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

Overview

• This study confirms the presence of West Nile Virus (WNV) causing neurological disease in a mule from southern Brazil using immunohistochemical and molecular techniques.
• The research provides the first detailed pathological evidence of WNV-associated polioencephalomyelitis in equids from this region, highlighting the virus’s spread beyond previously known areas in Brazil.

Background

• West Nile fever is a zoonotic disease caused by West Nile Virus, an arbovirus transmitted primarily by mosquitoes.
• WNV affects a variety of hosts including humans, mammals, and birds, often leading to neurological symptoms and sometimes death.
• Prior to this study, WNV investigations in Brazil were primarily serological or molecular, focused on northern and southeastern regions and involving humans, birds, and equids.
• Reports of WNV-related neurological disease in horses globally are limited, making this study significant in understanding disease manifestation in equids.

Case Description

• The subject was a mule from Paraná in southern Brazil that died after showing neurological symptoms during an outbreak affecting horses and related animals.
• The central nervous system (CNS) tissue was examined through:
  • Histopathological analysis
  • Immunohistochemical (IHC) staining
  • Molecular testing for viral genetic material

Pathological Findings

• Histopathology showed:
  • Lymphocytic polioencephalomyelitis — inflammation in the gray matter of the brain and spinal cord primarily with lymphocytes
  • Choroiditis at the lateral ventricle — inflammation of the choroid plexus, which produces cerebrospinal fluid and forms the blood-CSF barrier
• These findings indicate a viral encephalomyelitis with immune cell infiltration causing CNS damage.

Immunohistochemical Analysis

• An IHC assay targeting the WNV glycoprotein E was performed on CNS tissue sections.
• This assay revealed intracytoplasmic presence of viral proteins in:
  • Neurons
  • Endothelial cells (lining blood vessels)
  • Ependymal cells (lining the brain ventricles)
• Positive IHC staining localized virus directly within cells involved in CNS structure and function, providing strong evidence of infection at the site of pathology.
• Detection of viral antigens in the choroid plexus suggests it may be a portal for virus entry into the CNS.

Molecular Testing and Phylogenetics

• Molecular amplification targeted the NS5 gene region, a conserved segment within the Flavivirus genus, confirming the presence of viral RNA in cerebrospinal fluid and brain tissue.
• Phylogenetic analysis classified the virus strain within West Nile Virus Lineage Ia, consistent with strains known to cause neurological disease.
• Other infectious agents causing neurological diseases in equids, including rabies virus, were ruled out by molecular testing.

Significance and Conclusions

• This study confirms that WNV infection can cause severe neurological disease in equids in southern Brazil, expanding the known geographic distribution of the virus in the country.
• Detection of viral antigen in the choroid plexus supports hypotheses about how the virus crosses into the CNS, an important insight for understanding pathogenesis.
• The findings underscore the importance of surveillance and diagnostic efforts in regions previously not known to harbor WNV.
• Reports like this help raise awareness among veterinarians and public health officials regarding potential risks to animal and human health.
• Overall, the study adds valuable pathological and molecular documentation of WNV-associated polioencephalomyelitis in equids, informing future research and control efforts.