This thesis describes three phases of our studies on the biology of human coronavirus, 229E: the development of a plaque assay system, studies on the effect of Actinomycin D on the replication of this virus, and the isolation and preliminary characterization of an internal component of the virion. A medium was developed which permitted the production of plaques by 229E on monolayers of L 132 cells in closed containers which could be incubated in regular incubators, a result which we were not able to duplicate using published formulations. This assay system exhibited a linear dose-response curve over a wide range of virus concentrations and was applicable to the assay of this virus in several cell lines; in addition, it could be used for the assay of other viruses. Several other features of the assay are also discussed briefly. The replication of 229E in L 132 cells was found to be inhibited in the presence of Actinomycin D, a potent inhibitor of DNA-dependent RNA synthesis. While the drug has adverse effects on the host cell, several arguments have been brought forth which argue in favor of a specific effect on virus replication; the replication of vesicular stomatitis virus was unimpeded at 10 times the 50% inhibitory level of AMD for 229E, and largely unaffected at 100 times the mean inhibitory dose; in addition the cells were not themselves greatly affected and the time course of drug sensitivity appeared to indicate a temporally discrete period of AMD susceptibility. The sensitivity of AMD was shown to be dependent on the virus input used to initiate the replicative cycle. Using disruption by the detergent Nonidet P-40, we were able to isolate, in sucrose gradients, a subviral particle. This particle, a nucleoprotein, has a density of 1.27 and appears linear in the electron microscope. It is suggested that this structure probably represents the viral nucleocapsid. Some further aspects of its relation to virus morphology are discussed.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/10659 |
| Date | January 1977 |
| Creators | Kennedy, Douglas A. |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 153 p. |
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