Complete genome of a Puumala virus strain from Central Europe

Abstract

Puumala virus (PUUV) is one of the predominant hantavirus species in Europe causing mild to moderate cases of haemorrhagic fever with renal syndrome. Parts of Lower Saxony in north-western Germany are endemic for PUUV infections. In this study, the complete PUUV genome sequence of a bank vole-derived tissue sample from the 2007 outbreak was determined by a combined primer-walking and RNA ligation strategy. The S, M and L genome segments were 1,828, 3,680 and 6,550 nucleotides in length, respectively. Sliding-window analyses of the nucleotide sequences of all available complete PUUV genomes indicated a non-homogenous distribution of variability with hypervariable regions located at the 3'-ends of the S and M segments. The overall similarity of the coding genome regions to the other PUUV strains ranged between 80.1 and 84.7 % at the level of the nucleotide sequence and between 89.5 and 98.1 % for the deduced amino acid sequences. In comparison to the phylogenetic trees of the complete coding sequences, trees based on partial segments revealed a general drop in phylogenetic support and a lower resolution. The Astrup strain S and M segment sequences showed the highest similarity to sequences of strains from geographically close sites in the Osnabrück Hills region. In conclusion, a primer-walking-mediated strategy resulted in the determination of the first complete nucleotide sequence of a PUUV strain from Central Europe. Different levels of variability along the genome provide the opportunity to choose regions for analyses according to the particular research question, e.g., large-scale phylogenetics or within-host evolution.

Spatiotemporal dynamics of Puumala hantavirus associated with its rodent host, Myodes glareolus

“…; Plyusnin and Sironen ; Zhang and Holmes ; Sheikh Ali et al. ), a more precise estimation of the contribution of de novo variation to temporal variation in PUUV population diversity will have to await dedicated analyses.…”

Orthohantaviruses, Emerging Zoonotic Pathogens

“…The N protein contains the major antigenic site located between 7 aa and 94 aa and a hyper variable region between 232 aa and 275 aa [24]. In some viruses, the nucleotide sequence identity of the S segment shows high diversity above the identity limit accepted for PUUV lineages [24,65,118]. This could be explained by the accumulation of the silent point mutations in the PUUV genome.…”

Isolation and characterization of new Puumala orthohantavirus strains from Germany

“…Autologous VLPs represent a useful tool to generate highly efficient immune responses against a variety of viruses and for the generation of monoclonal antibodies in particular [20]. PUUV strain Astrup [21] GPC-derived VLPs were generated in this study as previously described for Maporal orthohantavirus [22].…”

“…The enriched cDNA libraries were quantified with the NEBNext Library Quantification Kit (New England Biolabs, Ipswich, MA, USA), pooled in equimolar amounts, and sequenced with a 600 cycle MiSeq Reagent Kit v3 (Illumina, San Diego, CA, USA) using paired-end sequencing (2 × 300 cycles) on a MiSeq sequencer (Illumina, San Diego, CA, USA). The resulting reads were trimmed and assembled against the known complete genome of strain Astrup from the Osnabrück region [21] with Geneious R11.1.5 (https ://www.genei ous.com). For sequences lacking the 5′ and 3′ ends of the M segment, RNA ligation was done using T4 RNA Ligase (Thermo Fisher Scientific, Waltham, MA, USA) and subsequent in vitro transcription with a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…For expression and generation of VLPs in HEK293 cells, a codon-optimized synthetic gene of the PUUV GPC of the strain Astrup [21] was purchased (GeneArt, Regensburg, Germany). The gene encoding the glycoproteins was PCR amplified using primer pair O GRS 101/O GRS 102 (aat-taaGGT ACC TCC AGA GGC GAC ACC CGG AACC and aattattAAG CTT TCA GGG CTT GTG TTC TTT GG) and the PCR product and the acceptor vector pHAN-1 (Roman-Sosa, unpublished) were digested with the restriction endonucleases KpnI and HindIII.…”