Biochemical and genetic studies of wild type and attenuated vaccine strains of bluetongue virus

Typescript (photocopy).A biochemical, and genetic examination of the tetravalent, attenuated vaccine for bluetongue virus (BTV) developed at Texas A&M was undertaken. These studies were aimed at (i) identifying reliable vaccine-associated markers, (ii) determining the genetic stability of the vaccine, and (iii) examining the molecular basis of attenuation in BTV. Comparative growth studies showed that maximal yields of all strains were obtained when cell monolayers were harvested early after the onset of cytopathic effects. One-step growth curves showed an altered growth kinetics for type 13 V. The four V strains were temperature-sensitive (ts) for growth at 39° and for plaque formation at 37°. Recombination tests indicated that the ts lesions of the vaccine strains were on different reassorting genome segments. Electrophoretic comparisons of the genome RNAs, proteins, and protease digests of individual proteins species from homotypic W:V pairs revealed numerous differences. Only changes in segment 2 and its product were seen in all four W:V pairs. Changes in genome segment 2 of 10V, 11V, and 13V were detected by both direct electrophoretic analysis of genomic RNAs and partial protease analyses of the segment 2 gene product. Changes in genome segment 2 of 17V could only be inferred from the heat lability of the segment 2 product. Analyses of mixed infections between heterotypic BTV strains revealed that reassortment of genome segments between V strains and W strains, occurred at high frequency. These analyses also allowed the tentative assignment of the ts lesions of 10V and 17V to genome segments 4 and 5, respectively. This study has yielded significant results relative to the original aims. Several differences in the biological and biochemical properties of the vaccine strains that may serve as reliable vaccine markers were identified. The results of this study have been used to construct the hypothesis that mutations in genome segment 2 of the vaccine strains are responsible for the attenuated phenotype of the vaccine. If the hypothesis that attenuating mutations are located in genome segment 2 of each vaccine strain is correct, the tetravalent attenuated vaccine will be genetically stable. Finally, these results suggest that genome segment 2 of BTV encodes a product that is important in determining the virulence phenotype of the virus