Immunohistochemical and molecular confirmation of West Nile Virus associated polioencephalomyelitis in a mule from Southern Brazil

West Nile fever is a zoonotic arboviral disease caused by the West Nile Virus (WNV), responsible for deaths in humans, mammals, and birds with associated neurological manifestations. All previous investigations of WNV from Brazil were based primarily on serological and molecular analyses and in humans, equids, and birds in the northern and southeastern regions of the country. This study describes the pathological and molecular findings observed in a mule, from the state of Paraná, southern Brazil, that died during an outbreak involving equids with clinical manifestations of a neurological disease. The central nervous system (CNS) of a mule that died after presenting clinical manifestations of a neurological disease was evaluated by histopathological, immunohistochemical (IHC), and molecular analyses. Histopathology revealed lymphocytic polioencephalomyelitis in most areas of the CNS evaluated and choroiditis at the lateral ventricle. An IHC assay based on the WNV glycoprotein E demonstrated positive intracytoplasmic immunoreactivity within neurons, endothelial cells, and ependymal cells from several parts of the CNS with histopathological evidence of disease. Molecular testing amplified the NS5 region of the Flavivirus genus from the cerebrospinal fluid and brain of the mule. The phylogenetic analysis revealed that the strain detected in this animal clustered within the WNV Lineage Ia. Additionally, rabies was not identified and the principal infectious neurological disease agents of equids were discarded by molecular testing. These findings confirmed that this animal was infected by WNV and developed a related neurological syndrome. Additionally, this report represents the few confirmed demonstrations of WNV-associated neurological disease in horses worldwide. Furthermore, the detection of intralesional antigens within the choroid plexus may suggest a possible entry of WNV into the CNS of equids. The detection of WNV in southern Brazil indicates the dissemination of this virus to other geographical regions of this continental nation.