Gut microbiota dysbiosis associated with plasma levels of Interferon-γ and viral load in patients with acute hepatitis E infection.

Few studies have focused on the effect of hepatitis E virus (HEV) infection on gut microbiota. To explore the relationship between changes in gut microbiota and inflammatory factors and viral load, we conducted a comparative study of 33 patients with acute hepatitis E (AHE) patients and 25 healthy controls (HCs) using high-throughput 16S ribosomal ribonucleic acid gene sequencing. Shannon and Simpson's indices showed no significant differences in bacterial diversity between the AHE and HCs groups. Proteobacteria, Gammaproteobacteria, and Enterobacteriaceae were most abundant in the AHE group, which contributed to the difference between the gut microbiota of the AHE and HCs groups, and the same difference between the HEV-RNA-positive and HEV-RNA-negative groups. Functional prediction analysis showed that ribosome, purine metabolism, and two-component system were the top three pathways. Compared with the AHE group with normal interferon (IFN)-γ, Proteobacteria, Gammaproteobacteria, Xanthomonadaceae, and Enterobacteriaceae were more abundant in the high-IFN-γ group. The abundance of Gammaproteobacteria was positively correlated with the level of serum alanine transaminase and total bilirubin. The abundance of Gammaproteobacteria could discriminate AHE patients from HCs, and could better predict the severity of AHE patients. We believe that our findings will contribute toward a novel treatment strategy for AHE.

Evaluation of oral fluids for surveillance of foodborne and zoonotic pathogens in pig farms.

The use of oral fluid (OF) to detect zoonotic pathogens in pigs has been only scarcely assessed. We evaluated OF as a potential specimen for detection by culture of methicillin-resistant Staphylococcus aureus (MRSA) and Yersinia enterocolitica, and the detection of antibodies against Salmonella spp. and hepatitis E virus (HEV) using commercial ELISAs. Samples from 33 pig farms were collected at the beginning and end of the fattening period. Results of the OF samples were compared with the results of serum samples and nasal swabs from individual pigs and pen floor fecal samples, using the Cohen kappa (κ) and the McNemar test. For Salmonella spp. antibodies, OF samples were negative, although the corresponding serum samples were positive. The detection of HEV antibodies in sera and OF had agreement at the first sampling, and poor and significant agreement at the second sampling (κ = 0.185, McNemar p = 0.238; κ = 0.088, McNemar p < 0.001). At both sampling times, the detection of MRSA in nasal swabs and OF showed agreement (κ = 0.466, McNemar p = 0.077; κ = 0.603, McNemar p = 1); agreement was seen for the detection of Y. enterocolitica in fecal and OF samples (κ = 0.012, McNemar p = 0.868; κ = 0.082, McNemar p = 0.061, respectively). According to the McNemar test, the use of pen-based OFs is more feasible for the detection of MRSA and Y. enterocolitica by culture than is detection of antibodies by commercial ELISA.

Altered faecal microbiota on the expression of Th cells responses in the exacerbation of patients with hepatitis E infection.

Fulminant hepatitis E may lead to acute liver failure (ALF). Perturbations of intestinal microbiota are related to severe liver disease. To study the correlations between faecal microbiota and the occurrence and exacerbation of hepatitis E virus (HEV) infection, we characterized 24 faecal samples from 12 patients with acute hepatitis E (AHE) and 12 patients with HEV-ALF using high-throughput sequencing. We found both the alpha and beta diversity indices showed no significant differences between the AHE and HEV-ALF groups. Several predominant taxa were significantly different between the AHE and HEV-ALF groups. Most notably, the HEV-ALF group had increased levels of Gammaproteobacteria, Proteobacteria, Xanthomonadceae and Stenotrophomonas, but reduced levels of Firmicutes, Streptococcus, Subdoligranulum and Lactobacillus, compared with the AHE group. The levels of Lactobacillaceae and Gammaproteobacteria could be used to distinguish patients with HEV-ALF from those with AHE. In addition, the level of Th lymphocytes was significantly lower in the HEV-ALF group than in the AHE group. The relative abundances of Lactobacillaceae and Gammaproteobacteria were positively correlated with Th lymphocytes, serum international normalized ratio (INR) and hepatic encephalopathy severity. Moreover, surviving patients had higher levels of Lactobacillus mucosae than deceased patients. Our study demonstrated that the presence of altered faecal microbiota is associated with exacerbation of HEV infection; this finding may be useful for exploring the interactions among faecal microbiota, immune responses, mechanisms of infection and progression in patients with HEV, as well as for the development of novel diagnostic and therapeutic strategies.

Phylogenetic analysis of Hepatitis E virus strains isolated from slaughter-age pigs in Colombia.

Hepatitis E virus (HEV) is a zoonotic virus that causes acute hepatitis in humans, and can be transmitted via the fecal-oral route. Pigs are considered to be a reservoir for this infection-mainly where the disease is not endemic. In a previous study conducted in Antioquia, which is a region in Colombia where the production and consumption of pork meat is higher than in the rest of the country, the presence of anti-HEV IgG-type antibodies was reported in slaughter-age pigs. Aiming to identify the HEV genotype circulating in swine, animal liver, and feces samples from five swine cattle slaughterhouses located in six different sub-regions of Antioquia were collected. A nested RT-PCR (nRT-PCR) was used in order to amplify the HEV ORF-1 (170bp) and ORF-2 (348, and 958bp). The amplicons yielded in this study were sequenced, and a molecular phylogeny analysis based on the maximum likelihood method, including HEV sequences reported in several countries, was performed. Phylogeny analysis revealed that HEV amplification fragments from Antioquia's pigs were grouped in three clades within the sub-genotype 3a without a specific geographical structure, and were also genetically related to Japanese and American HEV sequences. This analysis provides the first approach on the genetic diversity and circulation dynamics of HEV in Colombian herds.

Case Report Associated with Aspergillosis and Hepatitis E Virus Coinfection in Himalayan Griffons.

This study involved a death which occurred in four Himalayan griffons housed in Beijing zoo, China. Based on pathogen identification and the pathological changes observed, we did characterize the fungi and Hepatitis E virus (HEV) in four dead Himalayan griffons. Pathological changes were severe. Membranous-like material was observed on the surface of the internal organs. Spleen was necrotic. Focal lymphocyte infiltration in the liver and many sunflower-like fungi nodules were evident in the tissues, especially in the kidney. PCR was used to identify the pathogen. Based on the 18SrRNA genomic sequence of known fungi, the results confirmed that all four dead Himalayan griffons were infected with Aspergillus. At the same time the detection of HEV also showed positive results. To the best of our knowledge, this work appears to be the first report of concurrent presence of Aspergillosis and Hepatitis E virus in rare avian species.

Extended Microbiological Characterization of Göttingen Minipigs in the Context of Xenotransplantation: Detection and Vertical Transmission of Hepatitis E Virus.

Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pigs are currently favoured as donor animals for xenotransplantation of cells, including islet cells, or organs. To reduce the xenotransplantation-associated risk of infection of the recipient the pig donor should be carefully characterised. Göttingen minipigs from Ellegaard are often used for biomedical research and are regularly tested by their vendor for the presence of numerous bacteria, fungi, viruses and parasites. However, screening for some pathogens transmittable to humans had not been performed.The presence of microorganisms was examined in Göttingen Minipigs by PCR methods. Since zoonotic transmission of porcine hepatitis E virus HEV to humans has been demonstrated, extended search for HEV was considered as a priority. RNA from sera, islet and other cells from 40 minipigs were examined for HEV using different real-time reverse transcription (RT)-PCRs, among them two newly established. In addition, sera were examined by Western blot analysis using two recombinant capsid proteins of HEV as antigens. HEV RNA was not detected in pigs older than one year including gilts, but it was detected in the sera of three of ten animals younger than 1 year. Furthermore, HEV was also detected in the sera of three sows six days after delivery and their offspring, indicating vertical transmission of the virus. PCR amplicons were cloned, sequenced and the viruses were found to belong to the HEV genotype (gt) 3/4. Anti-HEV immunoglobulins G were detected in one sow and maternal antibodies in her six day old piglet. Since Göttingen minipigs were negative for many xenotransplantation-relevant microorganisms, they can now be classified as safe. HEV may be eliminated from the Ellegaard herd by selection of negative animals and/or by treatment of the animals.

Hepatitis E virus infection.

Hepatitis E virus (HEV) infection is a worldwide disease. An improved understanding of the natural history of HEV infection has been achieved within the last decade. Several reservoirs and transmission modes have been identified. Hepatitis E is an underdiagnosed disease, in part due to the use of serological assays with low sensitivity. However, diagnostic tools, including nucleic acid-based tests, have been improved. The epidemiology and clinical features of hepatitis E differ between developing and developed countries. HEV infection is usually an acute self-limiting disease, but in developed countries it causes chronic infection with rapidly progressive cirrhosis in organ transplant recipients, patients with hematological malignancy requiring chemotherapy, and individuals with HIV. HEV also causes extrahepatic manifestations, including a number of neurological syndromes and renal injury. Acute infection usually requires no treatment, but chronic infection should be treated by reducing immunosuppression in transplant patients and/or the use of antiviral therapy. In this comprehensive review, we summarize the current knowledge about the virus itself, as well as the epidemiology, diagnostics, natural history, and management of HEV infection in developing and developed countries.

Hepatitis E virus infection causing isolated fetal ascites: a case report.

Maternal hepatitis infection, excepting hepatitis E, causing isolated fetal ascites with variable outcome has been reported previously. We present a case of maternal hepatitis E virus (HEV) infection causing isolated fetal ascites which resolved spontaneously during pregnancy and resulted in a term live-born baby with anti-HEV seropositivity. A 39-year-old primigravida woman was diagnosed with acute HEV infection at 15 weeks of gestation. Ultrasound at 19 weeks showed significant fetal ascites with abdominal calcifications. Fetal karyotype did not show any abnormality. Cord blood was positive for anti-HEV IgM and negative for other intrauterine infections. Ultrasound at 25 weeks showed partial resolution of fetal ascites with complete resolution at 30 weeks. She delivered a healthy baby at 38 completed weeks, with normal liver enzymes at birth and 1-month follow-up.

[Autochthonous hepatitis E-virus infection as cause of acute hepatitis in Germany - a case report]. / Autochthone Hepatitis-E-Virus-Infektion als Ursache der akuten Hepatitis in Deutschland - eine Kasuistik.

There is an increasing body of evidence that hepatitis E virus (HEV) triggers acute hepatitis not only in tropical and subtropical regions of Asia, Africa, and America with low sanitary standards but also in highly industrialized countries. We here report on two patients from Thuringia (Germany) with a HEV infection without a recent stay abroad. All other common causes of hepatitis were excluded. Transaminases were significantly increased in both cases, while icterus could be proven in one patient, only. Both patients fully recovered in the long-term course. Epidemiological and phylogenetic data from viral analyses suggest that HEV infection has to be considered as a zoonosis. It is likely that viral transmission from animals to humans occurs through insufficiently cooked meat or entrails, e. g., from pigs or wild animals. In summary, HEV infection is a relevant differential diagnosis in acute non-A/B/C viral hepatitis. Further studies are required for the identification of other transmission pathways, pathogen reservoirs as well as novel concepts for prophylaxis, especially for patients at risk for hepatic diseases.

Hepatitis E en niños / Hepatitis E in children

La hepatitis E, conocida previamente como hepatitis no A, no B, de transmisión enteral, es una infección causada por el virus de la hepatitis E (VHE), cuyas características clínicas y epidemiológicas son las de una hepatitis aguda. Pertenece a la familia Hepaviridae. El VHE setransmite principalmente por la vía fecal-oral, la mayoría de las epidemias se pueden asociar conbrotes que tienen su origen en el agua, alta densidad de población y deficientes condiciones sanitarias. El cuadro clínico es el de una hepatitis aguda típica, sin embargo, tiene un amplio espectro de presentación clínica. Es común encontrar marcadores serológicos para VHE positivos en niños con hepatitis viral aguda por otros virus hepatótropos, especialmente el virus B y C. El diagnóstico de infección por VHE se puede hacer por detección serológica y/o molecular. La teórica viabilidad de una vacuna frente alVHEestá basada en diversas evidencias.

Hepatitis E, formerly known as hepatitis non A, non B, enteral transmission, is an infection causedby the hepatitis E virus (HEV), clinical and epidemiological characteristics are those of acute hepatitis. Hepaviridae belongs to the family. HEV is transmitted primarily through fecal-oral route,most outbreaks have been associated with outbreaks that have their origin in water, high population density and poor sanitation. The clinical picture is that of a typical acute hepatitis, however, has a broad spectrum of clinical presentation. Commonly found positive for HEV serological markers in children with acute viral hepatitis other hepatotropic viruses, especially viruses B and C. The diagnosis of HEV infectionan be done by serological detection and/or molecular. The theoretical feasibility of a vaccineagainstHEVis based on different evidence.

Hepatitis E: pasado, presente y futuro / Hepatitis E virus: present and future

El Virus de la Hepatitis E (HEV) de transmisión entérica, está ampliamente diseminado en muchos países, tropicales y subtropicales, desde el año 1982. HEV tiene múltiples vías de transmisión, por ejemplo, agua o alimentos contaminados con materia fecal, de persona a persona y a través de crustáceos crudos o mal cocidos. Existen también evidencias a favor de la probable transmisión sexual del virus...

El espectro creciente de las hepatitis virales / The growing spectrum of viral hepatitis

El conocimiento de las hepatitis se remonta por lo menos al siglo VIII, pero éste ha avanzado vertiginosamente en los últimos 30 años gracias a factores como la aplicación de técnicas de inmunoquímica, microscopía electrónica, estudios epidemiológicos tanto clínicos como experimentales, técnicas de ingeniería genética, etc. Este trabajo revisa aspectos históricos de estas afecciones, las lateraciones histológicas hepáticas y manifestaciones clínicas que desencadenan. También describe las características principales de los virus A, B, C y E, la respuesta inmune que produce cada uno de ellos, su forma de infectar y la evolución, complicaciones y posibilidades de prevención de la enfermedad que desencadenan. Se comentan los resultados de los tratamientos con antivirales e interferón para las infecciones crónicas por virus B y C

[An experimental infection in lambs by the hepatitis E virus]. / Eksperimental'naia infektsiia iagniat virusom gepatita E.

Lambs were experimentally infected with a pool of 10% hepatitis E (HE) patient fecal suspension containing HE virus (HEV) isolates Osh-225 and Osh-228 which caused an infection closely resembling experimental HE in primates. Clinical manifestations consisted of acute biochemical and histological hepatitis, virus shedding in feces, the presence of virus-like particles in the peripharyngeal lymph nodes and the contents of small intestine, of HEV RNA in the parenchymal organs of lambs. Susceptibility of lambs to HEV derived from the infected piglets and the possibility of passaging the piglets' HEV in lambs was demonstrated. This passaging led to shortening of the incubation period.

Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys.

The infectivity titer of a standard stock of the SAR-55 strain of hepatitis E virus (HEV) was determined in cynomolgus macaques (Macaca fascicularis) and the effect of dose on the course of the infection was examined by weekly monitoring of alanine aminotransferase (ALT) and anti-HEV levels. Antibody to HEV (anti-HEV) was measured with ELISAs based on ORF-2 recombinant antigens consisting of either a 55 kDa region expressed in insect cells or shorter regions expressed as fusion proteins in bacteria. The ELISA based on the 55 kDa antigen was generally more sensitive. The infectivity titer of SAR-55 was 10(6) cynomolgus 50% infectious doses per gram of feces. The infectivity titer corresponded to the HEV genome titer of the inoculum as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Anti-HEV IgM was detected in only a portion of the animals that had an anti-HEV IgG response. Biochemical evidence of hepatitis was most prominent in animals that were inoculated with the higher concentrations of virus and the incubation period to seroconversion was prolonged in animals that received the lower doses.

Seroepidemiology of water-borne hepatitis in India and evidence for a third enterically-transmitted hepatitis agent.

Many epidemics of water-borne hepatitis have occurred throughout India. These were thought to be epidemics of hepatitis A until 1980, when evidence for an enterically transmitted non-A, non-B hepatitis was first reported. Subsequently, hepatitis E virus was discovered and most recent epidemics of enterically transmitted non-A, non-B hepatitis have been attributed to hepatitis E virus infection. However, only a limited number of cases have been confirmed by immuno electron microscopy, polymerase chain reaction, or seroconversion. In the present study we have performed a retrospective seroepidemiologic study of 17 epidemics of water-borne hepatitis in India. We have confirmed that 16 of the 17 epidemics were caused at least in part by serologically closely related hepatitis E viruses. However, one epidemic, in the Andaman Islands, and possibly a significant minority of cases in other epidemics, appears to have been caused by a previously unrecognized hepatitis agent.

Detection of the negative strand of hepatitis E virus RNA in the livers of experimentally infected rhesus monkeys: evidence for viral replication.

Hepatitis E virus (HEV), the causative agent of enteric non-A, non-B hepatitis, is a positive-stranded RNA virus. Because of the virus's inability to grow in culture, several nonhuman primates have been used for the propagation of HEV. Using strand-specific reverse transcription-polymerase chain reaction (RT-PCR), we demonstrate the presence of negative-stranded HEV RNA replicative intermediates in the livers of infected animals. This constitutes the first direct evidence of HEV replication in the liver of the infected animals and reinforces the validity of such a model to study HEV infection, disease pathogenesis, and immunity.

Enzyme immunoassay for the detection of antibody to hepatitis E virus based on synthetic peptides.

Five synthetic peptides were prepared based on the nucleotide sequence of open reading frames 2 and 3 encoded in the hepatitis E virus (HEV) genome and were used to develop an enzyme immunoassay (EIA) for the detection of anti-HEV activity in sera. Three different approaches were employed to ascertain the optimal preparation of these peptides as an immunodiagnostic reagent, including (1) a mixture of unconjugated peptides, (2) conjugating individual peptides to bovine serum albumin (BSA) followed by mixing each conjugate at various concentrations, and (3) mixing the peptides before conjugation with BSA to create an artificial antigen complex. The third method was superior in discriminating anti-HEV activity in sera previously tested by Western blot (WB). A frequency distribution of optical density values demonstrated that the peptide-based EIA was able to readily discriminate anti-HEV positive sera from sera devoid of anti-HEV activity. To confirm anti-HEV activity a neutralization test was developed using a mixture of 5 unconjugated peptides. With the exception of sera containing high levels of anti-HEV activity, all sera were neutralized greater than 50%. Strong sera required a higher dilution before a 50% neutralization was achieved. The sensitivity of the WB compared to EIA was 89.5% with and overall concordance of 94.8%. The peptide-EIA was used to determine anti-HEV activity in sera collected from various populations worldwide. In six outbreaks of ET-NANB hepatitis in various geographic regions, anti-HEV activity was demonstrated in 78-100% of cases. The peptide-EIA also detected anti-HEV activity in 14 out of 14 follow-up sera obtained 4-6 months after onset of disease and in 2 of 2 of these patients 5 yr after the acute episode. Anti-HEV activity was found in 8.5% of sera obtain from a healthy population residing in an HEV endemic region and 0.5% in two non-endemic regions (P < 0.001). These data demonstrate that a synthetic peptide-based EIA is sensitive for detecting anti-HEV activity in the sera of patients with acute hepatitis E, convalescents, and among healthy individuals.

Rhesus monkeys infected with hepatitis E virus (HEV) from the former USSR are immune to subsequent challenge with an Indian strain of HEV.

Two rhesus monkeys (M. mullata) of approximately two years of age were inoculated intravenously with a 10% suspension of hepatitis E virus (HEV) positive stool from Kirghistan as evidenced by immuno-electron microscopy. Evidence of HEV infection was demonstrated by rise in serum alanine transaminase (ALT) levels and seroconversion of these monkeys to anti-HEV after 1-1/2 months post-inoculation as evidenced by immunoblot. One year after the primary inoculation, these monkeys were challenged with an Indian strain of HEV. No rise in serum ALT levels was noted during an observation period of 6 months. The same inoculum produced HE in two rhesus monkeys. The results showed that strains from India and Kirghistan were antigenically closely related and rhesus monkeys infected with one strain of virus were immune to another strain.

Partial sequence comparison of eight new Chinese strains of hepatitis E virus suggests the genome sequence is relatively stable.

Partial genomic sequences representing 420 nucleotides of a nonstructional region, 480 nucleotides of the putative RNA polymerase region, and 540 nucleotides of the structural region of epidemic-associated Chinese strains of hepatitis E virus (HEV) were obtained by direct sequencing of PCR-amplified DNA. Comparison with previously published HEV sequences showed a clear relatedness of all Chinese strains to each other and to a Pakistani strain (Sar-55). All eight Chinese strains examined had very similar sequences (98.5-99.8% homology) in the regions examined and were much closer to the Pakistani strain (Sar-55) (97.9-98.4% homology) than to the Burmese strain (92.5-93.3% homology). Sequence comparisons of the three genomic regions in the Chinese strains indicated that the RNA polymerase region was much more conserved than the other nonstructural region or the structural region. HEV isolates from three remote geographic regions of China had sequences closely related to each other.