Different Outcomes of Experimental Hepatitis E Virus Infection in Diverse Mouse Strains, Wistar Rats, and Rabbits

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but autochthonous cases of zoonotic genotype 3 (HEV-3) infection also occur in industrialized countries. In contrast to swine, rats, and rabbits, natural HEV infections in mice have not yet been demonstrated. The pig represents a well-established large animal model for HEV-3 infection, but a suitable small animal model mimicking natural HEV-3 infection is currently missing. Therefore, we experimentally inoculated C57BL/6 mice (wild-type, IFNAR−/−, CD4−/−, CD8−/−) and BALB/c nude (nu/nu) mice, Wistar rats, and European rabbits with a wild boar-derived HEV-3 strain and monitored virus replication and shedding, as well as humoral immune responses. HEV RNA and anti-HEV antibodies were detected in one and two out of eight of the rats and all rabbits inoculated, respectively, but not in any of the mouse strains tested. Remarkably, immunosuppressive dexamethasone treatment of rats did not enhance their susceptibility to HEV infection. In rabbits, immunization with recombinant HEV-3 and ratHEV capsid proteins induced protection against HEV-3 challenge. In conclusion, the rabbit model for HEV-3 infection may serve as a suitable alternative to the non-human primate and swine models, and as an appropriate basis for vaccine evaluation studies

A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)–specific monoclonal antibody (mAb), the Escherichia coli–expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli–expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient–derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. Key points • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells

Epidemiology of the Hepatitis E Virus in Reservoir Hosts

Background: Hepatitis E virus (HEV) is the etiological agent of an acute self-limiting hepatitis in humans worldwide. The main route of infection is by ingestion of food or water contaminated with the virus. In Germany, several hundred human cases are reported each year, while preliminary studies suggest a high infestation rate of herds of domestic pig (Sus scrofa domesticus) and sounders of wild boar (Sus scrofa). Autochthonous cases are originating mainly from zoonotic transmission from domestic pig and wild boar, but other animals may also be involved. Recently, a novel strain of HEV (ratHEV) had been found in Norway rats (Rattus norvegicus) in Germany, that could contribute to human epidemiology. Therefore, the aim of this study was to assess the seroprevalence of both HEV and the novel ratHEV in human, domestic pig and rat. For each of the three mammal species, an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was established, that based on an Escherichia coli-expressed carboxy-terminal segment (GT3-Ctr, amino acid (aa) 326–608) of the capsid protein of the autochthonous genotype 3 (GT3), derived from a wild boar from Germany. In parallel, a segment from ratHEV homologous to GT3-Ctr was also expressed in E. coli (ratHEV-Ctr, aa315–599) and was used in the ELISA. Hence, the established tests detect antibodies directed against HEV GT3 when using GT3-Ctr as antigen and ratHEV when using ratHEV-Ctr. Results: The GT3-based in-house human IgG test was validated using a commercial assay and showed high specificity and sensitivity. The average human population (represented by a panel of blood donors from Berlin and Brandenburg) reached a seroprevalence of 12.3% (37/301) with the in-house ELISA. A panel of forestry workers from Brandenburg had an even higher seroprevalence of 21.4% (119/555). Furthermore, ratHEV-specific antibodies could be detected in several sera of forestry workers. The novel ratHEV-based rat IgG ELISA could not be compared to similar tests, however, parallel testing with GT3-Ctr and statistical inference allowed conclusion of a seroprevalence. Rats trapped from several sites in Germany had an overall seroprevalence of 24.5% (36/147). The sera were reactive exclusively with ratHEV-Ctr. As with the in-house ELISA for human sera, the porcine IgG test was validated using a commercial assay, yielding high specificity and sensitivity. A panel of domestic pigs from ten federal states of Germany showed a seroprevalence of 42.7% (383/898) when tested with the in-house ELISA. Reactivity with ratHEV was present, but seemed to be caused mostly by cross-reactivity to GT3-Ctr. Conclusion: The HEV seroprevalence observed for human sera of the average population of Germany is among the highest in Europe and has been confirmed recently by other authors. The high seroprevalence found in forestry workers suggests that they should be counted as a risk group for HEV infection. Populations of rats have been shown to be infested heavily with ratHEV, as rats from all trapping sites situated within cities had a high prevalence for ratHEV exclusively and no serum reacted exclusively with GT3-Ctr. Seroprevalence in domestic pigs was demonstrated to be distributed evenly across federal states and districts. However, a vast difference of infestation could be detected in different herds, suggesting either differences in husbandry conditions, or an external source of infection that acts locally only. The rare but exclusive reactivity of human sera with ratHEV as well as the high cross-reactivity of swine sera with ratHEV suggests that viral strains other than the ones already known may contribute to cases of hepatitis E.Grundlage: Das weltweit vorkommende Hepatitis E virus (HEV) ist die Ursache für eine akut auftretende, selbstlimitierende Hepatitis beim Menschen. Hauptsächlich erfolgt die Infektion durch die orale Aufnahme von mit Virus kontaminierten Nahrungsmitteln und Wasser. In Deutschland werden jährlich mehrere hundert Fälle von Hepatitis E im Menschen gemeldet, auch von einer hohen Durchseuchung der Haus- und Wildschweinpopulationen (Sus scrofa domesticus und Sus scrofa) wird berichtet. Autochthone Erkrankungen in Deutschland werden primär durch zoonotische Übertragung von Haus- und Wildschweinen verursacht, doch andere Übertäger könnten ebenfalls beteiligt sein. Ein 2010 beschriebener HEV-Stamm aus der Wanderratte (Rattus norvegicus), Ratten-HEV, könnte für die Epidemiologie der Hepatitis E ebenfalls von Bedeutung sein. Daher war das Ziel dieser Studie die Abschätzung der Seroprävalenz von HEV (sowohl Ratten-HEV als auch humanpathogenes HEV) in Mensch, Hausschwein und Wanderratte. Für jede der drei Säugetierspezies wurden indirekte Immunoglobulin G (IgG) Enzymimmunoassays (ELISAs) etabliert, die auf einem Escherichia coli-exprimierten carboxyterminalen Segment des Kapsidproteins basieren (GT3-Ctr, Aminosäureposition (aa) 326– 608). Der dafür verwendete Stamm gehört dem in Deutschland autochthonen Genotyp 3 (GT3) an und entstammt einem Wildschwein aus Deutschland. Parallel dazu wurde für den ELISA auch ein zu GT3-Ctr homologes und in E.coli exprimiertes Segment des Ratten-HEV verwendet (Ratten-HEV-Ctr, aa315– 599). Infolgedessen erfassen die etablierten serologischen Tests Antikörper gegen GT3, wenn sie GT3-Ctr als Antigen verwenden, und Antikörper gegen Ratten-HEV beim Einsatz von Ratten-HEV-Ctr als Antigen. Ergebnisse: Der auf GT3 basierende in-house Human-IgG-Test wurde mithilfe eines kommerziell erhältlichen Tests validiert und erwies sich als hoch spezifisch und sensitiv. Die Durchschnittsbevölkerung (repräsentiert durch eine Gruppe von Blutspendern aus Berlin und Brandenburg) zeigte mit dem in-house ELISA eine Seroprävalenz von 12,3% (37/301). Eine Gruppe Waldarbeiter aus Brandenburg zeigte eine Seroprävalenz von 21,4% (119/555). Desweiteren wurden Ratten-HEV-spezifische Antikörper in einigen Waldarbeitern gefunden. Der neuartige, auf Ratten-HEV basierende Ratten-IgG-ELISA konnte nicht mit ähnlichen Tests verglichen werden. Durch parallele Testung mit GT3-Ctr-Antigen konnte jedoch auf eine Seroprävalenz geschlossen werden. Ratten von verschiedenen Fangorten innerhalb Deutschlands wiesen eine durchschnittliche Seroprävalenz von 24,5% (36/147) auf. Die Seren zeigten ausschließlich Reaktivität mit Ratten-HEV-Ctr. Wie beim Human-IgG-ELISA wurde auch der Schweine-IgG-ELISA mittels eines kommerziellen Tests validiert und erreichte eine hohe Sensitivität und Spezifität. Ein Serumpanel von Hausschweinen aus insgesamt zehn deutschen Bundesländern erreichte im in-house-ELISA eine Seroprävalenz von 42,7% (383/898). Wenige Seren reagierten auch mit Ratten-HEV-Ctr, was aber größtenteils auf eine Kreuzreaktivität mit GT3-Ctr zurückgeführt werden kann. Schlussfolgerung: DiebeobachteteSeroprävalenzderdeutschenDurchschnittsbevölkerunggehört zu den bislang höchsten in Europa und konnte von anderen Autoren bestätigt werden. Die höchste Seroprävalenz wurde für Waldarbeiter bestimmt und lässt vermuten, daß es sich bei diesen um eine Risikogruppe für HEV-Infektionen handelt. Deutsche Rattenpopulationen zeigten eine hohe Durchseuchung mit Ratten-HEV. Die Tiere von allen innerstädtischen Fangorten wiesen eine hohe Reaktivität ausschließlich mit Ratten-HEV-Ctr auf; kein Serum reagierte mit GT3-Ctr allein. Die Seroprävalenz in Hausschweinen ist gleichmäßig auf alle getesteten Bundesländer und Landkreise verteilt. Im Vergleich einzelner Betriebe zeigte sich jedoch ein starker Unterschied in der Durchseuchung. Die Ursachen hierfür könnten entweder Unterschiede in der Haltung, oder eine externe, lediglich lokal wirkende Infektionsquelle sein. Aufgrund der ausschließlichen Reaktivität einiger Humanseren mit Ratten-HEV-Ctr und wegen der hohen Kreuzreaktivität weniger Hausschweinseren mit Ratten-HEV-Ctr liegt der Schluss nahe, daß weitere, bisher unbekannte HEV-Stämme bei humanen HEV-Infektionen eine Rolle spielen könnten

Optical Genome Mapping in Routine Human Genetic Diagnostics—Its Advantages and Limitations

In recent years, optical genome mapping (OGM) has developed into a highly promising method of detecting large-scale structural variants in human genomes. It is capable of detecting structural variants considered difficult to detect by other current methods. Hence, it promises to be feasible as a first-line diagnostic tool, permitting insight into a new realm of previously unknown variants. However, due to its novelty, little experience with OGM is available to infer best practices for its application or to clarify which features cannot be detected. In this study, we used the Saphyr system (Bionano Genomics, San Diego, CA, USA), to explore its capabilities in human genetic diagnostics. To this end, we tested 14 DNA samples to confirm a total of 14 different structural or numerical chromosomal variants originally detected by other means, namely, deletions, duplications, inversions, trisomies, and a translocation. Overall, 12 variants could be confirmed; one deletion and one inversion could not. The prerequisites for detection of similar variants were explored by reviewing the OGM data of 54 samples analyzed in our laboratory. Limitations, some owing to the novelty of the method and some inherent to it, were described. Finally, we tested the successful application of OGM in routine diagnostics and described some of the challenges that merit consideration when utilizing OGM as a diagnostic tool

Hepeviridae: An expanding family of vertebrate viruses

The hepatitis E virus (HEV) was first identified in 1990, although hepatitis E-like diseases in humans have been recorded for a long time dating back to the 18th century. The HEV genotypes 1–4 have been subsequently detected in human hepatitis E cases with different geographical distribution and different modes of transmission. Genotypes 3 and 4 have been identified in parallel in pigs, wild boars and other animal species and their zoonotic potential has been confirmed. Until 2010, these genotypes along with avian HEV strains infecting chicken were the only known representatives of the family Hepeviridae. Thereafter, additional HEV-related viruses have been detected in wild boars, distinct HEV-like viruses were identified in rats, rabbit, ferret, mink, fox, bats and moose, and a distantly related agent was described from closely related salmonid fish. This review summarizes the characteristics of the so far known HEV-like viruses, their phylogenetic relationship, host association and proposed involvement in diseases. Based on the reviewed knowledge, a suggestion for a new taxonomic grouping scheme of the viruses within the family Hepeviridae is presented

Natural and experimental hepatitis E virus genotype 3 - infection in European wild boar is transmissible to domestic pigs

International audienceHepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialised countries. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with domestic pig and wild boar. However, little is known about the course of HEV infection in European wild boar and their role in HEV transmission to domestic pigs. To investigate the transmissibility and pathogenesis of wild boar-derived HEVgt3, we inoculated four wild boar and four miniature pigs intravenously. Using quantitative real-time RT-PCR viral RNA was detected in serum, faeces and in liver, spleen and lymph nodes. The antibody response evolved after fourteen days post inoculation. Histopathological findings included mild to moderate lymphoplasmacytic hepatitis which was more prominent in wild boar than in miniature pigs. By immunohistochemical methods, viral antigens were detected mainly in Kupffer cells and liver sinusoidal endothelial cells, partially associated with hepatic lesions, but also in spleen and lymph nodes. While clinical symptoms were subtle and gross pathology was inconspicuous, increased liver enzyme levels in serum indicated hepatocellular injury. As the faecal-oral route is supposed to be the most likely transmission route, we included four contact animals to prove horizontal transmission. Interestingly, HEVgt3-infection was also detected in wild boar and miniature pigs kept in contact to intravenously inoculated wild boar. Given the high virus loads and long duration of viral shedding, wild boar has to be considered as an important HEV reservoir and transmission host in Europe

Different Outcomes of Experimental Hepatitis E Virus Infection in Diverse Mouse Strains, Wistar Rats, and Rabbits

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but autochthonous cases of zoonotic genotype 3 (HEV-3) infection also occur in industrialized countries. In contrast to swine, rats, and rabbits, natural HEV infections in mice have not yet been demonstrated. The pig represents a well-established large animal model for HEV-3 infection, but a suitable small animal model mimicking natural HEV-3 infection is currently missing. Therefore, we experimentally inoculated C57BL/6 mice (wild-type, IFNAR−/−, CD4−/−, CD8−/−) and BALB/c nude (nu/nu) mice, Wistar rats, and European rabbits with a wild boar-derived HEV-3 strain and monitored virus replication and shedding, as well as humoral immune responses. HEV RNA and anti-HEV antibodies were detected in one and two out of eight of the rats and all rabbits inoculated, respectively, but not in any of the mouse strains tested. Remarkably, immunosuppressive dexamethasone treatment of rats did not enhance their susceptibility to HEV infection. In rabbits, immunization with recombinant HEV-3 and ratHEV capsid proteins induced protection against HEV-3 challenge. In conclusion, the rabbit model for HEV-3 infection may serve as a suitable alternative to the non-human primate and swine models, and as an appropriate basis for vaccine evaluation studies

Hepatitis E virus seroprevalence of domestic pigs in Germany determined by a novel in-house and two reference ELISAs

International audienceAutochthonous hepatitis E virus (HEV) infections by zoonotic transmission of genotype 3 (GT3) have been reported increasingly from industrialized countries. In this paper the development and validation of an IgG ELISA for the detection of HEV-specific antibodies in domestic pigs is described. Comparison of the diagnostic value ofEscherichia coli-expressed HEV-GT3 capsid protein (CP) derivatives revealed a carboxyterminal derivative as most suitable. Validation of the in-house assay using a commercially available IgG ELISA revealed a high diagnostic specificity and sensitivity. The average HEV seroprevalence of domestic pigs from Germany and the federal state Baden-Wuerttemberg determined by the in-house test was 42.7% and 50.3%, respectively. The seroprevalence in different districts of Baden-Wuerttemberg ranged from 34.9% to 60%, but from 0% to 100% between different herds. These data were compared to those achieved by two commercially available ELISA kits and an in-house ratHEV-based ELISA. In conclusion, the CP-based in-house test proved sensitive and specific, indicating that the ORF3-encoded protein might be dispensable for diagnostics. The novel assay also allowed a parallel analysis by a homologous ratHEV-derived antigen. Thus, the novel IgG ELISA represents a useful tool for future standardized seroprevalence studies in domestic pigs from Germany and other regions of Europe. (C) 2013 Elsevier B.V. All rights reserved