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. , ABSTRACT 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.

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