| Viruses are obligatory parasites, they need access to susceptible and permissive host cells in order to replicate and propagate. Induction of cellular antiviral responses during infection can, in some cases, inhibit viral replication and terminate infection spread. Improved understanding of how antiviral responses are induced in cells can facilitate the development of antiviral therapies. To facilitate advances and gain insights into how cellular responses can mitigate viral propagation, we are developing in vitro experimental methodologies. In this thesis, we demonstrate that focal infections can be used to visualize and quantify the dynamics of infection spread in cell populations. Thus far, we have investigated the spread of two viruses: a RNA virus, vesicular stomatitis virus (VSV), and a DNA virus, human cytomegalovirus (HCMV). Results from VSV studies suggested that while the rate of infection spread generally correlated with the rate of virus replication, induction of cellular antiviral activities, specifically interferon signaling, can in some cases be the overriding factor. A mutant strain of VSV that lacked the ability to shut-off host interferon production failed to spread beyond the site of inoculation even though its one-step growth was comparable to wildtype. We also observed antagonistic interactions between different cellular antiviral pathways. Simultaneous activation of interferon signaling and nitric oxide production resulted in larger infected areas than induction of interferon signaling alone. In HCMV experiments, cell density and incubation with serum, as oppose to interferon signaling, were the dominant factors in dictating infection spread. HCMV spread rate was dramatically increased in monolayers plated at high cell densities and those incubated with serum. Incubation with serum also altered the pattern of HCMV gene expression in focal infections. In control monolayers, a clear wave of viral immediate early 2 (IE2) protein expression was observed in advance of late-stage viral glycoprotein B. By contrast, irregular foci of IE2 expression were randomly distributed in monolayers incubated with serum. In summary, we have visualized and quantified the infection spread of two different virus systems: VSV and HCMV. Further, the results demonstrate that focal infections can be used as a model system to investigate complex virus-host interactions in vitro. |
