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.

A549 and PLC/PRF/5 cells can support the efficient propagation of swine and wild boar hepatitis E virus (HEV) strains: demonstration of HEV infectivity of porcine liver sold as food.

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 × 10(4) copies per well and found that the HEV progeny replicated as efficiently as human HEV strains, with a maximum load of ~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 × 10(2) to 1.1 × 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 × 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.

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.

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 x 10(4) copies per well and 100% at >or=3.5 x 10(4) 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.

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 >or=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).

[Treatment by danaparoid sodium for portal venous thrombosis].

We report a case of hepatitis B type liver cirrhosis with portal venous thrombosis in which danaparoid sodium was very effective. The portal venous thrombosis in this case disappeared 2 weeks commencing after administration of danaparoid sodium. The patient had not adverse effects or complications such as hemorrhage, and the clinical course was good. We consider that danaparoid sodium is an anticoagulant unlikely to cause adverse effects such as hemorrhage, and that it might be effective for treatment of portal venous thrombosis. We intend to examine the indications of treatment with danaparoid sodium, clarify the best administration method, and establishment of maintenance therapy by investigating more cases.

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

Recent studies revealed that hepatitis E virus (HEV) genomes are more variable than previously thought and well-conserved regions suitable for designing universal primers are limited. In this study, based on alignment of 70 full-length HEV sequences of genotypes 1-4, a part of the ORF2/ORF3 overlapping region was found to be the best target region for PCR amplification of various HEV strains. Using the newly designed primers, an RT-PCR method (ORF2/3-137 PCR) that amplifies a 137-nucleotide (nt) sequence within the ORF2/ORF3 overlapping region and is capable of amplifying all known HEV sequences was developed. When compared with the previous RT-PCR method (ORF2-457 PCR) that amplifies a 457 nt ORF2 sequence, ORF2/3-137 PCR was two to three times more sensitive than ORF2-457 PCR upon testing serial dilutions of three HEV RNA-positive serum samples. The ORF2/3-137 PCR assay could detect viraemia in five patients with acute or fulminant hepatitis E 3-14 days longer than ORF2-457 PCR after disease onset. All 41 ORF2-457 PCR-positive serum samples of various genotypes tested positive for HEV RNA by the ORF2/3-137 PCR assay. Since the amplicons of ORF2/3-137 PCR contain variable sequences, a phylogenetic tree of the ORF2/3-137 products could clearly distinguish the different HEV genotypes.

Groove pancreatitis: report of a case and review of the clinical and radiologic features of groove pancreatitis reported in Japan.

We report a case of groove pancreatitis in which a hypoechoic mass between the duodenum and pancreas head was clearly imaged, and narrowing of the supra-ampullary area of the duodenum and bile duct stenosis were also found. The diagnosis was confirmed by surgery. Microscopic examination showed extensive scarring between the duodenum and pancreas head. Protein plugs were found in Santorini's duct. We consider that the disturbance of the pancreatic juice outflow in Santorini's duct is one of the important pathogenic factors in the development of groove pancreatitis. Therefore, we emphasize the finding of Santorini's duct in the differential diagnosis of groove pancreatitis.

A case in which danaparoid sodium was effective for portal venous thrombosis developed after endoscopic injection sclerotherapy for esophageal varices.

We report a case of hepatitis C type liver cirrhosis with portal venous thrombosis in which danaparoid sodium was very effective. The patient developed portal venous thrombosis, esophageal ulcer, and esophageal stenosis at the same time after sclerotherapy. Since it was confirmed by abdominal computed tomography that there was no portal venous thrombosis before sclerotherapy, development of the thrombosis was considered to be associated with sclerotherapy. The patient was treated with balloon dilation therapy for esophageal stenosis, and with anticoagulation therapy using danaparoid sodium for portal venous thrombosis. The portal venous thrombosis disappeared 4 weeks after the treatment. Despite the condition of esophageal ulcer being caused by sclerotherapy, the patient was safely treated without any adverse effects and complications, and the clinical course has been good. It was indicated that danaparoid sodium was an anticoagulant unlikely to cause adverse effects such as hemorrhage and might be an effective drug for treatment of portal venous thrombosis.

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.

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 x 10(3) to 1.7 x 10(7) 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 x 10(4) copies/ml for 10 of the 11 patients, while for the remaining patient (patient 1) it was markedly high, 2.0 x 10(7) 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 10(7) 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.

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

Hepatitis E in industrialized countries has not been well studied. To define the possible risk factors for transmission of hepatitis E virus (HEV) and for the severe form of hepatitis E in Japan, we investigated the clinical and virological characteristics of hepatitis E in 32 patients who contracted the mild (n=23) or severe form (n=9) of domestically acquired hepatitis E between 1996 and 2004 in Hokkaido, where hepatitis E is most prevalent in Japan. Nine patients with the severe form of hepatitis E included two patients with fulminant hepatitis E and seven patients who were diagnosed with severe acute hepatitis in which hepatic encephalopathy did not appear during the course of the illness despite low plasma prothrombin activity (or=20 mg/dl). At least 25 patients (78%) had consumed uncooked or undercooked pig liver and/or intestine 1-2 months before the onset of hepatitis E. When compared with the seven patients with HEV genotype 3, the 25 patients with HEV genotype 4 had a higher peak alanine aminotransferase (ALT) level (P=0.0338) and a lower level of lowest prothrombin activity (P=0.0340). The severe form of hepatitis E was associated with the presence of an underlying disease (56% [5/9] vs. 17% [4/23], P=0.0454). The study suggests that zoonotic food-borne transmission of HEV plays an important role in the occurrence of hepatitis E in Hokkaido, Japan, and that the HEV genotype and the presence of an underlying disease influence the severity of hepatitis E.

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 Kerckring's 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.

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.