ABSTRACT Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state of the art telemetry to measure critical physiological parameters. Following challenge, all parameters were rapidly and profoundly altered, and all nonhuman primates (NHPs) met the euthanasia criteria. In this study, we performed the first comprehensive pathology investigation of tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61-68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation of EEEV clinical disease course and pathogenesis in a NHP model and will provide significant insights into the evaluation of countermeasure. Author Summary EEEV is an arbovirus endemic in parts of North America and is able to produce fatal encephalitis in humans and domesticated animals. Despite multiple human outbreaks during the last 80 years, there are still no therapeutic or vaccines to treat or prevent human disease. One critical obstacle in the development of effective countermeasure is the lack of insights into EEEV pathogenesis in a susceptible animal host. We recently conducted a study in cynomolgus macaques to investigate the disease course by measuring clinical parameters relevant to humans. Following infection, these parameters were rapidly and profoundly altered leading to severe disease. In this study, we examined the potential mechanisms that underlie pathogenesis to cause severe disease. The virus was present in many parts of the brain and spinal cord, however, little or no pathological lesions as well as active virus replication were observed. Additionally, neurons were the predominant target of EEEV and virus transport was facilitated by axonal transport system to spread neuron-to-neuron throughout the brain and spinal cord. These data show that EEEV likely hijacks host cell transport system to rapidly spread in the brain and local/global neuronal dysfunction rather than death is the principal cause of severe disease.

Eastern Equine Encephalitis Virus Rapidly Infects and Disseminates in the Brain and Spinal Cord of Infected Cynomolgus Macaques Following Aerosol Challenge