This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Study of viral spread in 3D cultures. The most relevant system for modeling viral spread would be in solid cell masses. As shown in the preliminary data, we have been able to image individual infected cells in solid masses by reconstituting infected and uninfected cells into spheres on Matrigel. We will first carry out analysis on AdEGFPuci-infected 293 cells. We will first infect a monolayer of 293 cells with AdEGFPuci, and 6 hr after infection, the infected cells will be trypsinized. The infected single cell suspensions will be mixed with an excess of uninfected 293 cells, and plated onto Matrigel matrices to allow formation of cell aggregates and spheres. The spheres will be monitored daily for appearance of green fluorescent cells and fluorescent Z-stack montages will be recorded and compiled for any sphere that initially show one infected cell. A confocal microscope suitable for these experiments is available in the campus optical biology shared resource. It seems possible that the rate of virus spread may be more rapid in 3D structures than in monolayer, or that the number of secondarily infected cells may be larger. It will also be possible to determine the effects of initial MOI on spread through 3D cultures.
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