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 reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocol and high-throughput sequencing. The investigation resulted in the detection of RusV RNA exclusively in 50 of 396 (12.6 per cent) 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. The histopathological evaluation did not reveal any hints of encephalitis in the brains of infected animals despite the detection of viral RNA in neurons by in situ hybridization (ISH). The positive association between the 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 analyses indicated evolutionary processes of RusV mostly in local reservoir populations. A comparison of complete genome sequences from all detected RusV lineages demonstrated a high level of amino acid and nucleotide sequence variability within a part of the p150 peptide of the non-structural polyprotein and its coding sequence, respectively. The location of this region within the RusV genome and its genetic properties were comparable to the hypervariable region of the rubella virus. The broad range of detected RusV spillover 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. Future studies have to prove a putative co-evolution scenario for RusV in the yellow-necked field mouse reservoir.

Mystery of fatal 'staggering disease' unravelled: novel rustrela virus causes severe meningoencephalomyelitis in domestic cats.

'Staggering disease' is a neurological disease entity considered a threat to European domestic cats (Felis catus) for almost five decades. However, its aetiology has remained obscure. Rustrela virus (RusV), a relative of rubella virus, has recently been shown to be associated with encephalitis in a broad range of mammalian hosts. Here, we report the detection of RusV RNA and antigen by metagenomic sequencing, RT-qPCR, in-situ hybridization and immunohistochemistry in brain tissues of 27 out of 29 cats with non-suppurative meningoencephalomyelitis and clinical signs compatible with'staggering disease' from Sweden, Austria, and Germany, but not in non-affected control cats. Screening of possible reservoir hosts in Sweden revealed RusV infection in wood mice (Apodemus sylvaticus). Our work indicates that RusV is the long-sought cause of feline 'staggering disease'. Given its reported broad host spectrum and considerable geographic range, RusV may be the aetiological agent of neuropathologies in further mammals, possibly even including humans.

Revisiting Rustrela Virus: New Cases of Encephalitis and a Solution to the Capsid Enigma.

Rustrela virus (RusV; species Rubivirus strelense) is a recently discovered relative of rubella virus (RuV) that has been detected in cases of encephalitis in diverse mammals. Here, we diagnosed two additional cases of fatal RusV-associated meningoencephalitis in a South American coati (Nasua nasua) and a Eurasian or European otter (Lutra lutra) that were detected in a zoological garden with history of prior RusV infections. Both animals showed abnormal movement or unusual behavior and their brains tested positive for RusV using specific reverse transcription quantitative PCR (RT-qPCR) and RNA in situ hybridization. As previous sequencing of the RusV genome proved to be very challenging, we employed a sophisticated target-specific capture enrichment with specifically designed RNA baits to generate complete RusV genome sequences from both detected encephalitic animals and apparently healthy wild yellow-necked field mice (Apodemus flavicollis). Furthermore, the technique was used to revise three previously published RusV genomes from two encephalitic animals and a wild yellow-necked field mouse. When comparing the newly generated RusV sequences to the previously published RusV genomes, we identified a previously undetected stretch of 309 nucleotides predicted to represent the intergenic region and the sequence encoding the N terminus of the capsid protein. This indicated that the original RusV sequence was likely incomplete due to misassembly of the genome at a region with an exceptionally high G+C content of >80 mol%. The new sequence data indicate that RusV has an overall genome length of 9,631 nucleotides with the longest intergenic region (290 nucleotides) and capsid protein-encoding sequence (331 codons) within the genus Rubivirus. IMPORTANCE The detection of rustrela virus (RusV)-associated encephalitis in two carnivoran mammal species further extends the knowledge on susceptible species. Furthermore, we provide clinical and pathological data for the two new RusV cases, which were until now limited to the initial description of this fatal encephalitis. Using a sophisticated enrichment method prior to sequencing of the viral genome, we markedly improved the virus-to-background sequence ratio compared to that of standard procedures. Consequently, we were able to resolve and update the intergenic region and the coding region for the N terminus of the capsid protein of the initial RusV genome sequence. The updated putative capsid protein now resembles those of rubella and ruhugu virus in size and harbors a predicted RNA-binding domain that had not been identified in the initial RusV genome version. The newly determined complete RusV genomes strongly improve our knowledge of the genome structure of this novel rubivirus.