COLD ADAPTATION OF HUMAN ROTAVIRUSES

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Three human rotavirus cell-culture isolates (strains D [VP7:1, VP4:1A], DS-1 [VP7:2, VP4:1B], and 202 [VP7:4, VP4:1B]) as well as two human x human rotavirus reassortants (Wa x P [VP7:3, VP4:1A] and Wa x DS-1 [VP7:2, VP4:1A]) have been successfully adapted to grow in primary African Green monkey kidney cells at the suboptimal temperature of 30 degrees C, 28 degrees C, or 26 degrees C. These rotavirus strains were cold-adapted in order to determine whether cold-adapted (ca) mutants selected by this strategy are attenuated and suitable for use in a live virus vaccine. The D and the Wa x P strains were passaged 10 times either at 28 degrees C or 26 degrees C and are being plaque-purified. Analysis of the efficiency of plaque formation at various temperatures established that the 26 degrees C cold-adapted D strain is indeed a ca mutant; it produced plaques at 26 degrees C, whereas its parent did not. In addition, this mutant is also temperature sensitive having a shut-off temperature for plaque formation of 37 degrees C. The DS-1 strain (passaged 12 times at 28 degrees C) and Wa x DS-1 reassortant (passaged 10 times at 28 degrees C) are also being plaque-purified. Selected human rotaviruses including the D, Wa x P, and Wa x DS-1 have successfully been adapted to growth in DBS-FRhL-2 cells, which is a cell substrate suitable for preparation of vaccines for use in humans.