Abstract Rustrela virus (RusV, species Rubivirus strelense ) is a recently discovered relative of rubella virus (RuV) that has been detected in cases of encephalitis across a wide spectrum of mammals, including placental and marsupial animals. Here we diagnosed two additional cases of fatal RusV-associated meningoencephalitis in a South American coati ( Nasua nasua ) and a Eurasian otter ( Lutra lutra ) that were detected in a zoological garden with history of prior RusV infections. Both animals showed abnormal movement or unusual behaviour and their brains tested positive for RusV using specific RT-qPCR and RNA in situ hybridization. As previous sequencing of RusV 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. Virus-to-host sequence ratio and thereby sequence coverage improved markedly using the enrichment method as compared to standard procedures. 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 &gt;80 mol%, which could not be resolved even by enormous sequencing efforts with standard methods. The updated capsid protein amino acid sequence now resembles those of RuV and ruhugu virus in size and harbours a predicted RNA binding domain that was not encoded in the original RusV genome version. The new sequence data indicate that RusV has the largest overall genome (9,631 nucleotides), intergenic region (290 nucleotides) and capsid protein-encoding sequence (331 codons) within the genus Rubivirus .

Revisiting rustrela virus – new cases of encephalitis and a solution to the capsid enigma