Genetic dynamics of HIV-1 : Recombination, drug resistance and intrahost evolution
Abstract: A striking characteristic of HIV is the enormous capacity of genetic variation. Frequent mutations, deletions, insertions and recombination events create a population of genetically related but non-identical viruses that is under constant change and ready to adapt to environmental changes. The great genetic variability allows the virus to escape the host immune system, develop drug resistance and escape candidate vaccines. Therefore, knowledge about the genetic variation of HIV-1 is important for the design of drugs and vaccines, and for the understanding of the natural history and pathogenesis of the infection. In this thesis, the genetic dynamics of HIV-1 was investigated from different perspectives with special focus on recombination, drug resistance and intra-host evolution. A new pyrosequencing assay was developed that could rapidly screen for the presence of drug resistance mutations in the protease gene of HIV-1. The method was robust and sensitive compared to conventional sequencing. Further development of the assay may provide a new tool for efficient monitoring of the development of drug resistance mutations in HIV-1 patients. The prevalence of drug resistance mutations in HIV-1 from newly diagnosed Swedish patients was investigated by sequencing of the protease and RT genes. Among 100 patient samples, 6 carried mutations that conferred intermediate to strong drug resistance, indicating transmission of drug-resistant virus from antiviral treated patients. In addition, subtype-specific amino acid patterns were found that might be important to consider when patients infected with different subtypes are treated. Several intersubtype recombinant HIV-1 genomes originating from Central and West Africa were characterised by full-length sequencing and recombination analysis. Two genomes were the first representatives of a new circulating recombinant form called CRF13-cpx, and two other genomes belonged to CRF11-cpx. Several unique recombinant genomes were identified as well as two other isolates that may represent an additional new CRF. The problems associated with the characterisation of complex recombinant genomes resulted in the development of a new metric called the branching index . The branching index can aid in the classification of problematic sequence fragments that show only distant relationship to a subtype despite a high bootstrap value. We believe that this new approach may be a useful tool for classification of complicated HIV-1 sequences. The intrahost evolution of HIV-1 was investigated by sequencing the env gene of an HIV-1 population in sequential samples from an asymptomatic drug-naive patient. Phylogenetic analysis of the sequence clones revealed a robust pattern of subpopulations. In addition, it was possible to distinguish a directed evolution from the intrasample diversity at a time interval of only two weeks. Calculations of nucleotide substitution rates indicated an underestimation of the genetic divergence at longer time intervals, suggesting that current nucleotide substitution models need to be improved.
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