Cytomegaloviruses are highly host specific, and murine cytomegalovirus (MCMV) has been widely used as a model for studying human cytomegalovirus (HCMV) infections to overcome the difficulty of experimentation with HCMV in vivo. The ability to manipulate the viral genome and introduce specific mutations into viral genes should facilitate the investigation of mechanisms governing cytomegalovirus host specificity. Our laboratory has utilized a modified MCMV genome lacking a SwaI site to construct recombinant MCMVs through homologous recombination. However, it was not known whether this modification of the MCMV genome by removing the SwaI site would affect the biological properties of MCMV, or whether the SwaI mutation could be reversed should the need arose. To this end, two recombinant bacterial artificial chromosomes, pMCMV_ETwt and pMCMV_ETSwa-*, were constructed and characterized by restriction endonuclease analysis, demonstrating that only the expected genome modifications were present. Recombinant viruses were then reconstituted in tissue culture and their biological properties were compared to the Smith strain of MCMV as well as the parental MCMV_EGFP virus. Viral DNA isolated from infected cells showed the expected restriction patterns for MCMV-ETwt and MCMV-ETSwa-. The expression of MCMV proteins M112-113, ppM44 and gB, representative of those expressed in the early, delayed early, and late phases of infection, respectively, did not differ significantly between the recombinant viruses or the Smith strain of MCMV. In addition, virus growth in permissive Balb/3T3 cells at both low and high multiplicities of infection, and semi-permissive COS-1 cells at a high multiplicity of infection showed growth kinetics or patterns of infection that were similar to the Smith strain of MCMV. Results from these preliminary experiments suggested that the modification of the MCMV genome by removal of the SwaI site did not appear to affect the biological properties of MCMV in vitro. Furthermore, analysis of MCMV_ETwt demonstrated that we could reconstitute the SwaI site in the MCMV genome if necessary, resulting in a virus that should be identical to the Smith strain of MCMV. Therefore, these results suggest that the SwaI- MCMV genome will enable easier construction of recombinant MCMVs with desired alterations in any region of the viral genome.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-07022008-115356 |
| Date | 02 July 2008 |
| Creators | Craggs, Megan Mari |
| Contributors | Warrington, Rob C., Wang, Hong, Tikoo, Suresh K., Roesler, William J., Misra, Vikram, Loh, Lambert, Khandelwal, Ramji L., Xiang, James |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-07022008-115356/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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