A nationwide molecular epidemiological study on hepatitis B virus in Indonesia: identification of two novel subgenotypes, B8 and C7.

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.

High prevalence of hepatitis B virus infection and inferior vena cava obstruction among patients with liver cirrhosis or hepatocellular carcinoma in Nepal.

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 HCC 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.

Identification of genotype 4 hepatitis E virus strains from a patient with acute hepatitis E and farm pigs in Bali, Indonesia.

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.

Epidemiology of hepatitis B, C, and E viruses and human immunodeficiency virus infections in Tahuna, Sangihe-Talaud Archipelago, Indonesia.

BACKGROUND/AIMS: The epidemiology of hepatitis B, C,E viruses (HBV, HCV, HEV) and human immunodeficiency virus(HIV) has been obscure in Indonesia, particularly remote areas. METHODS: We undertook serological surveys for HBV/HCV/HEV/HIV infections in the general population of Tahuna, the capital city of Sangihe-Talaud Archipelago,outlier in the northeastern part of Indonesia. RESULTS: Of 581 sera collected in April 2005, 1.4% was reactive for HBsAg,0.2% for anti-HCV, and 5.9% for anti-HEV, but none for HIV. All the HBsAg-positive sera were also positive for DNA, the nucleotide sequence of which is segregated within subgenotype C5. Most of the preschool children were positive for anti-HBs as a result of an HB immunization initiated in 1997. The titer of anti-HCV in the only individual detected was very low, with a negative result of HCV RNA detection,suggesting a nonspecific reaction. Anti-HEV was significantly more frequent in those over 30 years of age than in the younger age group (24 vs. 1.9%, p ! 0.0001). CONCLUSION: Thus, it seems that HCV and HIV have fortunately not made it as far as the Sangihe-Talaud Archipelago. Although HBV infection remains a major problem in adults (with the HBsAg-positive rate at 4.9%), HB immunization has begun to protect the younger generation.

Development and validation of an improved RT-PCR assay with nested universal primers for detection of hepatitis E virus strains with significant sequence divergence.

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 (nt) 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 457 nt 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.

Distinct changing profiles of hepatitis A and E virus infection among patients with acute hepatitis, patients on maintenance hemodialysis and healthy individuals in Japan.

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.

Serological and molecular studies on subclinical hepatitis E virus infection using periodic serum samples obtained from healthy individuals.

Subclinical 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 subclinical 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 subclinical HEV infection.

Possible risk factors for the transmission of hepatitis E virus and for the severe form of hepatitis E acquired locally in Hokkaido, Japan.

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 (or=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.

Characterization of seven genotypes (A to E, G and H) of hepatitis B virus recovered from Japanese patients infected with human immunodeficiency virus type 1.

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 plus 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 HB-JI260 (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.

Serological markers of hepatitis B, C, and E viruses and human immunodeficiency virus type-1 infections in pregnant women in Bali, Indonesia.

Except for hepatitis B virus (HBV), there have been few data on serological markers of hepatitis viruses such as hepatitis C virus (HCV) and E virus (HEV), and human immunodeficiency virus type-1 (HIV) in Bali, Indonesia. During 5 months from April to August 2003, sera were collected from 2,450 pregnant women at eight jurisdictions in Bali, and they were tested for markers of these viruses. Only one (0.04%) was positive for antibody to HCV, but none for antibody to HIV. Hepatitis B surface antigen (HBsAg) was detected in 46 (1.9%) at a prevalence significantly lower than that in 271 of the 10,526 (2.6%) pregnant women in Bali surveyed 10 years previously (P < 0.045). The prevalence of hepatitis B e antigen in pregnant women with HBsAg decreased, also, from 50% to 28% during the 10 years (P < 0.011). Antibody to HEV (anti-HEV) was examined in 819 pregnant women who had been randomly selected from the 2,450. The overall prevalence of anti-HEV was 18%, and there were substantial regional differences spanning from 5% at Tabanan district to 32% at Gianyar district. Furthermore, the prevalence of anti-HEV differed substantially by their religions. In the Sanglah area of Denpasar City, for instance, anti-HEV was detected in 20 of the 102 (20%) Hindus, significantly more frequently than in only 2 of the 101 (2.0%) Muslims (P < 0.001). Swine that are prohibited to Muslims, therefore, is likely to serve as a reservoir of HEV in Bali. In conclusion, HBV is decreasing, HCV and HIV have not prevailed, as yet, while HEV is endemic probably through zoonotic infection in Bali.

Prevalence of hepatitis E virus infection among hemodialysis patients in Japan: evidence for infection with a genotype 3 HEV by blood transfusion.

To investigate the prevalence of hepatitis E virus (HEV) infection among patients on maintenance hemodialysis, serum samples collected in January 2003 from 416 patients who had been undergoing hemodialysis for 7.6 +/- 6.3 (mean +/- standard deviation) (range, 0.3-26.0) years in a dialysis unit in Japan and serum samples that had been collected from these patients at the start of hemodialysis were tested for IgG antibodies to HEV (anti-HEV IgG) by an "in-house" enzyme-linked immunosorbent assay (ELISA). Overall, 39 patients (9.4%) had anti-HEV IgG in January 2003, and included 35 patients (8.4%) who had already been positive for anti-HEV IgG at the start of hemodialysis and 4 patients (1%) who seroconverted after initiation of hemodialysis. Periodic serum samples that had been collected from the four seroconverted patients were tested for HEV antibodies and HEV RNA. The four patients became positive for anti-HEV IgG in 1979, 1980, 1988, or 2003, and continued to be seropositive until the end of the observation period. Although anti-HEV IgM was not detectable in the four patients, three were infected transiently with apparently Japanese indigenous HEV strains of genotype 3. The patient who contracted HEV infection in 1979 had been transfused with 2 U of blood 21 days before the transient viremia: one of the two stored pilot serum samples had detectable HEV RNA with 100% identity to that recovered from the patient. Our study provides evidence of transfusion-transmitted HEV infection in Japan in 1979, and that the prevalence of de novo HEV infection during hemodialysis was low (1.1% or 4/374).

Prevalence of antibodies to hepatitis E virus among Japanese blood donors: identification of three blood donors infected with a genotype 3 hepatitis E virus.

Risk factors for acquiring hepatitis E among individuals in industrialized countries including Japan are not fully understood. We investigated whether Japanese blood donors with or without an elevated alanine aminotransferase (ALT) level are likely to have hepatitis E virus (HEV) infection. Serum samples were collected from 5,343 voluntary blood donors including 1,087 donors with elevated ALT of 61-966 IU/L and 4,256 donors with normal ALT (< or = 60 IU/L) at two Japanese Red Cross Blood Centers, and were tested for the presence of anti-HEV IgG by in-house enzyme-linked immunosorbent assay (ELISA). Overall, 200 donors (3.7%) were positive for anti-HEV IgG, including 32 (2.9%) with elevated ALT and 168 (3.9%) with normal ALT. Serum samples with anti-HEV IgG were further tested for the presence of anti-HEV IgM by in-house ELISA and for HEV RNA by reverse transcription (RT)-polymerase chain reaction (PCR). Three donors with ALT of 966, 62 or 61 IU/L were positive for anti-HEV IgM and HEV RNA. The HEV isolates obtained from the three viremic donors segregated into genotype 3, were 91.5-93.4% similar to each other in the 412 nucleotide sequence of open reading frame 2, and had the highest identity of 91.5-94.9% with the JRA1 isolate which was recovered from a Japanese patient with sporadic acute hepatitis E who had never been abroad, suggesting that these three HEV isolates are indigenous to Japan. This study suggests that a small but significant proportion of blood donors in Japan with or without elevated ALT are viremic and are potentially able to cause transfusion-associated hepatitis E.

Prevalence of antibodies to hepatitis E virus among apparently healthy humans and pigs in Bali, Indonesia: Identification of a pig infected with a genotype 4 hepatitis E virus.

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.

High prevalence of antibodies to hepatitis A and E viruses and viremia of hepatitis B, C, and D viruses among apparently healthy populations in Mongolia.

The prevalence of infection with hepatitis A virus (HAV), 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 HAV 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 1 subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype I, 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 1 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.

Genetic changes in hepatitis E virus of subtype 1a in patients with sporadic acute hepatitis E in Kathmandu, Nepal, from 1997 to 2002.

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 1a isolates was performed, they further segregated into five clusters, with two predominant clusters of 1a-2 and 1a-3: the annual frequency of cluster 1a-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 take over of HEV strains may contribute to the genetic variability of HEV in the community.

Molecular investigation of hepatitis E virus infection in patients with acute hepatitis in Kathmandu, Nepal.

One hundred fifty-four consecutive patients with sporadic acute hepatitis, who were seen at a city hospital in the Kathmandu valley of Nepal in 1997, were studied. IgM antibodies to hepatitis A virus were detected in four patients (3%), IgM antibodies to hepatitis B core in four patients (3%), hepatitis B surface antigen in 20 (13%), and hepatitis C virus RNA in four patients (3%). IgM antibodies to hepatitis E virus (HEV) (anti-HEV IgM) and HEV RNA were detected in 77 (50%) and 48 (31%), respectively. Consequently, 86 patients (56%) including nine HEV-viremic patients without anti-HEV IgM, were diagnosed with hepatitis E. The cause of hepatitis was not known in 53 patients (34%). All 48 HEV RNA-positive samples were genotyped as 1, and subtyped further as 1a in 17 (35%), 1c in 29 (60%), and mixed infection of 1a and 1c in 2 (4%). A seasonal difference in the prevalence of HEV subtypes was recognized. Before the rainy season (January to July), both 1a and 1c isolates were found: the intrasubtypic difference was up to 9.0% and 1.7%, respectively, in the 412-nucleotide sequence of open reading frame 2. During the rainy season (August), only 1c isolates (n = 17) with 99.5-100% identity were found; 13 of 17 isolates had the same sequence, being identical to the 3 isolates that emerged at the end of July. These results suggest that a particular HEV 1c strain spread widely during the rainy season and was implicated in a small epidemic in the Kathmandu valley in August 1997.

Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus.

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 (15 %) 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 > or = 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.

Persistence of hepatitis B viremia after recovery from acute hepatitis B: correlation between anti-HBc titer and HBV DNA in serum.

During the follow-up of 19 patients with self-limited acute hepatitis B for more than 2 years, clearance of hepatitis B surface antigen from the sera was observed in all patients within 6 months after disease onset, and the corresponding antibody (anti-HBs) appeared in 17 of the 19 patients within 12 months. However, upon performing nested polymerase chain reaction with the estimated sensitivity of 120-200 copies/ml, using two independent pairs of primers derived from the well-conserved sequences in the S gene or C gene region of the hepatitis B virus (HBV) genomes of all seven genotypes, HBV DNA was detected over a period of at least 12 months in serum samples obtained from five (26%) of the 19 patients, although it became undetectable in all five patients at 2-3 years after disease onset. The titer of antibody against hepatitis B core antigen (anti-HBc), assayed by the hemagglutination inhibition (HI, 2(N)) test, was significantly lower at the initial examination in the five patients who remained viremic for at least 12 months, than in the remaining 14 patients who cleared HBV DNA from their sera within 12 months after disease onset (10.6+/-2.7 vs. 13.6+/-0.7, P<0.02). Furthermore, these five patients showed a significantly lower rate of decrease of anti-HBc titer during the 12-month period after disease onset than the remaining 14 patients (55.0+/-32.6 vs. 91.0+/-7.9%, P<0.01). These results indicate that the initial titer and dynamics of anti-HBc may reflect the evolution of HBV viremia after clinical recovery from acute hepatitis B.

TT virus is distributed in various leukocyte subpopulations at distinct levels, with the highest viral load in granulocytes.

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 < 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.