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.

Identification of a novel hantavirus strain in the root vole (Microtus oeconomus) in Lithuania, Eastern Europe.

Hantaviruses are zoonotic pathogens that can cause subclinical to lethal infections in humans. In Europe, five orthohantaviruses are present in rodents: Myodes-associated Puumala orthohantavirus (PUUV), Microtus-associated Tula orthohantavirus, Traemmersee hantavirus (TRAV)/ Tatenale hantavirus (TATV)/ Kielder hantavirus, rat-borne Seoul orthohantavirus, and Apodemus-associated Dobrava-Belgrade orthohantavirus (DOBV). Human PUUV and DOBV infections were detected previously in Lithuania, but the presence of Microtus-associated hantaviruses is not known. For this study we screened 234 Microtus voles, including root voles (Microtus oeconomus), field voles (Microtus agrestis) and common voles (Microtus arvalis) from Lithuania for hantavirus infections. This initial screening was based on reverse transcription-polymerase chain reaction (RT-PCR) targeting the S segment and serological analysis. A novel hantavirus was detected in eight of 79 root voles tentatively named "Rusne virus" according to the capture location and complete genome sequences were determined. In the coding regions of all three genome segments, Rusne virus showed high sequence similarity to TRAV and TATV and clustered with Kielder hantavirus in phylogenetic analyses of partial S and L segment sequences. Pairwise evolutionary distance analysis confirmed Rusne virus as a strain of the species TRAV/TATV. Moreover, we synthesized the entire nucleocapsid (N) protein of Rusne virus in Saccharomyces cerevisiae. We observed cross-reactivity of antibodies raised against other hantaviruses, including PUUV, with this new N protein. ELISA investigation of all 234 voles detected Rusne virus-reactive antibodies exclusively in four of 79 root voles, all being also RNA positive, but not in any other vole species. In conclusion, the detection of Rusne virus RNA in multiple root voles at the same trapping site during three years and its absence in sympatric field voles suggests root voles as the reservoir host of this novel virus. Future investigations should evaluate host association of TRAV, TATV, Kielder virus and the novel Rusne virus and their evolutionary relationships.

Spatial and Temporal Evolutionary Patterns in Puumala Orthohantavirus (PUUV) S Segment.

The S segment of bank vole (Clethrionomys glareolus)-associated Puumala orthohantavirus (PUUV) contains two overlapping open reading frames coding for the nucleocapsid (N) and a non-structural (NSs) protein. To identify the influence of bank vole population dynamics on PUUV S segment sequence evolution and test for spillover infections in sympatric rodent species, during 2010-2014, 883 bank voles, 357 yellow-necked mice (Apodemus flavicollis), 62 wood mice (A. sylvaticus), 149 common voles (Microtus arvalis) and 8 field voles (M. agrestis) were collected in Baden-Wuerttemberg and North Rhine-Westphalia, Germany. In total, 27.9% and 22.3% of bank voles were positive for PUUV-reactive antibodies and PUUV-specific RNA, respectively. One of eight field voles was PUUV RNA-positive, indicating a spillover infection, but none of the other species showed evidence of PUUV infection. Phylogenetic and isolation-by-distance analyses demonstrated a spatial clustering of PUUV S segment sequences. In the hantavirus outbreak years 2010 and 2012, PUUV RNA prevalence was higher in our study regions compared to non-outbreak years 2011, 2013 and 2014. NSs amino acid and nucleotide sequence types showed temporal and/or local variation, whereas the N protein was highly conserved in the NSs overlapping region and, to a lower rate, in the N alone coding part.

Isolation and characterization of new Puumala orthohantavirus strains from Germany.

Orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. Here, we report the isolation of a Puumala orthohantavirus (PUUV) strain from bank voles caught in a highly endemic region around the city Osnabrück, north-west Germany. Coding and non-coding sequences of all three segments (S, M, and L) were determined from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RNA-dependent RNA polymerase (RdRP) of the two stable PUUV isolates. The PUUV strain from VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. In conclusion, this is the first isolation of a PUUV strain from Central Europe and the generation of glycoprotein-specific monoclonal antibodies for this PUUV isolate. The obtained virus isolate and GPC-specific antibodies are instrumental tools for future reservoir host studies.

Molecular Epidemiology of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in Wild, Captive and Laboratory Rats: Effect of Habitat on the Nasal S. aureus Population.

Rats are a reservoir of human- and livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). However, the composition of the natural S. aureus population in wild and laboratory rats is largely unknown. Here, 144 nasal S. aureus isolates from free-living wild rats, captive wild rats and laboratory rats were genotyped and profiled for antibiotic resistances and human-specific virulence genes. The nasal S. aureus carriage rate was higher among wild rats (23.4%) than laboratory rats (12.3%). Free-living wild rats were primarily colonized with isolates of clonal complex (CC) 49 and CC130 and maintained these strains even in husbandry. Moreover, upon livestock contact, CC398 isolates were acquired. In contrast, laboratory rats were colonized with many different S.aureus lineages-many of which are commonly found in humans. Five captive wild rats were colonized with CC398-MRSA. Moreover, a single CC30-MRSA and two CC130-MRSA were detected in free-living or captive wild rats. Rat-derived S. aureus isolates rarely harbored the phage-carried immune evasion gene cluster or superantigen genes, suggesting long-term adaptation to their host. Taken together, our study revealed a natural S. aureus population in wild rats, as well as a colonization pressure on wild and laboratory rats by exposure to livestock- and human-associated S.aureus, respectively.

Field vole-associated Traemmersee hantavirus from Germany represents a novel hantavirus species.

Vole-associated hantaviruses occur in the Old and New World. Tula orthohantavirus (TULV) is widely distributed throughout the European continent in its reservoir, the common vole (Microtus arvalis), but the virus was also frequently detected in field voles (Microtus agrestis) and other vole species. TULV and common voles are absent from Great Britain. However, field voles there harbor Tatenale and Kielder hantaviruses. Here we screened 126 field voles and 13 common voles from Brandenburg, Germany, for hantavirus infections. One common vole and four field voles were anti-TULV antibody and/or TULV RNA positive. In one additional, seropositive field vole a novel hantavirus sequence was detected. The partial S and L segment nucleotide sequences were only 61.1% and 75.6% identical to sympatrically occurring TULV sequences, but showed highest similarity of approximately 80% to British Tatenale and Kielder hantaviruses. Subsequent determination of the entire nucleocapsid (N), glycoprotein (GPC), and RNA-dependent RNA polymerase encoding sequences and determination of the pairwise evolutionary distance (PED) value for the concatenated N and GPC amino acid sequences confirmed a novel orthohantavirus species, tentatively named Traemmersee orthohantavirus. The identification of this novel hantavirus in a field vole from eastern Germany underlines the necessity of a large-scale, broad geographical hantavirus screening of voles to understand evolutionary processes of virus-host associations and host switches.

Genomic and spatial variability of a European common vole hepevirus.

Rodents host different orthohepeviruses, namely orthohepevirus C genotype HEV-C1 (rat hepatitis E virus, HEV) and the additional putative genotypes HEV-C3 and HEV-C4. Here, we screened 2,961 rodents from Central Europe by reverse transcription polymerase chain reaction (RT-PCR) and identified HEV RNA in 13 common voles (Microtus arvalis) and one bank vole (Myodes glareolus) with detection rates of 2% (95% confidence interval [CI]: 1-3.4) and 0.08% (95% CI: 0.002-0.46), respectively. Sequencing of a 279-nucleotide RT-PCR amplicon corresponding to a region within open reading frame (ORF) 1 showed a high degree of similarity to recently described common vole-associated HEV (cvHEV) sequences from Hungary. Five novel complete cvHEV genome sequences from Central Europe showed the typical HEV genome organization with ORF1, ORF2 and ORF3 and RNA secondary structure. Uncommon features included a noncanonical start codon in ORF3, multiple insertions and deletions within ORF1 and ORF2/ORF3, and the absence of a putative ORF4. Phylogenetic analysis showed all of the novel cvHEV sequences to be monophyletic, clustering most closely with an unassigned bird-derived sequence and other sequences of the species Orthohepevirus C. The nucleotide and amino acid sequence divergence of the common vole-derived sequences was significantly correlated with the spatial distance between the trapping sites, indicating mostly local evolutionary processes. Detection of closely related HEV sequences in common voles in multiple localities over a distance of 800 kilometers suggested that common voles are infected by cvHEV across broad geographic distances. The common vole-associated HEV strain is clearly divergent from HEV sequences recently found in narrow-headed voles (Microtus gregalis) and other cricetid rodents.

Norway and black rats in Europe: potential reservoirs for zoonotic arthropod-borne pathogens?

BACKGROUND: Norway rats (Rattus norvegicus) and black rats (R. rattus) are known to be cosmopolitan reservoirs for zoonotic agents. Nevertheless, little is known about prevalence and distribution of arthropod-borne pathogens in rats from Europe. Therefore, this survey focused on the detection of arthropod-borne pathogens. Spleen-derived DNA samples were available from 528 Norway rats and 74 black rats collected in several European countries. Further, these samples were processed by polymerase chain reaction for the detection of zoonotic pathogens such as Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis (CNM), Babesia spp. and Bartonella spp. eventually followed by sequencing. RESULTS: Babesia spp. was not detected. Four Norway rat samples were positive for A. phagocytophilum DNA and two for CNM. In 50 rat samples, Bartonella spp. DNA was detected (8.1%; 95% Confidence interval (CI) 6.2-10.61). Whereas B. tribocorum (n = 45) and B. grahamii (n = 1) were carried exclusively in Norway rats from Central Europe (Belgium, Germany), B. coopersplainsensis (n = 4) was detected only in black rats from southern European countries (Spain, Italy). CONCLUSIONS: Pathogenic Bartonella spp. DNA was found in black and Norway rats from Germany, Italy, Spain and Belgium for the first time. Bartonellae were found focally in zoos suggesting Norway rats as a possible reservoir for B. tribocorum and black rats as a reservoir for B. coopersplainsensis in Europe. These findings should raise awareness of pathogenic Bartonella spp. in Norway rats, especially in terms of pest management control in zoos. Norway and black rats seem not to be predominantly involved in the life cycle of the other examined arthropod-borne pathogens in Europe. © 2019 Society of Chemical Industry.

Hepatitis E virus in feral rabbits along a rural-urban transect in Central Germany.

Rabbit associated genotype 3 hepatitis E virus (HEV) strains were detected in feral, pet and farm rabbits in different parts of the world since 2009 and recently also in human patients. Here, we report a serological and molecular survey on 72 feral rabbits, collected along a rural-urban transect in and next to Frankfurt am Main, Central Germany. ELISA investigations revealed in 25 of 72 (34.7%) animals HEV-specific antibodies. HEV derived RNA was detected in 18 of 72 (25%) animals by reverse transcription-polymerase chain reaction assay. The complete genomes from two rabbitHEV-strains, one from a rural site and the other from an inner-city area, were generated by a combination of high-throughput sequencing, a primer walking approach and 5'- and 3'- rapid amplification of cDNA ends. Phylogenetic analysis of open reading frame (ORF)1-derived partial and complete ORF1/ORF2 concatenated coding sequences indicated their similarity to rabbit-associated HEV strains. The partial sequences revealed one cluster of closely-related rabbitHEV sequences from the urban trapping sites that is well separated from several clusters representing rabbitHEV sequences from rural trapping sites. The complete genome sequences of the two novel strains indicated similarities of 75.6-86.4% to the other 17 rabbitHEV sequences; the amino acid sequence identity of the concatenated ORF1/ORF2-encoded proteins reached 89.0-93.1%. The detection of rabbitHEV in an inner-city area with a high human population density suggests a high risk of potential human infection with the zoonotic rabbitHEV, either by direct or indirect contact with infected animals. Therefore, future investigations on the occurrence and frequency of human infections with rabbitHEV are warranted in populations with different contact to rabbits.

Serological evidence of hepatitis E virus infection in zoo animals and identification of a rodent-borne strain in a Syrian brown bear.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an emerging infectious disease of humans. HEV infections have also been described in various animal species. Whereas domestic pigs and wild boars are well-known animal reservoirs for HEV, the knowledge on natural HEV infection in zoo animals is scarce so far. Here, we analysed 244 sera from 66 mammal species derived from three zoos in Germany using a commercial double antigen sandwich ELISA. HEV-specific antibodies were detected in 16 animal species, with the highest detection rates in suids (33.3%) and carnivores (27.0%). However, RNA of the human pathogenic HEV genotypes 1-4 was not detected in the serum samples from suids or carnivores. Using a broad spectrum RT-PCR, a ratHEV-related sequence was identified in a sample of a female Syrian brown bear (Ursus arctos syriacus). Subsequent serum samples within a period of five years confirmed a HEV seroconversion in this animal. No symptoms of hepatitis were recorded. In a follow-up investigation at the same location, closely related ratHEV sequences were identified in free-living Norway rats (Rattus norvegicus), whereas feeder rats (Rattus norvegicus forma domestica) were negative for HEV-specific antibodies and RNA. Therefore, a spillover infection of ratHEV from free-living Norway rats is most likely. The results indicate that a wide range of zoo animals can be naturally infected with HEV or HEV-related viruses. Their distinct role as possible reservoir animals for HEV and sources of HEV infection for humans and other animals remains to be investigated.

Detection of rat hepatitis E virus in wild Norway rats (Rattus norvegicus) and Black rats (Rattus rattus) from 11 European countries.

Rat hepatitis E virus (HEV) is genetically only distantly related to hepeviruses found in other mammalian reservoirs and in humans. It was initially detected in Norway rats (Rattus norvegicus) from Germany, and subsequently in rats from Vietnam, the USA, Indonesia, China, Denmark and France. Here, we report on a molecular survey of Norway rats and Black rats (Rattus rattus) from 12 European countries for ratHEV and human pathogenic hepeviruses. RatHEV-specific real-time and conventional RT-PCR investigations revealed the presence of ratHEV in 63 of 508 (12.4%) rats at the majority of sites in 11 of 12 countries. In contrast, a real-time RT-PCR specific for human pathogenic HEV genotypes 1-4 and a nested broad-spectrum (NBS) RT-PCR with subsequent sequence determination did not detect any infections with these genotypes. Only in a single Norway rat from Belgium a rabbit HEV-like genotype 3 sequence was detected. Phylogenetic analysis indicated a clustering of all other novel Norway and Black rat-derived sequences with ratHEV sequences from Europe, the USA and a Black rat-derived sequence from Indonesia within the proposed ratHEV genotype 1. No difference in infection status was detected related to age, sex, rat species or density of human settlements and zoological gardens. In conclusion, our investigation shows a broad geographical distribution of ratHEV in Norway and Black rats from Europe and its presence in all settlement types investigated.

Survey for zoonotic pathogens in Norway rat populations from Europe.

BACKGROUND: The Norway rat Rattus norvegicus is an important reservoir of various zoonotic pathogens, such as cowpox virus and Leptospira, but also for agents of no or unknown zoonotic potential. We describe a survey of 426 Norway rats originating from five European countries and different habitats for Leptospira spp., rickettsiae, orthopoxvirus (OPV), avian metapneumovirus subtypes A and B (aMPV) and rat polyomavirus (rat PyV). RESULTS: Leptospira DNA was detected in 60 out of 420 (14.3%) rats, and Rickettsia DNA was found in three out of 369 (0.8%) rats investigated. PCR-based typing resulted in the identification of L. interrogans sequence type 17, which corresponds to the serogroup Icterohaemorrhagiae, and Rickettsia helvetica respectively. Rat PyV DNA was detected in 103 out of 421 (24.5%) rats. OPV DNA and aMPV RNA were detected in none of the rats, but OPV-specific antibodies were detected in three out of 388 (0.8%) rats. The frequency of single Leptospira and rat PyV infections and coinfections was, independent of sex, greater for adults compared with juveniles/subadults and greater at rural sites compared with urban areas. CONCLUSIONS: Study results indicate a broad geographical distribution of Leptospira DNA in rats within Europe, underlining the need to investigate further the potential mechanisms leading to increased prevalence in rural habitats and to assess the relevance to public health. In contrast, rickettsia and OPV infections rarely occurred in wild rat populations. The potential influence of rat PyV on the susceptibility to infections with other pathogens should be investigated in future studies. © 2016 Society of Chemical Industry.