title: Response of Mammalian Macrophages to Challenge with the Chlorovirus Acanthocystis turfacea Chlorella Virus 1.
abstract: It was recently reported that 44% of the oropharyngeal samples from the healthy humans in a study cohort had DNA sequences similar to that of the chlorovirus ATCV-1 (Acanthocystis turfacea chlorella virus 1, family Phycodnaviridae) and that these study subjects had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened interleukin-6 (IL-6), nitric oxide (NO), and ERK mitogen-activated protein (MAP) kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral PFU counts in murine RAW264.7 cells and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after a virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h, and the percentage increased 2- to 3-fold over 72 h. Immunoblot assays with rabbit anti-ATCV-1 antibody detected a 55-kDa protein consistent with the viral capsid protein from 1 to 72 h and increasing de novo synthesis of a previously unidentified 17-kDa protein beginning at 24 h. Emergence of the 17-kDa protein did not occur and persistence of the 55-kDa protein declined over time when cells were exposed to heat-inactivated ATCV-1. Moreover, starting at 24 h, RAW264.7 cells exhibited cytopathic effects, annexin V staining, and cleaved caspase 3. Activation of ERK MAP kinases occurred in these cells by 30 min postchallenge, which preceded the expression of IL-6 and NO. Therefore, ATCV-1 persistence in and induction of inflammatory factors by these macrophages may contribute to declines in the cognitive abilities of mice and humans.
  
  
  OBJECTIVE
  Virus infections that persist in and stimulate inflammatory factors in macrophages contribute to pathologies in humans. A previous study showed that DNA sequences homologous to the chlorovirus ATCV-1 were found in a significant fraction of oropharyngeal samples from a healthy human cohort. We show here that ATCV-1, whose only known host is a eukaryotic green alga (Chlorella heliozoae) that is an endosymbiont of the heliozoon Acanthocystis turfacea, can unexpectedly persist within murine macrophages and trigger inflammatory responses including factors that contribute to immunopathologies. The inflammatory factors that are produced in response to ATCV-1 include IL-6 and NO, whose induction is preceded by the activation of ERK MAP kinases. Other responses of ATCV-1-challenged macrophages include an apoptotic cytopathic effect, an innate antiviral response, and a metabolic shift toward aerobic glycolysis. Therefore, mammalian encounters with chloroviruses may contribute to chronic inflammatory responses from macrophages.
authors.affiliation: Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA Department of Oral Biology, University of Nebraska Medical Center, Lincoln, Nebraska, USA tpetro@unmc.edu ddunigan2@unl.edu.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Petro
authors.affiliation: Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: Irina V
authors.initials: IV
authors.lastname: Agarkova
authors.affiliation: Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: You
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Stanley Division of Developmental Neurovirology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Yolken
authors.affiliation: Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Van Etten
authors.affiliation: Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA tpetro@unmc.edu ddunigan2@unl.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Dunigan
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology; D015153:Blotting, Western; D036022:Capsid Proteins / Q000096:biosynthesis; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D003072:Cognition Disorders / Q000276:immunology / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D015151:Immunoblotting; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000821:virology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism; D017862:Phycodnaviridae / Q000276:immunology*; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D017304:Annexin A5; D000914:Antibodies, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D015850:Interleukin-6; D009569:Nitric Oxide; D053148:Caspase 3