長嶋 茂雄

© 2017, Japanese Society of Gastroenterology. Hepatitis E, which is caused by hepatitis E virus (HEV), is a public health concern in Japan, where the zoonotic food-borne transmission of HEV from domestic pigs and wild boars plays an important role. A 44-year-old Japanese man with autochthonous sporadic acute hepatitis E was admitted with general fatigue and moderate liver dysfunction. In the present study, two distinct HEV strains were recovered from the patient, who had consumed the raw or undercooked pig liver and intestine two or three times per week for 3 months before the disease onset. The recovered HEV strains were segregated into two clusters within subgenotype 3b, the open reading frame (ORF)1 and ORF2 sequences of which each showed ~10% difference, indicating HEV mixed infection. Because most notified patients with clinical HEV infection in Japan are diagnosed based on the detection of IgA-class HEV antibodies and because serum samples from only a limited number of HEV-infected patients are subjected to HEV RNA detection and nucleotide sequencing, it is very likely that patients with HEV mixed infection remain largely overlooked. The identification of sources of autochthonous HEV infection remains an important goal. Continued efforts to trace the sources of acute or chronic autochthonous HEV infection are warranted.

The viral factors associated with the development of fulminant hepatitis B are not fully understood. We recently found four unique mutations [G to A at nucleotide 1742 (G1742A), C1766T, T1768A and T1809C] in the basal core promoter (BCP) region of a genotype A hepatitis B virus (HBV) strain (FH) obtained from a 53-year-old man with fatal fulminant hepatitis. To elucidate the association of the mutations of the FH genome with the disease, we constructed a 1.3-fold FH genome and its five variants by replacing one or two mutated nucleotides with wild-type nucleotide(s) via site-directed mutagenesis, and transfected human hepatoma cells (HepG2/C3A) with the constructs. There were no discernible differences between FH and two variants (FH_A1742G and FH_C1809T) with regard to viral replication and protein expression. However, in comparison to three other variants (FH_T1766C, FH_A1768T and FH_T1766C/A1768T) with wild-type nucleotide(s) at 1766 and/or 1768, the FH genome exhibited a 2.5-5-fold enhancement of viral replication by heightened pregenomic RNA synthesis and a 1.5-2.5-fold reduction in the hepatitis B e antigen (HBeAg) synthesis by the downregulation of the precore mRNA level. An immunofluorescence analysis revealed the increased and predominant cytoplasmic localization of the core protein in the FH genome. The present study demonstrates that the C1766T/T1768A mutations in the BCP region of genotype A HBV enhance viral replication, downregulate HBeAg expression and are responsible for the predominant localization of the core protein in the cytoplasm, which are likely associated with the development of fulminant hepatitis.

Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101-644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV. (C) 2016 Elsevier B.V. All rights reserved.

Despite the high endemicity of hepatitis A virus (HAV) in Mongolia, the genetic information on those HAV strains is limited. Serum samples obtained from 935 patients with acute hepatitis in Ulaanbaatar, Mongolia during 2004-2013 were tested for the presence of HAV RNA using reverse transcription-PCR with primers targeting the VP1-2B region (481 nucleotides, primer sequences at both ends excluded). Overall, 180 patients (19.3%) had detectable HAV RNA. These 180 isolates shared 94.6-100% identity and formed four phylogenetic clusters within subgenotype IA. One or three representative HAV isolates from each cluster exhibited 2.6-3.9% difference between clusters over the entire genome. Cluster 1 accounted for 65.0% of the total, followed by Cluster 2 (30.6%), Cluster 3 (3.3%), and Cluster 4 (1.1%). Clusters 1 and 2 were predominant throughout the observation period, whereas Cluster 3 was undetectable in 2009 and 2013 and Cluster 4 became undetectable after 2009. The Mongolian HAV isolates were closest to those of Chinese or Japanese origin (97.7-98.5% identities over the entire genome), suggesting the evolution from a common ancestor with those circulating in China and Japan. Further molecular epidemiological analyses of HAV infection are necessary to investigate the factors underlying the spread of HAV and to implement appropriate prevention measures in Mongolia. J. Med. Virol. 88:622-630, 2016. (c) 2015 Wiley Periodicals, Inc.

We describe a 92-year-old man who developed acute hepatitis E living in the mountains in Tottori, Japan. He had no history of travel abroad and blood transfusion as well as consumption of animal meat/viscera and raw seafood within three months before the disease onset. However, he had a peculiar habit of drinking diluted bile of dried gall bladders obtained from wild boars. Although leftover bile suspensions were not available, seven (39%) of the 18 stored gall bladders had detectable hepatitis E virus (HEV) RNA at the viral load of up to 4.6×105 copies/ml in 10% bile suspension. The wild boar HEV isolates shared 99.0-99.8% nucleotide sequence identities with a subgenotype 3a HEV strain recovered from the index patient. These results suggest that dried gall bladders obtained from HEV-infected wild boars may have been a source of HEV infection in this patient.

Background: Recently, chronic hepatitis E has been increasingly reported in organ transplant recipients in European countries. In Japan, the prevalence of hepatitis E virus (HEV) infection after transplantation remains unclear, so we conducted a nationwide cross-sectional study to clarify the prevalence of chronic HEV infection in Japanese liver transplant recipients. Methods: A total of 1893 liver transplant recipients in 17 university hospitals in Japan were examined for the presence of immunoglobulin G (IgG), IgM and IgA classes of anti-HEV antibodies, and HEV RNA in serum. Findings: The prevalence of anti-HEV IgG, IgM and IgA class antibodies was 2.9% (54/1893), 0.05% (1/1893) and 0% (0/1893), respectively. Of 1651 patients tested for HEV RNA, two patients (0.12%) were found to be positive and developed chronic infection after liver transplantation. In both cases, HEV RNA was also detected in one of the blood products transfused at the perioperative period. Analysis of the HEV genomes revealed that the HEV isolates obtained from the recipients and the transfused blood products were identical in both cases, indicating transfusion-transmitted HEV infection. Interpretation: The prevalence of HEV antibodies in liver transplant recipients was 2.9%, which is low compared with the healthy population in Japan and with organ transplant recipients in European countries; however, the present study found, for the first time, two Japanese patients with chronic HEV infection that was acquired via blood transfusion during or after liver transplantation.

Reactivation of a former hepatitis B virus (HBV) infection can be triggered by immunosuppressive therapy, diseases associated with an immunocompromised state, organ transplantation or the withdrawal of antiviral drugs. Despite the absence of such risk factors, a spontaneous reactivation of HBV replication occurred in two elderly patients with resolved or occult HBV infection. A 73-year-old male underwent coronary artery bypass grafting in October 2008, and was negative for HBsAg but positive for anti-HBs. In July 2009, his serum became positive for HBsAg, HBeAg and HBV DNA (6.4 log copies/ml; genotype C), but negative for anti-HBc IgM, with abrupt elevation of the liver enzymes. The entire genomic sequence of HBV recovered from this patient revealed no mutations in the core promoter and precore regions that interfere with HBeAg production. A 76-year-old male with a history of endoscopic mucosal resection for esophageal cancer in 2002 and an initial diagnosis of diabetes mellitus in 2009, at which time he was negative for HBsAg. He was found to be positive for HBsAg in September 2012 during a laboratory examination performed prior to the resection of recurrent esophageal cancer, despite a low HBV load (2.1 log copies/ml). Three months later, without the administration of any anticancer drugs, the HBV DNA (genotype B) level increased to 5.1 log copies/ml. A precore G1896A variant with high quasispecies diversity was recovered from the patient. Aging, surgical stress and complication of disease(s) associated with compromised immunity, such as cancer, arteriosclerosis and diabetes mellitus may trigger spontaneous HBV reactivation. J. Med. Virol. 87:589-600, 2015. (c) 2015 Wiley Periodicals, Inc.

Autochthonous hepatitis E is increasingly being recognized in industrialized countries, including Japan. Although neurological abnormalities have been sporadically reported as an extrahepatic manifestation of hepatitis E virus (HEV) infection, it is rare and has not been reported in Japan. The present study aimed to characterize a total of 20 patients consecutively diagnosed with sporadic acute

A 71-year-old (C1I) and 69-year-old (C2I) Japanese female contracted fulminant hepatitis B after 50 and 49 years of marriage, respectively. Both index cases exhibited high levels of anti-HBc IgM antibodies (24.2 and 31.5 S/CO, respectively), suggestive of acute hepatitis B virus (HBV) infection, although they had no discernible risk factors for HBV infection, except for chronically HBV-infected spouses with detectable HBV DNA (3.3 log copies/ml [C1S: 72-year-old] and 7.2 log copies/ml [C2S: 71-year-old]). The HBV genotype/subgenotype was identical in each couple (B/B1 or C/C2). The HBV isolates from the index cases and spouses shared a nucleotide sequence identity of 99.5% and 99.7%, respectively, over the entire genome, and these four isolates had the highest nucleotide sequence identity of only 97% to HBV isolates deposited in DNA databases. Phylogenetic trees confirmed a close relationship of the HBV isolates between C1I and C1S and between C2I and C2S, supported by a high bootstrap value of 100% within each couple, indicating the transfer of HBV infection between spouses. These four isolates shared a precore mutation of G1896A known to be associated with fulminant hepatitis B. Although the history of sexual contact within a reasonable incubation period was obscure for one stable, monogamous couple (C1I and C1S), the other couple had a monogamous sexual relationship within six months prior to disease onset. This study indicates that two elderly Japanese patients with fulminant hepatitis B acquired HBV infection via interspousal (most likely sexual) transmission during long-lasting marriages. J. Med. Virol. 86:1851-1860, 2014. (c) 2014 Wiley Periodicals, Inc.

Aim: To characterize hepatitis E in Mie prefecture and to investigate whether raw pig liver sold as food in Mie is contaminated with hepatitis E virus (HEV) strains similar to those recovered from patients. Methods: Seventeen patients with sporadic acute hepatitis E treated from 2004 to 2012 were studied. A total of 243 packages of raw pig liver from regional grocery stores were tested for the presence of HEV RNA. The partial genomic sequences of human and swine HEV isolates were determined and subjected to the phylogenetic analyses. Results: The HEV isolates recovered from the 17 patients segregated into genotype 3 (n = 15) and genotype 4 (n = 2), and 15 genotype 3 isolates further segregated into 3e (n = 11) and 3b (n = 4). Pig liver specimens from 12 (4.9%) of the 243 packages had detectable HEV RNA. All 12 swine HEV isolates were grouped into genotype 3 (3a or 3b). Although no 3e strains were isolated from pig liver specimens, two 3b swine strains were 99.5-100% identical to two HEV strains recovered from hepatitis patients, within 412-nt partial sequences. Conclusion: The 3e HEV was prevalent among hepatitis E patients. HEV RNA was detected in approximately 5% of pig liver sold as food. The presence of identical HEV strains between hepatitis patients and pig liver indicated that pigs play an important role as reservoirs for HEV in humans in Mie. Further studies are needed to clarify the source of 3e HEV in the animal and environmental reservoirs.

Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5%, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.

A 67-year-old male living in Tsu city, Mie prefecture, Japan was referred to our hospital for further examination of acute liver injury and was diagnosed as having clinical hepatitis E virus (HEV) infection in January 2010. The HEV strain (HE-JA11-1701) isolated from the patient belonged to genotype 3 and European-type subgenotype 3e. It was presumed that the patient had been infected from a wild boar (Sus scrofa leucomystax) because he consumed meat/viscera from a wild boar that he had captured himself as a hunter approximately 2 months before disease onset. A specimen of the boar meat/viscera that the patient had ingested was not available. However, the HE-JA11-1701 strain was 99.8% identical within the 412-nucleotide sequence of the open reading frame 2 region to a HEV strain (JBOAR012-Mie08) that had been recovered from a wild boar captured near the patient's hunting area in 2008. A phylogenetic analysis confirmed that the two HEV strains had a close genetic relationship and were segregated into subgenotype 3e, supported by a high bootstrap value of 99%. Of note, the HE-JA11-1701 and JBOAR012-Mie08 strains were remotely related to the 3e strains reported in Japan and European countries, with a nucleotide difference of 7.9-13.9%, reinforcing the uniqueness of the 3e strains obtained in the present study. These results strongly support our speculation that the patient developed acute hepatitis E via consumption of HEV-infected boar meat/viscera. Genetic analyses of HEV strains are useful for tracing infectious sources in sporadic cases of acute hepatitis E.

Although rat hepatitis E virus (HEV) has been identified in wild rats, no cell culture systems for this virus have been established. A recent report suggesting the presence of antibodies against rat HEV in human sera encouraged us to cultivate rat HEV in human cells. When liver homogenates obtained from wild rats (Rattus rattus) in Indonesia were inoculated onto human hepatocarcinoma cells, the rat HEV replicated efficiently in PLC/PRF/5, HuH-7 and HepG2 cells, irrespective of its genetic group (G1-G3). The rat HEV particles released from cultured cells harbored lipid-associated membranes on their surface that were depleted by treatment with detergent and protease, with the buoyant density in sucrose shifting from 1.15-1.16 g/ml to 1.27-1.28 g/ml. A Northern blotting analysis revealed genomic RNA of 7.0 kb and subgenomic RNA of 2.0 kb in the infected cells. The subgenomic RNA of G1-G3 each possessed the extreme 5'-end sequence of GUAGC (nt 4933-4937), downstream of the highly conserved sequence of GAAUAACA (nt 4916-4923). The establishment of culture systems for rat HEV would allow for extended studies of the mechanisms of viral replication and functional roles of HEV proteins. Further investigation is required to clarify the zoonotic potential of rat HEV. (C) 2014 Elsevier B.V. All rights reserved.

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.

Although a consensus classification system for hepatitis E virus (HEV) genotypes is currently unavailable, HEV variants (JBOAR135-Shiz09 and wbJOY_06) from wild boars (Sus scrofa leucomystax) have provisionally been classified into two novel genotypes (5 and 6). While performing a survey of HEV infections among 566 wild boars that were captured in Japan between January 2010 and August 2013, we found 24 boars (4.2%) with ongoing HEV infections: 13 had genotype 3 HEV, 10 had genotype 4 HEV and the remaining boar possessed a novel HEV variant (designated wbJNN_13). The entire wbJNN_13 genome comprised 7247 nucleotides excluding the poly(A) tail, and was highly divergent from known genotype 1 to 4 HEV isolates derived from humans, swine, wild boars, deer, mongoose and rabbits by 22.4-28.2%, JBOAR135-Shiz09 and wbJOY_06 by 19.6-21.9% and rat, ferret, bat and avian HEV isolates by 40.9-46.1% over the entire genome. Phylogenetic trees confirmed that wbJNN_13 is distantly related to all known HEV isolates. A Simplot analysis revealed no significant recombination among the existing HEV strains. These results indicate the presence of at least three genetic lineages of presumably boar-indigenous HEV strains. Further studies to fully understand the extent of the genomic heterogeneity of HEV variants infecting wild boars are warranted. (C) 2013 Elsevier B.V. All rights reserved.

Rat hepatitis E virus (HEV) strains have recently been isolated in several areas of Germany, Vietnam, the United States, Indonesia and China. However, genetic information regarding these rat HEV strains is limited. A total of 369 wild rats (Rattus rattus) captured in Central Java (Solo) and on Lombok Island, Indonesia were tested for the presence of rat HEV-specific antibodies and RNA. Overall, 137 rats (37.1%) tested positive for rat anti-HEV antibodies, while 97 (26.3%) had rat HEV RNA detectable on reverse transcription-PCR with primers targeting the ORF1-ORF2 junctional region. The 97 HEV strains recovered from these viremic rats were 76.3-100% identical to each other in an 840-nucleotide sequence and 75.4-88.4% identical to the rat HEV strains reported in Germany and Vietnam. Five representative Indonesian strains, one from each of five phylogenetic clusters, whose entire genomic sequence was determined, were segregated into three genetic groups (a German type, Vietnamese type and novel type), which differed from each other by 19.5-23.5 (22.0 +/- 1.7)% over the entire genome. These results suggest the presence of at least three genetic groups of rat HEV and indicate the circulation of polyphyletic strains of rat HEV belonging to three distinct genetic groups in Indonesia. (C) 2013 Elsevier B.V. All rights reserved.

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.

Rabbit hepatitis E virus (HEV) strains have recently been isolated in several areas of China and in the US and France. However, the host range, distribution and zoonotic potential of these HEV strains remain unknown and their propagation in cultured cells has not yet been reported. A total of 211 4-month-old rabbits raised on a farm in Inner Mongolia were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 121 rabbits (57.3%) tested positive for anti-HEV antibodies, and 151 (71.6%) had detectable HEV RNA. The 174 HEV strains recovered from these viremic rabbits, including two distinct strains each from 23 rabbits, differed from each other by up to 13.6% in a 412-nucleotide (nt) sequence within ORF2, and were 89.3-95.9% identical to the reported rabbit HEV strains in other provinces of China. Three representative Inner Mongolian strains, one each from three phylogenetic clusters, whose entire genomic sequences were determined, shared 79.6-96.7% identities with reported rabbit HEV strains within the entire or 242- to 1349-nt partial genomic sequence. Rabbit HEV strains recovered from liver tissues of rabbits with a high HEV load propagated efficiently in human cell lines (A549 and PLC/PRF/5 cells), suggesting the potential zoonotic risk of rabbit HEV. (c) 2012 Elsevier B.V. All rights reserved.

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 a parts per thousand yen2.0 x 10(4) copies per well and found that the HEV progeny replicated as efficiently as human HEV strains, with a maximum load of similar to 10(8) copies/ml. However, the HEV load in the culture medium at 30 days post-inoculation differed markedly by inoculum, ranging from 1.0 x 10(2) to 1.1 x 10(7) copies/ml upon inoculation at a lower load of approximately 10(5) 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 x 10(4) 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.

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), Cl (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. (C) 2011 Elsevier B.V. All rights reserved.

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wildtype ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4% of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6%, respectively, while the overexpression of wildtype Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles. © 2011 SGM.

Rotaviruses with the P[8] VP4 genotype are a major cause of acute infantile diarrhea. The P[8] genotype is classified into two genetically distinct subtypes, P[8]a and P[8]b. Most of the P[81 strains belong to subtype P[8]a, whilst P[8]b strains are rare. To date, the whole genomes of a few P[8]a strains have been analyzed, whilst there are no reports on full genomic analysis of the P[8]b strains. To determine the genetic makeup of the rare P[8]b strains and their overall genetic relatedness to the P[8]a strains, the present study analyzed the full genomes of a human G9P[8]b strain, MMC38, and a G1P[8]b strain, MMC71, detected in Bangladesh in 2005. By nucleotide sequence identities and phylogenetic analyses, strains MMC38 and MMC71 exhibited a human rotavirus Wa-like genotype constellation. Except for the VP4 gene, all the genes of strains MMC38 and MMC71 were closely related to cognate genes of the contemporary and more recent human Wa-like G1P[8]a, G9P[8]a, G11P[8]a, G11P[25], G12P[6] and G12P[8]a strains, including those from Bangladesh. Therefore, strains MMC38 and MMC71 possessed the genetically distinct P[8]b VP4 gene on a common human Wa-like genetic backbone, pointing towards their possible origin from reassortment events between common human Wa-like strains and unidentified rotavirus strains possessing the rare P[8]b-like VP4 gene. Since strains with this stable Wa-like genetic backbone can spread rapidly, and it is not certain as to whether the current rotavirus vaccines will be equally efficacious against the P[8]b strains as the P[8]a strains, proper detection of P[8]b strains and their whole genomic analyses might be of public health significance. To our knowledge, the present study is the first report on full genomic analysis of the rare P[8]b rotavirus strains. (C) 2011 Elsevier BM. All rights reserved.

To study human rotaviruses in Cuban children up to 5 years old with acute diarrhea, a total of 64 faecal samples from two pediatric hospitals between 2006 and 2008 were analyzed. Thirty-nine samples (60.9%) were found positive for rotaviruses by enzyme-linked innmunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE), while four (6.2%) exhibited discordant results (ELISA positives/PAGE negatives). All the positive samples were genotyped by a reverse transcription-polymerase chain reaction (RT-PCR). The most prevalent G and P types were G1 (60.5%) and P[8] (90.6%), respectively. Among the rotaviruses detected, two long RNA patterns were identified by PAGE (L/A and L/B). The predominant genotype in 2006 and 2007 was G1/P[8] with detection rates 71.4% and 80%, respectively. In contrast, G9/P[8] was found at the highest rate (78.5%) in 2008. The phylogenetic analysis of VP7 genes of the ten representative strains indicated that Cuban G1/P(8) rotaviruses were close to those reported in the Americas, and G9/P[8] rotaviruses were located in the lineage of the emerging G9 strains spreading worldwide. This is the first molecular epidemiologic study of rotaviruses in Cuba, highlighting the current increase of the G9 rotavirus which has been recognized globally as an emerging genotype. The study reinforces the need for a systematic surveillance of the molecular epidemiology of rotaviruses. J. Med. Virol. 83:738-744, 2011. (C) 2011 Wiley-Liss, Inc.

Picobirnaviruses (PBV) are small, non-enveloped viruses with a bisegmented double-stranded RNA genome. In this study a PBV strain, PBV/Horse/India/BG-Eq-3/2010, was identified in the faeces of a 10 month old weaned female foal with diarrhoea in January 2010 from Kolkata, India. Surprisingly, sequence comparison and phylogenetic analysis of a short stretch of the RNA dependent RNA polymerase gene revealed close genetic relatedness (> 98% nucleotide identity) to a human genogroup I PBV strain (Hu/GPBV1) detected earlier from the same part of India. Our observations together with earlier findings on genetic relatedness between human and animal PBV warrant further studies on zoonotic potential.

Background: Picobirnaviruses (PBVs) associated with viral gastroenteritis were reported from humans and several animal species to date. PBVs belonging to family Picobirnaviridae under proposed order Diplornavirales are small, non-enveloped, with bisegmented dsRNA genome. Methods: PBV was detected by polyacrylamide gel electrophoresis (PAGE) and silver staining. Confirmatory RT-PCR using primer pair PicoB25 (+) and PicoB43 (-) for genogroup I PBV and PicoB23(+) and PicoB24(-) for genogroup II PBV, resulted in amplicons of 201bp and 369bp respectively. The amplicons of genogroup I PBV were cloned and sequenced; amplicon of genogroup II PBV was directly sequenced. Further, the phylogenetic relationship and genetic diversity of strains from Kolkata was compared with hitherto reported PBV strains. Results: In PAGE, a faecal specimen showed three sets of PBV with large profile bisegmented genomic RNA with slight variation in migration pattern. Molecular cloning experiments confirmed that PBV/Human/INDIA/GPBV6/2007 had mixed infection comprising four different strains of PBV genogroup I [GPBV6C1P-GPBV6C4P] and one PBV genogroup II strain [GPBV6G2P]. Conclusion: Sequence comparison and phylogenetic analysis of gene segment 2 of GPBV6 clones (C1, C2, C3 and C4) revealed low nucleotide identities (59-63%) and distant genetic relatedness to other human and porcine genogroup I picobirnaviruses. The strain GPBV6G2P represents another PBV genogroup II strain after prototype strain 4-GA-91/USA as genogroup II PBVs have seldom been reported to date, except from Kolkata, India and Netherlands. We are reporting the first incidence of detection of multiple strain (mixed) infection of picobirnavirus [genogroups I and II] from a diarrhoeic child in a slum community of Kolkata, India.

During the 2004 surveillance of rotaviruses in Wuhan, China, a G4P[6] rotavirus strain R479 was isolated from a stool specimen collected from a 2-year-old child with diarrhea. The strain R479 had an uncommon subgroup specificity 1+11, and analysis of the VP6 gene suggested that it was related to porcine rotaviruses. In the present study, full-length nucleotide sequences of all the RNA segments of R479 were determined and analyzed phylogenetically to identify the origin of individual RNA segments. According to the rotavirus genotyping system based on 11 RNA segments, the genotype of R479 was expressed as G4-P[6]-15-R1-C1-M1-Al-N1-T7-E1-H1. This genotype includes the porcine-like VP6 genotype (15) and bovine-like NSP3 genotype (T7). Phylogenetic analysis revealed that R479 genes encoding VP1, VP2, VP3, VP6, VP7, VP8*, NSP1, NSP4, and NSP5 were more closely related to those of porcine rotaviruses than human or other animal rotaviruses. In contrast, it was remarkable that the NSP3 gene of R479 was genetically closely related to only a bovine rotavirus strain UK. The NSP2 gene of R479 was also unique and clustered with only the G5P[8] human strain IAL28 and G3P[24] simian strain TUCH. These results suggested that R479 may be a reassortant virus having the NSP3 gene from a bovine rotavirus in the genetic background of a porcine rotavirus, with an NSP2 gene related to the porcine-human reassortant strain IAL28. To our knowledge, R479 is the first porcine bovine reassortant rotavirus isolated from a human. J. Med. Virol. 82:1094-1102, 2010. (C) 2010 Wiley-Liss Inc

The genus, Picobirnavirus (PBV), Spanish 'pico' = 'small', birna for 'bipartite RNA' genome, belongs to the family Picobirnaviridae under the proposed order Diplornavirales. PBV infections have been reported from diarrhoeic animal species and humans as well as from asymptomatic cases. The detection of Picobirnaviruses (PBVs) in diarrhoeic faecal specimens from children aged <5 years, suggestive of zoonotic transmission is being reported. 23 Picobirnavirus positive faecal specimens were detected by polyacrylamide gel electrophoresis (PAGE) and silver staining from a set of 1112 faecal specimens collected from an urban slum community in Kolkata between July and October 2007. The Picobirnaviruses showed either large profile (n = 22) or small profile (n = 1) for their bisegmented genomic double-stranded RNA (dsRNA). 13/23 positives were amplified by reverse transcription polymerase chain reaction (RT-PCR) as 201 bp amplicon with genogroup I primers [PicoB25(+) and PicoB43(-) specific for RNA dependent RNA polymerase (RdRp) gene fragment encoded by genomic segment 2] and seven amplicons were sequenced [GPBV1-5, 7 and 8]. Sequence analyses showed that four PBV strains [GPBV1-3 and 8) resembled different clones of porcine PBV strains (D4, D6 and C10) reported in 2008 from Hungary and two PBV strains [GPBV4 and 7] resembled human PBV strains (P597, Kolkata and 2-GA-91, USA) with the maximum nucleotide (nt) identity ranging from 78% to 92%. One strain GPBV5 clustered with human PBVs and porcine PBVs that were reported from Hungary, Venezuela and Argentina showing close homology to human-like PBVs. Therefore, the close monitoring of their global spread as well as in-depth molecular characterization is essential for better understanding of emerging PBV strains. (C) 2010 Elsevier B.V. All rights reserved.

In our previous study, a novel P[8] subtype, i.e., P[8]b was identified for human rotavirus strains MMC38 and MMC71 detected in Bangladesh, of which the P types could not be determined by conventional RT-PCR genotyping methods. In the present study, a modified multiplex RT-PCR method was developed to detect P[8]b as well as common human rotavirus P types. With this method, P[8]b was detected in three strains among the 26 rotavirus specimens which had been judged as mixed P types in the previous study in Bangladesh. The VP4 nucleotide sequences of these strains showed more than 98.9% identities to those of strains MMC38 and MMC71. The newly designed RT-PCR method was considered as useful for identifying P[8]b and avoiding misclassification by the conventional RTPCR genotyping methods.

The study evaluated the antibiotic resistance patterns of Helicobacter pylori strains against metronidazole and clarithromycin in a hospital in Havana, Cuba. Eighty-five percent, 22.5%, and 10% of 40 H. pylori strains investigated were resistant to metronidazole, ciprofloxacin, and clarithromycin respectively but all were susceptible to amoxicillin and tetracycline. RdxA truncation was found only in metronidazole-resistant strains. In such strains, reported are eight and two novel mutations in the rdxA and frxA genes respectively. Two-point mutations in the 23S rRNA genes of clarithromycin-resistant strains were detected. A high prevalence of metronidazole resistance was found in Cuban H. pylori strains. Mutations in the rdxA gene may contribute more significantly than frxA gene to the high level of resistance to metronidazole. This study supports the need to continue monitoring the antibiotic susceptibility in H. pylori in Cuba to guide the treatment of such infection.

We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6')- aph(2 ''), aph(3'), ant(6), ant(3 '')(9), aph(2 '')- Id, aph(2 '')-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6')-Ie-aph(2 '')-Ia, aph(3'), ant(6), ant(3 '')(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U. S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits.

In Yangon, Myanmar, a human group B rotavirus was first detected in 2007 in a stool specimen from a sporadic case of acute gastroenteritis in an adult. The strain was designated as MMR-B1. The full-length sequences of the MMR-B1 genes encoding VP7, VP4 (VP5* and VP8*), VP6, and NSP4 were determined for genetic characterization. These four MMR-B1 genes showed considerable higher sequence identities (97.2-98.4%) to those of group B rotaviruses detected in India (CAL-1 in 1998) and Bangladesh (Bang373 and Bang544 in 2000 and 2001, respectively) than to those of Chinese strains (90.7-93.6%) (ADRV and WH-1 in 1982 and 2002, respectively). Phylogenetically, the four genes of MMR-B1 were clustered into the Indian-Bangladeshi lineage. Although the deduced amino acid sequences of MMR-B1 were similar to those of strains CAL-1 and Bang373, several amino acids in VP8* were found to be different from those of the group B rotaviruses described previously. The first detection in Myanmar of a human group B rotavirus suggested endemic distribution or expansion of the group B rotavirus of the Indian-Bangladeshi lineage in Southeast Asia. J. Med. Virol. 8 1: 1968-1974, 2009. (C); 2009 Wiley-Liss, Inc.

Rotaviruses are the major cause of severe dehydrating gastroenteritis in children Worldwide. In this study, we report a positive role of cellular chaperone Hsp90 during rotavirus infection. A highly specific Hsp90 inhibitor, 17-allylamono-demethoxygeldanamycin (17-AAG) was used to delineate the functional role of Hsp90. In MA104 cells treated with 17-AAG after viral adsorption, replication of simian (SA11) or human (KU) strains was attenuated as assessed by quantitating both plaque Forming units and expression of viral genes. Phosphorylation of Akt and NF kappa B observed 2-4 hpi with SA11, was strongly inhibited in the presence of 17-AAG. Direct Hsp90-Akt interaction in virus infected cells was also reduced in the presence of 17-AAG. Anti-rotaviral effects of 17-AAG were due to inhibition of activation of Akt that was confirmed since, PI3K/Akt inhibitors attenuated rotavirus growth significantly. Thus, Hsp90 regulates rotavirus by modulating cellular signaling proteins. The results highlight the importance of cellular-proteins during rotavirus infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies. (C) 2009 Elsevier Inc. All rights reserved.

Prevalence of high-level gentamicin resistance genes aac(6')-Ie-aph(2 '')-Ia and aph(2 '')-Ie, which encode distinct aminoglycoside-modifying enzymes, was analyzed for a total of 1128 clinical isolates of enterococci obtained in a Japanese hospital during a period between 1997 and 2007. The aac(6')-Ie-aph(2 '')-Ia was detected in 40.1%, 12.9%, and 3.6% of Enterococcus faecalis, E. faecium, and other enterococcal species, respectively, and aph(200)-Ie was detected in 3.3% of E. faecium. During the study period, detection rate of aac(6')-Ie-aph(2 '')-Ia in E. faecium increased from 4% (1997-1998) to 28% (2006-2007), whereas generally constant in E. faecalis. By the analysis of IS256-flanking patterns of aac(6')-Ie-aph(2 '')-Ia, truncated forms of Tn5281 lacking IS256 at the 3'-end, 5'-end, and both ends of aac(6')-Ie-aph(2 '')-Ia were identified in 4.6%, 32.4%, and 34.2% of E. faecalis strains, respectively, while the composite Tn5281-like element with IS256 at both sides was detected in 28.7% of the strains. A truncated form of Tn5281 lacking IS256 at the 5'-end was predominant in other enterococcal species. Among 14 E. faecalis and 10 E. faecium strains harboring aac(6')-Ie-aph(2 '')-Ia, 8 and 6 different sequence types (STs) were identified by multilocus sequence typing, respectively. Some E. faecalis STs (ST4, ST16, ST64, and ST223) were found in more than one strain, and ST4 and ST64 were associated with different IS256-flanking patterns. STs of five among six E. faecium strains with aac(6')-Ie-aph(2 '')-Ia (ST78, ST203, and ST418) belonged to the clonal complex (CC) 17, which is known as globally emerging lineage of vancomycin-or ampicillin-resistant E. faecium clones. E. faecium strains with aph(2 '')-Ie were classified into newly assigned STs, ST426, and its single locus variant ST427, which also belonged to CC17. Therefore, it was suggested that E. faecium of CC17 is prone to acquire high-level gentamicin resistance genes, and aph(2 '')-Ie is distributed to specific E. faecium clones that are distinct from those having aac(6')-Ie-aph(2 '')-Ia.

The G1 and G9 rotavirus strains MMC71 and MMC38 (subgroup II, NSP4 genogroup B), respectively, isolated from children in Bangladesh, were analyzed genetically. Full-length VP4 genes of these strains had 98.9% identity to each other and showed 83.9-89.4% identity to those of the P[4] and P[8] rotaviruses. Phylogenetic analysis of VP4 nucleotide sequences revealed that strains MMC38 and MMC71 were located in a lineage of P[8] strains. However, the cluster was highly divergent from the previously established P[8] strains. The VP8* portions of strains MMC38 and MMC71 showed more than 93.9% nucleotide sequence identity to OP354-like P[8] strains, and these strains were clustered into the same lineage. These findings indicate that the VP4 of these strains should be classified into a subtype of the P[8] genotype (P[8]b) that is distinct from that of common P[8] rotaviruses (P[8]a).

Deduced amino acid sequence and phylogenetic analyses of a group A rotavirus G9P[6] strain (designated as mcs/13-07), detected from a 3-year-old child in Eastern India, revealed a VP8* closely related to porcine P[6] strains (P[6] sublineage 1D), and the VP7 clustered with G9 lineage-III strains. To our knowledge, this is the first report of human P[6] strain clustering in sublineage Id. Thus, to further characterize the evolutionary diversity of strain mcs/13-07, all gene segments were analyzed. VP6 and NSP4 exhibited genetic relatedness to Wa-like human subgroup II strains, while VP1-3, NSP1-3 and NSP5 were closely related to porcine strains. Based on the new classification system of rotaviruses, mcs/13-07 revealed a G9-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1 genotype with close similarity to human Wa-like and porcine Gottfried strains. Therefore, considering the porcine-like or porcine origin of multiple gene segments, it might be tempting to assume that strain mcs/13-07 represents a rare instance of whole-virus transmission from pig to human, after which the virus evolved with time. Alternatively, it is possible that strain mcs/13-07 resulted from multiple reassortment events involving human subgroup II and porcine P[6] strains. Nevertheless, detection of strain mcs/13-07 provides further evidence for complex interspecies transmission events, which are frequent in developing countries.

We studied the role of osteopontin (OPN) in host responses against rotavirus (RV) infection. OPN knockout (OPN-KO) suckling mice were more susceptible to RV (strain EW) infection and showed prolonged diarrhea duration compared to wild-type (WT) suckling mice. OPN in the small intestine of WT mice was expressed after 48 h post-infection. On day 2 postinfection, mRNA levels of interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-15 in OPN-KO mice were lower than in WT mice, although mRNA expression of Th-1- and Th-2-related cytokines in the small intestine were nearly the same between OPN-KO and WT mice. These results suggested that OPN is involved in innate responses against RV infection.