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 during a 15 minutes tick bite, and enter the CNS to cause encephalitis (10% fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodesticks 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-b(IFNb) and IFN-stimulated genes, with delayed IFNbsecretion 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.

Age-dependent Powassan Virus Lethality is Directed by Glial Cell Activation and Divergent Neuroinflammatory Cytokine Responses in a Murine Model

“…In these studies, lethality varied by strain, dose, kinetics, and suggested requirements for tick saliva in directing neurovirulence (29,33) . Our analysis was performed using mice 10-50 weeks of age and POWV LI9, a strain from circulating Ixodes ticks isolated in VeroE6 cells (21,42) . Footpad inoculation of POWV LI9 into 10-50 week old C57BL/6 mice resulted in 82% lethality in 50 week old mice, with lethality sequentially reduced with age to 7.1% in 10 week old mice (Figure 1B,E).…”

“…Like SP, this may reflect LI9 persistence (27) , or result from persistent auto-amplifying inflammatory CNS responses. Whether the use of very young mice or POWV SP (27) , serially passaged in murine brains (25) , account for differences in murine persistence may be revealed using POWV reverse genetics (42) .…”

“…VeroE6 cells (ATCC CRL 1586) were grown as previously described in Dulbecco's modified Eagle's medium at 37°C in 5% CO 2 . POWV strain LI9 (GenBank accession number: MZ576219) was isolated from infected Ixodes scapularis ticks via inoculation on Vero E6 cells, and stocks passaged 3-4 in VeroE6 cells as previously described (21,42,115) . POWV LI9 was adsorbed onto 60% confluent VeroE6 monolayers for 1 h. Monolayers were PBS washed and grown in DMEM 5% FBS and POWV titers were determined by serial dilution and quantifying infected VeroE6 cell foci 1 dpi by immunoperoxidase staining with anti-POWV hyperimmune mouse ascites fluid (HMAF; 1:5,000…”

“…In a 2020 survey, 2% of I. scapularis ticks in Long Island, NY were POWV positive, and POWV strain LI9 was isolated in Vero cells directly from Ixodes ticks, without murine neuroadaptation (21,41) . POWV LI9 nonlytically infects VeroE6 cells, spreading cell-to-cell in the presence of neutralizing antibodies (21,42) , and in vitro, infects primary human brain microvascular endothelial cells (hBMECs) that form the blood-brain-barrier (21) . Basolateral spread from polarized hBMECs suggests a mechanism for POWVs to enter neuronal compartments (21) , although in vivo CNS entry mechanisms for POWV have not been assessed.…”

“…Basolateral spread from polarized hBMECs suggests a mechanism for POWVs to enter neuronal compartments (21) , although in vivo CNS entry mechanisms for POWV have not been assessed. C57BL/6 mice subcutaneously inoculated with POWV LI9 seroconvert and produce crossreactive POWV neutralizing antibodies (21,42) .…”