Abstract
A reliable working methodology for the reverse transcription (RT) and polymerase chain reaction (PCR) amplification of rubella virus (RV) RNA was established. The effect of magnesium concentration and RNA concentration on the yield and specificity of PCR products was investigated. Factors involved in the design of efficient primers for PCR were also studied. RT primers designed to specifically anneal to the RV genome were shown to increase the yield of PCR product when compared to RT-PCRs in which the RT reaction was primed by random hexamers. Using the RT-PCR technology, nonstructural (NS) protein coding regions of the wild-type strain Thomas and the vaccine strain Cendehill were amplified, cloned and sequenced. In addition a region encompassing part of the 5' NC region and the start NS protein ORF, covering nucleotides 18 to 540, for the wild-type strains Thomas, RB-1 and Machado and vaccine strains Cendehill, RA27/3, HPV77.DE5 and TO-336 was amplified, cloned and sequenced. When the Cendehill and Thomas sequences were compared with the equivalent sequences in the Therien and M33 wild-type strains, three amino acids were found which were unique to the Cendehill vaccine strain. The sequences of part of the 5' NC and 5' end of the NS coding regions of the above strains were compared to the equivalent sequences in the Therien and M33 strains. One amino acid substitution was found which was unique to RA27/3 and a second was identified which was present in both the RA27/3 and TO-336 vaccine strains. Nucleotide substitutions found in an area of the 5' NC region which, it has been suggested, plays a key role in the initiation of translation and positive strand replication were also identified. The importance of all of these substitutions is discussed with particular reference to their possible roles in attenuation. The suitability of the NS RV RT-PCR system developed in the early stages of these studies was examined with regard to its use in the amplification and detection of RV in clinical samples. The results obtained were in total agreement with those obtained using an RT-PCR system which detects the El RV gene and also correlated well with other laboratory results. Possible future applications of the NS RV RT-PCR system were discussed. Results obtained in this thesis were discussed in the context of possible future molecular biology studies in this field.