Cloning viral dsRNA genomes : analysis and application

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.

Double-stranded RNA viruses occur in a large number of hosts in nature ranging from bacteria to mammals. Molecular studies of the double-stranded RNA viruses have greatly enhanced man's understanding of this large group of viruses as far as structure and function of their genes and epidemiology is concerned. However, one of the major prerequisites of obtaining this information is the ability to clone the genomes of these viruses for nucleotide sequencing and recombinant protein expression studies. In the dsRNA field, cloning viral genomes has historically been difficult and time consuming and created a bottleneck that hampered molecular studies. The main aim of this investigation was to optimise a method for cloning viral dsRNA genomes to the extent that it would be easy and fast as well as applicable to most dsRNA viruses. In this study a sequence-independent, oligo-ligation mediated dsRNA cloning procedure for large genes (up to 6.8 kb) was perfected and tailored for routine use to amplify and clone complete genome sets or individual genes. Complete genome sets could be amplified and cloned from as little as 1 ng dsRNA. The method was shown to be simple and efficient compared to other methods and is currently the only method that allows the amplification of complete genomes in a single PCR reaction. Complete gene sets of seven genomes from the Reovirus family, one from the Cystovirus family and one mycovirus, have been amplified and cloned. The full-length VP2 genes of all 9 AHSV and 24 BTV serotypes were also cloned. Phylogenetic analysis of VP2-genes revealed the same grouping of AHSVs and BTVs as serology. Several cloned genes of AHSV, rotavirus and EEV have been utilised for recombinant protein production establishing that the cloned cDNAs have full open reading frames. The nine AHSV VP2 genes have been developed as serotype-specific probes which allowed serotyping of AHSV isolates within 4 days compared to 2-4 weeks needed with the traditional serological serotyping. The new cloning procedure finally opens the bottleneck that hamstrung the development of complete repertoires of recombinant vaccines, molecular diagnostics and epidemiology to combat dsRNA viral diseases. It should now be possible to deliver on many of the expectations that were envisaged for dsRNA virus research and biotechnology since the advent of recombinant DNA technology.

Doctoral