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1.
Mulyanto Sulaiman Ngongu Depamede Made Sriasih Masaharu Takahashi Shigeo Nagashima Suljid Jirintai Tsutomu Nishizawa Hiroaki Okamoto 《Archives of virology》2013,158(1):87-96
One hundred sixteen rats (Rattus rattus) captured in Indonesia from 2011 to 2012 were investigated for the prevalence of hepatitis E virus (HEV)-specific antibodies and HEV RNA. Using an ELISA based on HEV genotype 4 with an ad hoc cutoff value of 0.500, 18.1 % of the rats tested positive for anti-HEV IgG. By nested RT-PCR, 14.7 % of the rats had rat HEV RNA, and none were positive for HEV genotype 1-4. A high HEV prevalence among rats was associated with lower sanitary conditions in areas with a high population density. Sixteen of the 17 HEV isolates obtained from infected rats showed >93.0 % nucleotide sequence identity within the 840-nucleotide ORF1-ORF2 sequence and were most closely related to a Vietnamese strain (85.9-87.9 % identity), while the remaining isolate differed from known rat HEV strains by 18.8-23.3 % and may belong to a novel lineage of rat HEV. These results suggest a wide distribution of rat HEV with divergent genomes. 相似文献
2.
Koji Ichiyama Kentaro Yamada Toshinori Tanaka Shigeo Nagashima Jirintai Masaharu Takahashi Hiroaki Okamoto 《Archives of virology》2009,154(12):1945-1951
Using RNA preparations extracted from PLC/PRF/5 cells transfected with infectious genotype 3 hepatitis E virus (HEV) cDNA
clones or inoculated with a fecal suspension containing a genotype 4 HEV, the 5′-terminal sequence of a 2.2-kb subgenomic
RNA of genotype 3 and 4 HEVs was determined. Despite an insertion of T after nucleotide 5116 or an ORF3-null mutation in genotype
4 HEV and/or one of the genotype 3 variants, it was found that the subgenomic RNA of genotype 3 and 4 HEVs initiates exclusively
at nucleotide 5122 with the common sequence of 5′-GC, which is identical to that of the prototype genotype 1 HEV. 相似文献
3.
Masaharu Takahashi Toshinori Tanaka Hideyuki Takahashi Yu Hoshino Shigeo Nagashima Jirintai Hitoshi Mizuo Yasuyuki Yazaki Tomofumi Takagi Masahiro Azuma Eiji Kusano Norio Isoda Kentaro Sugano Hiroaki Okamoto 《Journal of clinical microbiology》2010,48(4):1112-1125
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 × 104 copies per well and 100% at ≥3.5 × 104 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.Hepatitis E, an acute viral hepatitis caused by infection with hepatitis E virus (HEV), is a globally distributed human disease. In developing countries of Asia, Africa, and Latin America, where sanitation conditions are not well maintained, HEV infection is transmitted via the fecal-oral route through virus-contaminated water or food, with substantial mortality in pregnant women (7, 33). In industrialized countries, autochthonous hepatitis E is far more common than previously recognized and has a predilection for older men, in whom it causes substantial morbidity and mortality (5, 13, 31, 36, 44). HEV is the sole member of the genus Hepevirus within the family Hepeviridae (6). It is a single-stranded, positive-sense, polyadenylated RNA molecule of approximately 7.2 kb in size, with short 5′- and 3′-untranslated regions (53). The genomic RNA contains three open reading frames (ORFs). ORF1 encodes nonstructural proteins involved in virus replication and virus protein processing. ORF2 and ORF3 overlap, and the ORF2 and ORF3 proteins are translated from a single bicistronic subgenomic RNA (11, 16). ORF2 encodes a 660-amino-acid (aa) capsid protein. ORF3 encodes a small phosphorylated protein (113 or 114 aa) that is essential for viral infectivity in vivo and for virion release (10, 16, 60, 63). The viral capsid protein induces neutralizing antibodies after immunization (8, 14, 26, 51) or during the course of infection (40, 41).Four major genotypes (genotypes 1 to 4) of HEV have been identified in mammalian species. The viruses in genotypes 1 and 2 are maintained among humans only and are responsible for waterborne epidemics of HEV infection in developing countries. Genotype 3 HEV has been found worldwide, and genotype 4 HEV was isolated in Asia (23, 32, 39). Recent comprehensive molecular and serological studies have led to the consensus that hepatitis E is a zoonosis with a reservoir in pigs and, possibly, a range of other mammals (27, 43, 47, 48, 57, 65). Genotype 3 and 4 HEVs are considered to undergo zoonotic transmission (23, 32). Recently, HEV was found in farmed rabbits in China, possibly representing a novel genotype (66). These rabbits are reared for their fur, and there is, as yet, no evidence of transmission to humans.Recently, using inocula comprised of fecal suspensions with high HEV loads, originally obtained from Japanese patients who contracted domestic infection of genotype 3 HEV (the JE03-1760F strain) or genotype 4 HEV (the HE-JF5/15F strain), we developed an efficient cell culture system for HEV in PLC/PRF/5 and A549 cells, which yielded the highest HEV load of 108 copies/ml in the culture supernatant, and we successfully propagated six or more generations in serial passages of culture supernatant (22, 55, 56). Infection of humans with HEV via blood transfusion has been reported not only in developing countries (genotype 1) (1, 18) but also in industrialized countries, including Japan (genotypes 3 and 4) (2, 24, 25, 28, 54), suggesting that HEV in serum samples can also be propagated in cultured cells. Therefore, in the present study, we examined whether HEV strains in serum samples obtained from various patients with sporadic acute hepatitis E can replicate in PLC/PRF/5 and A549 cells and produce infectious progenies in culture media, in relation to HEV load, genotype, and coexistence of HEV antibodies. In addition, in an attempt to clarify why HEV strains in sera are infectious in cultured cells despite the presence of HEV antibodies, HEV particles in serum samples were characterized and compared with those in culture supernatant and feces. 相似文献
4.
Mulyanto Pancawardani P Depamede SN Wahyono A Jirintai S Nagashima S Takahashi M Nishizawa T Okamoto H 《Virus research》2012,163(1):129-140
Four novel subgenotypes (C6, C11, C12, and D6) of hepatitis B virus (HBV) were identified in Papua, a multiethnic province of Indonesia. To characterize the HBV strains in Papua, serum samples collected from 515 indigenous inhabitants (mean age: 26.6 ± 9.6 years) in a previously unexamined area, Nabire, located in northern Papua, were used in the present study. Among 46 samples whose 1.6-kilobase (kb) HBV DNA sequence was amplified, 38 (83%) were typeable into known subgenotypes [B3 (n = 4), C1 (n = 2), C5, (n = 1), C6 (n = 5), C12 (n = 13), and D6 (n = 13)]. An analysis of the full-length sequence of the eight remaining HBV/C isolates whose sequence was either unclassifiable or uncertain within the 1.6-kb sequence showed no significant evidence of recombination in six isolates, and inter-genotypic recombination in two isolates (NAB20 and NAB46). By pairwise comparisons and a maximum-likelihood phylogenetic analysis, six non-recombinant isolates were considered significantly remote from known HBV/C isolates of subgenotypes C1−C12, and were classifiable into four novel subgenotypes (tentatively designated C13-C16). NAB20 and NAB46 were hybrids of C13/B3 and C12/G, respectively, displaying recombination breakpoints in the 5′-terminus of the P gene. Notably, the distribution of presumably indigenous subgenotypes C11−C16 was associated with particular language speakers in Papua. 相似文献
5.
Mulyanto Sulaiman Ngongu Depamede Kiely Surayah Andreas Andhry Heru Tjahyono Jirintai Shigeo Nagashima Masaharu Takahashi Hiroaki Okamoto 《Archives of virology》2010,155(5):705-715
Six novel subgenotypes (B7, B8, C6, C8, C9, and D6) within three hepatitis B virus (HBV) genotypes (B–D) were recently identified
in Indonesia. To further characterize HBV in this country, 18 HBV-viremic samples obtained from blood donors in Nusa Tenggara,
Indonesia, were subjected to phylogenetic analysis of an 1.6-kb partial or full-length sequence. Thirteen HBV isolates were
classified into genotype B with four distinct subgenotypes [B3 (n = 2), B5 (n = 1), B7 (n = 4), and B8 (n = 6)], followed by 4 HBV isolates of genotype C (HBV/C); the remaining one isolate was of D (D1). As for the four HBV/C isolates,
one isolate segregated into subgenotype C1, and two into C2. The remaining HBV/C isolate [C0901177(NT3)] differed from reported
HBV/C isolates (C1–C9) by 4.6–7.7% over the entire genome and did not show evidence of recombination with any of the known
HBV genotypes/subgenotypes, justifying its conclusive assignment into a novel subgenotype (C10) within genotype C. 相似文献
6.
Takahashi H Tanaka T Jirintai S Nagashima S Takahashi M Nishizawa T Mizuo H Yazaki Y Okamoto H 《Archives of virology》2012,157(2):235-246
Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs and wild boars to humans,
causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. However, no efficient cell culture
system for swine and boar HEV strains has been established. We inoculated A549 cells with 12 swine and boar HEV strains of
liver, feces, or serum origin at an HEV load of ≥2.0 × 104 copies per well and found that the HEV progeny replicated as efficiently as human HEV strains, with a maximum load of ~108 copies/ml. However, the HEV load in the culture medium at 30 days post-inoculation differed markedly by inoculum, ranging
from 1.0 × 102 to 1.1 × 107 copies/ml upon inoculation at a lower load of approximately 105 copies per well. All progeny were passaged successfully onto A549 and PLC/PRF/5 cells. In sharp contrast, no progeny viruses
were detectable in the culture supernatant upon inoculation with 13 swine and boar HEV strains at an HEV load of <1.8 × 104 copies per well. The present study also demonstrates that swine liver sold as food can be infectious, supporting the risk
of zoonotic food-borne HEV infection. 相似文献
7.
Mulyanto Depamede SN Wahyono A Jirintai Nagashima S Takahashi M Okamoto H 《Journal of medical virology》2011,83(1):54-64
Two novel subgenotypes (C6 and D6) of hepatitis B virus (HBV) were identified recently in Papua, a multiethnic area of Indonesia. To characterize further the HBV strains in Papua, serum samples collected from 59 viremic subjects (44 males and 15 females; mean age: 30.0 ± 15.5 years) among indigenous inhabitants in Papua, were subjected to phylogenetic analysis of an 1.6-kb partial sequence. Forty-five samples (76%) had genotype C HBV (HBV/C) [C5 (n = 1), C6 (n = 40), and unclassifiable (n = 4)], while seven samples (12%) were HBV/D [D1 (n = 1) and D6 (n = 6)] and six samples (10%) were HBV/B [B2 (n = 1), B3 (n = 3), B7 (n = 1), and B8 (n = 1)]; the remaining sample possessed B3 and C6. An analysis of the full-length sequence of the four HBV/C isolates (NMB09122, NMB09124, NMB09075, and MRK89073) that were unclassifiable into any of the 10 known HBV/C subgenotypes (C1-C10) showed no significant evidence of recombination. Over the entire genome, the NMB09122 and NMB09124 isolates shared 99.8% identity and segregated into a cluster with a bootstrap value of 100%, differing from HBV/C1-HBV/C10 by 3.8-6.9% (mean, ≥4.0%), indicating that NMB09122 and NMB09124 can be classified into a novel subgenotype within genotype C (tentatively designated C11). The NMB09075 and MRK89073 isolates were 97.4% identical to each other and differed from known HBV/C isolates, including the C11 strains, by 4.0-7.2% (mean, ≥4.5%) over the entire genome, indicating that NMB09075 and MRK89703 can be classified into another novel HBV/C subgenotype (C12). The distribution of C11 and C12 seemed to be associated with particular language speakers in Papua. 相似文献
8.
Shigeo Nagashima Masaharu Takahashi Suljid Jirintai Tanggis Tominari Kobayashi Tsutomu Nishizawa Hiroaki Okamoto 《Archives of virology》2014,159(5):979-991
Our previous studies demonstrated that hepatitis E virus (HEV) requires the multivesicular body (MVB) pathway to release virus particles, suggesting that HEV utilizes the cellular ESCRT machinery in the cytoplasm, not at the cell surface, to be released from infected cells. In this study, we generated a murine monoclonal antibody (mAb) against the membrane-associated HEV particles to examine whether the membrane is derived from intracellular vesicles or the cell surface. An established mAb, TA1708, was found to capture the membrane-associated HEV particles, but not the membrane-dissociated particles or fecal HEV, in an immunocapture RT-PCR assay. Furthermore, digitonin treatment confirmed that the membrane on the surface of cell-culture-generated HEV particles was a lipid membrane. Double immunofluorescence staining revealed that mAb TA1708 specifically recognizes trans-Golgi network protein 2 (TGOLN2), an intracellular antigen derived from the trans-Golgi network. Supporting these findings, HEV particles with lipid membranes and ORF3 proteins on their surface were found abundantly in the lysates of HEV-infected cells. These results indicate that HEV forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and that the released HEV particles with a lipid membrane retain the antigenicity of TGOLN2 on their surface. 相似文献
9.
Molecular and serological survey of hepatitis E virus infection among domestic pigs in Inner Mongolia, China 总被引:1,自引:0,他引:1
Jinshan Jirintai Dugarjavin Manglai Masaharu Takahashi Shigeo Nagashima Hiroaki Okamoto 《Archives of virology》2010,155(8):1217-1226
To evaluate the prevalence and characteristics of swine hepatitis E virus (HEV) infection in Inner Mongolia, China, serum
samples obtained from 356 2- to 4-month-old pigs on 14 farms in Inner Mongolia were tested for the presence of anti-HEV antibodies
and HEV RNA. Overall, 186 pigs (52%) tested positive for anti-HEV antibodies, while 30 pigs (8%) had detectable HEV RNA levels.
The 30 HEV isolates recovered from the viremic pigs were phylogenetically classified into genotype 4 and differed from each
other by up to 15.3% in a 412 nt sequence within ORF2. The Inner Mongolian swine HEV strains were most similar to human or
swine HEV strains isolated in the other provinces of China but differed by 15.9–18.9% from those in Mongolia (formerly known
as Outer Mongolia). These results indicate that farm pigs in Inner Mongolia are frequently infected with markedly divergent
genotype 4 HEV strains that may be indigenous to China. 相似文献
10.
Distinct changing profiles of hepatitis A and E virus infection among patients with acute hepatitis in Mongolia: The first report of the full genome sequence of a novel genotype 1 hepatitis E virus strain
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Bira Tsatsralt‐Od Putu Prathiwi Primadharsini Tsutomu Nishizawa Hiroshi Ohnishi Shigeo Nagashima Masaharu Takahashi Suljid Jirintai Dulmaa Nyamkhuu Hiroaki Okamoto 《Journal of medical virology》2018,90(1):84-92
In January 2012, Mongolia started a hepatitis A vaccination program, which has not yet been evaluated. The first occurrence of autochthonous acute hepatitis E in 2013, caused by genotype 4 hepatitis E virus (HEV), suggests the need for a routine study to monitor its prevalence. One hundred fifty‐four consecutive patients who were clinically diagnosed with acute hepatitis between 2014 and 2015 in Ulaanbaatar, Mongolia were studied. By serological and molecular testing followed by sequencing and phylogenetic analysis, only one patient (0.6%) was diagnosed with acute hepatitis A, caused by genotype IA hepatitis A virus (HAV), and 32 (20.8%) patients were diagnosed with acute hepatitis E, caused by genotype 1 HEV. The 32 HEV isolates obtained in this study shared 99.5‐100% nucleotide identity and were grouped into a cluster separated from those of subtypes 1a to 1f. Upon comparison of p‐distances over the entire genome, the distances between one representative HEV isolate (MNE15‐072) and 1a‐1f strains were 0.071‐0.137, while those between 1b and 1c were 0.062‐0.070. In conclusion, the prevalence of acute hepatitis A has decreased in Mongolia since the start of the vaccination program, while the monophyletic genotype 1 HEV strain of a probably novel subtype has been prevalent. 相似文献