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

Inflammatory disorders of the central nervous system (CNS) across mammalian species are associated with significant morbidity, mortality, and long-term neurological deficits. These can be broadly categorized into infectious and immune-mediated disorders, yet frequently, the cause of encephalitis remains unknown. This lack of diagnosis creates uncertainty for clinicians, patients, and pet owners regarding origin, treatment, and prognosis. This is particularly true for non-suppurative, lymphohistiocytic encephalitides, a large group of histopathologically similar conditions often undiagnosable with conventional methods like immunohistochemistry (IHC), in situ hybridization (ISH), and polymerase chain reaction (PCR). One such enigmatic encephalitis is the feline ‘staggering disease,’ first described in the 1970s in Sweden. Characterized by hind leg ataxia, increased muscle tone, and a staggering gait, it also presents with a range of other neurological signs and behavioral changes. The disease progresses over days to weeks, sometimes longer, often leading to euthanasia. Histopathology reveals non-suppurative, lymphohistiocytic meningoencephalomyelitis with angiocentric immune cell infiltration. Despite its consistent clinical presentation and histopathology, and geographically associated occurrence, the causative agent has remained elusive for nearly five decades. Borna disease virus 1 (BoDV-1) was a prime suspect, but conclusive evidence of natural infection in cats with ‘staggering disease’ in Sweden has been lacking. Advances in metagenomics offer new tools for identifying previously unknown pathogens in unexplained encephalitides. One such recently discovered agent is rustrela virus (RusV), a rubella virus relative, identified in the brains of various mammals exhibiting lymphohistiocytic encephalitis. This study investigates the potential role of RusV in feline ‘staggering disease.’

The literature extensively documents feline ‘staggering disease,’ noting its consistent clinical and pathological features, suggesting a viral etiology. Early studies in Sweden and Austria detailed the clinical signs and histopathological findings, highlighting the non-suppurative, lymphohistiocytic meningoencephalomyelitis. For decades, Borna disease virus 1 (BoDV-1) was considered a strong candidate, with some studies suggesting a link through serological testing or detection of viral genetic material. However, these findings were often inconclusive, inconsistent across different diagnostic methods, or geographically inconsistent with known BoDV-1 endemic areas. The lack of definitive proof led to ongoing uncertainty regarding the cause of ‘staggering disease.’ The recent discovery of rustrela virus (RusV) in various mammals with encephalitis provided a new potential candidate pathogen. Previous studies on RusV infections described the clinical presentation and histopathological changes, which bear a striking resemblance to feline ‘staggering disease.’ This laid the groundwork for the current study, aiming to definitively establish RusV as the causative agent.

This study analyzed brain samples from 29 cats with clinical and histopathological features consistent with ‘staggering disease.’ The samples originated from Sweden, Austria, and Germany, representing both recent and historical cases dating back to the 1990s. A control group included brain samples from cats without encephalitis and those with other forms of encephalitis. The methodology employed a multi-pronged approach for pathogen detection: 1. **Bornavirus Detection:** Brain samples were tested for bornaviruses, including BoDV-1, using RT-qPCR and IHC. 2. **Metagenomic Sequencing:** Selected samples underwent metagenomic sequencing to identify potential novel pathogens. This involved RNA extraction, cDNA synthesis, library preparation, and sequencing using Ion Torrent and Illumina platforms. Target enrichment with panRubi myBaits panels (v2 and v3) was employed to enhance RusV detection. rRNA depletion was also used to improve the signal-to-noise ratio. De novo assembly and sequence annotation were performed to identify and characterize viral genomes. 3. **RusV-Specific Detection:** Following the initial metagenomic findings, additional methods were used to confirm RusV detection: RT-qPCR (using both a previously established assay and a newly designed panRusV assay targeting a highly conserved region), RNAscope in situ hybridization (ISH), and immunohistochemistry (IHC) using a newly generated monoclonal antibody targeting the RusV capsid protein. The specificity and sensitivity of these methods were carefully validated using positive and negative controls. 4. **Rodent Screening:** Brain samples from rodents (wood mice and yellow-necked field mice) from a region in Sweden were screened for RusV RNA using RT-qPCR and ISH. 5. **Phylogenetic Analysis:** Phylogenetic analysis of RusV sequences from cats and rodents, using both nucleotide and amino acid sequences, was used to investigate the genetic diversity and geographic distribution of RusV. 6. **Histopathological Examination:** Histological examination of brain and spinal cord tissues from cats was performed using standard staining techniques (H.E., Luxol Fast Blue-Cresyl Echt Violet) to assess the nature and extent of inflammatory lesions and their correlation with RusV detection.

The study found RusV RNA and/or antigen in 27 out of 29 cats with ‘staggering disease,’ consistently detected by at least two independent diagnostic methods (RT-qPCR, ISH, IHC, HTS). In contrast, RusV was not detected in any of the control cats without neurological disease or with other types of encephalitis. The newly identified RusV sequences clustered with other known RusV sequences, but showed considerable genetic variability (down to 75% nucleotide identity), resulting in three major phylogenetic clades representing Sweden, Austria, and Northeastern Germany. A highly conserved region at the 5' terminus of the genome allowed for the development of broadly reactive diagnostic tools. Histopathological examination revealed widespread, polio-predominant angiocentric lymphocytic and/or lymphohistiocytic infiltrates in the RusV-positive cats, consistent with previous descriptions of ‘staggering disease.’ RusV RNA and antigen were primarily located in neurons of the cerebral cortex, hippocampus, cerebellum, brainstem, and spinal cord. Importantly, the study identified RusV infection in wood mice (*Apodemus sylvaticus*) in Sweden, suggesting a potential reservoir host. The seasonal occurrence of ‘staggering disease’ (more cases in winter and spring) was consistent with the findings in the study, possibly reflecting fluctuations in rodent reservoir populations. Analysis of the data revealed that 21(77.8%) of the 27 cats with RusV infection were neutered or intact males, consistent with previous studies. The median age of the affected cats was 3.2 years with a range of 1.5 to 12.3 years. Notably, all affected cats had outdoor access.

This study provides compelling evidence linking RusV infection to feline ‘staggering disease.’ The consistent detection of RusV in affected cats, coupled with the absence of the virus in controls, strongly supports a causative role. The observed clinical course and histopathological lesions closely resemble those reported for other RusV-infected mammals. The discovery of RusV in wood mice points to a potential reservoir host, explaining the disease’s sporadic occurrence and geographic distribution. The significant genetic diversity of RusV emphasizes the importance of using broadly reactive diagnostic tools for accurate detection. The observed seasonal pattern of ‘staggering disease’ likely reflects fluctuations in reservoir host populations. The restricted geographic distribution of RusV clades suggests localized transmission, with cats serving primarily as incidental hosts. This pattern is consistent with other reservoir-bound viruses. This work refutes the hypothesis that BoDV-1 is the causative agent of ‘staggering disease’, especially given the absence of BoDV-1 detection in this study and the distinct distribution of BoDV-1 compared to the distribution of RusV infection in cats.

This study definitively identifies RusV as the causative agent of feline ‘staggering disease,’ resolving a long-standing mystery in veterinary medicine. The discovery of a potential reservoir host in wood mice provides crucial insights into the disease’s epidemiology. The development of broadly reactive diagnostic tools allows for wider surveillance and detection of RusV infections. Future research should focus on isolating RusV, establishing experimental infection models, developing serological assays, and investigating the zoonotic potential of RusV.

The study did not fulfill Koch's postulates by experimentally reproducing the disease in cats. While a strong association was established between RusV infection and ‘staggering disease,’ experimental infection would provide definitive proof of causality. The relatively small sample size, especially regarding the rodent samples, could limit the generalizability of the findings concerning reservoir hosts. The genetic variability of RusV, requiring the development of broadly reactive diagnostic tools, could have influenced the sensitivity and specificity of the assays used.