Ongoing subclinical infection of hepatitis E virus among blood donors with an elevated alanine aminotransferase level in Japan.

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/l 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 in-house 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 > or = 201 IU/l than among the 6591 donors with ALT of 61-200 IU/l (2.8% vs. 0.1%, P < 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-nucleotide sequence of open reading frame 2. This study suggests that approximately 3% of Japanese individuals with ALT > or = 201 IU/l have ongoing subclinical infection with various HEV strains.

HBV NAT positive [corrected] blood donors in the early and late stages of HBV infection: analyses of the window period and kinetics of HBV DNA.

BACKGROUND AND OBJECTIVES: The Japanese Red Cross (JRC) carries out nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) by using a multiplex (MPX) reagent. Screening is undertaken on serologically negative units. In this study we characterized HBV NAT-positive donations individually and analysed the window period and kinetics of HBV DNA, during acute infection, in follow-up studies. MATERIALS AND METHODS: Two hundred and seventy-seven HBV DNA-positive donations have been identified in Japan since the introduction of NAT screening of 50-donation minipools. The viral loads and genotypes of these HBV DNA-positive donations were characterized. The doubling time and half-life of HBV was estimated from the data of 123 follow-up donors. The sensitivity of the NAT system (based on 50-donation minipools) was compared with the sensitivities of the enzyme immunoassay (EIA) and the chemiluminescence immunoassay (CLIA). Samples that were CLIA negative, but with > 10(4) copies/ml of HBV DNA, were analysed by sequencing the hepatitis B surface antigen (HBsAg) region. RESULTS: Out of 277 HBV NAT-positive samples, 125 (45%) were found to have an increasing viral load and 45 (16%) a decreasing viral load. Forty per cent of HBV NAT-positive samples with an increasing viral load, and 33% of those with a decreasing viral load, were negative when tested by using the CLIA. No mutations related to escape mutants were found in the samples that were CLIA negative but with HBV DNA loads of > 10(4) copies/ml. The median HBV doubling time was 2.6 days (n = 93, 1.3-15.2 days) and the half-life was 1.6 days (n = 55, 0.9-6.3 days). Some kinetic difference was observed between genotypes A and B. CONCLUSIONS: HBV NAT screening detected HBV DNA in both early (the so-called serological window period) and late stages of acute HBV infection.

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.

Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food.

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.

Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99 % identity over the entire genome.

The full-length genomic sequences were determined of Japanese swine and human hepatitis E virus (HEV) isolates (swJ13-1 and HE-JA1, respectively) with 100 % identity in the partial sequence of open reading frame (ORF) 2 (ORF2, 412 nt). swJ13-1 was isolated from a 4-month-old farm pig born in Hokkaido, Japan, in 2002 and HE-JA1 was recovered from a 55-year-old patient who lived in Hokkaido and who had contracted sporadic acute hepatitis E in 1997. Both isolates consisted of 7240 nt, excluding the poly(A) tail, and contained three ORFs (ORFs 1-3) that encoded proteins of 1707, 674 and 114 aa. The overall nucleotide sequence identity between them was 99.0 % and the deduced amino acid sequence identities of ORFs 1-3 were 99.8, 100 and 100 %, respectively. The high degree of genomic similarity observed between swine and human HEV isolates in a restricted area of Japan further supports the finding that sporadic hepatitis E in Japan is a zoonosis.

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.

Molecular and serological characterization of sporadic acute hepatitis E in a Japanese patient infected with a genotype III hepatitis E virus in 1993.

Serum samples collected periodically from a 40-year-old Japanese woman who had not travelled abroad and who had contracted sporadic acute hepatitis E in 1993 were semi-quantitatively tested by enzyme immunoassay for IgM, IgA and IgG antibodies to hepatitis E virus (HEV). Anti-HEV IgM and IgA antibody levels were the highest (1 : 2400 dilution and 1 : 3400 dilution, respectively) on day 9 after the onset of hepatitis and then decreased rapidly in a parallel manner. Anti-HEV IgG antibody levels were the highest (1 : 17000 dilution) on day 145 and then decreased gradually but remained at high titres (1 : 2200 dilution) even 8.7 years after the onset of hepatitis. An HEV isolate, HE-JA10, recovered from the patient's serum at admission was closely related to a genotype III strain isolated in the United States (US1), with 92.2% identity over the full-length genome, and was most closely related to the JMY-Haw isolate of Japanese origin (95.4% identity).

Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan.

Among 87 patients who were previously treated for acute hepatitis of unknown etiology between 1992 and 2001 at five hospitals in Japan, 11 (13%) patients were positive for immunoglobulin M-class antibodies to hepatitis E virus (HEV) by enzyme immunoassay and had detectable HEV RNA by reverse transcription-PCR with two independent sets of primers derived from well-conserved genomic areas in open reading frames 1 and 2. Clinical HEV infection was significantly associated with male sex (9 of 11 versus 29 of 76 patients [P < 0.01]) and older age (52 +/- 11 [mean +/- standard deviation] versus 41 +/- 17 years [P < 0.05]), and its prevalence differed by geographic region (6 to 25%), with a higher rate in the northern part of Japan. At admission, the 11 patients with HEV-associated hepatitis had elevated alanine aminotransferase levels of 914 to 4,850 IU/liter, and all but 1 had elevated bilirubin levels of 1.5 to 24.0 mg/dl. The 11 HEV isolates were of genotype III or IV and were segregated into three groups with intergroup nucleotide differences of 9.5 to 22.0%. Phylogenetic analysis revealed that four isolates of genotype III were closely related to a Japanese isolate, while the other four isolates of the same genotype were nearest those from the United States. The remaining three isolates were close to known isolates of genotype IV in China and Taiwan but shared less than 88% identity with them. These results indicate that multiple genotypes of HEV cocirculate in Japan and contribute to the development of sporadic acute hepatitis, with the prevalence differing by age, sex, and geographic region.

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.