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.

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.

Bacterial cells have a subpopulation—persister cells—that have a low metabolism. Persister cells survive antimicrobial treatment and can regrow to cause chronic and recurrent infections.

Bacterial persister cells − a metabolically dormant subpopulation tolerant to antimicrobials − contribute to chronic infections and are thought to evade host immunity. In this work, we studied the ability of Pseudomonas aeruginosa persister cells to withstand host innate immunity. We found that persister cells resist MAC−mediated killing by the complement system despite being bound by complement protein C3b at levels similar to regular vegetative cells, in part due to reduced bound C5b - and are engulfed at a lower rate (10−100 fold), even following opsonization. Once engulfed, persister cells resist killing and, contrary to regular vegetative cells which induce a M1 favored (CD80+/CD86+/CD206−, high levels of CXCL-8, IL-6, and TNFα) macrophage polarization, they initially induce a M2 favored macrophage polarization (CD80+/CD86+/CD206+, high levels of IL−10, and intermediate levels of CXCL−8, IL−6, and TNF[&alpha]), which is skewed towards M1 favored polarization (high levels of CXCL−8 and IL−6, lower levels of IL−10) by 24 hours of infection, once persister cells awaken. Overall, our findings further establish the ability of persister cells to evade the innate host response and to contribute chronic infections.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that persistently infects patients, enters protected brain, placental, and testicular compartments, is sexually transmitted, and causes fetal microcephaly in utero. ZIKV persistently infects brain microvascular endothelial cells (hBMECs) that form the blood-brain-barrier and Sertoli cells that form testicular barriers, establishing reservoirs that enable viral dissemination. ZIKV persistence requires inhibiting interferon (IFN) responses that direct viral clearance. We found that ZIKV induces IFN-β and IFN-λ in hBMECs but post-transcriptionally inhibits IFN-β/λ expression. IFN-β/λ mRNAs contain AU-rich elements (AREs) in their 3′ untranslated regions which regulate protein expression through interactions with ARE binding proteins (ARE-BPs). We found that ZIKV infection of primary hBMECs induces the expression of the ARE-BP tristetraprolin (TTP) and that TTP is a novel regulator of endothelial IFN secretion. In hBMECs, TTP knockout (KO) increased IFN-β/λ mRNA abundance and IFN-β/λ secretion in response to ZIKV infection and inhibited viral persistence. In contrast, TTP expression dramatically reduced IFN-β/λ secretion in hBMECs. IFN-β/λ mRNA stability was not significantly altered by TTP and is consistent with TTP inhibition of IFN-β/λ translation. TTP is similarly induced by ZIKV infection of Sertoli cells, and like hBMECs, TTP expression or KO inhibited or enhanced IFN-β/λ mRNA levels, respectively. These findings reveal a mechanism for ZIKV induced TTP to promote viral persistence in hBMECs and Sertoli cells by post-transcriptionally regulating IFN-β/λ secretion. Our results demonstrate a novel role for virally induced TTP in regulating IFN secretion in barrier cells that normally restrict viral persistence and spread to protected compartments.