Rat hepatitis E virus: Geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus)

Abstract

Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals.

Introduction

The hepatitis E virus (HEV) was identified as the causative agent of an acute hepatitis in humans (for Ref. see Khuroo, 2011). Although the majority of clinical infections is characterised by a self-limited course, in rare cases a lethal outcome or chronic infections have been reported. Previously reported case fatality rates of 0.5–4% during outbreaks may represent an overestimation, as in population surveys the rates ranged from 0.07–0.6% (Aggarwal, 2011). Recently, chronic infections have been mainly documented for immunosuppressed solid organ transplant recipients, exclusively for genotype 3 (for Ref. see Schlosser et al., in press). In outbreak regions the disease is typically characterised by symptoms like jaundice, malaise, anorexia, abdominal pain, hepatomegaly, nausea and vomiting, fever and pruritus (Aggarwal, 2011). In Europe jaundice was found to be the most common symptom. Asthenia, fever, joint and muscle pains as well as abdominal pain are other common symptoms (Pavio and Mansuy, 2010).

The small non-enveloped virion of HEV has a spherical shape similar to that of other small round structured viruses. Sucrose gradient centrifugation purified virions were found to have a diameter of 32–34 nm (Bradley et al., 1988). Recent studies indicated the association of lipids with the surface of the virions (Takahashi et al., 2010). The capsid of the virion comprises the capsid protein (CP) of about 660 amino acid (aa) residues. This protein is encoded by the open reading frame (ORF) 2 and target for the main immune response. The gene product of ORF3 of 123 aa was also found to be associated with the surface of virions, but additionally represents a phosphoprotein associated with the cytoskeleton. The 5′ part of the genome contains the large ORF1 which encodes a polyprotein with different enzymatic functions. The genome of the virus is completed by a 5′ untranslated region with a cap modification and a 3′ untranslated region preceding the poly(A) tail (for review see Ahmad et al., 2011).

The usual way of transmission in developing countries is the faecal-oral route. This transmission route has caused large outbreaks in countries with low sanitation standards and was found to be associated with genotypes 1 and 2. On the contrary, during the recent decennia autochthonous hepatitis E cases were increasingly identified in industrialised countries in Europe, Asia and America. Interestingly, these infections were found to be caused by genotypes 3 and 4. Molecular epidemiological evidence and sequence similarities of swine and human HEV sequences confirmed the food-borne transmission of these genotypes from reservoir hosts (Meng et al., 1998, Wang et al., 2002, Li et al., 2005, Tei et al., 2003, Tei et al., 2004, Takahashi et al., 2004). Additional evidence for a zoonotic transmission of these genotypes came from experimental transmission of “human” HEV strains to pig and of “pig” HEV strains to primates (Balayan et al., 1990, Meng et al., 1998, Arankalle et al., 2006). Recently, a novel genotype has been detected in wild boar in Japan (Sato et al., 2011, Takahashi et al., 2011). In addition to the mammalian HEV genotypes avian strains have been identified as causative agents of the big liver and spleen disease and of the similar hepatitis-splenomegaly syndrome (Payne et al., 1999, Haqshenas et al., 2001). The avian strains of different geographical origin were suggested to represent a separate genus of HEV with at least three genotypes (Marek et al., 2010). Most recently a HEV-related agent was identified in different fish species (Batts et al., 2011).

Molecular studies demonstrated the presence of HEV RNA in tissues of wild boar (Sus scrofa), domestic pig (S. scrofa domesticus) and deer species and therefore confirming them as reservoir for zoonotic transmission (Meng, 2010). The detection of HEV-reactive antibodies in other mammalian species, such as cattle, sheep, goat, horse and cat, may indicate the existence of further susceptible potential reservoir animals (Vitral et al., 2005, Sakano et al., 2009, Geng et al., 2010). Recently, a putative novel HEV genotype was identified in rabbits from China which is broadly distributed in rabbit farms in China (Zhao et al., 2009, Geng et al., 2011). However, its closer similarity to genotype 3 (Geng et al., 2011) might be explained by a spillover event of a genotype 1–4 derived strain in the past (Johne et al., 2010b).

For a long time serological investigations suggested spillover infections of human genotypes 1–4 to rodents or the presence of an additional partially cross-reactive HEV-like agent in rodents. HEV-reactive antibodies have been detected in different rat species, i.e. Norway or brown rats (Rattus norvegicus), black rats (R. rattus) and other Rattus species, family Muridae, subfamily Murinae, from India, Japan and USA (Arankalle et al., 2001, Favorov et al., 2000, Kabrane-Lazizi et al., 1999, Hirano et al., 2003). In addition, anti-HEV antibodies were detected in other species of the Murinae subfamily, i.e. Bandicota bengalensis (Arankalle et al., 2001), Mus musculus, and representatives of the Cricetidae subfamilies Arvicolinae, Sigmodintinae and Neotominae, i.e. Myodes (Clethrionomys) gapperi, Oryzomys palustris and Sigmodon hispidus, and different Neotoma and Peromyscus species, respectively (Favorov et al., 2000).

The development of a novel broad-spectrum RT-PCR resulted in the first detection of a novel HEV-like agent in faecal samples from two Norway rats from Hamburg, northern Germany (Johne et al., 2010a). The determination of the entire nucleotide (nt) sequence of two additional strains from Norway rats from the same trapping site demonstrated a genome organisation (see Fig. 2A) and electron microscopic structure similar to previously identified HEV strains (Johne et al., 2010b). In addition, real-time RT-PCR and immunohistochemical studies suggested a hepatotropism of this novel virus. However, phylogenetic analyses and sequence comparisons showed marked differences to all other mammalian HEV strains as well as to the avian HEV strains. These differences prompted us to state ratHEV as a novel genotype of HEV (Johne et al., 2010b, Johne et al., 2011).

Here we describe the detection of novel ratHEV strains in three additional cities in Germany. The studies suggest a continuous presence of this virus in different Norway rat populations in Germany. Phylogenetic and sequence analyses indicated a local evolution of ratHEV in the corresponding rat populations. Serological investigations using recombinant CP derivatives of ratHEV and genotype 3 HEV demonstrated strong antigenic differences and revealed ratHEV-specific antibodies in rats from different areas of Germany.