Inactivated Wild-Type West Nile Virus (L2, B-956 Uganda)

Powassan virus (POWV) is a tick-borne flavivirus (TBFV) that causes severe encephalitis in humans with a case fatality rate of up to 11%. Survivors of severe encephalitis may experience long-term neurological sequelae that are debilitating and lifelong. In this study, researchers sought to characterize a primary human fetal brain neural stem cell system (hNSC) that can be differentiated into neuronal and astrocyte co-cultures to be used as an in vitro transformation system for infection with POWV and a comparative mosquito-borne flavivirus (MBFV), West Nile virus (WNV). Both viruses were able to infect both cell types in the co-culture, and WNV elicited a robust inflammatory response characterized by increased production of the cytokines IL-4, IL-6, IL-8, TNF-α, and IL-1β, as well as activation of apoptotic pathways. POWV infection resulted in a reduced cytokine response, as well as a detectable reduction in apoptosis, and POWV-infected neurons exhibited structural aberrations formed in dendrites. These abnormalities are consistent with previous findings that tick-borne encephalitis virus (TBEV)-infected mouse primary neurons formed lamellar membrane structures (LMS). In addition, these structural aberrations were also recapitulated in brain tissue from infected mice. These findings suggest that POWV is able to infect human primary neurons and astrocytes without inducing overt widespread apoptosis, while forming punctate structures reminiscent with LMS in primary human neurons and in vivo.

Here, researchers determined cell viability and apoptosis in POWV- and WNV-infected hNSC-derived neuron/astrocyte cocultures. Differentiated cocultures were infected with 1.0 or 0.1 MOI of WNV or POWV, and virus-induced apoptosis was measured by cleavage of caspase 3/7. Cell viability did not change significantly. However, apoptosis was significantly increased in control cells infected with MOCK and treated with 10 μM poly I:C. Similar levels of cell viability were detected for both viruses and MOIs (Figure 1a). However, an MOI of 1 of WNV caused an elevated level of caspase cleavage, comparable to Poly I:C, while POWV had similar levels of apoptosis to MOCK-infected cells (Figure 1b). There were no detectable differences between mock-infected cells and controls compared to heat-inactivated West Nile Virus (MNV) controls. Therefore, the changes observed in infected cells were due to viral replication rather than infection. This suggests that, in this coculture system, POWV does not directly induce cell destruction or apoptosis in infected neurons or astrocytes, whereas WNV infection activates apoptosis.

Figure 1. Cell viability and apoptosis in POWV- and WNV-infected hNSC-derived neuron/astrocyte co-cultures. (Nelson J, et al., 2022)