Inhibiting Powassan Virus Persistence and Spread to Neuronal Compartments

Technical Abstract Background: Powassan virus (POWV) is transmitted in tick saliva during a 15-minute tick bite and causes a 10% fatal encephalitis, with severe neurologic damage in 50% of patients. Vaccines, postexposure prophylactics, and therapeutics for POWV are urgently needed to prevent lethal, severely debilitating, POWV neuropathogenesis. Human brain endothelial cells (hBMECs) and pericytes form a neurovascular blood-brain-barrier (BBB) that normally restricts viral entry into neuronal compartments. POWV crosses the BBB, lytically infects neurons and severely damages the brain. We isolated POWV from ticks on Long Island, New York, and found that POWV-LI9 persistently and nonlytically infects primary hBMECs and pericytes. POWV-LI9 uniquely forms foci in liquid culture and is preferentially released basolaterally (12-fold) from hBMECs. This provides a mechanism of POWV transmission across the BBB into the CNS, and suggests that hBMEC-pericyte complexes foster POWV entry into neuronal compartments. BBB integrity is critically determined by crosstalk of secreted factors from hBMECs and pericytes that can be therapeutically targeted to prevent POWV spread to the CNS. We recently reported that hBMECs are persistently infected by neurovirulent Zika virus (ZV). For ZV to persist in hBMECs, ZV inhibits interferon (IFN-beta) and highly induces CCL5 secretion. CCL5 inhibitors clear ZV from hBMECs and are potential ZV therapeutics. Consistent with this, POWV is IFN-sensitive and highly induces CCL5 secretion from infected hBMECs and pericytes. These findings rationalize prophylactic and therapeutic targeting of hBMEC-pericyte complexes to inhibit POWV persistence and spread to the CNS. POWV-LI9 causes lethal neuropathology in C57BL/6 mice; however, we found that passage of POWV-LI9 in VeroE6 cells for 4 months resulted in an attenuated POWV (POWV-LI9-P) that is unable to infect hBMECs and fails to cause lethal neurovirulent disease in mice. Sequencing identified 11 mutations in attenuated POWV that permit us to define residues that block POWV spread to the CNS and to evaluate POWV-LI9-P as a potential vaccine that prevents WT POWV neurovirulence. Hypothesis: We hypothesize that hBMEC and pericyte responses required for POWV persistence are potential preventative and therapeutic targets for inhibiting POWV spread to the CNS and neurovirulence. Objective: (1) Resolve determinants of POWV persistence, spread, and neurovirulence in vitro and in vivo. (2) Define inhibitors of POWV persistence and spread in hBMECs and pericytes. (3) Evaluate prophylactic and therapeutic approaches for preventing POWV spread to the CNS and lethal neurovirulence. (4) Determine if attenuated recombinant POWVs protect mice from WT POWV neurovirulence. Specific Aims Aim 1: Define determinants of POWV spread and attenuation Aim 2: Therapeutically targeting POWV infected hBMECs and pericytes Aim 3: Prophylactic/therapeutic inhibition of POWV spread and neurovirulence in a murine model Study Design: In Aim 1 we define residues that confer an attenuated phenotype to POWV-LI9-P virus. We use reverse genetics to engineer attenuated recombinant POWVs and evaluate changes that define determinants of POWV persistence, pathogenesis, and spread to the CNS. In Aim 2 we evaluate therapeutic approaches to preventing POWV persistence and spread across model hBMEC-pericyte neurovascular complexes. We test the inhibitory potential of CCL5 and IFN inhibitors, antagonists of CCR3 and CCR5 receptors, and targeting pericyte-hBMEC responses for their ability to prevent POWV persistence and basolateral spread. In Aim 3 we assess potential pre- and postexposure therapeutics for preventing POWV infection, spread, and neurovirulence in a lethal murine POWV model. We determine if IFN, CCL5 KO, CCL5, Aim 2 resolved inhibitors and attenuated POWV-LI9-P immunization prevent lethal POWV infection, spread, and neuronal damage. These studies will reveal preventative and hBMEC prophylactic and therapeutic interventions that inhibit POWV spread to the CNS and neurovirulence in mice. Innovation. We evaluate attenuated POWV as a potential vaccine, and novel prophylactic and therapeutic approaches for preventing POWV neurovirulence using neurovascular complex targeting approaches, reverse genetics and our unique understanding of viral persistence and pathogenesis requirements. These studies and approaches applied to primary human BMECs/pericytes and murine animal models of POWV neurovirulence are conceptually and technologically innovative. We assess CRISPR KO, Transwell hBMEC/pericyte complexes, transcytosis assays and inhibitors in BSL3 and ABSL3 POWV infection settings to resolve therapeutics and preventative measures to block POWV spread to neuronal compartments. Impact: Proposed resolve determinants of POWV attenuation and neurovirulence and assess POWV therapeutics that prevent POWV spread across the BBB to neuronal compartments. Our findings are likely to reveal existing postexposure prophylactics and therapeutics that can be applied to inhibit POWV neurovirulence. Less