Fast-evolving RNA viruses are a leading cause of morbidity and mortality among human and animal populations, contributing significantly to both global health and economic burden. The advent and revolution of high-throughput sequencing has empowered phylogenetic analyses with increasing amounts of temporally and spatially sampled viral data. Moreover, the parallel advancement in molecular evolution and phylogenetic methods has provided investigators with a unique opportunity to gain detailed insight into the evolutionary and epidemiological dynamics of emerging viral pathogens. Using state-of-the-art statistical approaches, this thesis addresses some of the important but controversial questions in viral emergence. Chapter 2 introduces a new framework to quantify and investigate reassortment events in influenza A viruses. By developing a computationally efficient algorithm to calculate the largest common subtree for a pair of tree sets, which are estimated from diffe rent parts of the genome for the same taxa set, the level of phylogenetic incongruency due to reassortment can be appropriately ascertained. Chapters 3, 4 and 5 investigate the evolutionary origins of three diff erent viruses: the novel emergence and cross-species transmission of SARSCoV, the genesis and dissemination of the unique HCV circulating recombinant form, and the ancient divergence of all influenza viruses, respectively. Moreover, Chapter 4 presents an improved statistical framework, which provides more precise evolutionary estimates, by utilizing the hierarchical bayes approach to investigate recombination events in emerging RNA viruses. The last empirical study, presented in Chapter 6, applies the recently developed Bayesian phylogeography models to a large viral sequence dataset sampled from southern Viet Nam to examine the fine-scale spatiotemporal dynamics of endemic dengue in Southeast Asia. The work presented here reflects both the advancements made in sequencing technology and statistical phylogenetics, along with some of the challenges that remain in studying the emergence of fast-evolving RNA viruses. This thesis proposes new and improved solutions to these evolutionary problems, such as incorporating non-vertical evolution (i.e. homologous recombination and reassortment) into the phylodynamic framework, with the aim of facilitating future investigations of emerging viral diseases.

Identifying sites under evolutionary pressure and predicting the effects of substitutions at those sites are among the greatest standing problems in bioinformatics and computational biology. Moreover, the two problems have traditionally been separated by the enormous chasm that exists between molecular evolutionary biologists interested in the evolutionary process and theoretical chemists interested in free energy changes. As a result, identifying sites under selective pressure has most often left out any semblance of structural biology and biochemistry; likewise, theoretical chemistry tends to rely strictly on first principles calculations rather than thinking first about biologically simple and interpretable results. Here, I have tried to integrate these two intuitions with regard to protein function and evolution. First, I developed a model that implements structural measurements into a traditional structure-blind molecular evolutionary model. This structure-aware model performs significantly better at identifying sites under both purifying and diversifying selection than its structure-blind counter part. Second, I go further to understand the extent to which structural features of any kind can predict the evolutionary process. By comparing site-wise evolution between human and avian influenza, I find that structural features can account for 24% to 36% of the evolutionary pressure on influenza hemagglutinin. Third, I developed a computational method based on first principles molecular dynamics simulations to predict the biological effect of substitutions in the Machupo virus--Human receptor protein--protein interface. I found that relatively simple energetic proxies offer a reasonable substitute for rigorous free energy calculations; such simple proxies could allow non-experts to naively implement first principles methods without being forced to consider all possible degrees of freedom for post hoc calculations. / text

Philosophiae Doctor - PhD / The plasticity of single-stranded viral genomes permits the formation of secondary structures through complementary base-pairing of their component nucleotides. Such structures have been shown to regulate a number of biological processes during the viral life-cycle including, replication, translation, transcription, post-transcriptional editing and genome packaging. However, even randomly generated single-stranded nucleotide sequences have the capacity to form stable secondary structures and therefore, amongst the numerous secondary structures formed in large viral genomes only a few of these elements will likely be biologically relevant. While it is possible to identify functional elements through series of laboratory experiments, this is both excessively resource- and time-intensive, and therefore not always feasible. A more efficient approach involves the use of computational comparative analyses methods to study the signals of molecular evolution that are consistent with selection acting to preserve particular structural elements. In this study, I systematically deploy a collection of computationally-based molecular evolution detection methods to analyse the genomes of viruses belonging to a number of ssRNA viral families (Alphaflexiviridae, Arteriviridae, Caliciviridae, Closteroviridae, Coronavirinae, Flaviviridae, Luteoviridae, Picornaviridae, Potyviridae, Togaviridae and Virgaviridae), for evidence of selectively stabilised secondary structures. To identify potentially important structural elements the approach incorporates structure prediction data with signals of natural selection, sequence co-evolution and genetic recombination. In addition, auxiliary computational tools were used to; 1) quantitatively rank the identified structures in order of their likely biological importance, 2) plot co-ordinates of structures onto viral genome maps, and 3) visualise individual structures, overlaid with estimates from the molecular evolution analyses. I show that in many of these viruses purifying selection tends to be stronger at sites that are predicted to be base-paired within secondary structures, in addition to strong associations between base-paired sites and those that are complementarily co-evolving. Lastly, I show that in recombinant genomes breakpoint locations are weakly associated with co-ordinates of secondary structures. Collectively, these findings suggest that natural selection acting to maintain potentially functional secondary structures has been a major theme during the evolution of these ssRNA viruses.

High­-throughput sequencing has become pervasive in all facets of genomic analysis. I developed computational methods to analyze high­-throughput sequencing data and derive biological conclusions in two research areas -- transcriptional regulation in mammals and evolution of virus under immune pressure. To investigate transcriptional regulation, I integrated data from multiple experiments performed by the ENCODE consortium. First, my analysis revealed that Transcription Factors (TFs) prefer to bind GC-­rich, histone­-depleted regions. By comparing in vivo and in vitro nucleosome dynamics, I observed that while histones have an innate preference for binding GC-­rich DNA, TF binding overrides this preference and produces a negative correlation between GC content and histone enrichment. In the next project, I found that the binding events of multiple TFs co-­occur at genomic regions enriched in activating histone marks that are typically associated with gene enhancers and promoters, suggesting that these regions may be enhancers or have TSS-­distal transcription. Lastly, I used supervised machine ­learning techniques to train histone enrichment signals and sequence features to predict transcriptional enhancers to be validated in mouse-­transgenic assays. In a post­-clinical trial exploratory analysis of Hepatitis C Virus (HCV), I traced the evolutionary path of the envelope proteins E1 and E2 in HCV-infected liver transplant patients, in response to a novel antibody. I developed a systematic amino acid­-level analysis pipeline that quantifies differences in amino acid frequencies in each position between two time points. Upon applying this method across all positions in the E1/E2 region and comparing pre-­liver­-transplant and post­-viral­-rebound time points, mutations in two positions emerged as being key to antibody evasion. Both these mutations--N415K/D and N417S--were in the epitope targeted by the antibody, but surprisingly, did not co­-occur. In post­-rebound viral genomes that contain the N417S mutation but retain the wild-­type variant at 415, N-­linked glycosylation of 415 is another possible escape mechanism. Using the same analysis pipeline, I also identified additional candidate escape mutations outside the epitope, which could be potential therapeutic targets.

Viruses may acquire mutations that result in a tropism shift. RNA viruses, such as Coronaviruses (CoVs), are susceptible to tropism shifts. A tropism shift occurs when a virus alters the tissue or cell type it infects, which can have important implications for disease pathogenesis, virulence, transmission, and treatment control. Tropism shifts can occur after cross-species jumps, as well as result from within-host evolution. Beyond the human host, CoVs can be highly pathogenic to a wide variety of wildlife and companion animals. A spillover event from animals to humans, resulting in a tropism shift, has occurred at some point in the evolutionary history of all three highly pathogenic human CoVs: severe acute respiratory syndrome coronavirus (SARS-CoV), middle eastern respiratory syndrome coronavirus (MERS), and severe acute respiratory syndrome 2 (SARS-CoV-2). Therefore, studying the evolution of CoVs in non-human animals may be of critical importance for pandemic prevention. This was the focus of my dissertation, to apply state-of-the-art codon models of evolution to a variety of CoV viral sequences to identify how natural selection may alter viral proteins priming them for tropism shifts. Statistical codon models can infer both which codon sites and genes have been subject to positive or negative selection, effectively differentiating signal between random mutations and those that may impact fitness. These models may also compare selection at homologous sites between different phenotypes (i.e., Spike protein sequences isolated from the gastrointestinal tract and those from macrophages) to identify where selection is acting differently between the phenotypes. In chapter 2 I examined a CoV sequence isolated from hospitalized humans in Malaysia that resembled a Canine Coronavirus (CCoV) to investigate how natural selection had shaped the Spike protein sequence in related animal CoV sequences priming it to jump into humans. In chapter 3 I compared the natural selection signals at specific codon positions in the Spike protein from sequences isolated from two separate feline tropisms (gastrointestinal and macrophage) to identity which adaptive mutations may be associated with the tropism shift and subsequent shift in virulence. This was performed on Feline Coronavirus (FCoVs), where almost 90% of all wild and domestic cats are gastrointestinally infected with FCoVs, and infection becomes highly pathogenic as a result of the shift in tropism to the macrophages. Since intra-host evolution can impact tropism shifts, in Chapter 4 I performed a detailed high-throughout analysis of intra-host evolution of RNAseq data of Equine Coronavirus (ECoV), as well as natural selection analyses of related embecoviruses that have colonized the human host. Taken together, I report on novel signals of natural selection across viral proteins, with an emphasis on Spike, on a diverse set of CoV clades that shed light on the complexities of coronavirus evolution as it relates to tropism shifts. / Biology

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

No description available.

The Human immunodeficiency virus (HIV) pandemic has resulted in a heavy global disease burden, and clinically causes Acquired Immuno-Deficiency Syndrome (AIDS). The development of highly active antiretroviral therapy (HAART) has achieved remarkable control of the rapidly evolving HIV. However, HIV remains neither curable nor preventable by vaccine, and in the developing regions worst affected by HIV, HAART remains inaccessible to most patients. Furthermore, the change in both immunology and viral evolution during chronic HIV infection and its relation to AIDS pathogenesis remains unknown. Following the failure of recent HIV vaccines, it is believed that a better understanding of host-pathogen interaction is vital to advance therapeutic (vaccine and drug) design. In this thesis, I have performed an investigation of viral adaptation in response to different selection forces during advanced HIV infection and AIDS. The thesis first examined a case study that reveals the potential role of B cell-mediated neutralising antibody (NAb) in chronic HIV infection through the unexpected effect of B cell depletion agent, anti-CD20 (Rituximab). Here, longitudinal results have shown that viral load (VL), env gene diversity, and NAb sensitive strains increased during B cell and NAb depletion as a result of Rituximab administration, and reversed as B cells recovered. The study provides preliminary evidence to support the idea that NAb may be effective at suppressing HIV. The rest of the thesis focused on the cross-sectional cohort at Bloemfontein, South Africa (n=1491), a resource-limited region affected by the pandemic. Here, we used methods that include molecular and pretherapy drug resistance epidemiology, mathematical modelling, phylogenetically adjusted bioinformatics analysis and in vitro viral replication capacity (VRC) assay to study materials including cohort demography, plasma samples, CD4 cell count, VL, viral genetic sequences and host human leukocyte antigen (HLA) tissue types. Our analysis was further augmented by the additional data kindly contributed by our neighbouring Durban cohort collaborators (n=775), which also includes an IFN! ELISPOT assay that measures cytotoxic T lymphocyte (CTL) responses. Using the HIV pol sequencing data and phylogenetic analysis we confirmed that the local molecular epidemiology is similar to the circulating strains documented in the regional database. However, the pretherapy drug resistance mutation screening results have revealed an unexpected high incidence of drug-induced viral mutants in the AIDS patients with CD4 counts <100 cells/μl. According to mathematical modelling, this finding is attributable to additional sources of antiretroviral therapy exposure, which warrants public health caution. The investigation then focused on studying the changes in HLA class I mediated CTL selection and viral evolution as CD4 counts are reduced in AIDS. Interestingly we have noted evidence that suggest weakening CTL immune selection against gag during AIDS is associated with increased viral fitness (measured by VRC) and reversion of previous immune-escape mutations which conferred high fitness costs. In conclusion, this thesis compared different sources of host and drug mediated HIV selection and its implication for viral evolution. The identification of more bottleneck sites conferring high fitness costs to the selection of escape mutants is expected to be helpful in the design of future therapeutics (via vaccine, drug, immune therapy, or public health strategy). As we have learnt from the principle of combinational ARV, it would be desirable for a vaccine to select HIV at multiple sites of high escape-mutation fitness cost, hence offering protective effect.

No presente trabalho, investigamos a filodinâmica de três modelos virais diferentes, utilizando técnicas baseadas em verossimilhança e inferência bayesiana. Dois desses são flavivírus com genoma de RNA fita simples e senso positivo. O terceiro é um bunyavírus com genoma tri-segmentado de RNA fita simples com senso negativo. Estes diferentes modelos permitiram estudar diferentes mecanismos promotores de diversidade viral, reagrupamento de segmentos genômicos (shift) e mutação (drift), que atuam em diferentes granularidades. Descrevemos pela primeira vez o espalhamento geográfico das linhagens de vírus Zika (ZIKV) em um nível continental, assim como ocorrência de recombinação e associação entre padrões de glicosilação e vetores. Para o flavivírus da encefalite transmitida por carrapatos (TBEV), investigamos seu espalhamento e encontramos evidências que corroboram a hipótese de circulação viral restrita a focos na Europa central. As análises sobre o vírus da Febre da Grande Fenda Africana (RVFV) apontaram a ocorrência de reagrupamento de segmentos genômicos e também ajudaram a elucidar sua dispersão do leste do continente africano para o oeste, encontrando-se diversas introduções no Senegal e Mauritânia. Aparentemente, este vírus teve a entrada facilitada nesses países por uma região que funciona como um centro de dispersão (hub) por ser encontro de rotas migratórias de animais. Ademais, investigamos a ocorrência de rearranjos de segmentos genômicos de RVFV e também estudamos as diferenças nas dinâmicas evolutivas de cada segmento. / In this study, we investigated the phylodynamics of three different viral models, using techniques based on maximum likelihood and Bayesian inference methods. Two of these viruses are flaviviruses, whose genomes are formed by a single-stranded positive-sense RNA molecule. The third is a Bunyavirus with tri-segmented single-stranded RNA genome with negative sense. These different models allowed us to investigate two different mechanisms to promote viral diversity, (i) recombination of genomic segments (\"shift\") and (ii) mutation (\"drift\"), therefore exploring different levels of granularity of evolutionary process. We described for the first time the geographic spread of Zika virus (ZIKV) strains in a continental level, as well as, the occurrence of recombination and association between glycosylation patterns and vectors. For the other Flavivirus, tick-borne encephalitis virus (TBEV), we investigated its spreading and found evidences to support the hypothesis that viral circulation is very constrained by the foci in central Europe. The analyses about the Rift Valley Fever Virus (RVFV) revealed the occurrence of reassortment of genomic segments and their dispersal from eastern Africa to the west, with several introductions to Senegal and Mauritania. Apparently, the entry of RVFV in these countries was facilitated by the region of Kedougou, where several migratory routes of animals converge. This place maybe works as a hub to spread RVFV for West Africa. Moreover, we also investigated the differences in evolutionary dynamics of each genomic segment of RVFV.

Cette thèse menée à l'interface entre deux écoles doctorales, a permis d'étudier la diversité virale et de proposer l'utilisation de biomarqueurs pour répondre à des questions environnementales. La première partie présente l'étude et la caractérisation de familles de virus géants isolées au laboratoire Information Génomique et Structurale. La première, les Pandoravirus, découverte il y a 2 ans, remet en question la définition de virus, leur origine et leur mode d'évolution. Ce virus possède un génome dépassant 2,5 Mb codant pour plus de 2500 protéines, dont 90% complètement inédites. Une autre famille de virus, les Marseilleviridae, m'a permis de mesurer leur vitesse d'évolution. Les particularités de cette famille sont (i) des génomes extrêmement conservés et (ii) la séparation de la famille en différentes lignées en fonction de leur pourcentage d'identité nucléotidique. J'ai pu étudier l'évolution de cette famille, montrant que la majorité des gènes de ces virus sont conservés entre les lignées et sous pression de sélection, donc nécessaires à leur réplication. La deuxième partie avait pour but de valider l'utilisation de marqueurs biologiques afin de répondre à une question environnementale. Il s'agissait d'identifier l'origine de l'eau saumâtre d'une rivière souterraine. Pour cela nous avons recensé les communautés de procaryotes de différentes sources d'eau douce et d'eau de mer dans le but de les comparer aux communautés de la rivière souterraine. Cette démarche nous a permis de conclure sur l'origine à la fois marine et terrestre de l'eau de l'exsurgence souterraine. Des hypothèses sur le mode d'acheminement de ces eaux vers l'exsurgence sont également proposées. / This thesis which takes place between two doctoral schools, allowed us to study viral diversity and to suggest the use of a biomarker to answer environmental questions. The first part presents a work on the characterization of two giant virus families isolated in the IGS laboratory. The first family, Pandoraviruses, questions the definition of virus, their origin and the way they evolve. This virus’ genome is bigger than 2,5 Mb, which codes for more than 2,500 proteins, of which 90% were unknown before. The isolation of new members of this family could allow us to study their evolution. Another virus family, Marseilleviridae, allowed me to study their evolution speed. This family features are (i) to have highly conserved genomes and (ii) the family is separated in three lineages, according to their nucleotide identity percentage. I thus studied the evolution of this family, showing that most genes of these viruses are conserved between lineages and under selection pressure, therefore necessary for their replication. The second part describes a work to validate the use of a biologic marker in order to respond an environmental question. We tried to identify the origin of underground river brackish water flowing from a submarine karstic spring in Port-Mio, Cassis. For that we initiated a comparison of prokaryotic community from various springs of fresh water, sea water, and the underground river. This method, coordinated to biogeochemical data allowed us to identify biomarkers which are specific of each sample. We can then conclude that this brackish water finds an origin in both fresh and sea water. Hypothesis on the way these waters flows in the spring are also proposed.