Genotypic Polymorphisms of Hepatitis B Virus Provide Useful Information for Estimating Geographical Origin or Place of Long-term Residence of Unidentified Cadavers
Abstract
Increasing numbers of unidentified cadavers are a major problem. We have developed a new method for providing identification information that can determine the geographical origin or place of long-term residence of unidentified cadavers based on genotypic polymorphisms of hepatitis B virus (HBV) known to correlate with their geographical distribution. PCR of serum samples detected HBV DNA from four (3.9%) of 102 randomly selected Japanese forensic cadavers. Multiplex PCR did not detect multiple HBV genotypes from any single cadaver, confirming the absence of coinfection. Phylogenetic tree analysis based on a 485-bp mutant region of the HBV S gene successfully classified the HBV genotypes into A to J. Among ten HBV-infected cadavers, eight had genotype Ce/C2, a genotype prevalent in East Asia, and two had genotype Bj/B1, a Japanese-specific genotype. HBV genotypic polymorphisms correlate with the geographical distribution of the virus and thus provide important information for identifying unidentified cadavers infected with HBV.
Introduction
The large number of bodies that remain unidentified due to a lack of identifying information continues to be a major problem worldwide. In eight European countries, a total of 3,035 unidentified cadavers were found over a period of four years between 1994 and 1998, of which 2,228 were subsequently identified and 807 remained unidentified. In Japan, about 1,000 unidentified bodies are found every year, and as many as 12,853 unidentified cadavers accumulated between 1999 and 2008.
Information used for the identification of cadavers includes personal belongings, facial and physical characteristics, fingerprints, dental findings, and human DNA typing. A lack of such information may complicate identification, and therefore, efforts have been made to develop new methods for collecting information that can help identify cadavers. One of these methods estimates the geographical origin of a cadaver based on geographical differences in DNA polymorphisms of viruses that reside in humans latently and continuously over the long term. To date, estimation methods of geographical origin based on DNA polymorphisms have been developed for the JC, BK, Epstein-Barr, and varicella-zoster viruses. These viruses infect infants and children and establish lifelong latent infection. They also have DNA polymorphisms that correlate with their geographical distribution. In this study, based on the expectation that the use of DNA polymorphisms of multiple viruses will provide investigators with more information and thus help with the identification of cadavers, we focused on establishing a method for hepatitis B virus that has DNA polymorphisms correlated with geographical distribution.
Hepatitis B virus is present in the blood, serum, saliva, semen, and vaginal fluid of an infected individual and is transmitted to others through the skin and mucous membranes that come into contact with these fluids. Approximately 350 million people are persistently infected with HBV worldwide, although the prevalence of HBV varies across regions of the world; the prevalence is low in Europe and the United States and is high in Asia, with 75% of the world’s infected individuals living in Asia. Hepatitis B virus is classified into ten genotypes, A to J, based on 8% or more of differences detected on molecular phylogenetic analysis of the full genome. Each of the genotypes is further divided into region-specific subgenotypes, based on 4% or more of differences.
Hepatitis B virus genotype A is divided into subgenotypes Aa/A1, Ae/A2, and Ac/A3, which are predominantly distributed in South and East Africa, Europe and the United States, and West and Central Africa, respectively. Genotype B is divided into subgenotypes Bj/B1, Ba/B2, Ba/B3, Ba/B4, and Ba/B5, predominantly distributed in Japan, China and Taiwan, Indonesia, Vietnam, and the Philippines, respectively. Genotype C is most prevalent in Asia and is divided into subgenotypes Cs/C1, Ce/C2, C3, C4, and C5, which are distributed in Southeast Asia, East Asia, Polynesia, Aboriginal Australia, and the Philippines and Vietnam, respectively. Genotype D is widely distributed around the world, but is most prevalent in the Mediterranean region, Middle East, and India. Genotype E is distributed in West and Central Africa, and genotypes F and H are most frequently detected in South and Central America. Genotype G was discovered in France and the United States, and genotype I was discovered in Laos and is proposed on a tentative basis. Lastly, genotype J was isolated from an elderly Japanese man who had lived in Borneo during World War II and has not been isolated anywhere else in the world.
In this study, with the aim of developing a method for estimating the geographical origin or place of long-term residence of unidentified cadavers, we examined whether HBV DNA can be detected from forensic cadavers and present here the detection rates for the virus from Japanese cadavers. We also quantitated HBV DNA detected from cadavers by real-time polymerase chain reaction and determined the nucleotide sequences of a region-specific polymorphic region of HBV DNA to classify viral genotypes.
Materials and Methods
Samples
With approval of the Ethics Committee of Chiba University, serum samples were extracted from blood collected from 102 cadavers (64 men, 38 women; age range 5 months to 96 years; approximate time since death 1 to 5 days), randomly selected from 461 legal autopsy cases at the Department of Forensic Medicine, Chiba University between 2009 and 2010. All cadavers were Japanese and had no history of immunodeficiency or clear evidence of severe infection leading to death. Another six serum samples from HBV-infected cadavers between 2006 and 2008 were also subjected to analysis. The serum samples were stored at minus 20 degrees Celsius immediately after collection.
Separately, eight blood samples were obtained from hepatitis patients who gave informed consent and were used as control for HBV DNA quantification by real-time PCR. Hepatitis B virus genotype DNA was extracted from 100 microliters of serum using the QIAamp DNA Mini Kit according to the manufacturer’s instructions. Final DNA elution was performed with 40 microliters of AE buffer, and extracted DNA was stored at minus 20 degrees Celsius.
PCR
A 485-base pair mutant region of the HBV S gene was amplified by PCR using extracted DNA as the template. The sequence of the sense primer used was 5′-GTCTAGACTCGTGGTGGACTTCTCTC-3′ and that of the antisense primer was 5′-AAGCCANACARTGGGGGAAAGC-3′. PCR was performed in a volume of 50 microliters consisting of 0.2 micromolar each of the sense and antisense primers, 0.2 millimolar of dNTP, PCR buffer and 1.25 units of AmpliTaq Gold DNA polymerase. The amplification reaction was performed with GeneAmp PCR System 9700 under the following conditions: 95°C for 11 minutes, followed by 45 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for one minute, and then final extension at 72°C for ten minutes. The PCR-amplified product was subjected to 2% agarose gel electrophoresis, stained with ethidium bromide, then visualized by ultraviolet light to confirm amplification.
Multiplex PCR
Serum samples from the blood collected from ten HBV DNA-positive Japanese cadavers (four bodies in 2009–2010 and six bodies in 2006–2008) were subjected to multiplex PCR to determine whether multiple HBV genotypes (i.e., coinfection) can be detected from any single cadaver. Multiplex PCR can detect, with high sensitivity, genotypes A, B, C, D, E, and F and subgenotypes B1, B2, C1, and C2, which have been shown to establish coinfection. Individual primer pairs or combined primer pairs of each HBV genotype A to F were added into one PCR reaction, while individual primer pairs or combined primer pairs of HBV subgenotype B1, B2, C1, or C2 were added into another PCR reaction. Genotypes were determined based on the difference in the size of PCR amplification products. PCR was performed in a volume of 50 microliters consisting of 1.25 units of AmpliTaq Gold DNA polymerase, 0.2 micromolar each of the primers, 0.2 millimolar of dNTP, and PCR buffer. The sequences of the primers were those reported by Chen et al. PCR was performed under the following conditions: 95°C for 11 minutes, followed by 35 cycles at 94°C for 60 seconds, 58°C for 60 seconds, 72°C for 60 seconds, and then final extension at 72°C for ten minutes. The PCR-amplified product was subjected to 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized by ultraviolet light to confirm amplification.
Sequencing
PCR products were subjected to cycle sequencing using the Big Dye Terminator Cycle Sequencing Kit v3.1, and the same primers were used as for PCR amplification described above. Cycle sequencing was performed in a volume of ten microliters containing primers at a final concentration of 0.25 pmol per microliter. The cycling condition was 25 cycles at 96°C for 30 seconds, 50°C for 15 seconds, and 60°C for two minutes. Cycle sequencing products were purified with the QuickStep 2 PCR Purification Kit. DNA sequencing was performed using an ABI 310 DNA Analyzer.
Phylogenetic Tree Analysis
Sequence alignment was performed using the Clustal X program on the 485-base pair region of the S gene from the ten HBV DNA-positive Japanese cadavers and the 132 strains of HBV derived from DNA databases. Aligned sequences were analyzed by the neighbor-joining method to construct a phylogenetic tree, which was visualized using the NJplot program. To confirm the reliability of the phylogenetic tree, bootstrap resampling tests were carried out 1,000 times. After alignment analysis was completed, single nucleotide polymorphisms were identified and amino acid changes were selected that would allow for the classification of genotypes A to J after amino acid substitution.
HBV DNA Quantification
Hepatitis B virus DNA quantification by real-time PCR was performed for samples derived from the ten HBV DNA-positive Japanese cadavers and eight hepatitis patients, and the results were compared between the two groups. Real-time PCR was performed as reported by Lole et al. The sequence of the sense primer was 5′-TAGGAGGCTGTAGGCATAAATTGG-3′ and that of the antisense primer was 5′-GCACAGCTTGGAGGCTTGT-3′. The size of the amplification product was 109 base pairs. The sequence of the TaqMan MGB probe was 5′-TCACCTCTGCCTAATC-3′. PCR amplification was performed in a 50 microliter reaction mixture containing TaqMan Universal Master Mix, 0.7 micromolar each of the sense and antisense primers, 0.15 micromolar of TaqMan probe, and five microliters of template DNA. StepOnePlus real-time PCR systems were used for real-time detection of amplification products. The amount of HBV DNA was compared statistically between the cadaver and hepatitis patient groups using Student’s t-test.
Results
Detection Rate from Cadavers
Hepatitis B virus DNA was detected from the blood samples of four of 102 (3.9%) forensic cadavers. When liver samples from thirty cadavers, including the four HBV-positive cadavers, were subjected to HBV DNA detection, the viral DNA was detected only in the same four cadavers. The demographics and cause of death of the four positive cadavers were as follows: a 39-year-old man who died one day earlier from loss of blood; a 40-year-old man who died two days earlier from a fatal injury; a 66-year-old woman who died three days earlier following a traffic accident; and a 76-year-old man who died two days earlier from a fatal injury.
Determination of HBV Genotypes
Sequence alignment for the S gene region of the 132 reference strains of HBV genotypes A to J revealed 118 single nucleotide polymorphisms and 35 corresponding amino acid changes reflecting genotypic differences. From these amino acid changes, seven changes were selected based on which HBV genotypes can be easily distinguished. Phylogenetic tree analysis of 77 reference strains successfully classified HBV genotypes into A to J. Subtype analysis revealed that genotype B included a cluster consisting only of subgenotype Bj, which is rarely detected outside Japan, and genotype C included a cluster consisting only of subgenotype Cs, which is most prevalent in Southeast Asia, and subgenotype Ce, which is detected from Japan, South Korea, and part of China and included Japanese, Chinese, and Korean strains.
The HBV genotypes for the ten positive Japanese cadavers detected in this study were Bj/B1 for two cadavers and Ce/C2 for the remaining eight. In the phylogenetic tree analysis, the two cadavers were included in the same cluster as genotype Bj/B1 and the eight cadavers were included in the same cluster as genotype Ce/C2.
HBV DNA Quantification
Serum samples from HBV DNA-positive cadavers were subjected to HBV DNA quantification by real-time PCR. The copy number of HBV DNA ranged from 5 × 10^2 to 4.3 × 10^6 copies per milliliter, which was not significantly different from those detected in HBV-infected patients (p > 0.05).
Coinfection
Multiplex PCR of the serum samples from the ten HBV DNA-positive Japanese cadavers was used to determine the presence of multiple HBV genotypes in each cadaver. One PCR tube was used for the detection of genotypes A to F, and another was used for the detection of subgenotypes B1, B2, C1, and C2. The band was confirmed based on the difference in the size of PCR amplification products by genotypes. All samples showed only one band, demonstrating the absence of coinfection.
Discussion
Detection of HBV DNA from Cadavers
This study demonstrates that it is possible to detect HBV DNA from forensic cadavers. HBV DNA was detected in serum samples from only four of 102 (3.9%) forensic cadavers. Considering that the prevalence of HBV among the Japanese population is reported to be around one percent and given that HBV DNA was detected from liver samples of the same cadavers and that HBV antigen was also detected from the same cadavers, the present technique is capable of precisely detecting HBV DNA from cadavers without false positive or negative results. Unlike biological samples, samples from cadavers undergo deterioration and putrefaction, which promote nucleic acid degradation. However, the method described here allows for reliable detection and genotyping of A-485 HBV DNA from postmortem samples, providing valuable information for forensic identification.