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Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne viruses in the Americas that cause central nervous system (CNS) disease in humans and equids. In this study, we directly characterized the pathogenesis of VEEV, EEEV, and WEEV in cynomolgus macaques following subcutaneous exposure because this route more closely mimics natural infection via mosquito transmission or by an accidental needle stick. Our results highlight how EEEV is significantly more pathogenic compared to VEEV similarly to what is observed in humans. Interestingly, EEEV appears to be just as neuropathogenic by subcutaneous exposure as it was in previously completed aerosol exposure studies. In contrast, subcutaneous exposure of cynomolgus macaques with WEEV caused limited disease and is contradictory to what has been reported for aerosol exposure. Several differences in viremia, hematology, or tissue tropism were noted when animals were exposed subcutaneously compared to prior aerosol exposure studies. This study provides a more complete picture of the pathogenesis of the encephalitic alphaviruses and highlights how further defining the neuropathology of these viruses could have important implications for the development of medical countermeasures for the neurovirulent alphaviruses.
•A comparative pathology study of the encephalitic alphaviruses in non-human primates exposed subcutaneously is lacking.•The pathogenesis of the encephalitic alphaviruses in non-human primates is affected by different exposure routes.•EEEV is significantly more pathogenic in non-human primates compared to VEEV similarly to what is observed in humans.•EEEV is just as neuropathogenic by subcutaneous exposure as it was in previously completed aerosol exposure studies.•The neuropathology of the encephalitic alphaviruses has important implications for medical countermeasure development.