Biological Sciences, School of


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A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Biological Sciences, Under the Supervision of Professors Roy French and T. Jack Morris. Lincoln, Nebraska: August, 2011

Copyright 2011 Melissa Sue Bartels


Previous workers in the lab created an infectious clone of Wheat Streak Mosaic Virus (WSMV) designated S1RN. An additional infectious clone with a GUS insert was also created to permit easy observation of virus movement (Gus1RN). A single point mutation made within the HC-Pro region created a mutant of WSMV designated PS81 that was unable to cause a systemic viral infection, although it could infect small clusters of cells on inoculated leaves. A similar mutation was made in the Gus1RN clone and it was designated Gus46. These particular mutants were capable of reverting back to wild type WSMV at high frequency and able to cause a systemic infection. We used this system to assess the multiplicity of infection (MOI) of the virus. MOI was calculated using the PS81 mutant as a control and the Gus46 as the experimental virus used to track virus movement. Wheat plants were inoculated with either PS81 or Gus46 and systemic leaves were collected at 20 and 28 days post inoculation (dpi), respectively. No significant difference was determined in the reversion rate between Gus46 and PS81. The reversion rate of Gus46 was determined and the number of virus genomes with a specific substitution at a particular location per cell was estimated to calculate the MOI of WSMV. The MOI for WSMV was determined experimentally to be approximately 11 genomes. This data supports our hypothesis that only a very limited number of virions escape and move from cell-to-cell during plant virus infections.

The low MOI observed is a severe bottlenecking event for a virus population. This causes a low genetic diversity within the virus population. Narrow genetic bottlenecking during cell-to-cell movement causes a higher selective pressure on viruses and enables viruses to quickly select for adaptive genomes. This ability to select for adaptive genome might offset the negative outcome of bottlenecking, i.e. the loss of fitness by enabling a plant RNA virus to rapidly respond to environmental changes. Further research is necessary to fully understand and appreciate the role MOI has on virus population genetics.

Advisors: Roy French and T. Jack Morris