Yasuyuki Yazaki

Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation

ABSTRACT 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.

Prolonged Fecal Shedding of Hepatitis E Virus (HEV) during Sporadic Acute Hepatitis E: Evaluation of Infectivity of HEV in Fecal Specimens in a Cell Culture System

ABSTRACT To investigate the duration of fecal shedding and changing loads of hepatitis E virus (HEV) in feces and serum from patients with acute HEV infection, HEV RNA was quantitated in periodic serum and fecal specimens obtained from 11 patients with sporadic acute hepatitis E. All 11 patients had detectable HEV RNA in serum at admission, with the highest viral load being 1.9 × 103 to 1.7 × 107 copies/ml, and HEV viremia lasted until days 17 to 48 (mean, 28.3) after the onset of hepatitis. Even at the initial examination on days 10 to 29 (mean, 17.6), the HEV load in fecal supernatant was less than 5.7 × 104 copies/ml for 10 of the 11 patients, while for the remaining patient (patient 1) it was markedly high, 2.0 × 107 copies/ml on day 22. In addition, although HEV RNA in fecal supernatant continued to be positive until days 14 to 33 (mean, 22.4) for patients 2 to 11, that for patient 1 was detectable even on day 121. HEVs in fecal specimens obtained on days 22, 24, 26, 28, and 30, but not day 121, from patient 1 grew efficiently in PLC/PRF/5 cells, reaching the highest titer of up to 107 copies/ml in culture medium on day 50 postinoculation. The HEV genome recovered from patient 1 had 29 unique nucleotides that were not seen in any of the 25 reported HEV isolates of the same genotype over the entire genome, with six amino acid substitutions in the ORF1 protein.

A nationwide survey of hepatitis E virus infection in the general population of Japan.

To investigate nationwide the prevalence of hepatitis E virus (HEV) infection in the general population of Japan, serum samples were collected from 22,027 individuals (9,686 males and 12,341 females; age, mean ± standard deviation: 56.8 ± 16.7 years; range: 20–108 years) who lived in 30 prefectures located in Hokkaido, mainland Honshu, Shikoku, and Kyushu of Japan and underwent health check‐ups during 2002–2007, 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, 1,167 individuals (5.3%) were positive for anti‐HEV IgG, including 753 males (7.8%) and 414 females (3.4%), the difference being statistically significant (P < 0.0001). The prevalence of anti‐HEV IgG generally increased with age and was significantly higher among individuals aged ≥50 years than among those aged <50 years (6.6% vs. 2.7%, P < 0.0001). Although 13 individuals with anti‐HEV IgG also had anti‐HEV IgM and/or anti‐HEV IgA, none of them had detectable HEV RNA. The presence of HEV RNA was further tested in 50 or 49‐sample minipools of sera from the remaining 22,014 individuals, and three individuals without anti‐HEV antibodies tested positive for HEV RNA. The HEV isolates obtained from the three viremic individuals segregated into genotype 3 and were closest to Japan‐indigenous HEV strains. When stratified by geographic region, the prevalence of anti‐HEV IgG as well as the prevalence of HEV RNA or anti‐HEV IgM and/or anti‐HEV IgA was significantly higher in northern Japan than in southern Japan (6.7% vs. 3.2%, P < 0.0001; 0.11% vs. 0.01%, P = 0.0056; respectively). J. Med. Virol. 82:271–281, 2010.

Duodenal erosions, a common and distinctive feature of portal hypertensive duodenopathy

OBJECTIVE:The aim of the present study was to assess the presence of duodenal erosion and its clinical characteristics on endoscopy in patients with portal hypertension who had undergone endoscopic injection sclerotherapy and/or endoscopic variceal ligation for esophagogastric varices.METHODS:The subjects were 440 patients with portal hypertension, 450 with chronic hepatitis as a related control group, and 450 who underwent upper endoscopic examination as part of their routine physical examination as the controls. The underlying hepatic disease, hepatic function, and endoscopic findings of duodenal erosion among the patients with portal hypertension were studied.RESULTS:Duodenal erosion was found in 68 patients with portal hypertension (68 of 440, 15.5%), four patients with chronic hepatitis (four of 450, 0.9%), and two controls (two of 450, 0.4%). The incidence of duodenal erosion among the patients with portal hypertension was significantly higher than that in the other two groups (p < 0.01, p < 0.01, respectively). The lesions commonly observed in duodenitis are speckle erosions mainly located in the duodenal bulb. However, the most frequently seen form of duodenal erosion among the patients with portal hypertension extended from the superior portion to the descending portion, and tended to show a circular alignment along the Kerckrings folds. The patients with portal hypertension with reduced hepatic reserve capacity had more severe duodenal erosion. Endoscopic ultrasonography revealed thickening of the duodenal wall and proliferation of vascular structures within and around the wall. The histological findings of the duodenal erosion included edema and vascular dilation in the mucosal and submucosal layers.CONCLUSIONS:The location of duodenal erosion in patients with portal hypertension differs from that in patients with ordinary duodenitis. Duodenal erosion in patients with portal hypertension is considered to be one of the lesions of portal hypertensive duodenopathy.

Characteristics of 20 Patients with Autochthonous Acute Hepatitis E in Hokkaido, Japan: First Report of Bilateral Facial Palsy Following the Infection with Genotype 4 Hepatitis E Virus.

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 hepatitis E at a city hospital in Hokkaido, Japan, during 2001-2014, focusing on a patient complicated with neuropathy. Seventeen patients were infected with genotype 4 HEV, while the remaining three patients were with genotype 3 HEV. Although a 67-year-old male with severe hepatitis did not have predisposing factors associated with the development of neurological disorders, such as diabetes mellitus and the use of immunosuppressive agents, he developed bilateral peripheral facial palsy six days after admission. A neurological examination revealed the inability to smile, frown, close his eyes completely or puff out his cheeks. MRI brain scans were considered to be normal. Although it took 83 days after admission for the total bilirubin levels to normalize, his neurological symptoms resolved gradually within three weeks without any sequelae following conservative therapy. A full-length genomic analysis of the HEV strain (HE-JA30) isolated from the patient belonged to genotype 4 and was closest to that currently circulating in Hokkaido, Japan. This is the first report of HEV-associated neuropathy in Japan. While all of previous reports on HEV-related neuropathy involve genotype 3 HEV, the present report is unique in that genotype 4 HEV is responsible for the neuropathy.

Endoscopic Variceal Ligation of Bleeding Rectal Varices: A Case Report

The incidence of ectopic varices in the rectum is likely to increase with improvements in the treatment and survival of patients with portal hypertension. If a patient with portal hypertension suffers massive lower gastrointestinal hemorrhage, it is important to perform a detailed endoscopic examination, as there is a possibility of rectal varices. Although a standard therapy for rectal varices has not been established, we encountered a case of rectal varices that was successfully treated with endoscopic variceal ligation alone. Endoscopic variceal ligation is minimally invasive, safe, effective, simple and reliable. Endoscopic variceal ligation is promising as a possible first line of therapy for rectal varices.

Importance of cytotoxic T lymphocytes in the rejection of transplanted hepatocellular carcinoma.

Fischer rats became resistant to syngeneic hepatocellular carcinoma (FAA-HTC1) cells on repeated sensitization with mitomycin C-treated FAA-HTC1 cells. In contrast, FAA-HTC1 cells injected into the liver killed normal control Fischer rats within 2 months. Histopathological studies revealed massive accumulation of mononuclear cells in the tumor tissues of sensitized rats that rejected syngeneic FAA-HTC1 cells, whereas very few mononuclear cells were found in the tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were identified as mostly T cells, and two-thirds of these T cells were CD8-positive. Compared with the activity in control rats, the killer activity of mononuclear cells infiltrating tumors was significantly increased in the sensititized rats 7 days after tumor inoculation. Depletion of CD8(+) T cells significantly reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD16(+) cells reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from both control and sensitized rats. Furthermore, the CD16(+) cell-depleted fraction of mononuclear cells infiltrating tumors showed significant cytotoxicity against FAA-HTC1 cells, but failed to show cytotoxicity against other syngeneic tumor cells or allogeneic hepatoma cells.

Adrenal adenoma mimicking a submucosal tumor of the stomach

The widespread use of diagnostic imaging modalities has increased the incidental detection of adrenal masses. We experienced a case of adrenal adenoma that was detected incidentally on mass X‐ray examination of the stomach, with findings that were initially suggestive of a gastric submucosal tumor. Only a few cases of adrenal adenoma that resembles a gastric submucosal tumor on diagnostic imaging have been reported. In addition, only a few papers have described the endoscopic ultrasonography findings of adrenal tumors. As the preoperative endoscopic ultrasonography findings of the tumor in our patient correspond with the features of the cut surface of the resected tumor, endoscopic ultrasonography appears to be useful as a new imaging technique in the diagnosis of adrenal adenoma. As endoscopic ultrasonography is minimally invasive, it can be performed in an outpatient setting to collect useful information. As adrenal tumors can be visualized relatively easily, the presence of adrenal lesions should also be searched for when endoscopic ultrasonography is performed for biliary or pancreatic disorders.

7152 Clinical study of rectal varices-examination of characteristics of rectal varices having susceptibility to hemorrhage-.

[Backgroud and Aim] Since it is now possible for cases of portal hypertention to live for a long period of time accompanying the progress made in various treatments in recent years, there is a high possibility of rectal varices occupying an important position as a cause of lower digestive tract bleeding that occurs in these cases. There are many aspects relating to the etiology and pathology of rectal varices that remain unknown, however and a treatment has yet to be established.We conducted a clinical study of rectal varices to examine characteristic findings of varices having susceptibility to hemorrhage. [Subjects] The patients consisted of 28 cases of rectal varices among 413 cases of portal hyper-tension under observation at our institution (13 men, 15women, mean age: 62.8yr(range 46-78 ). [Results] Esophageal varices were observed in all 28 cases ,and hepatocellular carcinoma was observed in 4 cases. Bleeding from rectal varices was observed in 13 of 28 cases (46%), bleeding was treated by preservative hemostasis in 6 cases, surgical ligation in 3 , PSE in 1, combined EVL and EIS in 1, EIS alone in 1, and EVL alone in 1. When the 28 cases were examined according to the Child-Pughclassification, bleeding from rectal varices was observed in 6 of 16 classified as Child A, 7 of 11 classified as Child B, and 0 of 1 classified as Child C. Although the rate of bleeding tended to increase with exacerbation of hepatic function, there were no significant differences observed. In addition, an examination of the presence of complication by internal hemorrhoid revealed that, in contrast to internal hemorrhoid only being observed in 2 of 15 non-hemorrhagic cases, it was observed in 11 of 13 hemorrhagic cases,indicating a significant difference. [Conclusions] An examination of the characteristics of hemorrhagic rectal varices revealed that the rate of bleeding tends to increase with exacerbation of hepatic function. In addition, complication by internal hemorrhoid was significantly higher in hemorrhagic cases.With respect to the susceptibility to hemorrhage in rectal varices, it is believed that not only the morphology of the varix itself, but also numerous other factors, such as the status of hepatic function, other collateral circulation paths including the esophagus and stomach and the presence of internal hemorrhoid, are involved.

A case of extrahepatic portal obstruction and cavernous transformation.

症例は64歳女性.心窩部不快感あり,腹部超音波検査を行ったところ,肝外門脈は描出されず,肝門部に多数の数珠状の管状構造を認めた.CT, MRIでも門脈本幹は描出されず,門脈造影では,著明に拡張蛇行した求肝性の側副血行路を認めた.肝外門脈閉塞の原因は不明であった.経過観察中に食道内視鏡検査で,食道静脈瘤を認めたので,内視鏡的硬化療法を行ったところ著明に改善された.肝外門脈閉塞症で門脈の海綿状血管腫Cavemoud transformationを呈する成人例は稀である.また,それに伴う食道静脈瘤に対して内視鏡的硬化療法を行った報告も少ない.