We plan to continue our investigations into the genetics and pathogenesis of rotaviruses, with the long-term goal of defining, at a molecular level, the roles of the viral genes that control various aspects of pathogenesis. This information will be useful in formulation of strategies for the control of rotavirus-induced disease. In addition, these studies will provide basic information on the interaction of enteric viruses with the host, and may reveal unique pathogenic pathways characteristic of rotavirus infection. Research on the """"""""genetics of the rotaviruses"""""""" will focus on three main areas. (1) Studies of basic rotavirus genetics will include the following. We will systematically isolate a set of nonsense mutants (""""""""amber"""""""" chain terminating mutants) from the model rotavirus SA11. The amber mutant collection will be placed into genetic groups by reassortment tests and those groups reconciled with the existing groups of ts mutants. We will exploit the amber mutant collection with its good phenotypic properties to examine mutant groups for specific defects in protein synthesis, the synthesis of single-stranded and doubled-stranded RNA, the activity of virion-associated enzymes, and defects in morphogenesis. In vitro complementation experiments will be performed with the amber mutants to elucidate pathways of viral morphogenesis. (2) Studies of rotavirus variability will include the following. We will examine the ability of amber mutations to be suppressed by virus-coded suppressor genes, to determine if amber mutations are subject to the same suppression pathways as ts mutations. We will continue to carry persistently-infected permissive cell lines, to examine the evolution of the viral genome and antigens in this unique infection. (3) We will continue our examination of in vivo pathogenesis of the rotaviruses using a mouse model system. Reassortment of genome segments in immune hosts will be examined to determine if selection occurs via immunity to nonserotype-specific antigens. A new collection of rotavirus reassortants will be constructed using, as parents, the bovine-origin rotaviruses B223 and B641. These reassortants will be used as probes to identify viral determinants of passive immunity and protection, determinants of active immunity and protection, determinants of the ability to replicate in the intestine, and determinants of virulence. Finally, the potential of rotavirus amber mutants as immunogens will be examined.
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