title: Assessing the feasibility of using neural precursor cells and peripheral blood mononuclear cells for detection of bioactive Sindbis virus.
abstract: Viruses form a significant class of bio-threat agents. Currently, the only method to determine the bioactivity of viruses in vitro is to measure viral and cellular responses after co-incubation of cells with virus. Our goal is to find biomarkers for classification of agents, establishment of bioactivity, and/or prediction of disease outcomes. To begin development of a cell-based biosensor for detection of bioactive Sindbis virus (SV), our model analyte, we surveyed the outcomes of SV interaction with primary rat neural precursor cells (NPC) and human peripheral blood mononuclear cells (PBMC). Confocal fluorescence analysis of NPC treated with recombinant SV carrying green-fluorescent-protein (SV-GFP) showed that most cells were GFP positive by day 1 post inoculation. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of the nucleus showed nuclear condensation and fragmentation, and the percentage of TUNEL positive cells were higher in virus-treated cells than in mock-treated control. Also, there were less BrdU positive cells in virus-treated cells compared to control. Thus, SV infects NPC, decreases cellular proliferation, and induces cell death via apoptosis. PBMC were treated with SV- or UV-inactivated SV. By day 5 post infection, there were fewer adherent cells in SV-treated PBMC compared to UV-inactivated SV treated PBMC. However, the percentage of viable cells remained the same, and virus growth curves showed only clearance of virus. Thus, SV induces detachment of a subpopulation of PBMC while not killing most of the cells. Together, these results indicate that NPC and PBMC respond to bioactive SV inoculation, suggesting potential use as detectors of SV in cell-based biosensor paradigm. These studies also provide the rationale, time-scale, and phenotypic correlates for further studies with gene expression arrays.
authors.affiliation: Center for Bio/Molecular Science and Engineering, Code 6910, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: Dzung C
authors.initials: DC
authors.lastname: Thach
authors.forename: Kara M
authors.initials: KM
authors.lastname: Shaffer
authors.forename: Wu
authors.initials: W
authors.lastname: Ma
authors.forename: David A
authors.initials: DA
authors.lastname: Stenger
mesh_terms: D000818:Animals; D017209:Apoptosis; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002455:Cell Division; D002478:Cells, Cultured; D018920:Coculture Techniques; D005240:Feasibility Studies; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000473:pathology / Q000821:virology*; D009474:Neurons / Q000473:pathology / Q000821:virology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012845:Sindbis Virus / Q000302:isolation & purification* / Q000472:pathogenicity / Q000502:physiology* / Q000648:ultrastructure; D013234:Stem Cells / Q000473:pathology / Q000821:virology*; D014779:Virus Replication