Long terminal repeats (LTRs) are non-coding repeats flanking the protein-coding genes of LTR retrotransposons. The variability of LTRs poses a challenge in studying them. Hidden Markov models (HMMs), probabilistic models widely used in pattern recognition, are useful in dealing with this variability. The aim of this work was mainly to study LTRs of retroviruses and LTR retrotransposons using HMMs. Paper I describes the methodology of HMM modelling applied to different groups of LTRs from exogenous retroviruses (XRVs) and endogenous retroviruses (ERVs). The detection capabilities of HMMs were assessed and were found to be high for homogeneous groups of LTRs. The alignments generated by the HMMs displayed conserved motifs some of which could be related to known functions of XRVs. The common features of the different groups of retroviral LTRs were investigated by combining them into a single alignment. They were the short inverted terminal repeats TG and CA and three AT-rich stretches which provide retroviruses with TATA boxes and AATAAA polyadenylation signals. In Paper II, phylogenetic trees of three groups of retroviral LTRs were constructed by using HMM-based alignments. The LTR trees were consistent with trees based on other retroviral genes suggesting co-evolution between LTRs and these genes. In Paper III, the methods in Paper I and II were extended to LTRs from other retrotransposon groups, covering much of the diversity of all known LTRs. For the first time an LTR phylogeny could be achieved. There were no major disagreement between the LTR tree and trees based on three different domains of the Pol gene. The conserved LTR structure of paper I was found to apply to all LTRs. Putative Integrase recognition motifs extended up to 12 bp beyond the short inverted repeats TG/CA. Paper IV is a review article describing the use of sequence similarity and structural markers for the taxonomy of ERVs. ERVs were originally classified into three classes according to the length of the target site duplication. While this classification is useful it does not include all ERVs. A naming convention based on previous ERV and XRV nomenclature but taking into account newer information is advocated in order to provide a practical yet coherent scheme in dealing with new unclassified ERV sequences. Paper V gives an overview of bioinformatics tools for studies of ERVs and of retroviral evolution before and after endogenization. It gives some examples of recent integrations in vertebrate genomes and discusses pathogenicity of human ERVs including their possible relation to cancers. In conclusion, HMMs were able to successfully detect and align LTRs. Progress was made in understanding their conserved structure and phylogeny. The methods developed in this thesis could be applied to different kinds of non-coding DNA sequence element.

The use of the MinION sequencer (Oxford Nanopore) was tested on samples prepared to simulate infectious samples. The tested procedure is to simulate work with a sample with an unknown pathogen. Therefore, a metagenomic approach was chosen. Three kits were tested: Rapid Barcoding Sequencing, PCR Barcoding and Premium whole genome amplification. Each kit differed in duration, difficulty to prepare and in amplification of nucleic acids. In total it was chosen eight viruses with different genome lengths and with varying types of the genome (5,6 - 152 kb, ss/ds RNA, dsDNA). Ten samples were prepared to simulate different types of infection (respiratory, gastrointestinal tract and urine), and one sample contained pure water as a negative control. Before preparation of the library with Oxford Nanopore's kits, DNase/RNase treatment was used. The viral RNA was transcribed into DNA and in chosen samples were amplificated to reach a higher concentration of nucleic acids. Rapid barcoding sequencing kit detected all selected viruses with the highest number of viral reads (4403) with a length between 100 and 250 nt and quality coverage of viral genomes. PCR Barcoding kit detected five out of eight viruses, and the number of identified reads with a length of 100-200 nt distinctly decreased. Premium whole genome...

The central problem for researchers of HIV-1 evolution is explaining the apparent design of the virus for causing pandemic infection in humans: understanding how HIV-1 spreads is key to halting the pandemic. Current knowledge of how HIV-1 spreads from host to host is based upon experimental observation and indirect inferences informed by theory. The hypothesis of this thesis is that diversity of HIV-1 around the time of transmission is important for viral adaptation to a new human host, rather than intrinsic superiority of particular strains found in infectious fluids from human donor hosts, and that studying recombination is important for understanding this behaviour. To demonstrate the apparent randomness of transmission, I test the null-hypothesis that hard selection accounts for between-host viral divergence in a rare case study of contemporaneous infection. I explain how the experimental data that I have generated and the analyses I have carried out address certain basic assumptions and predictions about HIV-1 transmission and may inform current strategies for vaccine design. Specifically, my approach contributes to the current literature on HIV-1, by investigating an alternative hypothesis to the single virion theory of sexual transmission and by characterizing the role of recombination in a pseudodiploid virus following multiple-infection.