Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes

Abstract

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the Northeastern U.S., with a 2% prevalence in Long Island (LI) deer ticks ( Ixodes scapularis ). POWVs are transmitted in as little as 15 minutes of a tick bite, and enter the CNS to cause encephalitis (10% fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of spread cell to cell, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9, -LI41 and Lineage I POWV-LB, productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-β (IFNβ) and IFN-stimulated genes, with delayed IFNβ secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion a subset of POWV infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. Importance We isolated POWVs from LI deer ticks ( I. scapularis ) directly in VeroE6 cells and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculating VeroE6 cells with POWV containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell to cell spread. POWV-LI9, -LI41, and Lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower chamber pericytes, suggesting a mechanism for POWV transmission across BBB. POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB, and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Mechanisms of Neuroinvasion and Neuropathogenesis by Pathologic Flaviviruses

“…This mechanism is possible without rupturing the BBB because flaviviruses are able to infect some cell types without causing cytopathic effects [ 76 ]. TBEV, ZIKV, Usutu virus, and POWV, for example, were shown in vitro to infect and replicate in human brain microvascular endothelial cells (hBMECs), albeit with different efficiencies and timing [ 76 , 77 , 78 , 79 ]. After Powassan infection, the absence of barrier disruption and the enhanced titer at the basolateral side, correlating with the intracranial side of the BBB, indicated a preference for virus release at that side [ 79 ].…”

“…TBEV, ZIKV, Usutu virus, and POWV, for example, were shown in vitro to infect and replicate in human brain microvascular endothelial cells (hBMECs), albeit with different efficiencies and timing [ 76 , 77 , 78 , 79 ]. After Powassan infection, the absence of barrier disruption and the enhanced titer at the basolateral side, correlating with the intracranial side of the BBB, indicated a preference for virus release at that side [ 79 ]. Although there was no barrier disruption measured during the timepoints taken in this study, infection of hBMECs likely causes a release of inflammatory factors, leading to delayed, indirect barrier disruption via previously discussed mechanisms.…”

Establishment of a CPER Reverse Genetics System for Powassan Virus Defines Attenuating NS1 Glycosylation Sites and an Infectious NS1-GFP11 Reporter Virus

“…The emergence of POWV disease parallels the spread of tick vectors in North America (2,3,6,12,17) , and recent analysis revealed ~2% POWV prevalence in Long Island, NY deer ticks (18) . POWV strain LI9 was isolated from Long Island deer ticks by directly infecting VeroE6 cells, without murine neuroadaptation (5,10,13,19) . In epithelial cells, POWV LI9 nonlytically spreads cell-to-cell forming infected cell foci, and in mice LI9 is neurovirulent and elicits cross-reactive neutralizing POWV antibody responses (19) .…”

“…POWV strain LI9 was isolated from Long Island deer ticks by directly infecting VeroE6 cells, without murine neuroadaptation (5,10,13,19) . In epithelial cells, POWV LI9 nonlytically spreads cell-to-cell forming infected cell foci, and in mice LI9 is neurovirulent and elicits cross-reactive neutralizing POWV antibody responses (19) . How POWV spreads to neuronal compartments remains to be revealed, but the ability of POWVs to hemagglutinate (10) provides a conduit for hematogenous dissemination.…”

“…How POWV spreads to neuronal compartments remains to be revealed, but the ability of POWVs to hemagglutinate (10) provides a conduit for hematogenous dissemination. In vitro, POWVs infect primary human brain microvascular endothelial cells (hBMECs) and, without permeabilizing polarized monolayers, POWVs are basolaterally released from hBMECs (19) . Basolateral release from hBMECs provides a potential mechanism for POWVs to spread across the blood-brain-barrier (BBB) into protected neuronal compartments.…”