Index Introduction Materials and Methods Subject DNA extraction and analysis Reported complete DNA sequences of JCV Phylogenetic Analysis Results Discussion Acknowledgments References

Introduction

JC virus (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). Although JCV causes progressive multifocal leukoencephalopathy (PML) in immunocompromised patients (Walker, 1985), it is ubiquitous in the human population; it infects children asymptomatically, then persists in renal tissue throughout life (Padgett and Walker, 1973; Chesters et al., 1983; Tominaga et al., 1992; Kitamura et al., 1997). In most adults, renal JCV is not latent, but instead replicates and its progeny are excreted into urine (Kitamura et al., 1990, 1994; Agostini et al., 1996). JCV strains around the world can be classified into more than ten major genotypes, with each genotype occupying a unique geographical domain (Sugimoto et al., 1997; Yogo et al., 2004). Analysis of JCV genotypes has thus provided new insights into the origins of various ethnic groups throughout the world (e.g. Kitamura et al., 1998; Guo et al., 2001; Saruwatari et al., 2002; Miranda et al., 2004; Takasaka et al., 2004; Yogo et al., 2004).

Using phylogenetic analysis, Yogo et al. (2003) recently analyzed thirteen JCV isolates from the Ainu, an indigenous population living on Hokkaido, the northernmost island of Japan, and classified them into five genotypes: two (MX and MY-x) were first identified in the Ainu, two (EU-a/Arc and EU-c) were prevalent in northeastern Siberians and an Arctic tribe, and one (MY-b) was widespread among Hondo Japanese (i.e. contemporary Japanese, excluding the Ainu). Although two new genotypes were recognized in Yogo et al.’s (2003) study, the data size was too small for them to draw definite conclusions about the ethnic distribution of these genotypes. In the present study, we performed a detailed phylogenetic analysis of three unique JCV isolates, one identified by ourselves in an Ainu subject and two detected by Cui et al. (2004) in Koreans living in Seoul, South Korea. The latter two isolates were described as Type 2A2, which is characteristic of Native Americans (Cui et al., 2004).

Materials and Methods

Subject

A 70-year-old male Ainu who was born in Makubetsu, southern Hokkaido, and died of pneumonia was the subject of this study. A medico-legal autopsy was performed at his family’s request. His ethnic origin was found to be Ainu according to interviews with the family.

DNA extraction and analysis

About 5 ml of urine was collected from the subject’s bladder and was used as the source for our extraction of viral DNA, as described by Kitamura et al. (1990). A 610-bp IG region was amplified by means of polymerase chain reaction (PCR) using the P1 and P2 primers (Kunitake et al., 1995). The entire JCV DNA was cloned into pUC19 at the unique BamHI site, as described by Yogo et al. (1991), and the resultant clone, which carried the entire JCV DNA, was sequenced as described by Sugimoto et al. (2002a).

Reported complete DNA sequences of JCV

The complete JCV DNA sequences reported previously and used in this study are shown in Table 1, along with their GSDB, DDBJ, EMBL, and NCBI accession numbers.

Phylogenetic Analysis

The non-coding regulatory region of the JCV genome was excluded from our phylogenetic analysis, as this region is hypervariable, especially in JCV isolates derived from the brains of patients with PML (Yogo and Sugimoto, 2001). DNA sequences were aligned using the CLUSTAL W program (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 neighbor-joining (NJ) method (Saitou and Nei, 1987) in CLUSTAL W. Divergences were estimated using Kimura’s two-parameter method (Kimura, 1980). To assess the confidence limit of the branching patterns of the NJ tree, bootstrap probabilities (BPs) were estimated using 1000 bootstrap replicates (Felsenstein, 1985) in CLUSTAL W. BPs greater than 70% were considered to be significant (Hillis and Bull, 1993). A phylogenetic tree was then visualized using the TREEVIEW program (Page, 1996).

Results

As the urine sample from the Ainu subject gave positive amplification in the PCR process, we attempted to establish complete clones of the JCV DNA from the urine. We obtained a complete JCV DNA clone, which we designated ANF. We then sequenced this clone completely. Cui et al. (2004) recently reported various JCV isolates in Asia, including China, South Korea, and India. Among these isolates, two from Seoul (255A and 256A) attracted our attention because despite their Asian origin, they were described as a genotype (Type 2A2) formerly considered to be characteristic of Native Americans (Cui et al., 2004). We constructed an NJ phylogenetic tree from the complete DNA sequences of ANF, 255A, and 256A, together with 118 complete sequences that had been collected from around the world, including ten detected in the Ainu (Yogo et al., 2003). Based on the resulting tree (Figure 1), we conclude that ANF and two isolates (AN-9 and AN-13) that had previously been detected in the Ainu (Yogo et al., 2003) formed a distinct clade (designated MX), with a high bootstrap probability (100%). In contrast, the Korean isolates (255A and 256A) together with an isolate (AN-1) from an Ainu formed another distinct clade (MY-x) with a 100% BP (Figure 1). MY-x and the other MY subgroups (MY-a to MY-g) constituted a superclade, designated MY. As previously shown (Yogo et al., 2003), the MX and MY grouping has been demonstrated with a significantly high BP (83%; Figure 1).

View Details

Figure 1. Neighbor-joining (NJ) phylogenetic tree relating 118 complete JCV DNA sequences. A phylogenetic tree was constructed from complete sequences, excluding regulatory sequences, using the NJ method. The phylogenetic tree was visualized using the TREEVIEW program (Page, 1996). The tree was rooted using Af1 as the outer group, as this genotype probably represents the ancestral type (Pavesi, 2003). The symbols for sequences are shown in Table 1. The number at each node in the tree indicates the bootstrap probability (BP, %) obtained from 1000 replicates (only values >70% are shown for major clusters). Superclusters (Types A, B, and C), genotypes (Af1, Af2, EU-a, EU-b, EU-c, B1-a, B1-b, B1-c, B1-d, B2, CY, MX, MY, 2E, 8A, and 8B), intra-EU-a subgroups (EU-a/Arc and EU-a/JK), and intra-MY subgroups (MY-a to MY-g and MY-x) are indicated. Isolates from the Ainu (Yogo et al., 2003; this study) and from Koreans (Cui et al., 2004) are shown in white on black backgrounds.

Discussion

Yogo et al. (2003) detected MY-x and MX in only one or a few subjects. Therefore, the ethnic distribution of these genotypes required further study. In the present study, we demonstrated the presence of the MX genotype in a forensic subject whose origin was Ainu. MX was previously detected at two distant sites (Asahikawa and Shiraoi) on Hokkaido (Yogo et al., 2003). In the present study, we detected MX in a subject from another site on Hokkaido (i.e. Makubetsu), which suggests that the MX genotype of JCV is widespread among the Ainu, albeit at a lower frequency.

We found that two isolates from Seoul belonged to MY-x, a subgroup within genotype MY (Yogo et al., 2003). These isolates were previously described as belonging to the Native American subgroup of MY (named Type 2A2; Cui et al., 2004), but the result of our analysis suggests that this classification should be revised. Although MY-x was distributed in two distinct human populations (i.e. the Ainu and Koreans), further division of the MY-x isolates was not demonstrated. This suggests that peoples carrying the MY-x genotype migrated to the Korean Peninsula and Hokkaido relatively recently (i.e. not earlier than 10000 years ago).

Acknowledgments

This study was supported in part by grants from Japan’s Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labour and Welfare.