The role of specific rotavirus genes involved in conferring phenotypes characterized by either: (i) cold-adaptation (26 degrees C), or (ii) ability to grow at 39 degrees C (non-temperature sensitive) is being investigated. In the initial approach to this project, reassortants were generated by coinfection of AGMK or MA104 cells with cold-adapted (26 degrees C), temperature sensitive human rotavirus Wa x P and wild type human rotavirus DS-1 (non-cold-adapted, non-temperature sensitive). Progeny virus was selected without antibody pressure at 30 degrees C or 39 degrees C. In later experiments, such reassortants were generated and selected at 34 degrees C, a temperature permissive for both parental viruses. Some reassortants generated at 34 degrees C were subjected to antibody pressure against the VP7 serotype of DS-1 during selection. Reassortants were identified by PAGE of genomic RNA and were plaque purified at the respective temperatures used to generate the reassortant. Rotavirus reassortants containing the Wa x P (ca) or DS-1 parental genes for VP6 and VP7 were also confirmed by ELISA. Parental origin of each gene segment was assigned for each reassortant. Rotavirus gene segments from the Wa x P (ca) parental strain which were present in reassortants selected at 39 degrees C include genes 2, 5, 6, 7, 8, 9, and 11. The presence of wild type rotavirus DS-1 genes 3, 4, 5, 6, 7, 8, 9, or 11 did not abrogate the ca phenotype of viruses selected at 30 degrees C. Genetic reassortment at the permissive temperature of 34 degrees C, in combination with antibody pressure yielded (Wa x P) x DS-1 reassortants with various constellations of genes from the parental viruses. Each Wa x P ca gene is represented in at least one reassortant from this group. Reassortants from this group have been triply plaque purified and selected reassortants will be tested for ca and ts phenotypes.
