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 antibody prevalence in hunters from a district in Central Germany, 2013: a cross-sectional study providing evidence for the benefit of protective gloves during disembowelling of wild boars

Background: In Germany, 17 % of the general human population have antibodies to hepatitis E virus (HEV) (recomLine HEV-IgG/IgM immunoassay [Mikrogen GmbH]). Wild boars represent an animal reservoir for HEV genotype 3, which is the common genotype in Germany. We estimated the seroprevalence among hunters with contact to wild boars to identify factors that may be associated with past or present HEV infection. Methods: In 2013, the local veterinarian authority in a district in Central Germany attended meetings of hunters who provided blood specimens and completed a questionnaire collecting information on age, sex, hunting-related activities and consumption of wild boar meat. Specimens of wild boars were taken during drive hunts in this district during the season 2012/2013. All specimens were tested for HEV RNA and anti-HEV IgM and IgG antibodies. Log-binomial regression was used to estimate prevalence ratios (PR) for the hunters. Results: Of 126 hunters (median age 55; 94 % male) 21 % tested positive for anti-HEV IgG antibodies (95 % confidence interval [CI] 13–28 %) (recomWell HEV IgG assay [Mikrogen GmbH]). Anti-HEV prevalence was highest in the age group of the 70–79-year-olds (67 %; 95 % CI 39–95 %). Wild boars showed an average anti-HEV prevalence of 41 %. HEV RNA was detected in 4/22 (18 %) liver specimens and in 1/22 (4.5 %) muscle specimens. Most wild boars were tested positive for HEV RNA (3/10; 30 %) and HEV-specific antibodies (7/15; 47 %) in the southwestern part of the district. Hunters preferring this hunting ground had a lower anti-HEV prevalence when gloves were frequently used during disembowelling of wild boars compared to hunters using gloves never or infrequently (age-adjusted PR 0.12; 95 % CI 0.02–0.86). Conclusions: Hunters may benefit from wearing gloves when in contact with blood or body fluids of HEV animal reservoirs. Anti-HEV prevalence among the hunters of this study did not significantly differ from that of the general population suggesting that other factors play a major role in the epidemiology of HEV in Germany

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