Summary: Hepatitis E virus (HEV) is an important but extremely understudied human pathogen, causing acute and chronic
hepatitis E, high mortality during pregnancy, neurological sequelae, and foodborne hepatitis. According to WHO, estimated
20 million HEV infections occur each year, leading to >3.3 million cases and >44,000 deaths annually. HEV infection is
associated with numerous extrahepatic manifestations including a range of neurological sequelae, which is associated with
HEV-3 (mainly) and HEV-4 (lesser extent) infection and occur in ~5.5% of HEV-infected patients. HEV-associated
neurological sequelae is an emerging clinical problem, but the underlying mechanism(s) of HEV-associated extrahepatic
pathogenesis is unknown. In preliminary studies, we showed that HEV infects cells in the neurovascular unit (NVU)
including brain microvascular endothelial cells, microglia, and astrocyte, and that HEV causes pathological lesions in CNS
in infected pigs with significantly higher levels of proinflammatory cytokines IL-18 and TNF-¿ in pigs with detectable HEV
RNA in brains than in pigs with no detectable HEV RNA in brains. Our pilot study showed that gerbils are highly susceptible
to infection by HEV-1, HEV-3 and HEV-4. Our long-term goal is to delineate the mechanisms of HEV-associated
extrahepatic pathogenesis. In aim 1, we hypothesize that HEV induces extrahepatic neurological injuries via induction of
pyroptosis by infecting NVU cells to release proinflammatory cytokines leading to neuroinflammation and neurological
injuries. We aim to: (1) determine the effect of HEV-3-induced pyroptosis in NVU cells; (2) identify the mechanisms of
HEV-induced pyroptosis in NVU cells particularly via caspase 1-mediated canonical pathway; (3) identify HEV-specific
pyroptosis-associated proinflammatory cytokines in NVU cells; (4) determine whether HEV-induced pyroptosis in NVU
cells is HEV genotype-specific. We anticipate that HEV induces pyroptosis in microglia, astrocyte, and neuronal cells; that
HEV induces pyroptosis through caspase 1-mediated canonical pathway to produce proinflammatory cytokines to cause
acute cell death; that only HEV-3/HEV-4, but not HEV-1, induce pyroptosis in NVU cells. In aim 2, we hypothesize that
HEV induces neurological sequelae through injuries of neural cells via induction of pyroptosis in brain and spinal cord
tissues. We aim to: (1) infect gerbils with HEV-1, HEV-3 and HEV-4, respectively, to delineate the mechanism of
neuroinflammation and neurological injuries attributable to each HEV genotype; (2) define HEV-specific neurological
lesions in CNS and correlate neurological lesions with hepatic lesions; (3) identify the mechanism(s) of HEV-induced
pyroptosis via the caspase-1–mediated pyroptosis in brain by using a caspase 1 inhibitor (VX-765) in HEV-infected gerbils.
We expect that we will identify HEV-specific neuroinflammation and neurological lesions and types of neural cells involved
in pyroptosis, that HEV-induced neurological lesions in brain occur mainly in HEV-3/HEV-4, but not in HEV-1, infected
gerbils, that HEV induces pyroptosis in brains via the caspase 1-mediated canonical pathway which will involve in activation
of inflammasome to release proinflammatory cytokines IL-1ß, IL-18, and TNF-¿, and that treatment of HEV-infected
gerbils with caspase 1 inhibitor VX-765 will reduce the amounts of neuroinflammation and lesions in CNS tissues compared
to controls. The information will inform potential therapeutic targets of HEV-related neurological sequelae.