1997a, 1998a, b; Kato et al. 1997; Sugimoto et al. 2002a). Superclusters (Types A to C) and subtypes of JCV (Af1, Af2, EU-a and -b, B1-a to B1-d, B2, CY, MY, SC) and within-MY clades (a to g) are shown.
24
Fig. 2. Quartet-puzzling tree relating 90 complete JCV DNA sequences. The tree was constructed from the same set of complete sequences as analyzed in Fig. 1. The phylogenetic tree was visualized using the TREEVIEW 1.4 program. The horizontal length of each branch is proportional to the ML estimate of nucleotide
substitutions. The numbers at nodes indicate a quartet-puzzling support value (percentage) (only those ³50% are shown). The isolates analyzed are those described in the legend to Fig. 1. Superclusters, subtypes of JCV, and within-MY clades are shown (see the legend to Fig. 1).
25 Table 4. Synonymous nucleotide substitutions among clades within MYa Gene
MY-b vs MY-f MY-b vs MY-g MY-f vs MY-g
VP1 VP2 LTag
0.039 0.003 0.009
0.024 0.004 0.015
0.036 0.001 0.007
0.015
0.015
0.013
Meanb a
For three genes, the estimated numbers of synonymous substitutions per synonymous site are shown as averages of comparisons among ®ve MY-b isolates (HR-7, MY, SK-1, Tky-1, Tokyo-1), ®ve MY-f isolates (PE-1, -11, -12, -16, -21), and ®ve MY-g isolates (CN-1, -13, -15, -25, -28). b Calculated with weight for the length of each gene.
Relationships Among the Three Native American Groups Since the tripartite migration model was proposed by Greenberg et al. (1986), many genetic studies using human mtDNA and the Y chromosome have been conducted to examine the relationships among the three Native American groups, Amerinds, Na-Denes and Eskimo-Aleuts. Recent studies indicated close genetic anity among the three Native American groups, arguing against the tripartite model (Bailliet et al. 1994; Merriwether et al. 1995; Foster et al. 1996; Bonatto and Salzano 1997; Karafet et al. 1997; Lell et al. 1997). On the other hand, multiple migration hypotheses have been presented on the basis of sequence variations in human mtDNA and the Y chromosome (Torroni et al. 1992, 1993, 1994; Horai et al. 1993; Lell et al. 2002). The present and previous studies (Agostini et al. 1997b; Sugimoto et al. 1998, 2000; Stoner et al. 2000; Yogo et al. 2001; Fernandez-Cobo et al. 2002) reported that a subtype (MY or Type 2A) belonging to Type B is prevalent in not only various Amerindspeaking populations in North, Central, and South America but also Na-Dene-speaking populations in the United States and Canada. Furthermore, the present phylogenetic analysis of Japanese/Korean
and Native American MY sequences revealed that the MY sequences diverged into two Japanese/Korean (MY-a and -b) and ®ve Native American clades (MY-c to MY-g). Two of the Native American clades (MY-d and -g) contained isolates mainly from NaDenes and the others (MY-c, -e, and -f) contained isolates mainly from Amerinds (see Table 5). The NaDene clades were not clustered together, nor were the Amerind clades (Fig. 1). In other words, there was no distinction between Amerinds and Na-Denes in terms of indigenous JCVs, although they are linguistically distinguished from each other. We recently reported that an ethnic group (Nanai) living in the lower Amur River region carries a new subtype (designated EU-c) belonging to Type A and that other northeastern Siberians (Chukchis and Koryaks) and an Arctic people (Inuits) carry a clade (EU-a/Arc) within the EU-a subtype that also belongs to Type A (Sugimoto et al. 2002b). Thus, Eskimo-Aleuts carrying EU-a/Arc (Type A) and Amerinds/Na-Denes carrying MY (Type B) were distinguished in terms of indigenous JCV genotypes. Conclusions We reported the ®rst comprehensive study on the phylogenetic relationships among worldwide JCV isolates belonging to the subtype MY. We found that the MY isolates diverged into two Japanese/Korean and ®ve Native American clades with high bootstrap probabilities. Two of the Native American clades contained isolates mainly from Na-Denes and the others contained isolates mainly from Amerinds. The Na-Dene clades were not clustered together, nor were the Amerind clades. In contrast, the two Japanese/ Korean clades were clustered at a high bootstrap probability. The present ®ndings provided support for a close anity between Amerinds and Na-Denes. Furthermore, as Eskimo-Aleuts carry a unique genotype of JCV belonging to Type A, our ®ndings suggested a distinction between Amerinds/Na-Denes and Eskimo-Aleuts.
Table 5. Assignment of within-MY clades to ethnic groupsa Clade
Assigned ethnic groupa
Native American language
Isolatesb
MY-a MY-b MY-c MY-d MY-e MY-f MY-g
Japanese Japanese/Koreans Tarahumalans Navahos Mayans Aymara/Quechua Beaver/Dene Tha0
Ðc Ð Amerind Na-Dene Amerind Amerind Na-Dene
Aic-1, AT-8, JP-7, YI HR-7, MY, SK-1, SK-4, Tky-1, Tokyo-1 GU-8, ME-5, -14, and -16, #224 #225, #226 GU-4, -15, -21, and -25, ME-8 PE-1, -11, -12, -16, -21 CN-1, -13, -15, -25, and -28, #228, #229
a
Assigned to an ethnic group where all or most of the isolates found in each within-MY clade were detected. The geographic and ethnic origins of each isolate are listed in Table 2. Isolates that were not consistent with the present assignment are in boldface. c Not applicable. b
26 Acknowledgments. We are grateful to all urine donors. We thank Yuichiro Tabaru, Naoko Shinoda and Ryoko Takeda for their help in urine collection. 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. C.S. was supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists.
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