RNA viruses

The reovirus nonstructural proteins muNS and sigmaNS are thought to play a role in assortment. To produce large quantities of the reovirus nonstructural proteins, two recombinant vaccinia virus systems were used, one for expression of muNS and the other for expression of muNS. The reovirus gene is under the control of the phage T7 RNA polymerase promoter. A recombinant vaccinia virus containing the gene for T7 RNA polymerase was added and protein expression was determined. Protein expression was confirmed by infecting mouse fibroblast L929 cells, harvesting the cells and running an SDS-PAGE gel, followed by western blot, followed by Western-immunoperoxidase assay using a polyclonal antibody for the reovirus proteins, or by radioimmunoprecipitation followed by an SDS-PAGE gel. Purification of the recombinant proteins was accomplished by ammonium sulfate fractionation and column chromatography. The purified proteins will be utilized to further investigate the role of the reovirus nonstructural proteins in assortment.

In this study, we report the identification and characterization of a new Reovirus strain isolated from the spinal fluid of a human patient. Unlike other reovirus serotypes that can be isolated from fecal matter, this strain was isolated from the spinal fluid of a patient; which suggests it may be a more pathogenic strain. The new isolate was compared to the three known strains of mammalian reoviruses. We have found that the virus is not a reassortment of the other three serotypes. This was confirmed by examination of the migration rates of the genomic dsRNA segments of all four viruses. The new isolate demonstrated measurable differences in viral protein molecular weights, when compared to the proteins of the three known serotypes. The molecular weight of the sigma I protein of the new isolate, was found to be more then 3Kd smaller then sigma 1 obtained from the other three serotypes. The largest difference in protein molecular weights was found with mu1C, which was about 7Kd smaller. The growth rate of the new virus was very similar to that of the other three serotypes. Some differences were found in select cell lines.