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The generation of defective interfering rubella virus particles

University of British Columbia

Rubella virus (RV) has been propogated in murine (L-2) fibroblasts and viral titers determined using a modified hemadsorption assay. The titration results suggested that rubella virus populations may be comprised of standard and defective virions. RV stocks were examined for their ability to interfere with standard (low interference) rubella virus. High interference RV stocks could reduce standard RV infectivity by greater than 99%. A new purification procedure has been developed in order to examine the virions produced by high and low interference RV stocks. Rubella viral particles have been purified using a discontinuous renografin gradient followed by a continuous renografin gradient. These gradients allowed separation of intact purified virions from contaminating host membranous material. Greater than 90% of the total original infectivity was recovered as determined by the hemadsorption assay. Electron microscopic examination demonstrated the presence of intact rubella virions. Virions from high and low interference RV stocks have been purified, the RNA extracted from the purified virions, labeled with ¹²⁵I, electrophoresed on 5% polyacrylamide gels and subjected to autoradiography. Virions from low interference RV stocks were contained in one band at a density ρ = 1.19 gm/cc³ . The two single-stranded RNA molecules that could be extracted from these virions have molecular weights of 2.95 and 2.80 x 10⁶ daltons. Virions purified from high interference RV stocks were contained in at least three bands at densities ρ = 1.19, 1.17 and 1.15 . 3 gm/cc³. RNA molecules with molecular weights of 2.95 and 2.80 x 10⁶ daltons could be extracted from the virions banding at a density ρ = 1.19 gm/cc³. Virions banding at the lighter densities ρ = 1.17 and 1.15 gm/cc contained RNA molecules of 1.25 and 1.05 x 10⁶ daltons in molecular weight respectively. A fresh isolate of RV has been serially passaged in L-2 cells in order to observe the sequential generation of defective viral particles. The virions from each passage have been purified, the RNA isolated, labeled and analyzed by PAGE. The molecular weights of the RNA molecules isolated indicated the possible cyclic generation of defective viral particles.

Text

2010-03-29

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http://hdl.handle.net/2429/22855

Microbiology and Immunology

University of British Columbia

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