高橋 雅春

Mongolia is known for its high endemicity for hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) infections among apparently healthy individuals. However, there are little or no data on the prevalence and genotype distribution of HBV, HCV, and HDV among patients with chronic liver disease in Mongolia. Therefore, serum samples obtained in 2004 from 207 patients (age, mean +/- standard deviation, 51.0 +/- 11.9 years) including those with chronic hepatitis (n = 90), liver cirrhosis (n = 41), and hepatocellular carcinoma (n = 76) were tested for serological and molecular markers of HBV, HCV, and HDV infections. Of the 207 patients, 144 (69.6%), 106 (51.2%), and 117 (56.5%) tested positive for hepatitis B surface antigen (HBsAg) and/or HBV DNA, HCV RNA, and HDV RNA, respectively. Collectively, 172 patients (83.1%) were viremic for one or more of these viruses, including dual viremia of HBV/HDV (26.6%) or HBV/HCV (7.7%) and triple HBV/HCV/HDV viremia (30.0%). Of note, triple ongoing infection was significantly more frequent among patients with hepatocellular carcinoma than among those with chronic hepatitis (63.2% vs. 14.4%, P < 0.0001). One hundred sixty patients (77.3%) had a history of blood transfusion and/or surgery. The distribution of HBV genotypes among the 116 HBV-viremic patients was: A (0.9%), B (0.9%), C (6.0%), D (88.8%), and C plus D (3.4%). All 117 HDV isolates were classified into genotype 1. The 106 HCV RNA-positive samples were typed as genotype lb (92.5%), 2a (0.9%), or 1b plus 2a (6.6%); mixed infection of two distinct HCV genotypes was found exclusively in the patients with hepatocellular carcinoma.

Two (2.3%) of 87 wild-caught boars in Japan had detectable hepatitis E virus (HEV) RNA. The two boar HEV isolates (wbJTS1 and wbJYG1) obtained in the present study and a previously reported isolate (wbJSGi) whose partial sequence had been determined were sequenced over the entire genome, The wbJSG1, wbJTS1 and wbJYG1 isolates comprised 7225 or 7226 nt, excluding the poly(A) tail, and segregated into genotype 3. They differed by 8.5-11.2 % from each other and by 8.6-18.4 % from 17 reported genotype 3 HEV isolates, including one boar isolate, in the full-length sequence. When compared with 191 reported genotype 3 HEV isolates whose partial sequences were known, these three boar isolates were closer to Japanese isolates than to isolates of non-Japanese origin (89.2 +/- 2.6 vs 85.9 +/- 2.2 %; P < 0.0001). A proportion of wild boars in Japan are infected with markedly heterogeneous HEV strains that are indigenous to Japan and may serve as reservoirs of HEV.

Serum samples obtained from 289 first-time and 114 repeat donors at the Blood Center of Mongolia (MBC) were tested for serological and molecular markers of hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) infections. Among the 403 blood donors, 33 (8.2%), 21 (5.2%), and 27 (6.7%) tested positive for hepatitis B surface antigen (HBsAg) and/or HBV DNA, HCV RNA, and HDV RNA, respectively. Collectively, 55 donors were viremic for one or more of these viruses, and included 54 first-time donors (18.7%) and 1 repeat donor (0.9%) (P < 0.0001). One discrepant case with HBsAg detectable only at MBC was negative for HBsAg, HBV DNA and anti-HBc in this study. Four donors who were HCV-viremic in this study were negative for anti-HCV by the MBC method. Further efforts to increase the sensitivity and specificity of the currently-used tests are urgently required in Mongolia. Three donors who were positive for anti-HBc and anti-HDV but negative for HBsAg, had both HBV DNA and HDV RNA. This suggests that introduction of a new anti-HDV serological test is useful for not only HDV screening but also HBV screening of anti-HBc-positive, HBsAg negative donors, considering a possibility of viral interference by coexisting HDV.

Subdinical hepatitis E virus (HEV) infection among healthy individuals was studied serologically and molecularly. Serum samples collected at screening between March and April 2004 (or just before retirement) from 266 medical staff members (35 males, 231 females) who had been working for 8.8 +/- 8.5 (mean standard deviation, range, 0.3-35.1) years in a city hospital in Japan and serum samples that had been collected from these staff members at the start of employment were tested for IgA, IgM, and IgG antibodies to HEV (anti-HEV) by in-house enzyme-linked immunosorbent assays. Overall, six subjects (2.3%) tested positive for anti-HEV IgG at the screening; among them, four subjects (1.5%) had already been positive for anti-HEV IgG at the start of employment and two subjects (0.8%) seroconverted after initiation of employment. Periodic serum samples that had been collected from the two seroconverted subjects were tested for HEV antibodies and HEV RNA. The two subjects became positive for anti-HEV IgG in 1978 or 2003, respectively, with no discernible elevation in alanine aminotransferase (ALT) level, and continued to be seropositive up through the screening date. Although anti-HEV IgM was not detectable in the two subjects, one was infected transiently with Japan-indigenous HEV strain of genotype 3 and the other was positive transiently for anti-HEV IgA. The present study indicates that even an individual with subdinical HEV infection had evidence of transient viremia in the absence of ALT elevation and that anti-HEV IgA detection may be useful for serological diagnosis of recent subdinical HEV infection. (c) 2005 Wiley-Liss, Inc.

Hepatitis E in industrialized countries has not been well studied. To define the possible risk factors for transmission of hepatitis E virus (HEV) and for the severe form of hepatitis E in Japan, we investigated the clinical and virological characteristics of hepatitis E in 32 patients who contracted the mild (n = 23) or severe form (n = 9) of domestically acquired hepatitis E between 1996 and 2004 in Hokkaido, where hepatitis E is most prevalent in Japan. Nine patients with the severe form of hepatitis E included two patients with fulminant hepatitis E and seven patients who were diagnosed with severe acute hepatitis in which hepatic encephalopathy did not appear during the course of the illness despite low plasma prothrombin activity (<= 40%) and/or increased total bilirubin level (>= 20 mg/dl). At least 25 patients (78%) had consumed uncooked or undercooked pig liver and/or intestine 1-2 months before the onset of hepatitis E. When compared with the seven patients with HEV genotype 3, the 25 patients with HEV genotype 4 had a higher peak alanine aminotransferase (ALT) level (P = 0.0338) and a lower level of lowest prothrombin activity (P = 0.0340). The severe form of hepatitis E was associated with the presence of an underlying disease (56% [5/9] vs. 17% [4/23], P = 0.0454). The study suggests that zoonotic food-borne transmission of HEV plays an important role in the occurrence of hepatitis E in Hokkaido, Japan, and that the HEV genotype and the presence of an underlying disease influence the severity of hepatitis E. (C) 2005 Wiley-Liss, Inc

A previous study revealed a high prevalence of hepatitis B surface antigen (HBsAg) and hepatitis delta virus (HDV) RNA among 249 apparently healthy individuals (mean standard deviation age, 48.4 +/- 13.9 years; 126 males and 123 females) in Ulaanbaatar, Mongolia. To investigate further the prevalence of HDV infection there, the same serum samples obtained from the cohort were tested for the presence of immunoglobulin G (IgG) class antibody to HDV (anti-HDV) by a newly developed enzyme-linked immunosorbent assay using recombinant hepatitis delta antigen protein expressed in the pupae of silkworm as the antigen probe. Anti-HDV was detected in 42 persons (16.9%), among whom 22 (52.4%) were positive for HBsAg and 20 (47.6%) had detectable HDV RNA. Among 170 persons with anti-HBc in the absence of HBsAg, 20 (11.8%) tested positive for anti-HDV, and I of the 20 subjects was positive for HDV RNA. Of note, none of 55 anti-HBc-negative persons had anti-HDV, supporting the specificity of the anti-HDV assay. The optical density (OD) value of anti-HDV was significantly higher among HDV RNA-positive subjects (n = 21) than among HDV RNA-negative subjects (n = 21) (2.513 +/- 0.514 vs. 0.836 +/- 0.550, P < 0.0001). The present study confirmed the extremely high prevalence of HDV infection in Mongolia, and identified a person who was positive for both anti-HDV and HDV RNA despite negativity for HBsAg and HBV DNA probably due to viral interference. The anti-HDV assay may be useful for further epidemiological studies on HDV infection in larger cohorts in urban and rural areas of Mongolia, where elucidation of the transmission route of HDV is required urgently. (C) 2005 Wiley-Liss, Inc

To evaluate the usefulness of detection of antibodies to hepatitis E virus (HEV) to screen for viraemic pigs, serum samples obtained from 1425 1-6-month-old pigs in Japan were tested for swine HEV RNA and IgG, IgM and IgA classes of anti-HEV antibody. Fifty-five (5%) of the 1071 2-5-month-old pigs were positive for swine HEV RNA, but none of 218 1 -month-old pigs or 136 6-month-old pigs had detectable HEV RNA. The prevalence of anti-HEV IgG among the viraemic pigs (67%, 37/55) was similar to that among the non-viraemic pigs (55%, 757/1370) and the prevalence of anti-HEV IgM among the viraemic pigs and non-viraemic pigs was 7 and 3%, respectively. However, anti-HEV IgA was detected significantly more frequently among viraemic pigs than among non-viraemic pigs (55 vs 10%, P<0.0001). These results suggest that anti-HEV IgA is more useful than anti-HEV IgM to screen for viraemic pigs.

To investigate the prevalence of hepatitis B virus (HBV) genotypes and characteristics of HBV isolates among Japanese patients infected with human immunodeficiency virus type 1 (HIV), serum samples collected between September 1990 and March 2002 from 471 HIV-infected patients (age, 38.8 +/- 11.4 [mean +/- standard deviation] years; male, 90%) were tested for hepatitis B surface antigen (HBsAg) and HBV DNA. Positivity for HBsAg and HBV DNA was seen in 42 patients (8.9%), 41 of whom had contracted HIV infection through sexual activity and 1 had hemophilia. Genotypes of HBV were determined by comparative and phylogenetic analyses of the S gene sequence (396 nucleotides [nt]). The distribution of HBV genotypes among the 42 HBV-viremic patients was: A (50%), B (5%), C (24%), D (5%), E (2%), H (10%), A p I us D (2%), A plus G (2%). The hemophilia patient had HBV genotype D. Genotypes E, G, and H which had not been reported in Japan, were found in one patient each who had traveled to Zambia, the US, and South America, respectively. Genotypes A and D, which are rare in Japan, were found in patients who had no history of traveling abroad. The entire genome of the HB-JI411 (genotype E [3,212 nt]), HB-JI444G (genotype G [3,248 nt]), and HBJI260 (genotype H [3,218 nt]) isolates had the highest identity of 98.3%, 99.9%, and 98.5%, respectively, with reported HBV isolates of the same genotype. Most Japanese patients coinfected with HIV and HBV had HBV genotypes that are found rarely or had not been reported in Japan. (c) 2005 Wiley-Liss, Inc.

Serum samples collected from 68 patients (age, mean +/- the standard deviation [SD], 56.3 +/- 12.8 years) at admission who were subsequently molecularly diagnosed as having hepatitis E and from 2,781 individuals who were assumed not to have been recently infected with hepatitis E virus (HEV; negative controls; 52.9 +/- 18.9 years), were tested for immunoglobulin M (IgM) and IgA classes of antibodies to HEV (anti-HEV) by in-house solid-phase enzyme immunoassay with recombinant open reading frame 2 protein expressed in the pupae of silkworm as the antigen probe. The 68 patients with hepatitis E had both anti-HEV IgM and anti-HEV IgA. Among the 2,781 controls, 16 (0.6%) had anti-HEV IgM alone and 4 (0.1%) had anti-HEV IgA alone: these IgA/IgM anti-HEV-positive individuals were not only negative for HEV RNA but lack IgG anti-HEV antibody as well (at least in most of the cases). Periodic serum samples obtained from 15 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, and anti-HEV IgA. Although HEV RNA was detectable in the serum until 7 to 40 (21.4 +/- 9.7) days after disease onset, both IgM and IgA anti-HEV antibodies were detectable until 37, 55, or 62 days after disease onset in three patients and up through the end of the observation period (50 to 144 days) in 12 patients. These results indicate that detection of anti-HEV IgA alone or along with anti-HEV IgM is useful for serological diagnosis of hepatitis E with increased specificity and longer duration of positivity than that by RNA detection.

Background: Hepatitis E virus (HEV) infection is highly prevalent among domestic pigs in Japan. It has been reported that pig handlers such as farmers and veterinarians are at increased risk of contracting HEV infection. Pigs are regarded as the most acceptable candidate animals for xenotransplantation and, recently, they are being used as experimental animals. Methods: We investigated the prevalence of IgG class antibodies to swine HEV (anti-HEV) and HEV RNA among 152 2-month-old domestic pigs and 38 miniature pigs of 4 to 10 months of age that had been brought to our center for medical experiments from five swine farms (A-E) in Japan. Serum samples were tested for anti-HEV by in-house enzyme immunoassay, and for HEV RNA by reverse transcription-polymerase chain reaction using primers targeting the open reading frame 2 (ORF2) region. Results: One percent (one of 84), 6% (one of 16), and 38% (20 of 52) of the domestic pigs from farms A, B and C, respectively, had detectable HEV RNA, and the 22 HEV isolates recovered from the viremic pigs were 89.8 to 100% identical to each other in the 412-nucleotide sequence of ORF2 and segregated into three clusters within genotype 3. Although one pig from farm A had detectable HEV RNA reproducibly, the HEV isolate recovered from this pig was up to 100% similar to those recovered from pigs from farm C, and the sera from all 84 pigs from farm A were negative for anti-HEV. These results suggested that farm A is free from HEV infection. As the viremic pig from farm A had been raised for 1 month in a barn at our center before serum sampling, it is most likely that the pig acquired HEV infection in the barn at our center where HEV-viremic pigs from farm C had been reared for several days approximately 3 months earlier. The 38 miniature pigs from farms D and E were negative for both anti-HEV and HEV RNA. In an attempt to further investigate the prevalence of HEV infection, pigs that were being raised in four swine farms (farms A, C, D, and E) were tested for anti-HEV. Although 96 (86%) of the 112 pigs from farm C were positive for anti-HEV, none of the 48 pigs in farm A and 138 miniature pigs in farms D and E was positive for anti-HEV. Conclusions: These results suggest that three of the five swine farms tested were free from HEV, and that periodic testing for anti-HEV and HEV RNA of pigs used as experimental animals and pigs raised in swine farms from which pigs are purchased, is useful for providing HEV-free pigs to researchers who are engaged in studies using pigs.

Zoonotic transmission of hepatitis E virus (HEV) from captured wild deer or boars to humans has been suggested. Antibody to HEV was detected in 9% of 35 wild boars and 2% of 117 wild deer tested, and a presumably indigenous HEV of genotype 3 was isolated from a boar in Japan.

Cases of imported hepatitis E in industrialized countries infected with a genotype I hepatitis E virus (HEV) have been identified. We report a 56-year-old Japanese man who acquired infection with a genotype 4 HEV with 98.8% identity to a Vietnamese isolate after ingestion of uncooked shellfish while traveling in Vietnam.

In Indonesia where hepatitis E virus (HEV) is believed to be highly endemic, only three outbreaks of HEV transmission have been documented to date in restricted areas (West Kalimantan and East Java). A total of 1,115 serum samples collected from apparently healthy individuals in Bali, Lombok, and Surabaya in Indonesia in 1996 where epidemic HEV transmissions have never been reported, were tested for IgG class antibodies to HEV (anti-HEV). In Bali, anti-HEV was detected in 20% (54/276) of the tested population, in remarkable contrast with 4% (17/446) in Lombok and 0.5% (2/393) in Surabaya. On the other hand, antibodies to hepatitis A virus were highly prevalent in all three regions (95% in Bali 90% in Lombok, and 89% in Surabaya). Although the majority of the population in Indonesia is Moslem, Balinese people are mostly Hindu and have a habit of consuming pork. Therefore, serum samples were obtained from the 99 farm pigs in Bali and tested for anti-HEV and HEV RNA. The sera from 71 pigs (72%) were positive for anti-HEV and a 2-month-old pig had detectable HEV RNA. The swine HEV isolate recovered from the viremic pig was named SB66-Bali. The SB66-Bali isolate was most closely related to the genotype 4 isolates from China, India, Japan, and Taiwan, but shared only 82.6-90.0% identity in the common 241-412 nucleotides within open reading frame 2 (ORF2). These results indicate that a presumably indigenous HEV strain(s) is circulating in Bali, Indonesia and that HEV infection may occur via zoonosis even in developing countries. (C) 2004 Wiley-Liss, Inc.

Recent studies indicate that hepatitis E virus (HEV) infection occurs not only in developing countries but also in industrialized nations. However, the characteristics of domestic infections of hepatitis E in Japan are not fully understood. We analyzed serum samples from 34 patients who were seen at a city hospital in Sendai, Japan, between January 1997 and December 2002, and who had been given the diagnosis of sporadic acute hepatitis of non-A, non-B, non-C etiology. Among these 34 patients, 3 (9%; all men; aged 54, 59, and 61 years) were positive for both IgG and IgM anti-HEV antibodies and for HEV RNA. The HEV isolates (HE-JASI and HE-JAS3) obtained from case 1 and case 3, respectively, segregated into genotype III; they had the highest nucleotide sequence identity, of 99.5% and 99.0%, with HE-JA7 and HE-JA8, respectively, both of which had been isolated in Iwate, a neighboring prefecture of Sendai. In contrast, the remaining HEV isolate (HE-JAS2), obtained from case 2, segregated into genotype IV; it had the highest nucleotide sequence identity, of 99.8% and 99.3%, with JKK-Sap and HE-JA3, respectively, both of which had been isolated in Hokkaido, Japan, although case 2 had never been to Hokkaido. Our three patients with hepatitis E had not traveled abroad in the preceding 1 year, had had no contact with pigs, and no history of blood transfusion. These results indicate that HEV should be considered as an etiological agent of acute hepatitis of non-A, non-B, non-C etiology in Japan. The risk factor(s) for acquiring domestic HEV infection in Japan needs to be clarified in future studies.

The prevalence of infection with hepatitis A virus (RAV), HBV, HCV, HDV, and HEV was evaluated in 249 apparently healthy individuals, including 122 inhabitants in Ulaanbaatar, the capital city of Mongolia, and 127 age- and sex-matched members of nomadic tribes who lived around the capital city. Overall, hepatitis B surface antigen (HBsAg) was detected in 24 subjects (10%), of whom 22 (92%) had detectable HBV DNA. Surprisingly, HDV RNA was detectable in 20 (83%) of the 24 HBsAg-positive subjects. HCV-associated antibodies were detected in 41 (16%) and HCV RNA was detected in 36 (14%) subjects, none of whom was coinfected with HBV, indicating that HBV/HCV carriers account for one-fourth of this population. Antibodies to RAV and HEV were detected in 249 (100%) and 28 (11%) subjects, respectively. Of 22 HBV DNA-positive subjects, genotype D was detected in 21 subjects and genotype F was detected in I subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype 1, but these differed by 2.1 to 11.4% from each other in the 522-to 526-nucleotide sequence. Of 36 HCV RNA-positive samples, 35 (97%) were genotype 1b and I was genotype 2a. Reflecting an extremely high prevalence of hepatitis virus infections, there were no appreciable differences in the prevalence of hepatitis virus markers between the two studied populations with distinct living place and lifestyle. A nationwide epidemiological survey of hepatitis viruses should be conducted in an effort to prevent de novo infection with hepatitis viruses in Mongolia.

To investigate the genetic changes in hepatitis E virus (HEV) strains in the Kathmandu valley of Nepal, we compared the 412 nt sequence within open reading frame 2 of HEV among HEV isolates recovered from 16 patients in 1999, 14 patients in 2000 and 38 patients in 2002, and additional isolates recovered from 48 patients in 1997 whose nucleotide sequences have been previously published. All 116 HEV-viraemic samples were genotyped as 1 and subtyped further as 1a (n = 85, 73%), 1c (n = 29, 25 %) and mixed infection of 1a and 1c (n = 2, 2%): subtype 1c was detected only in 1997. Among the 1a isolates, nucleotide sequence identity with the representative 1a isolate of Ne131-1997 was 96.4 +/- 2.4% (mean +/- SD) in 1997, 93.9 +/- 1.7% in 1999, 92.2 +/- 1.0% in 2000 and 91.7 +/- 0.5% in 2002, indicating gradual diversification of HEV sequences. When phylogenetic analysis of the 87 subtype 1 a isolates was performed, they further segregated into five clusters, with two predominant clusters of 1a-2 and 1a-3: the annual frequency of cluster 1 a-2 isolates decreased from 63% in 1997, to 50% in 1999, to 7% in 2000 and no cases in 2002; cluster 1a-3 isolates were observed in all four years and its annual frequency increased from 5% in 1997 to 95% in 2002. Of the remaining three clusters, cluster 1a-1 was detectable only in 1997 and clusters 1a-4 and 1a-5 emerged in 2000 and 2002, respectively. These results indicate that genetic changes and takeover of HEV strains may contribute to the genetic variability of HEV in the community.

Among ten patients who contracted sporadic acute or fulminant hepatitis E between 2001 and 2002 in Hokkaido, Japan, nine (90 %) had a history of consuming grilled or undercooked pig liver 2-8 weeks before the disease onset. We tested packages of raw pig liver sold in grocery stores as food in Hokkaido for the presence of hepatitis E virus (HEV) RNA by RT-PCR. Pig liver specimens from seven (1(.)9%) of 363 packages had detectable HEV RNA. Partial sequence analyses revealed that the seven swine HEV isolates belonged to genotype III or IV. One swine HEV isolate (swJL145) from a packaged pig liver had 100 % identity with the HE-JA18 isolate recovered from an 86-year-old patient in Hokkaido. Two swine HEV isolates (swJL234 and swJL325) had 98(.)5-100% identity with the HE-JA4 isolate. obtained from a 44-year-old patient in Hokkaido. These results indicate that inadequately cooked pig liver may transmit HEV to humans.

Japanese patients with sporadic acute hepatitis E are infected with polyphyletic strains of hepatitis E virus (HEV). Hepatitis E is considered a zoonotic disease. Thus far in Japan, only three strains of swine HEV have been identified and an antibody study for HEV antibodies has not been done on Japanese pigs. To determine the prevalence of swine HEV infection in Japan and the extent of genetic variation among Japanese swine HEV strains, we tested serum samples obtained from 2500 pigs from 2 to 6 months of age at 25 commercial swine farms in Japan for the presence of IgG antibodies to HEV and swine HEV RNA. Anti-HEV antibodies were detected in 1448 pigs (58%). One-hundred-and-thirteen (115%)of the 750 3-month-old pigs and 24 (13%) of the 180 4-month-old pigs were positive for swine HEV RNA. The nucleotide sequence of a 412 bp region within open reading frame 2 of the 137 swine HEV isolates was determined. Sequence analyses revealed that the 137 isolates shared 76(.)6-100% nucleotide sequence identities and were classifiable into genotype III (93%) or IV (7%) and that the isolates from the same farm were greater than or equal to 97(.)1% similar to each other. Phylogenetic analysis showed that the Japanese swine and human HEV isolates segregated into four clusters, with the highest nucleotide identity being 94(.)4-100% between swine and human isolates in each cluster. These results indicate that swine HEV is widespread in the Japanese swine population and further support the hypothesis that swine serve as reservoirs for HEV infection.

Hepatitis E virus (HEV) is a major cause of acute hepatitis in many developing countries. HEV is transmitted principally by the fecal-oral route, and water-borne epidemics are characteristic of hepatitis E. Recently, there is growing consensus that HEV-associated hepatitis also occurs among individuals in industrialized nations who had no history of travel to endemic areas. Zoonotic spread of HEV has been suggested as human and swine HEV strains are closely related genetically and experimental cross-species infection of swine HEV to a chimpanzee and that of human HEV to swine have been demonstrated. This review describes the clinical, epidemiological and virological characteristics of domestic HEV infection in Japan, the genetic relatedness of Japanese human and swine HEV strains, and possible modes of HEV transmission, emphasizing that HEV should be considered in the diagnosis of acute or fulminant hepatitis of non-A, non-B, non-C etiology, even in patients who have not traveled abroad.<br>(Internal Medicine 42: 1065-1071, 2003)

Two distinct hepatitis E virus (HEV) isolates, designated HE-JI3 and HE-JI4, were identified in a single patient with acute hepatitis in Japan, who had not travelled abroad. The HEV load of HE-JI3 at admission was 10(2) copies/ml, but that of HE-JI4 was tenfold higher at 103 copies/ml. The viraemia of HE-JI4 persisted for up to 16 days from admission, whereas HE-JI3 disappeared at 9 days after admission. The entire nucleotide sequence of the HE-JI4 isolate and partial nucleotide sequences of open reading frames (ORFs) I and 2 of the HE-JI3 isolate were determined. The full-length nucleotide sequence of HE-JI4 consisted of 7171 nucleotides excluding the poly(A) tail and contained ORF1 encoding 1684 amino acids, ORF2 encoding 671 amino acids and ORF3 encoding 114 amino acids. Sequence and phylogenetic analyses of the HEV genomes indicated that HE-JI4 was most closely related to an HEV isolate (T1) of genotype IV with the same strategy for translation of ORF2 and ORF3, but which differed from it by 16-5% over the entire genome. The HE-JI3 isolate showed the highest nucleotide identity (88(.)6-95(.)1 %) to the genotype III HEVs, having higher identity to human and swine HEV isolates from the United States (US1, US2 and swUS1) than to those reported thus far from Japan (JRA1 and swJ570). The two co-infecting strains of HE-JI3 and HE-JI4 identified from the single patient shared only 80(.)1 % nucleotide identity. These results indicate that multiple genotypes of HEV co-circulate in Japan, and that genotype IV comprises a remarkably heterogeneous group of HEVs.

When TT virus (TTV) DNA was quantitated in whole blood and plasma aliquots from 27 viremic individuals by real-time detection PCR that can detect essentially all TTV genotypes, the TTV load was 6.9 +/- 3.5 (mean +/- standard deviation)-fold higher in the whole blood than in the plasma samples [P &lt; 0.002 (paired t test)]. To clarify the reason for this difference, peripheral blood cells of various types including red blood cells, granulocytes (CD15+), B cells (CD19+), T cells (CD3+), monocytes (CD14+), and NK cells (CD3-/CD56+) were separated at a purity of 95.4-99.5% from each of three infected individuals with relatively high TTV viremia, and their TTV viral loads were determined. Red blood cells were uniformly negative, but the other cell types were positive for TTV DNA at various titers. In all three patients, the highest TTV load was found in granulocytes (4.2 x 10(4)-3.1 x 10(5) copies/10(6) cells), followed by monocytes (1.4-2.2 x 10(4) copies/10(6) cells) and NK cells (5.4-6.5 x 10(3) copies/10(6) cells); B and T cells were positive, with a low viral load (6.7 x 10(1)-2.7 x 10(3) copies/10(6) cells). These results indicate that TTV is distributed in various peripheral blood cell types at distinct levels, with the highest viral load in granulocytes, and that a significant proportion of the TTV DNA in peripheral blood is not identified by the standard plasma/serum DNA detection methods. (C) 2002 Elsevier Science.