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Adventitious viruses persistently infect three commonly used mosquito cell lines.

ABSTRACT: Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.

SUBMITTER: Weger-Lucarelli J

PROVIDER: S-EPMC6764424 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Adventitious viruses persistently infect three commonly used mosquito cell lines.

Weger-Lucarelli James J Rückert Claudia C Grubaugh Nathan D ND Misencik Michael J MJ Armstrong Philip M PM Stenglein Mark D MD Ebel Gregory D GD Brackney Doug E DE

Virology 20180626

Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous ...[more]

PMID: 29957338

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Project description:Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are 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 cell to cell spread, 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 and -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 (IFN-β) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-β 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, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -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 the blood-brain barrier (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.

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Adventitious viruses persistently infect three commonly used mosquito cell lines.

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Adventitious viruses persistently infect three commonly used mosquito cell lines.

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