Continuous presence of genetically diverse rustrela virus lineages in yellow-necked field mouse reservoir populations in northeastern Germany

Rustrela virus (RusV; species Rubivirus strelense, family Matonaviridae) was discovered in different zoo animal species affected by fatal encephalitis. Simultaneous RusV RNA detection in multiple yellow-necked field mice (Apodemus flavicollis) suggested this rodent as a reservoir of RusV. Here we investigated 1,264 yellow-necked field mice and sympatric other small mammals from different regions in Germany for RusV RNA using an optimized real-time RT-qPCR protocol and high-throughput sequencing. The investigation resulted in the detection of RusV RNA exclusively in 50 of 396 (12.6%) yellow-necked field mice, but absence in other sympatric species. RT-qPCR-determined tissue distribution of RusV RNA revealed the highest viral loads in the central nervous system, with other tissues being only very rarely affected. Histopathological evaluation did not reveal any hints of an encephalitis in the brains of infected animals despite detection of viral RNA in neurons by in situ hybridization. The positive association of body mass of yellow-necked field mice and RusV RNA detection suggests a persistent infection. Phylogenetic analysis of partial E1 and full genome sequences showed a high diversification with at least four RusV lineages (1A-1D) in northeastern Germany. Moreover, phylogenetic and isolation-by-distance analysis indicated evolutionary processes of RusV mostly in local reservoir populations. Comparison of complete genome sequences from all detected RusV lineages demonstrated a high-level of nucleotide and amino acid sequence variability within a part of the p150 peptide of the non-structural polyprotein. The location of this region within the RusV genome and its genetic properties were comparable to the hypervariable region of rubella virus. The broad range of detected RusV spill-over hosts in combination with its geographical distribution in northeastern Germany requires the assessment of its zoonotic potential and further analysis of encephalitis cases in mammals. Additional future studies have to prove a putative co-evolution scenario for RusV in the yellow-necked field mouse reservoir.