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.

Assessing Genome-Wide Diversity in European Hantaviruses through Sequence Capture from Natural Host Samples.

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.