MOLECULAR PERSPECTIVES OF THE EPIDEMIOLOGY, EVOLUTION AND TAXONOMY OF HUMAN PAPILLOMAVIRUSES
In this thesis, we demonstrate that the issues of DNA virus evolution, taxonomy, and identification of papillomaviruses are inextricably linked to the wider issues of papillomavirus epidemiology. In our studies, we have designed experiments and databases for the reliable assessment of HPV genomes from lesions, through the use of Polymerase Chain Reaction (PCR) and nucleotide sequencing. Conservation of 30 amino acids in a selected 450 bp L1 fragment unequivocally identifies fragments derived from HPV genomes. Nucleotide sequence alignments allow calculation of their phylogenetic relatedness. Furthermore, through our development of a quantitative criterium by which the concept of type, variant and subtype may be defined, we have established that sequence divergences of these PCR fragments that exceed 10% from any known HPV type suggest a potential novel HPV status. Applying these techniques and developments to a comprehensive study of some 700 samples from Brazil, of which 180 were derived from American Indian patients in the Amazon, we identified three novel HPV genomes. The hope that these three isolates would represent HPV genomes endemic to America's aboriginal populations proved unfounded when reiated HPV genomes were detected in North America, South-East Asia and in non-Indian Brazilians as part of a continuing worldwide search for novel papillomaviruses. Our studies of variants and subtypes concentrated mainly on HPV-18 and the sequence analysis of its Long Control Region (LCR). Our work demonstrated that diversity within HPV-18 correlates with patterns of the evolution and spread of Homo sapiens (substantiating a postulated African origin), similar to that observed in parallel HPV-16 studies. Furthermore, outgroup rooting of the HPV-18 tree against its closest relative HPV-45, identified African HPV-18 variants as the root of the tree. We estimated that diversity within HPV-18 and probably within other HPV types evolved over a period of more than 200,000 years and that diversity between types evolved over several million yean Comprehensive studies of several other genomic segments of HPV-18 and HPV-45 involving selected African variants and the reference genomes, presented further evidence for the African root of HPV-18 and HPV-45 speciation events. Finally, comparisons of micro-evolutionary events observed within types and within variants suggest that very different pressures affect both type and variant evolution. We derived evidence suggesting that we are unlikely to identify many additional novel papillomavirus types which infect the cervical mucosa. None of the novel papillomaviruses isolated to date were found to be as abundant as HPV-16 or HPV-18 and we speculate that any others that may be identified would also be rare types. Thus we clearly demonstrate the synergism that exist between phylogenetic analysis and epidemiology.
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