QUANTIFYING VIRAL PROPAGATION IN VITRO: TOWARDS A METHOD FOR CHARACTERIZATION OF COMPLETE PHENOTYPES.

Authors: DUCA, K. - TUFTS UNIVERSITY; LAM, V. - UNIVERSITY OF WISCONSIN; KEREN, IRIS - MEDICAL COLLEGE OF OHIO; ENDLER, ELIZABETH - UNIVERSITY OF WISCONSIN; Letchworth, Geoffrey; NOVELLA, ISABEL - MEDICAL COLLEGE OF OHIO; YIN, JOHN - UNIVERSITY OF WISCONSIN

Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/2001
Publication Date: 11/20/2001
Citation: Duca, K.A., Lam, V., Keren, I., Endler, E.E., Letchworth III, G.J., Novella, I.S., Yin, J. 2001. Quantifying viral propagation in vitro: towards a method for characterization of complete phenotypes. Biotechnology Progress, 17:1156-1165.

Interpretive Summary: Viral infections are a complex interaction between the virus and the cells in which the virus grows. The purpose of this study was to establish a reliable method to observe the progression of viral infection and cell death over time. This study both developed a useful method and used the method to show that the profile of viral spread and cell death were dramatically different in two cell lines.

Technical Abstract: For a eukaryotic virus to successfully infect and propagate in cultured cells several events must occur: the virion must identify and bind to its cellular receptor, become internalized, uncoat, synthesize viral proteins, replicate its genome, assemble progeny virions, and exit the host cell. While these events are taking place, intrinsic host defenses activate in order to defeat the virus, e.g., activation of the interferon system, induction of apoptosis, and attempted elicitation of immune responses via chemokine and cytokine production. As a first step in developing an imaging methodology to facilitate direct observation of such complex host/virus dynamics, we have designed an immunofluorescence-based system that extends the traditional plaque assay, permitting simultaneous quantification of the rate of viral spread, as indicated by the presence of a labeled viral protein, and cell death in vitro, as indicated by cell loss. We propose that our propagation and cell death profiles serve as phenotypic read-outs, complementing genetic analysis of viral strains. As our virus/host system we used vesicular stomatitis virus (VSV) propagating in hamster kidney epithelial (BHK-21) and murine astrocytoma (DBT) cell lines. Viral propagation and death profiles were strikingly different in these two cell lines, displaying both very different initial titer and cell age effects. The rate of viral spread and cell death tracked reliably in both cell lines. In BHK-21 cells, the rate of viral propagation, as well as maximal spread, was relatively insensitive to initial titer and was roughly linear over several days. In contrast, viral plaque expansion in DBT cells was contained early in the infections with high titers, while low titer infections spread in a manner similar to the BHK-21 cells. The effect of cell age on infection spread was negligible in BHK-21 cells but not in DBTs. Neither of these effects was clearly observed by plaque assay.