Abstract
A reliable working methodology for the reverse transcription (RT) and amplification of flaviviral RNA was established. This incorporated a low level methyl mercury hydroxide viral RNA denaturation procedure. The effect of chemical inactivation of flaviviruses, prior to RT and amplification, using beta-propriolactone was also investigated. The following regions of the structural protein genes of viruses of the tick-borne encephalitis (TBE) complex of the Flaviviridae were reverse transcribed, anplified, cloned and sequenced: the envelope (E) protein gene, and part of the membrane protein gene of the Central European subtype, strain Kumlinge A52 (CEE Kumlinge) virus; the majority of the E protein gene of Turkish TBE (TTE) virus; and part of the structural protein coding regions of Langat virus strains TP21 and TP64 (LGT TP21 and TP64). The E protein gene of CEE Kumlinge virus was found to differ from another CEE strain, CEE Neudoerfl virus, by a single amino acid, although the viruses were isolated 12 years apart, in different countries. The possibility of the existence of strong selection pressures against antigenic variation is discussed. Furthermore, the amino acid substitutions in the attenuated LGT TP21 and TP64 viral sequences were related to earlier studies of the molecular basis of attenuation. Finally a study of TTE viral sequence suggested that a closer homology existed between TTE viorus and other CEE virus subtypes, than between TTE virus and another TBE complex virus, Louping I11, which like TTE virus, causes encephalomyelitis in sheep. The significance of these findings was discussed. Factors involved in the design of efficient primers for RT and amplification were studied. The suitability of three primers, YF7, TICK1, and RS1 as probes for the identification of TBE complex viruses was examined. Results obtained in this thesis were discussed in the context of possible future molecular biology studies in this field.