高橋 雅春

We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 and A549 cells, using fecal specimens from HEV-infected patients. Since transfusion-associated hepatitis E has been reported, we examined PLC/PRF/5 and A549 cells for the ability to support replication of HEV in various serum samples obtained from 23 patients with genotype 1, 3, or 4 HEV. HEV progenies emerged in culture media of PLC/PRF/5 cells, regardless of the coexistence of HEV antibodies in serum but dependent on the load of HEV inoculated (31% at 2.0 x 10(4) copies per well and 100% at >= 3.5 x 10(4) copies per well), and were successfully passaged in A549 cells. HEV particles in serum, with or without HEV antibodies, banded at a sucrose density of 1.15 to 1.16 g/ml, which was markedly lower than that for HEV particles in feces, at 1.27 to 1.28 g/ml, and were nonneutralizable by immune sera in this cell culture system. An immuno-capture PCR assay of HEV virions treated with or without detergent indicated that HEV particles in serum are associated with lipids and HEV ORF3 protein, similar to those in culture supernatant. By immunoprecipitation, it was found that >90% of HEV particles in the circulation exist as free virions not complexed with immunoglobulins, despite the coexistence of HEV antibodies. These results suggest that our in vitro cell culture system can be used for propagation of a wide variety of HEV strains in sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains.

To investigate nationwide the prevalence of hepatitis E virus (HEV) infection in the general population of Japan, serum samples were collected from 22,027 individuals (9,686 males and 12,341 females; age, mean +/- standard deviation: 56.8 +/- 16.7 years; range: 20-108 years) who lived in 30 prefectures located in Hokkaido, mainland Honshu, Shikoku, and Kyushu of Japan and underwent health check-ups during 20022007, and were tested for the presence of IgG, IgM, and IgA classes of antibodies to HEV (anti-HEV) by in-house ELISA and HEV RNA by nested RT-PCR. Overall, 1,167 individuals (5.3%) were positive for anti-HEV IgG, including 753 males (7.8%) and 414 females (3.4%), the difference being statistically significant (P<0.0001). The prevalence of anti-HEV IgG generally increased with age and was significantly higher among individuals aged >= 50 years than among those aged <50 years (6.6% vs. 2.7%, P<0.0001). Although 13 individuals with anti-HEV IgG also had anti-HEV IgM and/or anti-HEV IgA, none of them had detectable HEV RNA. The presence of HEV RNA was further tested in 50 or 49-sample minipools of sera from the remaining 22,014 individuals, and three individuals without anti-HEV antibodies tested positive for HEV RNA. The HEV isolates obtained from the three viremic individuals segregated into genotype 3 and were closest to Japan-indigenous HEV strains. When stratified by geographic region, the prevalence of anti-HEV IgG as well as the prevalence of HEV RNA or anti-HEV IgM and/or anti-HEV IgA was significantly higher in northern Japan than in southern Japan (6.7% vs. 3.2%, P<0.0001; 0.11% vs. 0.01%, P=0.0056; respectively). J. Med. Virol. 82:271-281, 2010. (C) 2009 Wiley-Liss, Inc.

We describe a case of interspousal transmission of hepatitis C virus (HCV) infection after 30 years of marriage which was confirmed by gene analysis. A 60-year-old man was referred to our hospital because of severe hepatic dysfunction. Laboratory findings showed that HCV-Ab titer and qualitative Amplicor HCV were both positive in low levels. Because the patient regularly consumes various health foods, it was initially difficult to rule out drug-induced hepatopathy, but the patient was diagnosed with acute hepatitis C when HCV antibody titer increased 4 months later. Because his wife also tested positive for HCV antibody, interspousal transmission was suspected, and gene analysis was performed. Both husband and wife had HCV 1b, and the base sequence homology of 1087 base pairs (bp) in the NS5B region was 98.6% (99.4% at the amino acid level). In addition, upon analysis of the E1 and E2 junctional region sequence (268 bp) including hypervariable region 1 (HVR-1), a close relationship (89.2-99.6%) between clones obtained from each spouse was observed, thus confirming that the source of infection was his wife. Thorough medical history taking suggested that sexual intercourse was the most likely route of infection. In previous large-scale clinical studies, the frequency of HCV infection between married couples has been extremely low, but it is important to obtain informed consent regarding the potential risk of infection. © 2009 Springer.

To compare the epidemiologic profiles of hepatitis A virus (HAV) and hepatitis E virus (HEV) infections in children in Mongolia, the prevalence of HAV and HEV infections was studied serologically and molecularly among 520 apparently healthy children 7-12 years of age (mean +/- standard deviation. 8.5 +/- 0.8 years) using seurm, samples obtained in 2004. total antibody against HAV (anti-HAV) was detected in 438 children (84.20%), whereas IgG antibody against HEV (anti-HEV IgG) was detected in only three subjects (0.6%). All three subjects with anti-HEV IgG were negative For anti-HEV IgM and HEV RNA. The presence of HAV RNA was tested in all 520 subjects, and one child (9-year-old girl) was found to have detectable HAV RNA (subgenotype IA). In conclusion, HEV infection was uncommon, but subclinical HAV infection was highly prevalent among children in Mongolia.

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (Delta ORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The Delta ORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of Delta ORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of Delta ORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the Delta ORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15-1.16 g ml(-1), in contrast with the Delta ORF3 mutant in culture supernatant, which banded at 1.27-1.28 g ml(-1), similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.

Hepatitis E virus (HEV) is one of the causative agents of acute or fulminant hepatitis. Viral factors may play a role in the pathogenesis of fulminant hepatitis E. We aimed to investigate the nucleotide substitutions of the HEV genome affecting the severity of hepatitis E. The comparison of 28 reported full-length nucleotide sequences of genotype 4 HEV showed that the substitution of C at nucleotide 5907 (C5907) was most closely associated with fulminant hepatitis (fulminant hepatitis, 100%; acute hepatitis, 39.1%; p = 0.0204). Analyzing the full-length sequences of 28 genotype-4 and 11 genotype-3 HEV retrievable from DNA databases and 35 partial sequences recovered from patients with acute or fulminant hepatitis, we show that the presence of both U3148 and C5907 is associated with fulminant hepatitis in patients with HEV of genotype 4 (p = 0.0042) and genotype 3 or 4 (p = 0.0009), and that the prothrombin activity is significantly lower in patients infected with HEV carrying U3148 and C5907 than in those without the substitutions (p = 0.0069). U3148 and C5907 are silent substitutions that do not change amino acid. However, since U3148 is located at the RNA helicase domain and C5907 is located within the capsid gene, the secondary structure of the HEV RNA genome carrying U3148 and C5907 may be favorable for translation of the viral proteins. C5907 was associated with high HEV load (>= 10(5) copies/ml) at initial examination (p = 0.0427). We propose that U3148 and C5907 are associated with the severity of hepatitis E.

A 23-year-old nurse (HC-IP) developed acute hepatitis C. Intrafamilial transmission of hepatitis C virus (HCV) was suspected initially because her parents were carriers of HCV of the same genotype (1b) as that of Patient H&IP. However, the HCV isolate from Patient HC-IP and those from her parents shared identities of only 92.4-92.7% in the 1,087-nucleotide (nt) sequence within the NS5B region. It was then suspected that she contracted HCV infection during medical practice. Sixteen patients with antibodies to HCV (anti-HCV) were hospitalized 1-3 months before she became positive for anti-HCV. Upon analysis of stored serum samples, 14 of the 16 patients were found to be positive for HCV RNA, and 9 of the 14 viremic patients had genotype 1b HCV. Although the shared identities between the HCV isolate from Patient HC-IP and those from eight of the nine patients were merely 90.6-93.9% within the 1,087-nt NS5B sequence, the HCV isolate from the remaining one patient (HC-P12) was 99.7% identical to that from Patient HC-IP. Upon analysis of the E1 and E2 junctional region including hypervariable region 1 (283 nt), there was a close relationship (99.3-100%) between clones obtained from Patients HC-IP and HC-P12. Although the nurse HC-IP had a finger injury, she took care of Patient HC-P12, a 70-year-old man with HCV-related cirrhosis and recurrent epistaxis, occasionally without wearing protective gloves. This study indicates the occurrence of HCV transmission by exposure of non-intact skin to blood in health care settings. J. Med. Virol. 81:1363-1370,2009. (C) 2009 Wiley-Liss, Inc.

Background: The risk of patient-to-patient transmission of hepatitis C virus (HCV) during endoscopy remains controversial. Using molecular approaches, we examined the possibility of patient-to-patient transmission of HCV in three patients who developed acute hepatitis C 1-6 months after examination by upper gastrointestinal endoscopy (UGIE) in a hospital endoscopy unit in Japan. Methods: For the source of HCV infection, we used frozen sera obtained from potential candidates who underwent UGIE earlier than three index patients on the same days in the same unit. HCV genotype was determined by multiplex polymerase chain reaction (PCR) with genotype-specific primers. The 1087-nucleotide (nt) sequence of the NS5B region of the HCV genome was compared between index patients and their HCV-viremic candidates. Results: The three index patients were exclusively infected with HCV of genotype 1b. Among a total of 60 candidate patients who underwent UGIE earlier than the index patients, 14 were positive for anti-HCV, of whom 12 had detectable HCV-RNA (1b, n = 9; 2a, n = 1; 2b, n = 2) on sera collected during each UGIE. Shared identity within the 1087-nt NS5B sequence was less than 95.0% between index patients and HCV/1b-infected candidates (n = 3, 1 and 5, respectively). None of the remaining 46 candidates who were negative for anti-HCV at UGIE examination tested positive for HCV-RNA, nor seroconverted to anti-HCV on their sera, which most likely excludes the possibility of HCV viremia despite the anti-HCV-negative serology at UGIE examination. Conclusion: The present study suggests that patient-to-patient transmission of HCV during UGIE is infrequent.

Upon phylogenetic analysis of a partial S gene sequence [396 nucleotides (nt)], 928 hepatitis B virus (HBV) strains obtained from 899 viremic subjects in 28 major cities on 15 islands of Indonesia in 1989-2007 segregated into four HBV genotypes. Genotype B was predominant (66%), followed by genotype C (26%), genotype D (7%), and genotype A (0.8%). Comparative and phylogenetic analyses of the 396-nt S gene sequence of 928 HBV isolates and whole genomic sequences of 25 selected HBV isolates revealed a total of 14 subgenotypes within genotypes A-D: two (A1 and A2) in genotype A (HBV/A), five (B2, B3, B5, B7, and a novel subgenotype, tentatively designated B8) in HBV/B, five (C1, C2, C5, C6, and another novel subgenotype, C7) in HBV/C, and two (D1 and D3) in HBV/D. The distribution of HBV genotypes/subgenotypes, including B8 and C7, seems to be associated with ethnological origins in Indonesia.

We developed an efficient cell culture system for genotype 4 hepatitis E virus using the HE-JF5/15F strain recovered from a fulminant hepatitis patient. The sixth-passage virus in the culture supernatant reached 1.5 x 108 copies/ml at 10 days postinoculation and possessed 10 nucleotide mutations with four amino acid changes.

Recent studies have shown that indigenous hepatitis E virus (HEV) strains cause hepatitis E in industrialized countries. We aimed to clarify the characteristics of HEV infection in sporadic hepatitis patients during the last decade in Miyagi, northeast Japan. We analyzed 94 serum samples obtained from acute or fulminant hepatitis patients of non-A, non-B, and non-C etiology between 1999 and 2008. Antibody to HEV (anti-HEV) was assayed, and patients who were positive for IgM- and/or IgA-class anti-HEV were diagnosed with hepatitis E. HEV RNA was tested in these patients, and phylogenetic analysis was performed. The occurrence of hepatitis E was compared with that of hepatitis A. Eight acute hepatitis patients (8.5%) were diagnosed with hepatitis E, and HEV RNA was detectable in seven patients. Five isolates of HEV were segregated into genotype 3 and the remaining two isolates into genotype 4. The year of the occurrence of hepatitis E was distributed almost equally from 1999 to 2008, whereas the cases of acute hepatitis A (n = 16) have decreased markedly in the last several years. In 2004-2008, the occurrence of hepatitis E was greater than that of hepatitis A (five cases vs. one case). As for seasonality, hepatitis E occurred more frequently from September to December than hepatitis A (five cases vs. four cases), although less frequently from January to April (one case vs. seven cases). The occurrence of hepatitis E has not decreased during the last decade in northeast Japan, in contrast to hepatitis A.

Humans are frequently infected with three anelloviruses which have circular DNA genomes of 3.6-3.9 kb [Torque teno virus (TTV)], 2.8-2.9 kb [Torque teno mini virus (TTMV)] and 3.2 kb [a recently discovered anellovirus named Torque teno midi virus (TTMDV)]. Unexpectedly, human TTMDV DNA was not detectable in any of 74 chimpanzees tested, although all but one tested positive for both human TTV and TTMV DNA. Using universal primers for anelloviruses, novel variants of TTMDV that are phylogenetically clearly separate from human TTMDV were identified from chimpanzees, and over the entire genome, three chimpanzee TTMDV variants differed by 17.9-20.3 % from each other and by 40.4-43.6 % from all 18 reported human TTMDVs. A newly developed PCR assay that uses chimpanzee TTMDV-specific primers revealed the high prevalence of chimpanzee TTMDV in chimpanzees (63/74, 85%) but low prevalence in humans (1/100). While variants of TTV and TTMV from chimpanzees and humans were phylogenetically interspersed, those of TTMDV were monophyletic for each species, with sequence diversity of <33 and <20 % within the 18 human and three chimpanzee TTMDV variants, respectively. Maximum within-group divergence values for TTV and TTMV were 51 and 57 %, respectively; both of these values were substantially greater than the maximum divergence among TTMDV variants (44 %), consistent with a later evolutionary emergence of TTMDV. However, substantiation of this hypothesis will require further analysis of genetic diversity using an expanded dataset of TTMDV variants in humans and chimpanzees. Similarly, the underlying mechanism of observed infrequent cross-species infection of TTMDV between humans and chimpanzees deserves further analysis.

A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed ill this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 10(7) copies ml(-1) on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 10(6) copies ml(-1) at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins.

We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 cells, using a genotype 3 HEV (JE03-1760F strain). Thirteen generations of consecutive passages of culture supernatant were successfully carried out in PLC/PRF/5 cells, with the highest HEV load reaching 10(8) copies/ml in the culture medium. Based on continuous release of progenies into culture medium, 50% tissue culture infectivity doses were estimated to be 2.0 x 10(3) Copies for wild-type JE03-1760F and 1.4 x 10(2) copies for p 13 (progeny in the thirteenth passage). Earlier appearance and greater increase in the yield of progenies in the culture supernatant were evident in p13 compared with wild-type. The cell culture-produced variants in primary propagation (p0)and consecutive passages (p5 [fifth passage], p10 [tenth], and p13)differed from the wildtype virus by 1. 9, 18, and 19 nucleotides (nt), respectively, over the entire genome of 7226 nt, excluding the poly(A) tail. Three of five non-synonymous Mutations in p13 were shared by a variant (fifth passage) in another series of passages of JE03-1760F. These results suggest that adaptation of HEV variants to growth in vitro is associated with a limited number of mutations similar to hepatitis A virus. (C) 2008 Elsevier B.V. All Fights reserved.

Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of > 10(6) copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm(3) in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.

Although many extrahepatic manifestations have been described in patients with acute or chronic hepatitis B, there are few reports about neurological disorders. We describe a 55-year-old man who contracted acute hepatitis B virus (HBV) infection and transverse myelitis. His neurological findings were gradually reduced along with the recovery from hepatitis. The cerebrospinal fluid (CSF) was revealed to be positive for HBsAg and HBV DNA. Full-length sequences of HBV in his serum and CSF were determined, and it was revealed that these two isolates had Mutations at nucleotide (m) 1762/1764 in the core promoter region and nt 1896 in the precore region. They were identical to each other except for two ambiguous codes at nt 2020 and 2631 in the CSF isolate. After cloning of the amplicons, substitutions at nt 2020 and 2631 were found in 6 (38%) of the 16 CSF clones. One clone of the 6 CSF clones had an additional substitution at nt 2119. These substitutions were not found in 16 serum clones. The presence of HBV clones unique to CSF suggests that HBV was a possible causative agent of the myelitis. (C) 2008 Elsevier B.V. All rights reserved.

Nine murine monoclonal antibodies (mAbs) generated against a recombinant ORF2 protein (amino acids 111-660) of a genotype 4 hepatitis E virus (HEV) strain recognized four sets of epitopes by pairwise competitive ELISA. One mAb (H6225) was able to capture HEV efficiently regardless of genotype and was tested for its ability to neutralize a genotype 3 HEV strain (JE03-1760F) in a recently developed cell culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). When PLC/PRF/5 cells were inoculated with HEV (4.0 x 10(5) or 4.0 x 10(6) copies/ml) incubated with 100 mu g/ml of a negative control mAb, HEV RNA in the culture medium continued to be detectable after day 14 or 12 post-inoculation (dpi), respectively. However, when cells were inoculated with the two distinct concentrations of HEV that had been mixed with 100 mu g/ml of H6225, the harvested culture supernatants were negative for HEV RNA throughout the 60-day observation period. Upon prior mixing of the virus with 10 mu g/ml of H6225, HEV RNA in culture supernatant continued to be undetectable until 46 or 28 dpi, respectively. In conclusion, one mAb (H6225) against HEV capsid protein that can efficiently neutralize HEV in vitro was obtained in the present study.

Background and objective: IPF is an independent risk factor for lung cancer, but the mechanism of this association has not fully been elucidated. The role of Torque teno virus (TTV) in respiratory disease is poorly understood, although it has been shown that infection with TTV is associated with the activity and prognosis of IPF. This study aimed to investigate the prevalence and titre of TTV DNA among patients with IPF and lung cancer. Methods: The presence of TTV DNA was determined by PCR in the sera of patients with both lung cancer and IPF (n = 22), patients with IPF only (n = 35), and patients with lung cancer only (n = 142). Results: TTV DNA was detectable in all patients with both IPF and lung cancer, in 94.3% of the patients with IPF only and 97.2% of the patients with lung cancer only. The TTV DNA titre in the patients with IPF and lung cancer was significantly higher than that in the patients with IPF only or lung cancer only. The percentage of TTV-positive patients with a high TTV titre in the IPF and lung cancer group was significantly higher than that in the IPF only group. Conclusions: These findings are the first report on the association between TTV and the complication of lung cancer in IPF and suggest that TTV infection might be associated with the development of lung cancer in IPF.

We recently identified a novel human virus classifiable into a third group in the genus Anellovirus, tentatively designated torque teno midi virus (TTMDV), with a circular DNA genome of 3.2 kb and genomic organization resembling those of torque teno virus (TTV) (3.8 to 3.9 kb) and torque teno mini virus (TTMV) (2.8 to 2.9 kb). TTMDV was characterized by extreme genetic diversity similar to the TTV and TTMV genomes. Taking advantage of universal and virus species-specific primers derived from a highly conserved area located just downstream of the TATA box of the TTV, TTMDV, and TTMV genomes, a PCR method with simultaneous amplification of the genomic DNAs of these three anelloviruses in the first round and subsequent differential amplifications of these viruses in the second round was developed. High prevalence of TTMDV viremia was seen in adults (75/100 [75%]), comparable with the prevalences of TTV viremia (100%) and TTMV viremia (82%). Although none of 10 cord blood samples had detectable TTV, TTMDV, and TTMV DNAs, the prevalences of these three anelloviruses increased with the number of months after birth of the individual and reached 100% for individuals at one year of age. Dual or triple infection of TTV, TTMDV, and/or TTMV was seen in 10 (47.6%) of 21 infants 9 to 180 days of age and more frequently among infants 181 to 364 days of age (20/23 [86.9%]), comparable with the 93.1% (243/261) prevalence among subjects 1 to 81 years of age, indicating early acquisition of dual or triple anellovirus infection during infancy.

67歳の男性が結婚40年後にC型急性肝炎を発症した．その妻（68歳）がC型慢性肝炎患者であり，C型肝炎ウイルス（HCV）RNAが高力価陽性（>5,000 KIU/ml）であった．両者のHCV遺伝子型はともに1b型で，NS5B領域の1,087塩基長の配列において99.7%の一致率を示した．それに対し，これまでに報告されているHCV/1b株との一致率は最高でも96.7%に過ぎなかった．分子系統樹解析によっても，夫婦の持つHCV株は一つのクラスター（bootstrap値：100%）を形成し，同一株である可能性が強く示唆された．詳細な病歴聴取を行ったが，性交渉（月1, 2回）以外の感染経路はいずれも否定された．高齢夫婦間の感染には加齢に伴う生体側因子が関与していると考えられるが，高齢化社会を迎え，HCV感染者頻度の高い高齢者層でかかるHCV感染が起こる可能性を念頭に置く必要があると考え報告する．<br>

Background: Little is known about the prevalence of hepatitis B virus (HBV) DNA and the genotype distribution among patients with liver diseases in Nepal, where obstruction of the hepatic portion of the inferior vena cava (IVCO) is common. The aim of the present paper was to assess the roles of HBV infection and IVCO in liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Nepal. Methods: Serum samples from 121 patients (89 male, 32 female; age, 55.0 +/- 13.6 years) with or without IVCO consisting of 70 LC patients and 51 HCC patients in Nepal, were tested for HBV-DNA. Results: The HBV-DNA was detected in 68 patients (56%) including 20 hepatitis B surface antigen (HBsAg)-negative patients: 33 LC patients (47%) and 35 HCC patients (69%) had detectable HBV-DNA (P = 0.0303). Among the 89 patients with IVCO, HBV-DNA was detected in HCC patients significantly more frequently than in LC patients (80% vs 43%, P = 0.0005). The frequency of HBV viremia was significantly higher among CC patients with IVCO than those without (80% vs 44%, P = 0.0236), and that of HBV viremia with IVCO was significantly higher among HCC patients than among LC patients (55% vs 27%, P = 0.0153). The HBV genotypes A and D were predominant, and genotype A was significantly more frequent among HCC patients than among LC patients (22% vs 6%, P = 0.0090). Among HCC patients, those with genotype A HBV were significantly younger than those with genotype D (43 +/- 13 vs 57 +/- 12 years, P = 0.0252). Conclusion: Hepatitis B virus alone (especially genotype A) or in concert with IVCO may be responsible for development of HCC in Nepal.

To investigate the duration of fecal shedding and changing loads of hepatitis E virus (HEV) in feces and serum from patients with acute HEV infection, HEV RNA was quantitated in periodic serum and fecal specimens obtained from 11 patients with sporadic acute hepatitis E. All 11 patients had detectable HEV RNA in serum at admission, with the highest viral load being 1.9 x 10(3) to 1.7 x 10(7) copies/ml, and HEV viremia lasted until days 17 to 48 (mean, 28.3) after the onset of hepatitis. Even at the initial examination on days 10 to 29 (mean, 17.6), the HEV load in fecal supernatant was less than 5.7 X 10(4) copies/ml for 10 of the 11 patients, while for the remaining patient (patient 1) it was markedly high, 2.0 X 10(7) copies/ml on day 22. In addition, although HEV RNA in fecal supernatant continued to be positive until days 14 to 33 (mean, 22.4) for patients 2 to 11, that for patient I was detectable even on day 121. HEVs in fecal specimens obtained on days 22, 24, 26, 28, and 30, but not day 121, from patient 1 grew efficiently in PLC/PRF/5 cells, reaching the highest titer Of Up to 107 copies/ml in culture medium on day 50 postinoculation. The HEV genome recovered from patient 1 had 29 unique nucleotides that were not seen in any of the 25 reported HEV isolates of the same genotype over the entire genome, with six amino acid substitutions in the ORF1 protein.

Recently, we identified a novel human virus with a circular DNA genome of 3.2 kb, tentatively designated as torque teno midi virus (TTMDV), with a genomic organization resembling those of torque teno virus (TTV) of 3.8-3.9 kb and torque teno mini virus (TTMV) of 2.8-2.9 kb. To investigate the extent of genomic variability of TTMDV genomes, the full-length sequence was determined for 15 TTMDV isolates obtained from viremic individuals in Japan. The 15 TTMDV isolates comprised 3175-3230 bases and shared 67.0-90.3% identities with each other, and were only 68.4-73.0% identical to the 3 reported TTMDV isolates over the entire genome. TTMDV possessed a genomic organization with four open reading frames (ORF1-ORF4) with characteristic sequence motifs and stem and loop structures with high GC content, similar to TTV and TTMV. The total of 18 TTMDV genomes differed by up to 60.7% from each other in the amino acid sequence of ORF1 (658-677 amino acids), but segregated phylogenetically into the same cluster, which was distantly related to the TTVs and TTMVs. These results indicate that TTMDV with a circular DNA genome of 3.2 kb, has an extremely high degree of genomic variability, and is classifiable into a third group in the genus Anellovirus.

Hepatitis E is rare in Japan but is occurring more frequently than previously thought. To investigate whether de novo subclinical infection of hepatitis E virus (HEV) has recently increased in Japan, HEV RNA was assayed in serum samples obtained from 4019 Japanese voluntary blood donors with alanine aminotransferase (ALT) of &gt;= 61 IU/l, who are likely to have ongoing HEV infection, during 1991-2006. The overall rates of IgG-class antibody to HEV (anti-HEV IgG), anti-HEV IgM/IgA and HEV RNA among 3185 donors in 2004-2006 were comparable with those among 594 donors in 1998 (5.3 vs. 5.2%, 0.2 vs. 0.5%, and 0.2 vs. 0.3%, respectively). Among blood donors with ALT &gt;= 201 IU/l in three groups according to the year of blood collection (1991-1995 [n = 156], 1996-1999 [n = 116] and 2004-2006 [n = 116], there were no appreciable differences in the prevalence of anti-HEV IgG (5.8, 4.3, and 6.6%, respectively), anti-HEV IgM/IgA (1.9, 3.4, and 3.3%, respectively) and HEV RNA (1.3, 3.4, and 3.3%, respectively). The eleven HEV isolates obtained in the present study differed from each other by 1.7 - 22.8% in the ORF2 sequence and segregated into genotype 3 or 4. The occurrence rate of subclinical infection with divergent HEV strains has essentially remained unchanged during 1991 - 2006 in Japan.

Mongolia is highly endemic for hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) infections among apparently healthy adults. However, the age-specific prevalence of ongoing HBV, HCV, and HDV infections among children in Mongolia remains unknown. Therefore, samples obtained from a total of 655 apparently healthy children of 0.3-15 years of age (307 boys and 348 girls; age, mean- +/- standard deviation [SD], 8.4 +/- 4.2 years) living in Mongolia, between October 2005 and January 2006, were tested for serological and molecular markers of HBV, HCV, and HDV infections. Although 88.7% of the 655 children studied were immunized against hepatitis B, 64 (9.8%) tested positive for hepatitis B surface antigen (HBsAg) and/or HBV DNA and 13 (2.0%) for HDV RNA. Twenty-seven children (4.1%) had detectable HCV RNA. Collectively, 82 (12.5%) were viremic for one or more of these viruses, including eight children with dual viremia of HBV/HCV and one child with triple HBV/HCV/HDV viremia. When children without anti-HBc, anti-HCV and anti-HDV IgG (n=510) served as a control, a history of hospitalization was significantly associated with HBV viremia (P &lt; 0.0001), anti-HBc positivity (P &lt; 0.0001), and HCV viremia (P=0.0001). HBsAg mutation was found in 18 (31.6%) of the 57 children with viremia, including those at amino acid position 126, 127, 129, 131, 134, 143 or 144. There were no significant differences in the frequency of HBsAg mutation in relation to age, sex, and hepatitis B vaccination status of the children, suggesting that HBsAg mutation plays a limited role in failure of vaccination in Mongolia.

Although no outbreaks of hepatitis E have been reported in Mongolia, a significant proportion of the general population had antibodies to hepatitis E virus (HEV). To investigate whether pigs are possible reservoirs of HEV in Mongolia, serum samples obtained from 243 2- or 3-month-old pigs on four swine farms surrounding Ulaanbaatar, the capital city of Mongolia, were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 223 pigs (91.8%) tested positive for anti-HEV, while 89 pigs (36.6%) had detectable HEV RNA. The 89 HEV isolates obtained from the viremic pigs were 78.7-100% identical to each other, and 80.9-85.9% similar to the prototype genotype 3 HEV isolate (US1) in the 412-nucleotide (nt) sequence within open reading frame 2. They were classified into two novel phylogenetic groups within genotype 3, differing by 16.4-21.3%. The swMN06-A1288 and swMN06-C1056 isolates, representing each of the two clusters within genotype 3, had a genomic length of nucleotides (nt) 7,222 nt and 7,223 nt, respectively, excluding the poly(A) tail, and shared only 81.6% over the entire genome. Upon comparison with the 25-reported genotype 3 HEV isolates over the entire genome, swMN06-A1288 had identities of merely up to 84.9%, while swMN06-C1056 of only up to 85.9%. Phylogenetic analysis confirmed the remote relatedness of the Mongolian swine isolates to the genotype 3 HEV isolates reported thus far. These results indicate that farm pigs in Mongolia are frequently infected with presumably indigenous HEV strains of genotype 3 and could be a source of HEV infections in humans in Mongolia.

A previous study revealed that antibodies to hepatitis E virus (HEV) (anti-HEV) are highly prevalent among healthy individuals and farm pigs in Bali, Indonesia, and suggested that HEV infection may occur via zoonosis among Balinese people. However, there were no reports of acute hepatitis E in Bali. To elucidate whether Balinese HEV strains recovered from infected humans and pigs have significant sequence similarity, serum samples obtained from 57 patients (age, mean standard deviation, 31.1 +/- 11.9 years) with sporadic acute hepatitis and from one hundred and one 2- or 3-month-old farm pigs in Bali were tested for anti-HEV and HEV RNA. Among the 57 patients, 2 (3.5%) had high-titer IgM/IgA class anti-HEV antibodies and one of them had detectable HEV RNA (BaliE03-46). Overall, 58 pigs (57.4%) tested positive for anti-HEV,while 5 pigs (5.0%) had detectable HEV RNA. Based on the 412-nucleotide sequence within open reading frame 2, the BaliE03-46 isolate and the 5 swine HEV isolates recovered from the viremic pigs were phylogenetically classified in genotype 4, but were only 77.3-90.8% identical to the genotype 4 HEV isolates reported thus far in China, India, Japan, Taiwan, and Vietnam. The BaliE03-46 isolate of human origin shared high identities of 97.3-98.3% with 4 of the 5 Balinese swine isolates, but differed by 16.1% from the remaining swine isolate. These results suggest that indigenous HEV strains of genotype 4 with marked heterogeneity are circulating in Bali, Indonesia, and that pigs are reservoirs of HEV for Balinese people who have a habit of ingesting uncooked pigs.

In the process of searching for the recently described small anelloviruses 1 and 2 (SAVs) with the genomic DNA length of 2.2 or 2.6 kb in human sera, we isolated a novel virus with its genomic organization resembling those of torque teno virus (TTV) of 3.8-3.9 kb and torque teno mini virus (TTMV) of 2.8-2.9 kb. The entire genomic sequence of three isolates (MD1-032, MD1-073 and MD2-013), which comprised 3242-3253 bases, and exhibited 76-99% identities with the SAVs within the overlapping sequence, was determined. Although the MD1-032, MD1-073 and MD2-013 isolates differed by 10-28 % from each other over the entire genome, they segregated into the same cluster and were phylogenetically distinguishable from all reported TTVs and TTMVs. These results suggest that SAVs are deletion mutants of the novel virus with intermediate genomic length between those of TTV and TTMV and that the novel virus can be classified into a third group of the genus Anellovirus.

Ongoing subclinical infection of hepatitis E virus (HEV) has not been fully studied. In the present study, serum samples were collected from 6700 voluntary blood donors with an elevated alanine aminotransferase (ALT) level of 61-476 IU/I at a Japanese Red Cross Blood Center, and were tested for the presence of IgG, IgM and IgA classes of antibodies to HEV (anti-HEV) by inhouse ELISA and HEV RNA by nested RT-PCR. Overall, 479 blood donors (7.1%) were positive for anti-HEV IgG, including 8 donors with anti-HEV IgM and 7 donors with anti-HEV IgA. Among the nine donors with anti-HEV IgM and/or anti-HEV IgA, six had detectable HEV RNA. The presence of HEV RNA was further tested in 10-sample minipools of sera from the remaining 6691 donors, and three donors including one without anti-HEV IgG were found to be positive for HEV RNA. When stratified by ALT level, the prevalence of HEV RNA was significantly higher among the 109 donors with ALT &gt;= 201 IU/I than among the 6591 donors with ALT of 61-200 IU/I (2.8% vs. 0.1%, P &lt; 0.0001). The HEV isolates obtained from the nine viremic donors segregated into genotype 3, shared a wide range of identities of 85.6-98.5% and were 87.3-93.9% similar to the Japan-indigenous HEV strain (JRA1), in the 412-nucleoticle sequence of open reading frame 2. This study suggests that approximately 3% of Japanese individuals with ALT &gt;= 201 IU/I have ongoing subclinical infection with various HEV strains. J. Med. Virol. 79: 734-742, 2007. (C) 2007 Wiley-Liss, Inc.

To elucidate the extent of genomic heterogeneity of human hepatitis A virus (HAV) strains and to characterize genotype III HAV strains over the entire genome, the full-length sequence of three subgenotype IIIA isolates (HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F) and one IIIB isolate (HAJ85-IF) was determined. The HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F genomes which comprised 7463 or 7464 nt excluding the poly(A) tail, were closest to a reported nearly entire sequence of a MA isolate (NOR-21) with identities of 94.4-97.8% over the entire ORF sequence, and the HAJ85-1 genome (7462 nt) to HA-JNG06-90F of IIIB with an identity of 98.6%. The phylogenetic trees constructed based on the complete ORF sequence or the 168-nt VP1/2A junction sequence and comparative analysis with reported HAV isolates suggested the presence of three distinct clusters within IIIA represented by HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F. The extreme 5' end sequences of IIIA and IIIB were well-conserved, beginning with the sequence UUCAAGAGGG. A single base deletion of G at nt 20, which is involved in the formation of a small loop in domain 1, was characteristic of both IIIA and IIIB. Conserved and divergent amino acid sequences as well as amino acids unique to genotype III, IIIA or IIIB were recognized. (C) 2007 Elsevier B.V. All rights reserved.

Although the potential significance of hepatitis B surface antigen (HBsAg) mutants for failure of immunization has been studied in some endemic countries, whether the "a" determinant variants are responsible for vaccine failure in Mongolia remains unknown. Fifty-nine HBsAg-positive children (age: 8.8 +/- 0.9 years) who had been observed during the nationwide survey of vaccinated cohorts conducted in 2004 were subjected to molecular analyses of hepatitis B virus (HBV). Partial S gene sequences encoding amino acids (aa) 40-171 of HBsAg were determined in 57 children (96.6%) who had detectable HBV DNA. Phylogenetic analysis of the S gene sequences revealed that genotype D accounted for 93.0% and genotype A for 5.3%. Only one child (1.7%) had HBVs of genotypes A and D. HBsAg mutations were found in 17 (29.8%) children ranging from 1 to 4 aa per subject (mean +/- SD, 1.6 +/- 0.9 aa). Pro127Thr and Thr118Ala were the most common substitutions, which occurred in 6 (10.5%) and 3 (5.3%) subjects, respectively; none had Gly145Arg. There were no significant associations in the prevalence of HBsAg mutations with age, sex, residential area, or vaccination status against hepatitis B. Analysis of the deduced amino acid sequence of the entire preS1/preS2/S gene revealed that eight genotype D isolates and one genotype A isolate were quite similar to previously-reported wild-type isolates, suggesting that they are essentially wild-type, but not vaccine-induced mutants. In conclusion, the results demonstrate that hepatitis B surface gene mutants do not play a significant role in vaccination failure in Mongolia.

Using a faecal suspension with high load of Hepatitis E virus (HEV) (2.0 x 10(7) copies ml(-1), genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4 x 10(4) copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1 x 10(5) copies ml(-1) on day 60. When 8.6 x 10(5) copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6 x 107 copies ml-1 on day 60. HEV incubated at temperatures higher than 70 degrees C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 degrees C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.

Although all eight genotypes of hepatitis B virus (HBV) strains are circulating in Japan, no cases of acute hepatitis with foreign HBV strains of genotype H have thus far been reported in Japan. Here, we report a 35-year-old Japanese patient with severe acute hepatitis who was domestically infected with genotype H HBV. On admission, he had a high HBV load of 1.0 x 10(9) copies/ml, elevated levels of total bilirubin (7.0 mg/dl) and alanine aminotransferase (3606 IU/l), and reduced prothrombin activity of 39.0%. The HB-JAIW05 isolate obtained in the present study was composed of 3215 nucleotides and had the highest similarity of 99.7% with the reported genotype H HBV isolate recovered from a Japanese blood donor. The HB-JAIW05 isolate had neither precore (A1896) nor core promoter (T1762/A1764) mutations. However, upon comparison with the consensus sequence of ten reported HBV isolates of the same genotype, the HB-JAIW05 isolate had 17 nucleotide substitutions including five missense mutations in the P gene, which may be related to vigorous replication of HBV in this case. He had no history of traveling abroad, but had had extramarital sexual contact with two Japanese women living in Iwate, Japan, 2 weeks and 2 months before the disease onset, respectively. Our results suggest that rare HBV genotypes such as H may be spreading in Japan via sexual contact. Further molecular epidemiological studies on HBV to clarify the exact changing profiles of de novo HBV infection in Japan in relation to genotype and genomic variability are warranted.

Among six known subgenotypes (IA, IB, IIA, IIB, IIIA, and IIIB) of human hepatitis A virus (HAV), the complete genomic sequence has not been determined for IIIB. In this study, the full-length genomic sequence of a 11113 HAV isolate (HA-JNG06-90F) recovered from a Japanese patient who contracted sporadic hepatitis A in 1990, was determined. The HA-JNG06-90F genome, which comprised 7462 nt excluding the poly(A) tail, was related most closely to NOR-21 of subgenotype IIIA with an identity of 89.1%, and was only 82.6-83.4% similar to human HAV isolates of genotypes I and II over the entire genome. Comparison of full-length genomic sequences of 20 reported isolates and HA-JNG06-90F generated optimal results for separation of different levels: the nucleotide identities were 80.786.6% at the genotype level, 89.1-91.9% at the subgenotype level, and 94.6-99.7% at the isolate level. Similar ranges of nucleotide identity were observed when comparing partial nucleotide sequences of the VP1-213 (481 nt; primer sequences at both ends excluded) and 3C/3D (590 nt) regions, which were amplifiable by PCR with primers designed from well-conserved areas of the HAV genome. All 66 samples with IgM-class HAV antibodies tested positive for HAV RNA by both VP1-213 (481 nt)-PCR and 3C/3D (590 nt)-PCR: subgenotype assignment was concordant in all samples tested (IA [n = 61], 113 [n = 1], IIIA [n = 2] and IIIB [n = 2]). These results suggest that two broadly reactive PCRs using primers derived from the VP1-2B and 3C/3D regions, respectively, may be applicableto universal detection and phylogenetic analysis of various HAV strains.

To compare the epidemiologic profiles of hepatitis A virus (HAV) and hepatitis E virus (HEV) infections in children in Mongolia, the prevalence of HAV and HEV infections was investigated serologically and molecularly among 717 apparently healthy individuals of 0-20 years of age (mean standard deviation, 8.6 +/- 4.9 years) using serum samples obtained between October 2005 and January 2006. Total antibody against HAV (anti-HAV [total]) was detected in 494 (68.9%) of the 717 subjects, while IgG antibody against HEV (anti-HEV IgG) was detected in only five subjects (0.7%) (P &lt; 0.0001). All five subjects who had anti-HEV IgG, were negative for anti-HEV IgM and HEV RNA. Anti-HAV was detectable in 24 (75.0%) of the 32 infants aged 7 days to 6 months, but not in any of the 8 infants aged 7 to &lt; 12 months. The prevalence of anti-HAV was 19.5% (17/87) in the age group of 1-3 years, and it increased to 50.0% (69/138) in the age group of 4-6 years, and further to 81.4% (105/129) in the age group of 7-9 years. Of note, 97.2% of the subjects in the age group of 16-20 years had antiHAV. The presence of HAV RNA was tested in all 717 subjects, and three children of 1, 4, or 8 years of age were found to have detectable HAV RNA (subgenotype IA). No subject had a history of hepatitis or jaundice. In conclusion, HEV infection was uncommon, but HAV infection lacking overt clinical features was prevalent among children in Mongolia.

患者は30歳男性．2006年1月から4カ月間中国の雲南省とチベット自治区，ネパール，およびタイの順に旅行し，タイに移動後間もなく褐色尿と黄疸に気づき帰国した．患者血清からはE型肝炎ウイルス（HEV）RNAが検出され，遺伝子型は1型と判定された．取材旅行であったことより日々の飲食を全て記録しており，生ものや生水の摂取は旅行中一切無かったが，ネパールでの水かけ祭「ホーリー」に参加し，泥水を誤飲したことが感染契機になったとみられた．さらに，1型の既知全クローンとの比較から，感染したHEV株は2005年12月にネパールで分離されたHEV株と100%の一致率を示し，ネパールで感染したことがHEVクローンの遺伝子解析から裏付けられた．ネパールでは2006年春においても同種株の流行が続いていたことが推測された．<br>

Recent studies revealed that hepatitis E virus (HEV) genomes are more variable than previously thought and well-conserved regions suitable for designing universal primers are limited. In this study, based on alignment of 70 full-length HEV sequences of genotypes 1-4, a part of the ORF2/ORF3 overlapping region was found to be the best target region for PCR amplification of various HEV strains. Using the newly designed primers, an RT-PCR method (ORF2/3-137 PCR) that amplifies a 137-nucleotide (m) sequence within the ORF2/ORF3 overlapping region and is capable of amplifying all known HEV sequences was developed. When compared with the previous RT-PCR method (ORF2-457 PCR) that amplifies a 457nt ORF2 sequence, ORF2/3-137 PCR was two to three times more sensitive than ORF2-457 PCR upon testing serial dilutions of three HEV RNA-positive serum samples. The ORF2/3-137 PCR assay could detect viraemia in five patients with acute or fulminant hepatitis E 3-14 days longer than ORF2-457 PCR after disease onset. All 41 ORF2-457 PCR-positive serum samples of various genotypes tested positive for HEV RNA by the ORF2/3-137 PCR assay. Since the amplicons of ORF2/3-137 PCR contain variable sequences, a phylogenetic tree of the ORF2/3-137 products could clearly distinguish the different HEV genotypes. (c) 2006 Elsevier B.V. All rights reserved.

Full-length sequences were determined for a human hepatitis E virus (HEV) isolate (HE-JA04-1911) and two swine HEV isolates (swJ8-6 and swJ12-4) that belong to one of three clusters within genotype 3 in Japan and are close to Spanish isolates according to their partial sequences. The three HEV isolates were 89.7-92.9% identical to each other, but only 80.7-83.0% similar to 21 HEV strains of the same genotype isolated in Canada, Kyrgyzstan, the USA and Japan over their entire genome. On comparison with HEV isolates whose partial sequence is known, the HE-JA04-1911, swJ8-5 and swJ12-4 isolates segregated into a phylogenetic cluster consisting of human and swine HEV isolates in Japan and the UK, with identities of 89.8-100% and 87.9-92.4%, respectively. Genotype 3 HEV isolates were found to be markedly heterogeneous. The UK-isolate-like HEV strains in Japan may have originated from the UK via the importation of pigs since 1900.

To compare the epidemiologic profiles of hepatitis A virus (HAV) and hepatitis E virus (HEV) infections in Japan, the prevalence of clinical or subclinical HAV and HEV infections was investigated serologically and molecularly among 128 consecutive patients (age, mean +/- standard deviation, 37.5 +/- 14.7 years) who contracted acute hepatitis between 1989 and 2005 in a city hospital, and among 416 hemodialysis patients (60.1 +/- 12.6 years) and 266 medical staff members (34.6 +/- 11.4 years) at the same hospital, using stored periodic serum samples collected since the start of hemodialysis or employment, respectively. Between 1989 and 1995, among 93 patients with acute hepatitis, 51 (54.8%) were diagnosed with hepatitis A and only one patient with hepatitis E. Between 1996 and 2005, however, among 35 patients, only 3 (8.6%) were diagnosed with hepatitis A and 2 (5.7%) with hepatitis E. Although subclinical HEV infection was recognized in four hemodialysis patients (one each in 1979, 1980, 1988, and 2003) and two medical staff members (1978 and 2003) in previous studies, none of the 191 hemodialysis patients who had been negative for anti-HAV at the start of hemodialysis contracted HAV infection during the observation period of 7.6 +/- 6.4 years. Only one (0.4%) of the 246 medical staff members who had been negative for anti-HAV at the start of employment acquired hepatitis A during the observation period of 7.9 +/- 8.0 years: none had subclinical HAV infection. Clinical or subclinical HEV infection has occurred rarely during the last three decades, while HAV infection has markedly decreased at least since 1996.

The prevalence and risk factors for hepatitis delta virus (HDV) infection among Mongolian school children were assessed by detecting the antibody against HDV and HDV RNA, and through structured interviews. The study subjects consisted of 181 children with the past or ongoing hepatitis B virus infection who were investigated during the nationwide serosurvey conducted in 2004. The prevalence of antibody to HDV was 6.1%, with the proportion of 13.6% among hepatitis B surface antigen (HBsAg)-positive subjects, all of whom had HDV RNA. Multivariate logistic regression analyses showed that injections (&gt; 11 times) (odds ratio [OR] = 8.31, 95% confidence interval [CI] = 1.28-54.07) and blood sampling (&gt; 3 times) (OR = 5.34, 95% CI = 1.12-25.53) in health care settings, hospitalization (&gt; 3 times) (OR = 6.20, 95% CI = 1.18-32.71), and cohabitating with patients with chronic hepatitis (OR = 4.57, 95% CI = 1.26-16.55) predicted the seropositivity for antibody to HDV. These results suggest that parenteral exposures in health care settings and household transmission are the main routes of HDV transmission among Mongolian children.

One hundred ten consecutive patients (60 males and 50 females; age, mean standard deviation [SD], 22.6 +/- 6.4 years; range 16-48 years) who were clinically diagnosed with sporadic acute hepatitis between December 2004 and January 2005 in Ulaanbaatar, Mongolia, were studied. IgM antibodies to hepatitis A virus were detected in 18 patients (16.4%), IgM antibodies to hepatitis B core (anti-HBc IgM) in 38 patients (34.5%) including two patients with concurrent hepatitis delta virus (HDV) infection, and hepatitis C virus RNA in nine patients (8.2%). There were 30 hepatitis B virus (HBV) carriers who had detectable hepatitis B surface antigen and antibodies to HDV but were negative for anti-HBc IgM, suggesting that they acquired type D acute hepatitis due to superinfection of HDV on a background of chronic HBV infection. None had IgM antibodies to hepatitis E virus (HEV). Consequently, 16.4, 32.7, 6.4, 1.8, and 27.3% of the patients were diagnosed as having acute hepatitis of type A, B, C, type B+D (HBV/HDV coinfection), and type D (superinfection of HDV), respectively. The cause of hepatitis was not known in the remaining 17 patients (15.5%). All 18 HAV isolates were geno-typed as IA, all 9 HCV isolates were genotyped as 1b, and all 32 HDV isolates were classified into genotype I. The distribution of HBV genotypes among the 67 HBV isolates was A (1.5%, n=1) and D (98.5%, n =66). The present study indicates that de novo infections of HAV, HBV, HCV, and HDV are prevalent among young adults in Mongolia. (c) 2006 Wiley-Liss, Inc.

It was suggested that hepatitis E virus (HEV) genotype 4 is associated more closely with the severity of hepatitis E than genotype 3, although the virological basis remains unknown. The aim of this study was to examine whether genomic differences among genotype 4 HEVs are responsible for the development of fulminant hepatitis. Full-length sequences of genotype 4 HEVs from three patients with fulminant hepatitis and six patients with acute self-limited hepatitis were determined. The sequences were analyzed with those of 13 genotype 4 HEV isolates whose entire nucleotide sequence is known. Analysis of 22 full-length sequences (fulminant hepatitis, 5; acute hepatitis, 17) revealed that C at nt 1816 and U at nt 3148 (U3148), both of which do not change the amino acid sequences, were significantly associated with fulminant hepatitis (P=0.0489, respectively). When partial nucleotide sequences containing nt 1816 or nt 3148 were determined in 16 additional HEV isolates of genotype 4, a closer association between U3148 and fulminant hepatitis (P=0.0018) was observed. The comparison of 86 HEV isolates of all four genotypes showed that U3148 had a stronger association with fulminant hepatitis than other nucleotides at nt 3148 (P=0.0006). Patients infected with HEV with U3148 had a significantly lower value of the lowest prothrombin activity (P=0.0293). Nt 3148 is located within the RNA helicase domain, and 22-nt sequence including nt 3148 was well conserved among all genotypes. A silent substitution of U3148 in HEV may be associated with the development of fulminant hepatitis. Further studies are needed to clarify the underlying mechanism.

愛知県内で捕獲された野生イノシシの肉・内臓を摂食し，約1∼2カ月後にE型肝炎を発症した症例を2005年と2006年に2例ずつ合計4例経験した．いずれも中高年男性であり，3例に飲酒歴を認め1例に薬剤服用歴を認めた．4例いずれも著明な肝逸脱酵素の上昇を示し，2例で劇症化が懸念されステロイドパルス治療を施行，うち1例は血漿交換療法を施行した．分離されたHEV株の遺伝子型はいずれも4型であり，互いに98.8∼99.8%の塩基配列一致率を示すと同時に，最近愛知県内の野生イノシシから分離されたHEV株とも99.1∼100%の一致率を示した．一方，これまでに北海道を中心に他県で分離されている4型HEV株との一致率は85.4%∼89.3%に過ぎなかった．以上の結果より，野生イノシシを感染源とする土着HEVの感染が愛知県内で広がりつつあることが危惧され，感染予防の観点から，早急の感染実態の調査・把握が重要である．<br>

IFN治療著効後に血中HCV RNAが再陽性化したC型慢性肝炎の2例を経験した．症例1は54才男性，HCV genotype 2a. 1992年にIFN&alpha;2bの26週投与で著効と判定された．以後肝機能は正常であったが，12年後に肝機能の増悪と共にHCV RNAが陽性化した．HCV RNAは1カ月後に陰性化し肝機能も正常化したが，9カ月後に再度HCV RNAが陽転した．症例2は68才男性，HCV genotype 1b. 2003年にIFN&beta;2週後&alpha;2b/Ribavirin併用療法22週治療で著効と判定された．治療終了後2年2カ月目に，肝機能は正常のままHCV RNAが陽性化し持続した．治療前後におけるHCV NS5B領域の塩基配列の検討より，残存したHCVの再燃であると考えられた．以上のようにIFN著効例であってもHCV RNAが再出現する症例があり，注意深い経過観察が必要であると考えられた．<br>