{"doi":"10.1101/2021.09.30.462684","title":"Powassan Viruses Spread Cell to Cell During Direct Isolation from <i>Ixodes</i> Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes","abstract":"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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":226972,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9487,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":675540,"name":"Santiago Sánchez-Vicente","orcid":"0000-0002-4193-4251","position":1,"is_corresponding":false},{"id":830792,"name":"Nicholas Saladino","orcid":null,"position":2,"is_corresponding":false},{"id":556884,"name":"Elena E. Gorbunova","orcid":null,"position":3,"is_corresponding":false},{"id":556883,"name":"William R. Schutt","orcid":null,"position":4,"is_corresponding":false},{"id":698330,"name":"Megan C. Mladinich","orcid":"0000-0002-6041-4550","position":5,"is_corresponding":false},{"id":830356,"name":"Grace E. Himmler","orcid":"0000-0002-3100-6371","position":6,"is_corresponding":false},{"id":830357,"name":"Jorge L. Benach","orcid":"0000-0001-9375-6058","position":7,"is_corresponding":false},{"id":824705,"name":"Hwan Keun Kim","orcid":"0000-0003-2095-9705","position":8,"is_corresponding":false},{"id":556391,"name":"Erich R. Mackow","orcid":"0000-0001-9156-1222","position":9,"is_corresponding":false},{"id":556390,"name":"Jonas N. Conde","orcid":"0000-0001-5895-1114","position":0,"is_corresponding":true}],"reference_count":88,"raw_metadata":null,"created_at":"2026-07-18T23:54:38.004707Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}