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Geographical identification of cadavers by human parasites
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Forensic Science International: Genetics 2 (2008) 83–90 www.elsevier.com/locate/fsig
Review
Geographical identification of cadavers by human parasites Hiroshi Ikegaya * National Research Institute of Police Science, Kashiwa, Chiba, 277-0882 Japan Received 7 September 2007; accepted 10 October 2007
Abstract Increasing numbers of unidentified cadavers have recently become an important forensic problem in many countries. To identify such cadavers, DNA typing method is widely used. However, as this technique requires reference DNA samples, a method that would quickly narrow down possible candidates for the cadavers is needed to enable rapid identification. Unfortunately, no really reliable methods suitable for this purpose have been available; however, methods using the human parasites, JC virus, BK virus and EB virus, have been reported. These new methods narrow down the candidates by elucidating geographic origins. Though not detectable in all cases, results using such methods with several parasites have enabled us to estimate geographic origins of unidentified cadavers with a high detection rate. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Geographical identification; JC virus; BK virus; EB virus; Human parasites; Genotype
Contents 1. 2.
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4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JC virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Definition of geographical origin . . . . . . . . . . . . . . . . . . 2.2. Utilization of genotype distribution in small areas . . . . . . 2.3. JCV DNA chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BK virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Distribution of BKV genotypes worldwide . . . . . . . . . . . 3.2. Merits of BKV genotyping for geographical identification. 3.3. BKV DNA chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EB virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Increasing numbers of unidentified cadavers have become an important forensic problem in many areas worldwide because of the increasing internationalization of social interactions and occurrences of large-scale disasters [1]. In Japan, more than 100,000 people go missing annually [2] and more than 16,000 bodies remain unidentified. In England, more
* Tel.: +81 4 7135 8001; fax: +81 4 7133 9159. E-mail address: [email protected]. 1872-4973/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2007.10.184
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than 210,000 people go missing each year [3]. Generally, dental charts, fingerprints, and human DNA are used for individual identification. However, even if DNA typing of an unidentified cadaver has been performed, it remains impossible to promptly identify the individual if there are no candidates to be checked against. Therefore, a method that quickly narrows down possible identities of unidentified cadavers by providing geographical information would be extremely valuable. However, there are no effective ways to estimate human geographical origins using human DNA or morphological character analysis. However, a geographical identification method using the human parasite JC
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virus that would effectively narrow down the searching area for human DNA identification was firstly reported in 2002 [4]. 2. JC virus The 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 [5]. JCV was first isolated in 1971 from the brain of a patient with progressive multifocal leukoencephalopathy (PML) [6]. This virus is ubiquitous in the human population, infecting children asymptomatically [7]. After primary infection, JCV persists in the renal tissue of most adults, excreting its progeny virus in the urine. The same viral strains are maintained in the kidneys and urine throughout life despite a host’s relocation [8]. Using phylogenetic analyses of viral DNA sequences, JCV strains worldwide can be classified into more than 30 Table 1 Old world distribution of three virus genotypes
a
Genotypes are indicated according to reports [9,62]. Genotypes are indicated according to the report [35]. c Genotypes are indicated according to the report [47], no study. b
genotypes, consisting of 18 main ones. Each genotype occupies distinct domains in different parts of the world (Table 1) [9]. These characteristics of the JCV genotype were thus considered favorable for determining the geographical origins of unidentified cadavers. JCV DNA was initially used in an attempt to detect geographical origins from 64 kidneys and 36 urine samples taken from forensic autopsies [4]. DNA samples extracted from 200 mg renal tissue and 5 ml urine were used as templates for PCR amplification of a 610 bp region (IG region) [10] of the viral genome usually used for determining viral genotype. JCV DNA was detected in 29 cases (45%) and in 11 cases (33%), respectively, with the detection rate being much higher in adults. The actual places of origin of all samples matched with those estimated from the distribution areas of the JCV genotypes. The detection rate was not related to the cause of death and was hardly affected by post-mortem decomposition;
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indeed, it was detected from cases up to 10 months after death. Tests also succeeded in severely burned and highly corrupted cases. Sampling from renal tissue has also been reported [11]; in 36 autopsy cases three samples were taken from each kidney for JCV detection. As a result, the IG sequences of JCV were identical not only among the specimens delivered from the same kidney but also among those delivered from both kidneys of the same cadaver. These results exclude superinfections of JCV and confirmed the accuracy of this method. Moreover, detection of JCV from formalin-fixed and paraffin-embedded tissue is also reported [12]. In that report, eight forensic cases had been stored for one year under different conditions: frozen at 80 8C, formalin-fixed and paraffinembedded, and soaked in 5% formalin. PCR amplified IG fragments were cloned and sequenced. In the formalin-fixed, paraffin-embedded samples, not only the original samples but also those with 1% of base substitution were detected and no genotype change was found. From the formalin-soaked samples, the original sequences and those with more than 1% of substitution causing genotype change were detected. Therefore, the genotype can be determined with specimens in a frozen state or formalin-fixed for a short time. The limits of detection for the amount of urine and detection from urine stains have also been reported [13]; using a PCR method JCV DNA is detectable from 50 ml of urine. When 200 ml of urine was used for detection, 54.9% was JCV positive, which was almost the same detection rate shown in other reports that used 50 ml of urine samples for analysis. The detection of JCV DNA from urine stains that had been prepared 3 months earlier using 100 ml of JCV positive urine was also attempted; JCV DNA was detected in 68.8%. Therefore, this method can be used in urine stain samples left at a scene as well as in a small amount of urine taken from autopsies. 2.1. Definition of geographical origin As JCV has migrated and evolved along with humans, estimating geographical origins using a JCV method may be similar to ethnicity estimated using human DNA methods like SNP typing [14]. However, today it is usual for people to grow up in areas unrelated to that of birth, or to frequently move worldwide given the highly developed nature of modern transportation systems. JCV genotypes are much more related to the area where a person grew up than they are to race. That may be because even if a virus of foreign genotype comes into other area with a host, it usually does not infect the host’s descendants or other children. The virus of the major genotype in an area is the one that will usually infect them. Therefore, the geographic origin estimated with this method does not indicate a racial origin but rather the location the person grew up in. For instance, Japanese Okinawa was occupied by United States forces for more than 20 years. During that period, many Japanese-American children were also born to Japanese mothers and American fathers. However, almost all the people born in this period have the gentype that distributes in Okinawa [15] because most of these children grew up in the Japanese
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community. If we were to use human DNA or another morphlogical analysis, the results would be mixed. Therefore, in addition to the racial information obtained by other methods including human DNA analysis, geographical information from viral genotyping enables us to make a much more detailed profiling. In this case, the profiling would be a JapaneseAmerican who grew up in a Japanese area. In case of an immigration country, like U.S., each genotype may be maintained in each immgration community [16]. 2.2. Utilization of genotype distribution in small areas JCV genotypes are especially rich in East Asia; those include CY, MY, B1-a, B1-b, SC and 2E genotypes. In this area, Japan is the only country in which JCV genotype distribution has been surveyed in detail [17]; 98.5% of the genotypes were CY and MY. MY distributes mainly in the north of Japan, and CY mainly in the south. From the difference in the distribution of the two genotypes, we can statistically estimate from which area a cadaver originated and the probability that a cadaver originated from the place at which it was found [18]. However, even in Europe, where there are only three main genotypes, EUa, EU-b and B1-c, there are different distribution patterns among the three. EU-a mainly distributes in the whole of Europe, whereas EU-b and B1-c distribute in relatively southern Europe [9]. Therefore, as genotype distribution could be investigated in detail in Japan, it might also be possible to determine the origin of cadavers in Europe. 2.3. JCV DNA chip The JCV method has now become an integral part of police investigations in Japan, as it is effective in aiding cadaver identification by narrowing down the geographical areas of origin. However, despite the advantages of this method, viral genotypes must be obtained using a lengthy process of viral DNA extraction, PCR amplification, cloning, sequencing and phylogenetic analysis. This work is time consuming and requires highly qualified staff. In an attempt to develop a rapid but precise method to detect JCV genotypes for human geographical identification, a JCV DNA chip was developed [19]. A total of 54 probes that recognize genotype-specific SNP sites and are designed to distinguish 12 main JCV genotypes are chemically fixed on a 3 mm square silicon chip (Toyokohan, Yamaguchi, Japan) [20]. The principle of the DNA chip method is that we use PCR amplification to type the hypervariable region of the virus genome with fluorescent dye combined with dCTP. The amplified fragments are then hybridized to type-specific probes fixed on a DNA chip, and the fluorescent signals of fragments hybridized with type-specific probes are scanned by a detector. The 610-bp IG region of the JCV genome usually used for genotyping [10] was PCR amplified from the extracted DNA samples using Cy-5 dCTP (GE Healthcare, Buckinghamshire, UK). Two microliters of PCR amplified reaction mixture was hybridized with the chip for 30 min and scanned with a fluorescent scanner. Geographic origin was estimated from the
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obtained fluorescent patterns that indicated specific genotypes. With this chip we are able to discriminate 12 main JCV genotypes distributed in distinct areas of the world. Compared with the conventional method [4], the DNA chip method showed almost the same sensitivity (template DNA level at 0.001 pg/ml) and high reproducibility (R2 > 0.99). Moreover, the method can be applied to samples of blood, urine (>50 ml) and renal tissue (>20 mg) as well as urine and blood stains (>4 cm in diameter). This procedure requires no special knowledge or skill and involves only the application of 2 ml of PCR reaction mixture on the chip. It takes as little as four hours from collecting samples to yielding results and requires just a portable PCR system and hybridization machine. If security against contamination is ensured, it can be used in outside environments as well as inside ones. Thus, this method would be very effective in locations where there are many unidentified bodies following large-scale disasters. 3. BK virus Geographical identification using JCV has already been used in actual police investigations to facilitate identification in Japan. However, even if the positive rate is high, the JCV method does not always succeed in detecting where people are from. Its detection rate from urine or kidneys is around 50% [4,13]. Therefore, to improve its diagnostic value, other markers were needed. Another marker, the BK virus (BKV), is reported as available for determining geographical origins of unidentified cadavers [21]. Taxonomically, BKV belongs to the same family of Polyomaviridae as JCV. BKV was first detected from a renal transplant patient in 1971 [22]. Serological studies revealed that this virus is also ubiquitous in humans [23]. Primary infection with this virus usually occurs in childhood and the seroprevalence rate reaches the adult rate (65–90%) at age 5–10 years [24]. BKV is also known to persist in the kidneys [25,26]. It is rarely found in the urine of immunocompetent individuals, whereas it can be generally detected in the urine of immunocompromised hosts, such as renal transplant and HIVinfected patients [24]. A partial VP1 gene of the BKV sequence contains nucleotide substitutions responsible for antigenic diversity [27]. It is suspected that certain BKV genotypes may be related to the development of clinical conditions like cancer and hemorrhagic cystitis [24,28]. However, by using RFLP analysis or sequencing analysis of PCR-amplified fragments, BKV strains have been classified into four subtypes, I–IV, which corresponded well to groups based on serological assays [29]. The VP1 gene, probably responsible for serotypic differences, and complete genome were analyzed using restriction [29] or phylogenetic analysis [30–33] to classify BKV isolates obtained from several countries including England, Ireland, Tanzania, the United States, Finland and Japan [31–35]. These analyses were performed to establish possible correlations between BKV subgroups and these geographically different human populations. Using geographical correlations, estimations of the geographical origins of
unidentified cadavers in forensic medicine was investigated [21]. In this report, a 287-bp typing region of BKV, usually used for the identification of BKV genomes [29], and the IG region of JCV were amplified by PCR from kidney samples of 36 Japanese forensic cases [21]. The typing region of BKV was amplified in 11 cases (30.5%), and the IG region of JCV in 17 (47.2%). There were four cases (11.1%) in which both viral DNAs were amplified. The detection rates of the two viruses were not related to a specific cause of death, and BKV was also detected from cadavers severely burned. In such cases, it is practically impossible to identify or to estimate the origin from the appearance. As BKV and JCV persist in the kidneys, which are located deep in the body and are surrounded by thick adipose tissue, it is possible to detect them from burned bodies. JCV was detected mainly from subjects older than 30 years of age. On the other hand, BKV was detected from both young and old subjects. Both viruses were efficiently amplified up to 72 h after death. When time after death was divided into two categories of earlier or later than 24 h after death, there was no statistical difference in the detection rate between the two viruses. Thus these viruses appear stable in terms of postmortem changes. 3.1. Distribution of BKV genotypes worldwide The genotypes of the detected BKVs are usually Types I and IV. Types II and III are rarely detected. Type I distributes worldwide and type IV mainly distributes in Asia. There are four subtypes of Type I: subtypes Ia, Ib-1, Ib-2 and Ic. These subtypes mainly distribute in Africa, Southeast Asia, Europe and Northeast Asia, respectively. The distribution of the subtypes is shown in Table 1 [35]. Reported genotypes detected from urine of living subjects in Japan are Types I (79%), III (2.5%), and IV (18.3%) [33]. Most detected Type I strains were Type Ic, a subtype of Type I. Type Ic strains are mainly found in the Far East, including Japan, but are very rarely found in other areas such as West Asia, Europe, and Africa. In this report, the genotypes of BKV and JCV of 24 Japanese subjects completely agreed with their distribution area. 3.2. Merits of BKV genotyping for geographical identification The BKV method provides more approximate geographic information than does the JCV method [9,35]. For example, although Japan is a small country JCV can divide the Japanese population into two, but BKV cannot [17,33]. However, BKV was sometimes detected from cadavers in whom JCV was not detected. It was reported that geographic origins were able to be estimated in 17 cases using the JCV method, and the seven additional cases were located using the BKV method [21]. In total, estimations of geographic origins were successful in 24 of 36 cases (69.8%) with a combination of the two methods. In four of the 24 positive cases (16.7%) both JCV and BKV DNA were detected [21]. In cases from which type CY of JCV and genotype IV of BKV are detected, it is highly likely that the cadaver would be from northern China, because the BKV of
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type IV distributes mainly in continental Asia and the JCV of type CY distributes from northern China to West Japan. Thus, as the distribution areas of genotypes between BKV and JCV are not the same, it is possible to narrow down the geographic area, where the cadaver originates from. Therefore, if the BKV and JCV methods are used in combination, we should be able to estimate the geographic origins of a much higher number of cadavers. 3.3. BKV DNA chip A JCV DNA chip was developed for rapid and easy diagnosis [19]. This DNA chip takes little time to yield results and shows both high specificity and reproducibility. It makes the investigation of unidentified cadavers fast and easy by rapidly narrowing down the search area for human geographical identification. Recently, another chip, similar to the JCV, BKV DNA one, was developed for the same purpose [36]. A total of 17 probes recognizing genotype-specific SNP sites were designed to distinguish 7 main BKV genotypes and were chemically fixed on a 3 mm square silicon DNA chip (Toyokohan) [19]. The principle of the DNA chip method is almost the same as that of the JCV DNA chip. The 287-bp typing region of the BKV genome, which is usually used for genotyping [29], was PCR amplified using Cy-5 dCTP (GE Healthcare). Two microliters of PCR amplified reaction mixture was hybridized with the chip for 30 min and scanned with a fluorescent scanner. Geographic origin was estimated from the fluorescent patterns obtained that indicated the specific genotypes. With this chip we are able to discriminate seven main BKV genotypes distributed in distinct areas of the world. Compared with a conventional method [21], our new DNA chip method showed almost the same sensitivity (template DNA level at 0.001 pg/ml) and high reproducibility (R2 > 0.90). The reaction conditions are almost the same between the JCV and BKV DNA chip methods. Thus it is possible to use them at a same time for diagnosis. Therefore, in addition to the JCV DNA chip, the BKV DNA chip will contribute to rapid diagnoses of geographical origins of unidentified cadavers. 4. EB virus The distribution of the BKV and JC virus (JCV) genotypes are related to various worldwide geographical regions, suggesting that they would be useful for geographical identification of unidentified cadavers [4,13,18,19,21,36]. However, most of the samples obtained from crime scenes are not renal tissues or urine, but bloodstains. Unfortunately, BKVand JCVare rarely detectable from blood samples [37,38]. If other persistent parasites would be detectable in human blood, and if genotypes were distributed in geographically distinct areas, such parasites would be highly useful for forensic investigations. Epstein-Barr virus (EBV) is one that is ubiquitous in healthy populations throughout the world. It has infected more than 90% of the world’s adult population [39]. However, EBV is an
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etiological agent of infectious mononucleosis and several neoplasmas such as nasopharyngeal carcinoma (NPC) and African Burkitt’s lymphoma [40]. These EBV associated tumors show considerable geographical variation. Burkitt’s lymphoma is endemic to equatorial Africa and Papua New Guinea, while NPC is common in Southeast Asia, Mediterranean Africa, and Inuit populations [41]. It was originally suspected that particular oncogenic EBV strains cause specific tumors in genetically susceptible populations. Though many researchers have attempted to identify such viruses, viral strains are only related to geographic area, not to diseases [42–45]. This geographical diversity is seen mainly within the genes for Epstein-Barr Virus Nuclear Antigen 3 (EBNA3), EBNA4, and Latent Membrane Protein 2A (LMP2A) of the EBV gene [46]. Based on these above findings, the forensic application of EBV genotype has recently been reported [47]. In that report, two hundred micro-milliliters of whole blood samples from 56 Japanese cases were collected during forensic autopsies. A 447bp hyper variable region of the EBV LMP2A gene was PCRamplified. The PCR amplified fragment were sequenced and analyzed phylogenetically with worldwide reference data obtained from a database. Detection using an EBV detection kit was also done to confirm the detection. Among the 56 samples, the LMP2A region of the EBV genome was amplified in nine samples (16%). Using the EBV detection kit, EBV DNA was detected in 11 samples (18%). All of the LMP2A-amplified samples were detected with this method. At least two copies/ml of EBV DNA were also detected. However, the lowest detected concentration of EBV DNA was six copies/ml in LMP2A amplified samples. From those LMP-2A sequences, together with reference sequences obtained from previously reported sequences, a phylogenetic tree was constructed using the NJ method [48]. From the resulting phylogenetic tree, these EBV isolates from the various patient populations were classified into two major clusters, types I and II. Type I was sub-classified into three subclusters, Ia, Ib and Ic, and type II was also sub-classified into three sub-clusters, IIa, IIb and IIc. All the Japanese isolates were included in subtypes Ia or IIa. All the Asian reference isolates except for the isolate D6, which was included in Ic, were included in subtypes Ia or IIa. Mediterranean isolates, an Alaskan isolate and other African isolates were included in types Ib, Ic, IIb and IIc. The EBV detection rate in the results reported in this study was not so high. However, the EBV detection rates from blood taken as forensic samples were almost the same using the two methods. Wanger et al. [49] also reported that previously reported detection rates of EBV DNA from peripheral blood were very low, ranging from 0 to 54%; however, when they used purified B-cells, the detection rate became 93.8%. The low detection rate may mainly have been caused by post-mortem decomposition and DNA extraction from whole blood samples. If sample DNA was extracted from purified B-cells, the detection rate should improve. However, it is normally impossible to use samples taken under good conditions for analysis in the field of forensic medicine. Though the detection rate was not so high, EBV DNA was detectable from samples
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that had a post-mortem time under 48 h. As most deceased are found within 2–3 days after death, this detection method may therefore be acceptable in forensic cases. The results show that each EBV genotype has a geographic correlation (Table 1). Especially, all the Japanese isolates were only included in Asian clusters. Most of the isolates from other geographical regions included region-specific clades. Based on the findings, together with those reported previously, EBV genotype distribution is correlated to geographical area; if the EBV genotypes from various regions were obtained, it would be possible to estimate the geographic origins of unidentified cadavers and blood stains by detecting the EBV genotype. There are many studies reporting multiple EBV infection, especially in immunosuppressed patients, who are known to have a high level of oral EBV shedding [50,51], co-infection with multiple EBV genotypes [52–54], and temporal changes in EBV populations [55,56]. However, even in healthy individuals, multiple EBV infection is reported. Walling et al. [52] reported that about 22% of healthy individuals have multiple infections. Multiple infections may affect estimations of geographical origins of unidentified cadavers. However, even if several strains were detected from one individual, it may be categorized into a single genotype by phylogenetic analysis because those strains may infect hosts in the same geographic area. Thus, multiple infections may not affect the results of estimations of geographic origin. Further confirmation studies will of course be required. Thus it has been demonstrated that EBV markers may also be a very important in the field of forensic medicine. 5. Conclusion The recent trends of internationalization and more the complicated criminal activity resulting have made the identification of missing people very difficult. Genotyping of human parasites is a very simple and efficient way to estimate the geographic origin of human individuals and so helps narrow down the geographical search area for cadaver identification. However, this method is not useful for all cases and results are not always absolute. However, by using genotyping methods, we can initiate investigations of missing people in much more probable areas. Our modern highly developed transportation systems and social internationalization will mean that cases in which geographical information does not agree with racial information will greatly increase in the future. Most cadavers are found in the early stage of the post-mortem period and most still have soft tissue; therefore, in addition to the other methods for racial estimations, including morphological characteristics [57] and human DNA polymorphisms [58–60], the parasite method makes more detailed profiling and investigation of unidentified cadavers both more efficient and economical. As many parasites have co-evolved with humans, there will be many other parasites that can be used for geographic identification; HHV, HTLV-1, HBV, Candida albicans, Helicobacter pylori are possible candidates (Table 2). JCV is the virus for which its evolutional history has been almost perfectly synchronized to the history of modern human migrations [61]. If the geographical variation of
Table 2 Possible parasites that may be used for geographic identification Parasites
Referencesa
Human herpes virus 1 Human T-cell lymphotropic virus 1 Hepatitis B virus Candida species Helicobacter pylori
[63,64] [65–67] [68,69] [70,71] [72–75]
a These references indicate correlation between geographic area and genotype or species.
genotypes was not constructed from the migration of modern humans, it would be impossible to estimate human geographical origins. Even if a human is a present host to the parasite, the host might be changed once or more in its life history. In that case, it would be very difficult to estimate the host’s geographical origins from its genotype diversity. Therefore, it is obviously very important to study the evolutional history of these parasites. From such studies, by analyzing the genotype distribution of many types of human parasites, it will be possible to estimate human geographic origins more precisely and with a high confidence rate. References [1] C. Cattaneo, Unidentified cadavers and human remains in the EU: an unknown issue, Int. J. Legal Med. 113 (1999) N1–N3. [2] Ministry of Health, Labor and Welfare Japan. Vital Statics Japan, Vol. No. 3 (2006). [3] The National Missing Persons Helpline. http://www.missingpersons.org. [4] H. Ikegaya, H. Iwase, C. Sugimoto, Y. Yogo, JC virus genotyping offers a new means of tracing the origins of unidentified cadavers, Int. J. Legal Med. 116 (2002) 242–245. [5] C.N. Cole, S.D. Conzen, Polyomaviridae: the viruses and their replication, in: D.M. Knipe, P.M. Howley, D.E. Griffin, et al. (Eds.), Fields Virology, fourth ed., Lippincott, Philadelphia, 2001, pp. 2141–2173. [6] B.L. Padgett, D.L. Walker, G.M. ZuRhein, R.J. Eckroade, B.H. Dessel, Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy, Lancet 1 (7712) (1971) 1257–1260. [7] T. Kunitake, T. Kitamura, J. Guo, F. Taguchi, K. Kawabe, Y. Yogo, Parentto-child transmission is relatively common in the spread of the human polyomavirus JC virus, J. Clin. Microbiol. 33 (1995) 1448–1451. [8] T. Kitamura, C. Sugimoto, A. Kato, Persistent JC virus (JCV) infection is demonstrated by continuous shedding of the same JCV strains, J. Clin. Microbiol. 35 (1997) 1255–1257. [9] Y. Yogo, C. Sugimoto, H.Y. Zheng, H. Ikegaya, T. Takasaka, K. Kitamura, JC virus genotyping offers a new paradigm in the study of human populations, Rev. Med. Virol. 14 (2004) 179–191. [10] G.S. Ault, G.L. Stoner, Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences, J. Gen. Virol. 73 (1992) 2669–2678. [11] H. Ikegaya, H. Iwase, Y. Yogo, Detection of Identical JC virus DNA Sequences in Both Kidneys, Arch. Virol. 149 (2004) 1215–1220. [12] H. Ikegaya, H. Iwase, H.Y. Zheng, M. Nakajima, K. Sakurada, T. Takatori, M. Fukayama, Y. Yogo, JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues, J. Virol. Methods. 126 (2005) 37–43. [13] K. Sakurada, H. Ikegaya, H. Motani, H. Iwase, K. Sekiguchi, T. Akutsu, M. Yoshino, T. Takatori, I. Sakai, JC virus genotyping can be used to narrow down the native place of persons from urine stains, Jpn J. Forensic Sci. Technol. 10 (2) (2005) 111–117. [14] B. Sobrino, M. Brion, A. Carracedo, SNPs in forensic genetics: a review on SNP typing methodologies, Forensic Sci. Int. 154 (2–3) (2005) 181– 194.
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Forensic Science International: Genetics 2 (2008) 83–90 www.elsevier.com/locate/fsig
Review
Geographical identification of cadavers by human parasites Hiroshi Ikegaya * National Research Institute of Police Science, Kashiwa, Chiba, 277-0882 Japan Received 7 September 2007; accepted 10 October 2007
Abstract Increasing numbers of unidentified cadavers have recently become an important forensic problem in many countries. To identify such cadavers, DNA typing method is widely used. However, as this technique requires reference DNA samples, a method that would quickly narrow down possible candidates for the cadavers is needed to enable rapid identification. Unfortunately, no really reliable methods suitable for this purpose have been available; however, methods using the human parasites, JC virus, BK virus and EB virus, have been reported. These new methods narrow down the candidates by elucidating geographic origins. Though not detectable in all cases, results using such methods with several parasites have enabled us to estimate geographic origins of unidentified cadavers with a high detection rate. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Geographical identification; JC virus; BK virus; EB virus; Human parasites; Genotype
Contents 1. 2.
3.
4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JC virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Definition of geographical origin . . . . . . . . . . . . . . . . . . 2.2. Utilization of genotype distribution in small areas . . . . . . 2.3. JCV DNA chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BK virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Distribution of BKV genotypes worldwide . . . . . . . . . . . 3.2. Merits of BKV genotyping for geographical identification. 3.3. BKV DNA chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EB virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Increasing numbers of unidentified cadavers have become an important forensic problem in many areas worldwide because of the increasing internationalization of social interactions and occurrences of large-scale disasters [1]. In Japan, more than 100,000 people go missing annually [2] and more than 16,000 bodies remain unidentified. In England, more
* Tel.: +81 4 7135 8001; fax: +81 4 7133 9159. E-mail address: [email protected]. 1872-4973/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2007.10.184
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than 210,000 people go missing each year [3]. Generally, dental charts, fingerprints, and human DNA are used for individual identification. However, even if DNA typing of an unidentified cadaver has been performed, it remains impossible to promptly identify the individual if there are no candidates to be checked against. Therefore, a method that quickly narrows down possible identities of unidentified cadavers by providing geographical information would be extremely valuable. However, there are no effective ways to estimate human geographical origins using human DNA or morphological character analysis. However, a geographical identification method using the human parasite JC
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virus that would effectively narrow down the searching area for human DNA identification was firstly reported in 2002 [4]. 2. JC virus The 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 [5]. JCV was first isolated in 1971 from the brain of a patient with progressive multifocal leukoencephalopathy (PML) [6]. This virus is ubiquitous in the human population, infecting children asymptomatically [7]. After primary infection, JCV persists in the renal tissue of most adults, excreting its progeny virus in the urine. The same viral strains are maintained in the kidneys and urine throughout life despite a host’s relocation [8]. Using phylogenetic analyses of viral DNA sequences, JCV strains worldwide can be classified into more than 30 Table 1 Old world distribution of three virus genotypes
a
Genotypes are indicated according to reports [9,62]. Genotypes are indicated according to the report [35]. c Genotypes are indicated according to the report [47], no study. b
genotypes, consisting of 18 main ones. Each genotype occupies distinct domains in different parts of the world (Table 1) [9]. These characteristics of the JCV genotype were thus considered favorable for determining the geographical origins of unidentified cadavers. JCV DNA was initially used in an attempt to detect geographical origins from 64 kidneys and 36 urine samples taken from forensic autopsies [4]. DNA samples extracted from 200 mg renal tissue and 5 ml urine were used as templates for PCR amplification of a 610 bp region (IG region) [10] of the viral genome usually used for determining viral genotype. JCV DNA was detected in 29 cases (45%) and in 11 cases (33%), respectively, with the detection rate being much higher in adults. The actual places of origin of all samples matched with those estimated from the distribution areas of the JCV genotypes. The detection rate was not related to the cause of death and was hardly affected by post-mortem decomposition;
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indeed, it was detected from cases up to 10 months after death. Tests also succeeded in severely burned and highly corrupted cases. Sampling from renal tissue has also been reported [11]; in 36 autopsy cases three samples were taken from each kidney for JCV detection. As a result, the IG sequences of JCV were identical not only among the specimens delivered from the same kidney but also among those delivered from both kidneys of the same cadaver. These results exclude superinfections of JCV and confirmed the accuracy of this method. Moreover, detection of JCV from formalin-fixed and paraffin-embedded tissue is also reported [12]. In that report, eight forensic cases had been stored for one year under different conditions: frozen at 80 8C, formalin-fixed and paraffinembedded, and soaked in 5% formalin. PCR amplified IG fragments were cloned and sequenced. In the formalin-fixed, paraffin-embedded samples, not only the original samples but also those with 1% of base substitution were detected and no genotype change was found. From the formalin-soaked samples, the original sequences and those with more than 1% of substitution causing genotype change were detected. Therefore, the genotype can be determined with specimens in a frozen state or formalin-fixed for a short time. The limits of detection for the amount of urine and detection from urine stains have also been reported [13]; using a PCR method JCV DNA is detectable from 50 ml of urine. When 200 ml of urine was used for detection, 54.9% was JCV positive, which was almost the same detection rate shown in other reports that used 50 ml of urine samples for analysis. The detection of JCV DNA from urine stains that had been prepared 3 months earlier using 100 ml of JCV positive urine was also attempted; JCV DNA was detected in 68.8%. Therefore, this method can be used in urine stain samples left at a scene as well as in a small amount of urine taken from autopsies. 2.1. Definition of geographical origin As JCV has migrated and evolved along with humans, estimating geographical origins using a JCV method may be similar to ethnicity estimated using human DNA methods like SNP typing [14]. However, today it is usual for people to grow up in areas unrelated to that of birth, or to frequently move worldwide given the highly developed nature of modern transportation systems. JCV genotypes are much more related to the area where a person grew up than they are to race. That may be because even if a virus of foreign genotype comes into other area with a host, it usually does not infect the host’s descendants or other children. The virus of the major genotype in an area is the one that will usually infect them. Therefore, the geographic origin estimated with this method does not indicate a racial origin but rather the location the person grew up in. For instance, Japanese Okinawa was occupied by United States forces for more than 20 years. During that period, many Japanese-American children were also born to Japanese mothers and American fathers. However, almost all the people born in this period have the gentype that distributes in Okinawa [15] because most of these children grew up in the Japanese
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community. If we were to use human DNA or another morphlogical analysis, the results would be mixed. Therefore, in addition to the racial information obtained by other methods including human DNA analysis, geographical information from viral genotyping enables us to make a much more detailed profiling. In this case, the profiling would be a JapaneseAmerican who grew up in a Japanese area. In case of an immigration country, like U.S., each genotype may be maintained in each immgration community [16]. 2.2. Utilization of genotype distribution in small areas JCV genotypes are especially rich in East Asia; those include CY, MY, B1-a, B1-b, SC and 2E genotypes. In this area, Japan is the only country in which JCV genotype distribution has been surveyed in detail [17]; 98.5% of the genotypes were CY and MY. MY distributes mainly in the north of Japan, and CY mainly in the south. From the difference in the distribution of the two genotypes, we can statistically estimate from which area a cadaver originated and the probability that a cadaver originated from the place at which it was found [18]. However, even in Europe, where there are only three main genotypes, EUa, EU-b and B1-c, there are different distribution patterns among the three. EU-a mainly distributes in the whole of Europe, whereas EU-b and B1-c distribute in relatively southern Europe [9]. Therefore, as genotype distribution could be investigated in detail in Japan, it might also be possible to determine the origin of cadavers in Europe. 2.3. JCV DNA chip The JCV method has now become an integral part of police investigations in Japan, as it is effective in aiding cadaver identification by narrowing down the geographical areas of origin. However, despite the advantages of this method, viral genotypes must be obtained using a lengthy process of viral DNA extraction, PCR amplification, cloning, sequencing and phylogenetic analysis. This work is time consuming and requires highly qualified staff. In an attempt to develop a rapid but precise method to detect JCV genotypes for human geographical identification, a JCV DNA chip was developed [19]. A total of 54 probes that recognize genotype-specific SNP sites and are designed to distinguish 12 main JCV genotypes are chemically fixed on a 3 mm square silicon chip (Toyokohan, Yamaguchi, Japan) [20]. The principle of the DNA chip method is that we use PCR amplification to type the hypervariable region of the virus genome with fluorescent dye combined with dCTP. The amplified fragments are then hybridized to type-specific probes fixed on a DNA chip, and the fluorescent signals of fragments hybridized with type-specific probes are scanned by a detector. The 610-bp IG region of the JCV genome usually used for genotyping [10] was PCR amplified from the extracted DNA samples using Cy-5 dCTP (GE Healthcare, Buckinghamshire, UK). Two microliters of PCR amplified reaction mixture was hybridized with the chip for 30 min and scanned with a fluorescent scanner. Geographic origin was estimated from the
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obtained fluorescent patterns that indicated specific genotypes. With this chip we are able to discriminate 12 main JCV genotypes distributed in distinct areas of the world. Compared with the conventional method [4], the DNA chip method showed almost the same sensitivity (template DNA level at 0.001 pg/ml) and high reproducibility (R2 > 0.99). Moreover, the method can be applied to samples of blood, urine (>50 ml) and renal tissue (>20 mg) as well as urine and blood stains (>4 cm in diameter). This procedure requires no special knowledge or skill and involves only the application of 2 ml of PCR reaction mixture on the chip. It takes as little as four hours from collecting samples to yielding results and requires just a portable PCR system and hybridization machine. If security against contamination is ensured, it can be used in outside environments as well as inside ones. Thus, this method would be very effective in locations where there are many unidentified bodies following large-scale disasters. 3. BK virus Geographical identification using JCV has already been used in actual police investigations to facilitate identification in Japan. However, even if the positive rate is high, the JCV method does not always succeed in detecting where people are from. Its detection rate from urine or kidneys is around 50% [4,13]. Therefore, to improve its diagnostic value, other markers were needed. Another marker, the BK virus (BKV), is reported as available for determining geographical origins of unidentified cadavers [21]. Taxonomically, BKV belongs to the same family of Polyomaviridae as JCV. BKV was first detected from a renal transplant patient in 1971 [22]. Serological studies revealed that this virus is also ubiquitous in humans [23]. Primary infection with this virus usually occurs in childhood and the seroprevalence rate reaches the adult rate (65–90%) at age 5–10 years [24]. BKV is also known to persist in the kidneys [25,26]. It is rarely found in the urine of immunocompetent individuals, whereas it can be generally detected in the urine of immunocompromised hosts, such as renal transplant and HIVinfected patients [24]. A partial VP1 gene of the BKV sequence contains nucleotide substitutions responsible for antigenic diversity [27]. It is suspected that certain BKV genotypes may be related to the development of clinical conditions like cancer and hemorrhagic cystitis [24,28]. However, by using RFLP analysis or sequencing analysis of PCR-amplified fragments, BKV strains have been classified into four subtypes, I–IV, which corresponded well to groups based on serological assays [29]. The VP1 gene, probably responsible for serotypic differences, and complete genome were analyzed using restriction [29] or phylogenetic analysis [30–33] to classify BKV isolates obtained from several countries including England, Ireland, Tanzania, the United States, Finland and Japan [31–35]. These analyses were performed to establish possible correlations between BKV subgroups and these geographically different human populations. Using geographical correlations, estimations of the geographical origins of
unidentified cadavers in forensic medicine was investigated [21]. In this report, a 287-bp typing region of BKV, usually used for the identification of BKV genomes [29], and the IG region of JCV were amplified by PCR from kidney samples of 36 Japanese forensic cases [21]. The typing region of BKV was amplified in 11 cases (30.5%), and the IG region of JCV in 17 (47.2%). There were four cases (11.1%) in which both viral DNAs were amplified. The detection rates of the two viruses were not related to a specific cause of death, and BKV was also detected from cadavers severely burned. In such cases, it is practically impossible to identify or to estimate the origin from the appearance. As BKV and JCV persist in the kidneys, which are located deep in the body and are surrounded by thick adipose tissue, it is possible to detect them from burned bodies. JCV was detected mainly from subjects older than 30 years of age. On the other hand, BKV was detected from both young and old subjects. Both viruses were efficiently amplified up to 72 h after death. When time after death was divided into two categories of earlier or later than 24 h after death, there was no statistical difference in the detection rate between the two viruses. Thus these viruses appear stable in terms of postmortem changes. 3.1. Distribution of BKV genotypes worldwide The genotypes of the detected BKVs are usually Types I and IV. Types II and III are rarely detected. Type I distributes worldwide and type IV mainly distributes in Asia. There are four subtypes of Type I: subtypes Ia, Ib-1, Ib-2 and Ic. These subtypes mainly distribute in Africa, Southeast Asia, Europe and Northeast Asia, respectively. The distribution of the subtypes is shown in Table 1 [35]. Reported genotypes detected from urine of living subjects in Japan are Types I (79%), III (2.5%), and IV (18.3%) [33]. Most detected Type I strains were Type Ic, a subtype of Type I. Type Ic strains are mainly found in the Far East, including Japan, but are very rarely found in other areas such as West Asia, Europe, and Africa. In this report, the genotypes of BKV and JCV of 24 Japanese subjects completely agreed with their distribution area. 3.2. Merits of BKV genotyping for geographical identification The BKV method provides more approximate geographic information than does the JCV method [9,35]. For example, although Japan is a small country JCV can divide the Japanese population into two, but BKV cannot [17,33]. However, BKV was sometimes detected from cadavers in whom JCV was not detected. It was reported that geographic origins were able to be estimated in 17 cases using the JCV method, and the seven additional cases were located using the BKV method [21]. In total, estimations of geographic origins were successful in 24 of 36 cases (69.8%) with a combination of the two methods. In four of the 24 positive cases (16.7%) both JCV and BKV DNA were detected [21]. In cases from which type CY of JCV and genotype IV of BKV are detected, it is highly likely that the cadaver would be from northern China, because the BKV of
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type IV distributes mainly in continental Asia and the JCV of type CY distributes from northern China to West Japan. Thus, as the distribution areas of genotypes between BKV and JCV are not the same, it is possible to narrow down the geographic area, where the cadaver originates from. Therefore, if the BKV and JCV methods are used in combination, we should be able to estimate the geographic origins of a much higher number of cadavers. 3.3. BKV DNA chip A JCV DNA chip was developed for rapid and easy diagnosis [19]. This DNA chip takes little time to yield results and shows both high specificity and reproducibility. It makes the investigation of unidentified cadavers fast and easy by rapidly narrowing down the search area for human geographical identification. Recently, another chip, similar to the JCV, BKV DNA one, was developed for the same purpose [36]. A total of 17 probes recognizing genotype-specific SNP sites were designed to distinguish 7 main BKV genotypes and were chemically fixed on a 3 mm square silicon DNA chip (Toyokohan) [19]. The principle of the DNA chip method is almost the same as that of the JCV DNA chip. The 287-bp typing region of the BKV genome, which is usually used for genotyping [29], was PCR amplified using Cy-5 dCTP (GE Healthcare). Two microliters of PCR amplified reaction mixture was hybridized with the chip for 30 min and scanned with a fluorescent scanner. Geographic origin was estimated from the fluorescent patterns obtained that indicated the specific genotypes. With this chip we are able to discriminate seven main BKV genotypes distributed in distinct areas of the world. Compared with a conventional method [21], our new DNA chip method showed almost the same sensitivity (template DNA level at 0.001 pg/ml) and high reproducibility (R2 > 0.90). The reaction conditions are almost the same between the JCV and BKV DNA chip methods. Thus it is possible to use them at a same time for diagnosis. Therefore, in addition to the JCV DNA chip, the BKV DNA chip will contribute to rapid diagnoses of geographical origins of unidentified cadavers. 4. EB virus The distribution of the BKV and JC virus (JCV) genotypes are related to various worldwide geographical regions, suggesting that they would be useful for geographical identification of unidentified cadavers [4,13,18,19,21,36]. However, most of the samples obtained from crime scenes are not renal tissues or urine, but bloodstains. Unfortunately, BKVand JCVare rarely detectable from blood samples [37,38]. If other persistent parasites would be detectable in human blood, and if genotypes were distributed in geographically distinct areas, such parasites would be highly useful for forensic investigations. Epstein-Barr virus (EBV) is one that is ubiquitous in healthy populations throughout the world. It has infected more than 90% of the world’s adult population [39]. However, EBV is an
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etiological agent of infectious mononucleosis and several neoplasmas such as nasopharyngeal carcinoma (NPC) and African Burkitt’s lymphoma [40]. These EBV associated tumors show considerable geographical variation. Burkitt’s lymphoma is endemic to equatorial Africa and Papua New Guinea, while NPC is common in Southeast Asia, Mediterranean Africa, and Inuit populations [41]. It was originally suspected that particular oncogenic EBV strains cause specific tumors in genetically susceptible populations. Though many researchers have attempted to identify such viruses, viral strains are only related to geographic area, not to diseases [42–45]. This geographical diversity is seen mainly within the genes for Epstein-Barr Virus Nuclear Antigen 3 (EBNA3), EBNA4, and Latent Membrane Protein 2A (LMP2A) of the EBV gene [46]. Based on these above findings, the forensic application of EBV genotype has recently been reported [47]. In that report, two hundred micro-milliliters of whole blood samples from 56 Japanese cases were collected during forensic autopsies. A 447bp hyper variable region of the EBV LMP2A gene was PCRamplified. The PCR amplified fragment were sequenced and analyzed phylogenetically with worldwide reference data obtained from a database. Detection using an EBV detection kit was also done to confirm the detection. Among the 56 samples, the LMP2A region of the EBV genome was amplified in nine samples (16%). Using the EBV detection kit, EBV DNA was detected in 11 samples (18%). All of the LMP2A-amplified samples were detected with this method. At least two copies/ml of EBV DNA were also detected. However, the lowest detected concentration of EBV DNA was six copies/ml in LMP2A amplified samples. From those LMP-2A sequences, together with reference sequences obtained from previously reported sequences, a phylogenetic tree was constructed using the NJ method [48]. From the resulting phylogenetic tree, these EBV isolates from the various patient populations were classified into two major clusters, types I and II. Type I was sub-classified into three subclusters, Ia, Ib and Ic, and type II was also sub-classified into three sub-clusters, IIa, IIb and IIc. All the Japanese isolates were included in subtypes Ia or IIa. All the Asian reference isolates except for the isolate D6, which was included in Ic, were included in subtypes Ia or IIa. Mediterranean isolates, an Alaskan isolate and other African isolates were included in types Ib, Ic, IIb and IIc. The EBV detection rate in the results reported in this study was not so high. However, the EBV detection rates from blood taken as forensic samples were almost the same using the two methods. Wanger et al. [49] also reported that previously reported detection rates of EBV DNA from peripheral blood were very low, ranging from 0 to 54%; however, when they used purified B-cells, the detection rate became 93.8%. The low detection rate may mainly have been caused by post-mortem decomposition and DNA extraction from whole blood samples. If sample DNA was extracted from purified B-cells, the detection rate should improve. However, it is normally impossible to use samples taken under good conditions for analysis in the field of forensic medicine. Though the detection rate was not so high, EBV DNA was detectable from samples
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that had a post-mortem time under 48 h. As most deceased are found within 2–3 days after death, this detection method may therefore be acceptable in forensic cases. The results show that each EBV genotype has a geographic correlation (Table 1). Especially, all the Japanese isolates were only included in Asian clusters. Most of the isolates from other geographical regions included region-specific clades. Based on the findings, together with those reported previously, EBV genotype distribution is correlated to geographical area; if the EBV genotypes from various regions were obtained, it would be possible to estimate the geographic origins of unidentified cadavers and blood stains by detecting the EBV genotype. There are many studies reporting multiple EBV infection, especially in immunosuppressed patients, who are known to have a high level of oral EBV shedding [50,51], co-infection with multiple EBV genotypes [52–54], and temporal changes in EBV populations [55,56]. However, even in healthy individuals, multiple EBV infection is reported. Walling et al. [52] reported that about 22% of healthy individuals have multiple infections. Multiple infections may affect estimations of geographical origins of unidentified cadavers. However, even if several strains were detected from one individual, it may be categorized into a single genotype by phylogenetic analysis because those strains may infect hosts in the same geographic area. Thus, multiple infections may not affect the results of estimations of geographic origin. Further confirmation studies will of course be required. Thus it has been demonstrated that EBV markers may also be a very important in the field of forensic medicine. 5. Conclusion The recent trends of internationalization and more the complicated criminal activity resulting have made the identification of missing people very difficult. Genotyping of human parasites is a very simple and efficient way to estimate the geographic origin of human individuals and so helps narrow down the geographical search area for cadaver identification. However, this method is not useful for all cases and results are not always absolute. However, by using genotyping methods, we can initiate investigations of missing people in much more probable areas. Our modern highly developed transportation systems and social internationalization will mean that cases in which geographical information does not agree with racial information will greatly increase in the future. Most cadavers are found in the early stage of the post-mortem period and most still have soft tissue; therefore, in addition to the other methods for racial estimations, including morphological characteristics [57] and human DNA polymorphisms [58–60], the parasite method makes more detailed profiling and investigation of unidentified cadavers both more efficient and economical. As many parasites have co-evolved with humans, there will be many other parasites that can be used for geographic identification; HHV, HTLV-1, HBV, Candida albicans, Helicobacter pylori are possible candidates (Table 2). JCV is the virus for which its evolutional history has been almost perfectly synchronized to the history of modern human migrations [61]. If the geographical variation of
Table 2 Possible parasites that may be used for geographic identification Parasites
Referencesa
Human herpes virus 1 Human T-cell lymphotropic virus 1 Hepatitis B virus Candida species Helicobacter pylori
[63,64] [65–67] [68,69] [70,71] [72–75]
a These references indicate correlation between geographic area and genotype or species.
genotypes was not constructed from the migration of modern humans, it would be impossible to estimate human geographical origins. Even if a human is a present host to the parasite, the host might be changed once or more in its life history. In that case, it would be very difficult to estimate the host’s geographical origins from its genotype diversity. Therefore, it is obviously very important to study the evolutional history of these parasites. From such studies, by analyzing the genotype distribution of many types of human parasites, it will be possible to estimate human geographic origins more precisely and with a high confidence rate. References [1] C. Cattaneo, Unidentified cadavers and human remains in the EU: an unknown issue, Int. J. Legal Med. 113 (1999) N1–N3. [2] Ministry of Health, Labor and Welfare Japan. Vital Statics Japan, Vol. No. 3 (2006). [3] The National Missing Persons Helpline. http://www.missingpersons.org. [4] H. Ikegaya, H. Iwase, C. Sugimoto, Y. Yogo, JC virus genotyping offers a new means of tracing the origins of unidentified cadavers, Int. J. Legal Med. 116 (2002) 242–245. [5] C.N. Cole, S.D. Conzen, Polyomaviridae: the viruses and their replication, in: D.M. Knipe, P.M. Howley, D.E. Griffin, et al. (Eds.), Fields Virology, fourth ed., Lippincott, Philadelphia, 2001, pp. 2141–2173. [6] B.L. Padgett, D.L. Walker, G.M. ZuRhein, R.J. Eckroade, B.H. Dessel, Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy, Lancet 1 (7712) (1971) 1257–1260. [7] T. Kunitake, T. Kitamura, J. Guo, F. Taguchi, K. Kawabe, Y. Yogo, Parentto-child transmission is relatively common in the spread of the human polyomavirus JC virus, J. Clin. Microbiol. 33 (1995) 1448–1451. [8] T. Kitamura, C. Sugimoto, A. Kato, Persistent JC virus (JCV) infection is demonstrated by continuous shedding of the same JCV strains, J. Clin. Microbiol. 35 (1997) 1255–1257. [9] Y. Yogo, C. Sugimoto, H.Y. Zheng, H. Ikegaya, T. Takasaka, K. Kitamura, JC virus genotyping offers a new paradigm in the study of human populations, Rev. Med. Virol. 14 (2004) 179–191. [10] G.S. Ault, G.L. Stoner, Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences, J. Gen. Virol. 73 (1992) 2669–2678. [11] H. Ikegaya, H. Iwase, Y. Yogo, Detection of Identical JC virus DNA Sequences in Both Kidneys, Arch. Virol. 149 (2004) 1215–1220. [12] H. Ikegaya, H. Iwase, H.Y. Zheng, M. Nakajima, K. Sakurada, T. Takatori, M. Fukayama, Y. Yogo, JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues, J. Virol. Methods. 126 (2005) 37–43. [13] K. Sakurada, H. Ikegaya, H. Motani, H. Iwase, K. Sekiguchi, T. Akutsu, M. Yoshino, T. Takatori, I. Sakai, JC virus genotyping can be used to narrow down the native place of persons from urine stains, Jpn J. Forensic Sci. Technol. 10 (2) (2005) 111–117. [14] B. Sobrino, M. Brion, A. Carracedo, SNPs in forensic genetics: a review on SNP typing methodologies, Forensic Sci. Int. 154 (2–3) (2005) 181– 194.
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