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Seoul orthohantavirus (SEOV) is a rat-associated zoonotic pathogen with an almost worldwide distribution. In 2019, the first autochthonous human case of SEOV-induced hemorrhagic fever with renal syndrome was reported in Germany, and a pet rat was identified as the source of the zoonotic infection. To further investigate the SEOV reservoir, additional rats from the patient and another owner, all of which were purchased from the same vendor, were tested. SEOV RNA and anti-SEOV antibodies were found in both of the patient's rats and in two of the three rats belonging to the other owner. The complete coding sequences of the small (S), medium (M), and large (L) segments obtained from one rat per owner exhibited a high sequence similarity to SEOV strains of breeder rat or human origin from the Netherlands, France, the USA, and Great Britain. Serological screening of 490 rats from breeding facilities and 563 wild rats from Germany (2007-2020) as well as 594 wild rats from the Netherlands (2013-2021) revealed 1 and 6 seropositive individuals, respectively. However, SEOV RNA was not detected in any of these animals. Increased surveillance of pet, breeder, and wild rats is needed to identify the origin of the SEOV strain in Europe and to develop measures to prevent transmission to the human population.

In mammals, the zona pellucida glycoprotein 3 (ZP3) is considered a primary sperm receptor of the oocyte and is hypothesized to be involved in reproductive isolation. We investigated patterns of diversity and selection in the putative sperm-binding region (pSBR) of mouse ZP3 across Cricetidae and Murinae, two hyperdiverse taxonomic groups within muroid rodents. In murines, the pSBR is fairly conserved, in particular the serine-rich stretch containing the glycosylation sites proposed as essential for sperm binding. In contrast, cricetid amino acid sequences of the pSBR were much more variable and the serine-rich motif, typical of murines, was generally substantially modified. Overall, our results suggest a general lack of species specificity of the pSBR across the two muroid families. We document statistical evidence of positive selection acting on exons 6 and 7 of ZP3 and identified several amino acid sites that are likely targets of selection, with most positively selected sites falling within or adjacent to the pSBR.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole ( Myodes glareolus , syn. Clethrionomys glareolus ). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles ( Myodes rutilus , syn. Clethrionomys rutilus ). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species ( Hepacivirus F and Hepacivirus J ) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.

Tula orthohantavirus (TULV) is a rodent-borne hantavirus with broad geographical distribution in Europe. Its major reservoir is the common vole ( Microtus arvalis ), but TULV has also been detected in closely related vole species. Given the large distributional range and high amplitude population dynamics of common voles, this host-pathogen complex presents an ideal system to study the complex mechanisms of pathogen transmission in a wild rodent reservoir. We investigated the dynamics of TULV prevalence and the subsequent potential effects on the molecular evolution of TULV in common voles of the Central evolutionary lineage. Rodents were trapped for three years in four regions of Germany and samples were analyzed for the presence of TULV-reactive antibodies and TULV RNA with subsequent sequence determination. The results show that individual (sex) and population-level factors (abundance) of hosts were significant predictors of local TULV dynamics. At the large geographic scale, different phylogenetic TULV clades and an overall isolation-by-distance pattern in virus sequences were detected, while at the small scale (<4 km) this depended on the study area. In combination with an overall delayed density dependence, our results highlight that frequent, localized bottleneck events for the common vole and TULV do occur and can be offset by local recolonization dynamics.

Hepatitis E is an emerging viral disease that is the leading cause of viral hepatitis in the world. The vast majority of hepatitis E cases in developed countries are caused by zoonotic genotypes 3 and 4 of hepatitis E virus (HEV) for which pig and wild boar and to lesser extent rabbits are the main reservoir. According to recent reports rabbits are a source of human HEV infection and highlight the risk of zoonotic foodborne transmission. Here we report the molecular analysis of a novel HEV strain identified in a rabbit during a countrywide surveillance of rabbits and hares in Germany, 2016. The analysis of the complete genome reveals characteristics of a putative novel recombinant subtype of the species Orthohepevirus A within the clade of genotype 3 but not closely related to any known subtypes. Importantly, the genome of this strain possesses a nucleotide exchange in the overlapping region of open reading frames ORF2/ORF3 interfering with a broadly applied diagnostic real-time RT-PCR. In conclusion, a new type of HEV strain was identified in a German rabbit with atypical and novel sequence characteristics.

Research on the ecology and evolution of viruses is often hampered by the limitation of sequence information to short parts of the genomes or single genomes derived from cultures. In this study, we use hybrid sequence capture enrichment in combination with high-throughput sequencing to provide efficient access to full genomes of European hantaviruses from rodent samples obtained in the field. We applied this methodology to Tula (TULV) and Puumala (PUUV) orthohantaviruses for which analyses from natural host samples are typically restricted to partial sequences of their tri-segmented RNA genome. We assembled a total of ten novel hantavirus genomes de novo with very high coverage (on average >99%) and sequencing depth (average >247×). A comparison with partial Sanger sequences indicated an accuracy of >99.9% for the assemblies. An analysis of two common vole ( Microtus arvalis ) samples infected with two TULV strains each allowed for the de novo assembly of all four TULV genomes. Combining the novel sequences with all available TULV and PUUV genomes revealed very similar patterns of sequence diversity along the genomes, except for remarkably higher diversity in the non-coding region of the S-segment in PUUV. The genomic distribution of polymorphisms in the coding sequence was similar between the species, but differed between the segments with the highest sequence divergence of 0.274 for the M-segment, 0.265 for the S-segment, and 0.248 for the L-segment (overall 0.258). Phylogenetic analyses showed the clustering of genome sequences consistent with their geographic distribution within each species. Genome-wide data yielded extremely high node support values, despite the impact of strong mutational saturation that is expected for hantavirus sequences obtained over large spatial distances. We conclude that genome sequencing based on capture enrichment protocols provides an efficient means for ecological and evolutionary investigations of hantaviruses at an unprecedented completeness and depth.

Puumala virus (PUUV) causes mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS), and is responsible for the majority of hantavirus infections of humans in Fennoscandia, Central and Western Europe. Although there are relatively many PUUV sequences available from different European countries, little is known about the presence of this virus in Poland. During population studies in 2009 a total of 45 bank voles were trapped at three sites in north-eastern Poland, namely islands on Dejguny and Dobskie Lakes and in a forest near Mikołajki. S and M segment-specific RT-PCR assays detected PUUV RNA in three animals from the Mikołajki site. The obtained partial S and M segment sequences demonstrated the highest similarity to the corresponding segments of a PUUV strain from Latvia. Analysis of chest cavity fluid samples by IgG ELISA using a yeast-expressed PUUV nucleocapsid protein resulted in the detection of two seropositive samples, both being also RT-PCR positive. Interestingly, at the trapping site in Mikołajki PUUV-positive bank voles belong to the Carpathian and Eastern genetic lineages within this species. In conclusion, we herein present the first molecular evidence for PUUV in the rodent reservoir from Poland.