A study of structural characteristics and genetic interactions of closely related orbiviruses

Abstract

This thesis is concerned with a study of the closely related orbivirus serotypes, Wallal virus and Mudjinbarry virus. Several aspects of their structure and biology have been examined in order to establish the molecular basis of their serological relationship and to characterize the mechanism by which they recombine during mixed infections.Some structural characteristics of the genome of each virus were examined. By the nature of its base composition, thermal denaturation profile, and pattern of ribonuclease sensitivity, the genome of Wallal virus appeared to consist of double-stranded RNA. In base composition, the genome of Mudjinbarry virus was similar to that of Wallal virus. The genome of each virus was separated into ten segments by polyacrylamide gel electrophoresis. Distinct differences between corresponding genome segments were observed over a wide range of gel concentrations. The relative rates of migration of some segments of each virus were highly dependent on gel concentration, possibly due to unusual structural conformations in the RNA. Optimal conditions for electrophoretic resolution of corresponding segments of Wallal virus and Mudjinbarry virus were established.Methods for preparation of ribonuclease T1 fingerprints of orbivirus double-stranded RNA were devised. Corresponding genome segments of Wallal virus and Mudjinbarry virus generated distinctly different fingerprints. Only for one genome segment (segment 7) was a distinct relationship recognized by direct visual comparison of fingerprints. This segment may code for an antigen with group-specific serological reactivity. A closely related segment was also detected in a third serotype of this group of orbiviruses which was identified in this study.The ribonuclease T1 fingerprinting technique was applied to the analysis of previously described and newly derived recombinant orbiviruses. It was demonstrated unequivocally that genome segment reassortment is the primary mechanism of genetic recombination in orbiviruses. A combination of comparative gel electrophoresis and segment fingerprinting was found to be an effective method for determining the parental origin of each genome segment in recombinant orbiviruses. Loss of temperature-sensitive (ts) phenotype was shown to be consistently associated with reassortment of particular genome segments. Thus ts lesions characteristic of group X and group II Wallal serogroup mutants were determined to be respectively located in genome segment 2 and genome segment 1. No evidence of intragenic recombination was detected in any of the recombinant viruses analysed.Clonal variation in the structure of the Wallal virus genome was investigated. The prototype clone which had been used in this study was shown to be but one of many genetically distinct viruses which could be isolated from uncloned stocks. Different viruses in the pool were characterized by different patterns of segment electrophoretic mobility, differences in ribonuclease Ti fingerprints, and by serological methods. This heterogeneous mixture of viruses appeared to have been generated by segment reassortment between at least two serologically distinct orbiviruses that were propagated in a single host. This may have occurred either prior to or during early laboratory passage of the insect homogenate from which Wallal virus was isolated.The distinctive reactions of Wallal and Mudjinbarry viruses in serum-neutralization tests were confirmed. The hybrid structural characteristics of recombinant orbiviruses were used to identify those genome segments which confer serotype specificity in the serum-neutralization test. Only the origin of genome segment 2 and genome segment 5 influenced the specificity of the reaction. These segments appear to code for type-specific proteins which are probably located on the outer surface of virions.Proteins synthesized in cells infected with Wallal virus and Mudjinbarry virus were examined. For each virus, eight virus-coded polypeptides (PI- P VIII) were detected. In four of these polypeptides, reproducible differences in electrophoretic migration were detected between the two viruses. By comparison with the parental viruses, the origin of each of these proteins was established in a selected set of recombinant viruses of known genetic composition. By correlating the origins of polypeptides and genome segments in these viruses, specific coding relationships were identified. A protein encoded in genome segment 5, which bears type-specific determinants, was designated as polypeptide P V. Respective coding relationships were identified between genome segments 3,6, and 8 and virus-induced polypeptides P II, P VI, and P VII.