Prevalence of hepatitis E virus (HEV) Infection in wild boars and deer and genetic identification of a genotype 3 HEV from a boar in Japan

Zoonotic transmission of hepatitis E virus (HEV) from captured wild deer or boars to humans has been suggested. Antibody to HEV was detected in 9% of 35 wild boars and 2% of 117 wild deer tested, and a presumably indigenous HEV of genotype 3 was isolated from a boar in Japan.     

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. © 2009 Wiley‐Liss, Inc.     

Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV

Hepatitis E infection is associated with areas in which hepatitis E virus (HEV) infection is endemic. Acute infections in industrialized nations are usually linked to travel to endemic areas. Recently, an acute hepatitis infection in a patient from the United States (US), with no recent foreign travel history, was linked to a novel strain of HEV. Although a few additional cases have been reported from patients who have not traveled to endemic areas, the source of these infections has not been determined. The objective of this study was to identify additional HEV isolates from patients with acute infection who had no recent history of travel to areas where HEV is considered endemic, and to determine the genetic relationship between these and other HEV isolates. Viral RNA was isolated from serum and polymerase chain reaction (PCR) was performed using consensus primers based on a number of HEV isolates. HEV sequence in open reading frame (ORF) 1 and ORF2 was identified in three patients from nonendemic areas, one from Italy and two from Greece. Comparative and phylogenetic analyses were performed. The Greek and Italian isolates were significantly divergent from two isolates from the US and isolates identified previously from HEV-endemic regions. The Italian isolate was distinct from the two Greek isolates. In addition, the two Greek isolates were significantly divergent from each other. Phylogenetic analysis indicated that the Italian and two Greek isolates represent three new genotypes of HEV, distinct from the Burmese, Mexican, and US genotypes.     

Prior infection of pigs with a genotype 3 swine hepatitis E virus (HEV) protects against subsequent challenges with homologous and heterologous genotypes 3 and 4 human HEV

Hepatitis E virus (HEV) is an important human pathogen. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Genotypes 3 and 4 swine HEV is widespread in pigs and known to infect humans. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In this study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. The control group was inoculated and challenged with PBS buffer. Weekly sera from all pigs were tested for HEV RNA and IgG anti-HEV, and weekly fecal samples were also tested for HEV RNA. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia was not detected and only two pigs challenged with swine HEV had 1-week fecal virus shedding, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenges with homologous and heterologous genotypes 3 and 4 HEV. The results from this study have important implications for future development of an effective HEV vaccine.     

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 × 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.     

不同基因型戊型肝炎病毒重组抗原p166用于抗体检测的价值

目的探讨不同基因型和亚型戊型肝炎病毒（HEV）ORF2重组蛋白p166用于抗体检测的价值,为研发准确可靠的戊型肝炎诊断试剂提供新的途径.方法用等浓度的不同基因型和亚型HEV p166作为包被抗原,对血清标本进行酶联免疫吸附试验测定.用HEV多基因型通用性引物逆转录套式PCR（RT-nPCR）扩增标本中HEV RNA,并测序、分型.结果8种不同p166抗原对30份健康献血者血清无抗原性,对182份来自世界不同国家和地区的已知HEV抗体阳性血清和7份HEV实验感染动物血清标本均呈阳性反应,但所得血清抗体滴度的高低与所用抗原的基因型有明显关系.RT-nPCR检测的50份中国血清标本中,19份阳性,基因分型均为Ⅳ型,与Ⅳ型中国株p166抗原反应最好.而以同属于第Ⅲ基因型的猪HEV新西兰株和人HEV美国株重组p166检测血清标本,两者结果差异无统计学意义.以多基因型p166混合抗原建立的ELISA抗体检测法与两种市售试剂盒比较,前者敏感性高,特异性好.结论不同基因型和亚型的HEV重组蛋白p166对不同血清标本HEV抗体检测的敏感性高低不同,因此多基因型和亚型p166的混合抗原是HEV抗体检测的最佳抗原.     

Subcellular localization of hepatitis E virus (HEV) replicase

Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of approximately 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.     

Prevalence of hepatitis E virus infection among hemodialysis patients in Japan: evidence for infection with a genotype 3 HEV by blood transfusion

To investigate the prevalence of hepatitis E virus (HEV) infection among patients on maintenance hemodialysis, serum samples collected in January 2003 from 416 patients who had been undergoing hemodialysis for 7.6 +/- 6.3 (mean +/- standard deviation) (range, 0.3-26.0) years in a dialysis unit in Japan and serum samples that had been collected from these patients at the start of hemodialysis were tested for IgG antibodies to HEV (anti-HEV IgG) by an "in-house" enzyme-linked immunosorbent assay (ELISA). Overall, 39 patients (9.4%) had anti-HEV IgG in January 2003, and included 35 patients (8.4%) who had already been positive for anti-HEV IgG at the start of hemodialysis and 4 patients (1%) who seroconverted after initiation of hemodialysis. Periodic serum samples that had been collected from the four seroconverted patients were tested for HEV antibodies and HEV RNA. The four patients became positive for anti-HEV IgG in 1979, 1980, 1988, or 2003, and continued to be seropositive until the end of the observation period. Although anti-HEV IgM was not detectable in the four patients, three were infected transiently with apparently Japanese indigenous HEV strains of genotype 3. The patient who contracted HEV infection in 1979 had been transfused with 2 U of blood 21 days before the transient viremia: one of the two stored pilot serum samples had detectable HEV RNA with 100% identity to that recovered from the patient. Our study provides evidence of transfusion-transmitted HEV infection in Japan in 1979, and that the prevalence of de novo HEV infection during hemodialysis was low (1.1% or 4/374).     

Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV

Summary.  A novel virus of pigs, swine hepatitis E virus (swine HEV), was recently identified and shown to be antigenically and genetically related to human HEV. In the present study, we attempted to infect specific-pathogen-free (SPF) pigs experimentally with swine HEV or with human strains of HEV. Serum samples collected from naturally infected pigs were used as the source of swine HEV. Pigs inoculated intravenously with serum samples containing swine HEV seroconverted to anti-HEV 4 to 8 weeks postinoculation, and the virus spread to an uninoculated pig. Swine HEV was detected in nasal and rectal swab materials as early as 2 weeks postinoculation and for 4 to 8 weeks thereafter. Viremia appeared 4 to 6 weeks postinoculation and lasted 1 to 3 weeks. The inoculated pigs appeared clinically normal and serum liver enzymes were not significantly elevated. In contrast, pigs were not infected when inoculated intravenously with about 10⁵ monkey infectious doses of one of two human strains of HEV (Sar-55 or Mex-14). Received December 9, 1997 Accepted February 18, 1998     

Simultaneous detection of immunoglobulin A (IgA) and IgM antibodies against hepatitis E virus (HEV) Is highly specific for diagnosis of acute HEV infection

Serum samples collected from 68 patients (age, mean +/- the standard deviation [SD], 56.3 +/- 12.8 years) at admission who were subsequently molecularly diagnosed as having hepatitis E and from 2,781 individuals who were assumed not to have been recently infected with hepatitis E virus (HEV; negative controls; 52.9 +/- 18.9 years), were tested for immunoglobulin M (IgM) and IgA classes of antibodies to HEV (anti-HEV) by in-house solid-phase enzyme immunoassay with recombinant open reading frame 2 protein expressed in the pupae of silkworm as the antigen probe. The 68 patients with hepatitis E had both anti-HEV IgM and anti-HEV IgA. Among the 2,781 controls, 16 (0.6%) had anti-HEV IgM alone and 4 (0.1%) had anti-HEV IgA alone: these IgA/IgM anti-HEV-positive individuals were not only negative for HEV RNA but lack IgG anti-HEV antibody as well (at least in most of the cases). Periodic serum samples obtained from 15 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, and anti-HEV IgA. Although HEV RNA was detectable in the serum until 7 to 40 (21.4 +/- 9.7) days after disease onset, both IgM and IgA anti-HEV antibodies were detectable until 37, 55, or 62 days after disease onset in three patients and up through the end of the observation period (50 to 144 days) in 12 patients. These results indicate that detection of anti-HEV IgA alone or along with anti-HEV IgM is useful for serological diagnosis of hepatitis E with increased specificity and longer duration of positivity than that by RNA detection.     

Genotypic characterization of symptomatic hepatitis E virus (HEV) infections in Egypt

BackgroundHepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available.ObjectivesThe objectives were to determine the HEV genotype(s) currently circulating in Egypt.Study designAVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype.ResultsOf 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9–9.5% divergent from other genotype 1 isolates. ORF2 was 5.3–8.7% divergent from previously reported Egyptian isolates.ConclusionsThis study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.     

An overview: Rabbit hepatitis E virus (HEV) and rabbit providing an animal model for HEV study

Hepatitis E virus (HEV) is a single‐stranded, positive‐sense RNA virus and the causative agent of hepatitis E. The virus belongs to genus Orthohepevirus in the family Hepeviridae, which contains 4 major genotypes closely relating to humans. Genotypes 1 and 2 only infect humans whereas genotypes 3 and 4 HEV are harbored in a wide range of animal species worldwide and are zoonotic to humans. Recently, a novel animal strain of HEV has been isolated in farmed rabbits in China, and subsequently more strains were discovered in the rabbit populations in at least 7 other countries. Due to high sequence similarity to genotype 3 HEV, rabbit HEV (rHEV) has been assigned to genotype 3. Experimental study showed that rHEV could infect non‐human primate and human, which pose a direct threat to human. Further pathogenesis studies showed laboratory rabbits infected with rHEV and genotype 4 HEV could present similar signs of acute and chronic hepatitis E along with extra‐hepatic replication as observed in humans. High mortality and vertical transmission were reproduced in rHEV infected pregnant rabbits. Furthermore, rabbit model was also found suitable for evaluating HEV vaccine efficacy in order to manage zoonotic transmission. These data showed laboratory rabbits could serve as an alternative animal model for HEV study under the current circumstances that HEV propagation is limited in vitro. In general, this review aims at presenting comprehensive up‐to‐date information about rHEV strains and rabbit model for HEV studies.     

Generation and infectivity titration of an infectious stock of avian hepatitis E virus (HEV) in chickens and cross-species infection of turkeys with avian HEV

Avian hepatitis E virus (HEV), a novel virus identified from chickens with hepatitis-splenomegaly syndrome in the United States, is genetically and antigenically related to human HEV. In order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated, as HEV cannot be propagated in vitro. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock as a 10% suspension of positive fecal and bile samples in phosphate-buffered saline. The infectivity titer of this infectious stock was determined by inoculating 1-week-old SPF chickens intravenously with 200 microl of each of serial 10-fold dilutions (10(-2) to 10(-6)) of the avian HEV stock (two chickens were inoculated with each dilution). All chickens inoculated with the 10(-2) to 10(-4) dilutions of the infectious stock and one of the two chickens inoculated with the 10(-5) dilution, but neither of the chickens inoculated with the 10(-6) dilution, became seropositive for anti-avian HEV antibody at 4 weeks postinoculation (wpi). Two serologically negative contact control chickens housed together with chickens inoculated with the 10(-2) dilution also seroconverted at 8 wpi. Viremia and shedding of virus in feces were variable in chickens inoculated with the 10(-2) to 10(-5) dilutions but were not detectable in those inoculated with the 10(-6) dilution. The infectivity titer of the infectious avian HEV stock was determined to be 5 x 10(5) 50% chicken infectious doses (CID(50)) per ml. Eight 1-week-old turkeys were intravenously inoculated with 10(5) CID(50) of avian HEV, and another group of nine turkeys were not inoculated and were used as controls. The inoculated turkeys seroconverted at 4 to 8 wpi. In the inoculated turkeys, viremia was detected at 2 to 6 wpi and shedding of virus in feces was detected at 4 to 7 wpi. A serologically negative contact control turkey housed together with the inoculated ones also became infected through direct contact. This is the first demonstration of cross-species infection by avian HEV.     

Cross-protection of hepatitis E virus genotypes 1 and 4 in rhesus macaques

The purpose of this study was to determine cross-protection between HEV genotypes 1 and 4, which are prevalent in China. Fecal suspensions of genotypes 1 and 4 from patients, as well as genotype 4 from swine, were inoculated intravenously into rhesus macaques. Each inoculum contained 5 x 10(4) genome equivalents of HEV. After infection, serum and fecal samples were collected serially and the levels of alanine aminotransferase (ALT) and anti-HEV IgG and IgM in sera, and HEV RNA in fecal samples, were measured. Liver biopsies were carried out. All the infected monkeys (12/12) developed anti-HEV IgG and exhibited fecal shedding of virus. IgM was detected in 11 of 12, and ALT elevation occurred about 2-6 weeks post-inoculation in 10 of 12, infected monkeys. Hepatic histopathology was consistent with acute viral hepatitis and the ORF2 antigen of HEV was detected in the granular cytoplasm of hepatocytes by immunohistochemistry. After recovery from their initial HEV infection, the monkeys were challenged with a heterologous genotype or heterologous source of HEV and monitored for hepatitis and fecal shedding. Previous infection with HEV completely or partially protected against subsequent challenge with a heterologous virus, because 7 of 11 monkeys did not develop HEV infection or shed virus in the feces, and none of them developed hepatitis or exhibited ALT elevation or liver biopsy findings of hepatitis. In conclusion, previous HEV infection may give rise to cross-genotype and cross-host-species protection.     

The emergence of genotypes 3 and 4 hepatitis E virus in swine and humans: a phylogenetic perspective

To investigate whether there is any phylogenetic evidence to support the hypothesis that swine is the natural host of HEV genotypes 3 and 4, Bayesian analysis of 80 full-length genomic sequences of HEV was performed. The results showed that the strains of genotypes 3 and 4 from swine are paraphyletic with regard to strains of human origin, which are thus phylogenetically nested among the swine strains. Recognition of HEV genotypes 3 and 4 as viruses from swine or swine HEV can provide an evolutionary explanation to the observation of cross-species infection by genotypes 3 and 4 HEV.