Immunisation of pigs with recombinant HEV vaccines does not protect from infection with HEV genotype 3.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide. Up to now, no approved treatment nor a globally licensed vaccine is available. Several recombinant HEV vaccines have been developed to protect against HEV infection in humans, including the commercially available Hecolin vaccine, which are mainly based on HEV genotype 1. However, the efficacy of these vaccines against other HEV genotypes, especially genotype 3 is unknown. In this study, we evaluated the protective efficacy of Hecolin® and a novel genotype 3-based vaccine p239(gt3) against HEV-3 in a pig infection model. Pigs were divided into three groups: one group was vaccinated with Hecolin®, the second group was vaccinated with p239(gt3), and the control group received no vaccine. All pigs were subsequently challenged with HEV genotype 3 to assess the effectiveness of the vaccines. Although all immunised animals developed a high titer of neutralizing antibodies, the results showed that both vaccine applications could not provide complete protection against HEV (gt3) infection: Two out of four animals of the Hecolin® group displayed even virus shedding, and viral RNA could be detected in bile and/or liver of three out of four animals in both vaccination groups. Only one out of four animals in each group was fully protected. Neither Hecolin® nor the novel p239(gt3) vaccine provided sufficient protection against genotype 3 infection. While Hecolin® only partial protected pigs from HEV shedding, the novel p239(gt3) vaccine was at least able to prevent infected pigs from virus shedding. The results highlight the need for further development of HEV vaccines that exhibit broad protection against multiple HEV genotypes and the use of appropriate animal infection models.

Hepatitis E virus: Efficacy of pasteurization of plasma-derived VWF/FVIII concentrate determined by pig bioassay.

BACKGROUND: Hepatitis E virus (HEV) is the leading cause of acute hepatitis throughout the world. Increasing blood component transfusion-associated HEV infections highlight the need for reliable virus inactivation procedures for plasma derivatives from pooled plasma donations. STUDY DESIGN AND METHODS: An animal infection study was conducted to evaluate the efficiency of HEV inactivation by pasteurization during the manufacturing process of the von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate Haemate P/Humate-P (CSL Behring, Marburg, Germany). For this purpose, groups of pigs were inoculated with stabilized VWF/FVIII intermediate spiked with HEV-positive liver homogenate and exposed to increasing incubation times of 0, 3, 6, and 10 h at 60°C. Animals were evaluated for virus replication over 27 days and in a subsequent trial over 92 days. RESULTS: Virus replication was detected in animals up to the 6-h pasteurization group. In contrast, pasteurization for 10 h did not reveal virus detection when the observation period was 27 days. In an additional experiment using the 10-h pasteurized material, two individuals started virus excretion and seroconverted when the observation period was extended to 92 days. Based on the total infection rate (2 of 12) of the animals inoculated with the sample pasteurized for 10 h, a virus reduction factor of at least 4.7 log10 is calculated. CONCLUSION: This study demonstrates that pasteurization at 60°C for 10 h of an HEV-positive plasma derivative leads to the effective reduction of infectivity, resulting in a VWF/FVIII product with an appropriate margin of safety for HEV.

Hepatitis E virus persists in the ejaculate of chronically infected men.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infections are prevalent worldwide. Various viruses have been detected in the ejaculate and can outlast the duration of viremia, indicating replication beyond the blood-testis barrier. HEV replication in diverse organs, however, is still widely misunderstood. We aimed to determine the occurrence, features and morphology of HEV in the ejaculate. METHODS: The presence of HEV in testis was assessed in 12 experimentally HEV-genotype 3-infected pigs. We further tested ejaculate, urine, stool and blood from 3 chronically HEV genotype 3-infected patients and 6 immunocompetent patients with acute HEV infection by HEV-PCR. Morphology and genomic characterization of HEV particles from various human compartments were determined by HEV-PCR, density gradient measurement, immune-electron microscopy and genomic sequencing. RESULTS: In 2 of the 3 chronically HEV-infected patients, we observed HEV-RNA (genotype 3c) in seminal plasma and semen with viral loads >2 logs higher than in the serum. Genomic sequencing showed significant differences between viral strains in the ejaculate compared to stool. Under ribavirin-treatment, HEV shedding in the ejaculate continued for >9 months following the end of viremia. Density gradient measurement and immune-electron microscopy characterized (enveloped) HEV particles in the ejaculate as intact. CONCLUSIONS: The male reproductive system was shown to be a niche of HEV persistence in chronic HEV infection. Surprisingly, sequence analysis revealed distinct genetic HEV variants in the stool and serum, originating from the liver, compared to variants in the ejaculate originating from the male reproductive system. Enveloped HEV particles in the ejaculate did not morphologically differ from serum-derived HEV particles. LAY SUMMARY: Enveloped hepatitis E virus particles could be identified by PCR and electron microscopy in the ejaculate of immunosuppressed chronically infected patients, but not in immunocompetent experimentally infected pigs or in patients with acute self-limiting hepatitis E.

Spatial-Temporal Dynamics of Hepatitis E Virus Infection in Foxes (Vulpes vulpes) in Federal State of Brandenburg, Germany, 1993-2012.

Hepatitis E virus (HEV) is the main course for acute hepatitis in humans throughout the world. Human associated genotypes 1 and 2 as well as zoonotic genotypes 3 and 4 are grouped in the species Orthohepevirus A. In addition, a large variety of HEV-related viruses has been found in vertebrates including carnivores, rats, bats, and chickens, which were classified in species Orthohepevirus B-D. In 2015, partial genome sequences of a novel hepevirus were detected in feces of red foxes (Vulpes vulpes). However, no further information about virus circulation and the prevalence in foxes was available. We therefore assayed a unique panel of 880 transudates, which was collected from red foxes over 19 years (1993-2012) in Brandenburg, Germany, for HEV-related viral RNA and antibodies. Our results demonstrate a high antibody prevalence of HEV in red foxes, which oscillated annually between 40 and 100%. Molecular screening of the transudates revealed only a single RNA-positive sample, which was assigned to the carnivore species Orthohepevirus C based on the amplified partial sequence. These data indicate that the virus is circulating widely in the fox population and that foxes are carriers of this virus.

Co-infection of pigs with Hepatitis E and porcine circovirus 2, Saxony 2016.

Hepatitis E virus (HEV) is a recognized zoonotic disease; autochthonous infections in Europe are caused to a great extent by HEV genotype 3. Pigs and wild boar are the main reservoirs for this genotype and normally they develop no or only subclinical symptoms with mild histopathological lesions. However, co-infections with other pig pathogens can lead to severe cases in pigs, including liver hemorrhage and necrosis. During a monitoring program 2016 in Saxony, Germany, farmed pigs with various clinical outcomes including fatalities were analysed for HEV and concurrent infections. We could detect eight HEV infected pigs from which six were co-infected with porcine circovirus 2 (PCV2). Phylogenetic analysis revealed HEV sub-genotypes 3e and 3f as well as PCV2 genotypes 2b and 2d. A direct correlation of the co-infection to the course of disease could not be determined, but the results provide hints that the immune modulatory effects of PCV2 combined with HEV influence the disease pattern in pigs.

High sensitivity of domestic pigs to intravenous infection with HEV.

BACKGROUND: Hepatitis E virus (HEV) is one major cause of acute clinical hepatitis among humans throughout the world. In industrialized countries an increasing number of autochthonous HEV infections have been identified over the last years triggered by food borne as well as - to a much lower degree - by human to human transmission via blood transfusion. Pigs have been recognised as main reservoir for HEV genotype 3 (HEV-3), and zoonotic transmission to humans through undercooked/raw meat is reported repeatedly. The minimal infectious dose of HEV-3 for pigs is so far unknown. RESULTS: The minimum infectious dose of HEV-3 in a pig infection model was determined by intravenous inoculation of pigs with a dilution series of a liver homogenate of a HEV infected wild boar. Seroconversion, virus replication and shedding were determined by analysis of blood and faeces samples, collected over a maximum period of 91 days. A dose dependent incubation period was observed in faecal shedding of viruses employing a specific and sensitive PCR method. Faecal viral shedding and seroconversion was detected in animals inoculated with dilutions of up to 10- 7. This correlates with an intravenously (i.v.) administered infectious dose of only 6.5 copies in 2 ml (corresponding to 24 IU HEV RNA/ml). Furthermore the first detectable shedding of HEV RNA in faeces is clearly dose dependent. Unexpectedly one group infected with a 10- 4 dilution exhibited prolonged virus shedding for more than 60 days suggesting a persistent infection. CONCLUSION: The results indicate that pigs are highly susceptible to i.v. infection with HEV and that the swine model represents the most sensitive infectivity assay for HEV so far. Considering a minimum infectious dose of 24 IU RNA/ml our findings highlights the potential risk of HEV transmission via blood and blood products.

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.

Kinetics of maternally derived antibodies upon intramuscular vaccination against classical swine fever with Suvaxyn® CSF Marker (CP7_E2alf).

Classical swine fever (CSF) remains one of the most important viral diseases that impact on sustainable pig production world wide. To control the disease in either endemic situations or in case of large, high-impact contingencies, safe and highly efficacious live attenuated vaccines exist since decades. However, until recently, the available live vaccines did not allow a serological marker concept that would be important to circumvent long-term trade restrictions. Recently, a new live attenuated marker vaccine, Suvaxyn® CSF Marker (Zoetis), was licensed by the European Medicines Agency (EMA). To supplement the data that are necessary for the design of appropriate vaccination strategies, a trial was carried out with single "emergency-type" vaccination of two pregnant sows. Focus was laid on the kinetics of maternally derived antibodies (MDA) in the screening assays of their offspring that would be used in case of a CSF outbreaks, i.e. CSFV E2 and Erns antibody ELISA. Neutralization peroxidase linked antibody assays were carried out to allow a rough estimate of protection. Upon vaccination with Suvaxyn® CSF Marker 21days before farrowing, MDAs were measurable in all piglets born to the vaccinated sows. The E2 ELISA reactivities showed an almost linear decrease over 10 weeks after which all piglets were tested negative in the ELISA again. No problems were observed in DIVA assays (Erns antibodies) when heat-inactivated sera were used. The protective effect of MDA needs further investigations as the titers were found to be lower than reported for C-strain vaccines.

A competitive ELISA for species-independent detection of Crimean-Congo hemorrhagic fever virus specific antibodies.

Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans with case-fatality rates of up to 80%. CCHF is considered to be one of the major emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma function as vector as well as natural reservoir of CCHFV. Ticks feed on various domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs). Those animal species play an important role in the life cycle of the ticks as well as in amplification of CCHFV. Here we present a competitive ELISA (cELISA) for the species-independent detection of CCHFV-specific antibodies. For this purpose nucleocapsid (N) protein specific monoclonal antibodies (mAbs) were generated against an Escherichia coli (E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous and heterologous recombinant CCHFV antigens in ELISA and Western blot test and 20 of those 33 mAbs reacted additionally in an immunofluorescence assay with eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a prototype of the cELISA and nine of them competed with positive control sera of bovine origin. The cELISA was established by using the mAb with the strongest competition. For the validation, 833 sera from 12 animal species and from humans were used. The diagnostic sensitivity and specificity of the cELISA was determined to be 95% and 99%, respectively, and 2% of the sera gave inconclusive results. This cELISA offers the possibility for future large-scale screening approaches in various animal species to evaluate their susceptibility to CCHFV infection and to identify and monitor the occurrence of CCHFV.