Investigation of astrovirus and bornavirus in the cerebrospinal fluid of dogs clinically diagnosed with meningoencephalitis of unknown etiology

Abstract Background Non‐suppurative encephalitides in a variety of species, including humans and dogs, have been linked to infection by astroviruses and bornaviruses. Hypothesis/Objectives To determine whether or not ribonucleic acid of astroviruses or bornaviruses was present in the cerebrospinal fluid (CSF) of dogs with clinically diagnosed meningoencephalomyelitis of unknown etiology (MUE). Animals Twenty‐five client‐owned dogs evaluated by CSF analysis at a single university referral hospital. Methods Prospective case‐control study. Cerebrospinal fluid was collected from clinically diagnosed MUE and control cases and evaluated by reverse‐transcriptase polymerase chain reaction for the presence of astrovirus and bornavirus. Results Neither astrovirus nor bornavirus nucleic acids were identified in CSF collected from 20 clinically diagnosed MUE and 5 control cases. Conclusions and Clinical Importance The negative results of this investigation suggest that astrovirus and bornavirus are not commonly detectable in CSF of dogs with MUE.

Viruses are of particular interest as factors priming the development of autoimmunity. 2,9 Previous veterinary studies evaluated for the presence of several neurotropic DNA viruses, RNA viruses, and atypical bacterial pathogens associated with encephalitis in humans by polymerase chain reaction (PCR), serology, culture, immunohistochemistry, and metagenomics sequencing. 1,[6][7][8]10 Two studies did not detect viruses in either fresh frozen or formalin-fixed paraffin-embedded brain tissue of dogs with MUE, although neither astroviruses nor bornaviruses were screened for in either study. 1,6 Non-suppurative encephalitides have been linked to RNA viruses in the families Astroviridae and Bornaviridae, in a number of other species.
Recently, encephalitis in cattle, mink, swine, sheep, and immunocompromised humans has been associated with astroviruses. 6,[11][12][13][14][15][16][17][18] Retrospective evaluation of brain samples from cattle with European sporadic bovine encephalitis (ESBE), a non-suppurative polioencephalitis of previously undetermined etiology similar to MUE, confirmed the presence of astrovirus in 85% of samples. 18 Bornavirus encephalitis was originally recognized in horses and sheep but has since been diagnosed in a diverse host range including birds and other reptiles, rabbits, cattle, cats, squirrels, humans, and 2 dogs. 19,20 It is unknown if bornavirus encephalitis is more widespread in dogs. Dogs were excluded from the study if they were identified as a breed other than those listed in the inclusion criteria or had positive serum titers for Toxoplasma gondii, Neospora caninum, or Cryptococcus neoformans. Samples were also excluded if the dog was diagnosed with an alternate CNS disease associated with a pleocytosis. Such diseases included CNS neoplasia, fibrocartilaginous embolism, and compressive myelopathy associated with intervertebral disc disease. 26,27 Dogs were considered as negative controls if they met the inclu-

| Nucleic acid extraction and PCR
RNA was extracted from 100 μL of supernatant fluid of CSF using a commercial extraction kit (Qiagen RNeasy Kit, Qiagen) according the manufacturer's instructions. The extracted RNA was stored in duplicates as single-use aliquots at −80 C until PCR amplification. An internal control was performed on each sample to confirm for the presence of 18S (RNA) ( Table 1). For amplification of Astroviridae, a OneStep RT-PCR kit (Qiagen) was used as previously described with primers shown to amplify a both genera, Avastrovirus and mamastrovirus, of Astroviridae,

| Nucleic acid extraction and PCR
No amplification of either astroviruses or bornaviruses was identified from the CSF samples of any of the MUE or control cases. Previous work has focused on the examination of brain tissue in the quest for putative infectious agents. In a study of formalin-fixed paraffin-embedded (FFPE) brain tissue, PCR did not identify the presence of specific DNA viruses in 22 dogs diagnosed with NME, NLE, or GME. 6 Viral DNA or RNA was not identified utilizing broadly reactive PCR on fresh frozen sections of frontal lobe from 11 and 27 confirmed cases of GME and NME, respectively. 1 Several possible explanations exist for these results: the use of stored tissue samples which no longer carry the initiating agent of the disease process, the utilized assays lack sensitivity, or, more likely, these viruses were absent in the etiopathogenesis of MUE.

| DISCUSSION
Astroviruses have generally been associated with clinical signs of gastroenteritis and less commonly respiratory disease. [16][17][18] However, numerous reports across human and veterinary medicine have identified astroviruses as emerging neurotropic pathogens, particularly in immunocompromised transplant recipients humans, [11][12][13][14][16][17][18]33,34 A novel bovine astrovirus was identified by PCR in the brainstem, cerebellum, and spinal cord of steer diagnosed with an encephalomyelitis and ganglioneuritis of unknown origin. 13 Frequently diagnosed encephalitis in cattle with unresolved etiology, ESBE, was linked to an astrovirus. 18 Like GME, NME, and NLE, a definitive diagnosis of ESBE is based on histopathologic results. Investigators utilized RT-PCR and in situ hybridization on FFPE brain tissue of histologically confirmed ESBE cases to detect astrovirus in 12 of 14 cases (85%). 18 Most recently, astrovirus was identified by RT-PCR and in-situ hybridization in archived CNS samples of newly weaned paraplegic pigs in Hungary. 16 Given that the initiating etiology for MUE remains to be determined, it is reasonable to consider the possible role of astroviruses in its pathogenesis.
Bornavirus disease is associated with non-suppurative encephalomyelitis in horses and sheep in endemic areas of central Europe. 35 There are geographically widespread reports of bornavirus disease in diverse vertebrate hosts including 2 dogs. 19,32,35 The dogs with bornavirus were both diagnosed with non-suppurative encephalitis characterized by large perivascular lymphocytic cuffs with varying numbers of macrophages and plasma cells. 19,35 The GME histologic hallmark is perivascular cuffs composed of mixed lymphoid inflammatory cells, as the canine bornavirus. 25 Unlike GME but similar to NME and NLE, the lesion distribution of Borna disease in the 2 dogs was predominant in the gray matter but identified throughout the nervous system. 19,25,32,36 Given these similarities and the current unknown prevalence of bornavirus in the dog, this virus warranted further inquiry.
Several limitations apply to this work. Only a small number of cases were evaluated. The use of 20 clinically diagnosed MUE cases only allows to state with 95% confidence that the prevalence of PCR-detectable astrovirus in CSF of clinically diagnosed MUE cases is less than 10%. 28 Secondly, given that CSF samples were obtained antemortem from clientowned animals, histopathologic confirmation of GME, NME, or NLE was not available for the majority of cases. A confirmed diagnosis of GME or NME was available in 4 affected dogs that were euthanized due to the severity of the disease and whose owners approved a necropsy. Therefore, 16

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.