First isolation and genetic characterization of Puumala orthohantavirus strains from France
Puumala orthohantavirus (PUUV) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) named nephropathia epidemica (NE), regularly diagnosed in Europe. France represents the Western frontier of NE expansion in Europe with two distinct areas: the endemic area (Northeast) where PUUV circulates in rodent populations and where many cases of NE are detected in humans and non-endemic area (Southwest) where the virus is not detected and only a few human cases have been reported. The
... eported. The country is a pertinent target to study factors that influence the evolution of PUUV distribution. In this study, we describe for the first time the isolation of two PUUV strains from two distinct French geographical areas: Ardennes (endemic area) and Loiret (non-endemic area). To isolate PUUV efficiently, we selected wild rodents (Myodes glareolus, the specific reservoir of PUUV) from these areas that were seronegative for anti-PUUV IgG (ELISA) but associated with viral RNA load in lung (qRT-PCR). With this design, we are able to cultivate and maintain these two strains in VeroE6 cells but also to propagate efficiently and rapidly both strains in a bank vole colony. Complete coding sequences of S and M segments were determined by Sanger sequencing of RNA extracted from positive bank voles (naturally and experimentally infected) and from supernatant of Vero E6. For the M segment, nucleotidic sequences were 100% identical for both strains. For the S segment, the amino acid sequences from each strain revealed one mismatch between sequences obtained from tissue and from supernatant, revealing a bank vole and a cell signature. High throughput sequencing confirmed Sanger results, and provided a better assessment of the impact of isolation methods on intra-host viral diversity.