Phylogenetic analysis of within-host serially-sampled viral data

The primary goal of this dissertation is the study of patterns of viral evolution inferred from serially-sampled sequence data, i.e., sequence data obtained from strains isolated at consecutive time points from a single patient or host. RNA viral populations have an extremely high genetic variability, largely due to their astronomical population sizes within host systems, high replication rate, and short generation time. It is this aspect of their evolution that demands special attention and a different approach when studying the evolutionary relationships of serially-sampled sequence data. New methods that analyze serially-sampled data were developed shortly after a groundbreaking HIV-1 study of several patients from which viruses were isolated at recurring intervals over a period of 10 or more years. These methods assume a tree-like evolutionary model, while many RNA viruses have the capacity to exchange genetic material with one another using a process called recombination. A genealogy involving recombination is best described by a network structure. A more general approach was implemented in a new computational tool, Sliding MinPD, one that is mindful of the sampling times of the input sequences and that reconstructs the viral evolutionary relationships in the form of a network structure with implicit representations of recombination events. The underlying network organization reveals unique patterns of viral evolution and could help explain the emergence of disease-associated mutants and drug-resistant strains, with implications for patient prognosis and treatment strategies. In order to comprehensively test the developed methods and to carry out comparison studies with other methods, synthetic data sets are critical. Therefore, appropriate sequence generators were also developed to simulate the evolution of serially-sampled recombinant viruses, new and more through evaluation criteria for recombination detection methods were established, and three major comparison studies were performed. The newly developed tools were also applied to “real” HIV-1 sequence data and it was shown that the results represented within an evolutionary network structure can be interpreted in biologically meaningful ways

Spike-mediated viral membrane fusion is inhibited by a specific anti-IFITM2 monoclonal antibody

The early steps of viral infection involve protein complexes and structural lipid rearrangements which characterize the peculiar strategies of each virus to invade permissive host cells. Members of the human immune-related interferon-induced transmembrane (IFITM) protein family have been described as inhibitors of the entry of a broad range of viruses into the host cells. Recently, it has been shown that SARS-CoV-2 is able to hijack IFITM2 for efficient infection. Here, we report the characterization of a newly generated specific anti-IFITM2 mAb able to impair Spike-mediated internalization of SARS-CoV-2 in host cells and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Furthermore, the anti-IFITM2 mAb reduced HSVs- and RSV-dependent cytopathic effects, suggesting that the IFITM2-mediated mechanism of host cell invasion might be shared with other viruses besides SARS-CoV-2. These results show the specific role of IFITM2 in mediating viral entry into the host cell and its candidacy as a cell target for antiviral therapeutic strategies

Dissecting Human Antibody Responses Against Influenza A Viruses And Antigenic Changes That Facilitate Immune Escape

Influenza A viruses pose a serious threat to public health, and seasonal circulation of influenza viruses causes substantial morbidity and mortality. Influenza viruses continuously acquire substitutions in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These substitutions prevent the binding of pre-existing antibodies, allowing the virus to escape population immunity in a process known as antigenic drift. Due to antigenic drift, individuals can be repeatedly infected by antigenically distinct influenza strains over the course of their life. Antigenic drift undermines the effectiveness of our seasonal influenza vaccines and our vaccine strains must be updated on an annual basis due to antigenic changes. In order to understand antigenic drift it is essential to know the sites of antibody binding as well as the substitutions that facilitate viral escape from immunity. In this dissertation, we explore both the epitopes targeted in human antibody responses and how influenza viruses evade these responses. We first demonstrate that prior exposure shapes the sites targeted in human antibody responses, and show that many middle-age adults mounted an antibody response against H1N1 viruses that is focused against sites on HA conserved between contemporary strains and strains that circulated in early childhood. In addition, we demonstrate that a viral substitution in this epitope allows influenza viruses to evade neutralizing antibody responses. We next demonstrate that an H3N2 HA substitution introducing a glycosylation site prevents the binding of neutralizing antibodies present in a large number of individuals. Importantly, our egg-based vaccines lack this glycosylation due to culture-adaptive substitutions, but a vaccine containing this glycosylation motif more potently induced antibody responses against circulating strains. Finally, we identify and characterize antibodies that target conserved residues in the receptor-binding site (RBS) of HA. We demonstrate that in some individuals RBS antibodies in sera contribute to neutralization of antigenically distinct strains, even in the case of an antigenically mismatched vaccine. Overall, the work presented here helps address the complex interaction of influenza viruses and human immunity. Importantly, our work identifies shortcomings with our current process of vaccine strain selection and production and investigates epitopes of interest for universal influenza vaccine efforts

Genotypes of hepatitis B and C viruses in Nigeria

A small scale epidemiological survey of hepatitis B and C virus infection amongst healthy adult blood donors and children of pre-school age in Nigeria was carried out using various serological markers of hepatitis B virus (HBsAg, anti-HBs, HBeAg, anti-HBe), and hepatitis C virus infection (anti-HCV ELISA and RIBA tests). Between 10 and 11 % of the adults, and 16% of the children were HBsAg positive. A viraemia rate of 23.8% was found among the HBsAg positive adults, and 25% among the HBsAg positive children. Evidence of past exposure (anti-HBs) among adult blood donors was 41.24%. The subtype of HBV present in Nigeria was determined by amplifying and sequencing HBV DNA, encoding the antigenic determinant a and surrounding region. Two subtypes of HBV are present in Nigeria, adw & ayw. The subtype ayw was in the majority (7/10 of HBV sequenced). A multiple infection with both subtypes of HBV was found in one donor, another donor was infected with both wild type and a variant of ayw. HBV DNA encoding the pre-core and part of the core region of 7 donors was sequenced. One donor was infected with a precore mutant and another was infected with a further variant of HBV. The prevalence of anti-HCV detected by a second generation ELISA was 8% (16/200) of the adult donors, but most of them were negative on supplementary testing. None of the children tested was anti-HCV positive. 25% (4/16) of the anti-HCV positive sera were RIBA test positive and 12.5% (2/16) were indeterminate. Four RIBA positive samples and one negative were HCV-RNA positive, thus 31.25% (5/16) of the ELISA-positive sera were HCV RNA positive by reverse transcriptase polymerase chain reaction (RT-PCR). Three regions of HCV were sequenced, the 5'untranslated region (5'UTR), core and NS5. At least two HCV genotypes are present in Nigeria, type 1 and type 4. Phylogenetic analysis and pairwise comparison confirmed two new subtypes which were provisionally designated 1d and 4i

HIV infection in Angola : Molecular epidemiology, diagnosis and antibody neutralization

The UNAIDS fast-track goals established the need to reduce 75% of new HIV cases and deaths by 2020 and 90% by 2030, compared to 2010 [1]. However, it is estimated that no country in sub-Saharan Africa has reached the target of 75% reduction in new cases of HIV infection [2]. HIV-1 incidence in Angola increased 61.2% from 2010 to 2018 and the prevalence is now stable with 340,000 people living with HIV [2, 3]. Women and children are the most affected populations [3]. The aims of this thesis were: 1) to get a better understanding of the HIV-1 diversity, transmission dynamics and transmitted drug resistance (TDR) in Angola, 2) improve the early infant diagnosis (EID) in Angola and 3) characterize the neutralizing antibody responses against HIV-1 and assess the possible viral and host factors associated with it. The HIV epidemics in Angola is peculiar, since highly divergent forms of the virus circulates in the population. In this context our first objective was to assess HIV-1 diversity, transmission dynamics and prevalence of transmitted drug resistance (TDR) in Angola in 2009, in 139 drug naïve HIV-1 infected individuals and compare the results before ART scale-up (chapter 2). We saw an increase in genetic diversity between 2001 and 2009, with the prevalence of subtype A decreasing significantly while the prevalence of unique recombinant forms (URFs) increased 2-fold. Also, local U/H recombinants were newly identified. TDR mutation K103N was found in one (0.7%) patient. Overall, transmission of drug resistant strains was still negligible in Luanda in 2009 and the emergence of new URFs are consistent with a rising HIV-1 epidemics. Our second objective was to develop and validate a sensitive, simple and cheap qualitative proviral DNA PCR-based assay for early infant diagnosis (EID) in HIV-1-exposed infants (n=139) using dried blood spots (DBS) (chapter 3). We were able to successfully validate the assay, the limit of detection (LOD) using several integrase recombinant plasmids was 14 copies and clinical sensitivity and specificity were high. The percentage of HIV-1 mother-to-child-transmission (MTCT) between January 2012 and October 2014 was only 2.2%. In chapter 4 we performed the first detailed characterization of the neutralizing antibody response in 322 Angolan HIV-1 infected patients and identified its determinants. Remarkably, 56% of the individuals had broad cross-neutralizing activity. Also, cross-clade neutralization was positively associated with subtype C infection and negatively associated with CD4 counts and antibody binding titers against envelope C2V3C3 region was a good indicator of neutralization breadth and potency. In chapter 4 we concluded that development of broad and elite antibody neutralization against HIV-1 requires long-term and low-level envelope V3C3 stimulation from highly diverse subtype C isolates

Var gene diversity and their serological recognition by naturally exposed individuals

Plasmodium falciparum causes the worst form of human malaria and leads to 1-2 million deaths annually, most of them children below the age of 5 living in subsaharan Africa. Morbidity varies from asymptomatic infections with no symptoms to severe malaria accompanied by organ failure, severe anemia and coma. Most of these clinical presentations are associated with sequestration of infected red blood cells (iRBC) on host endothelium. By attaching the parasitized erythrocyte to host receptors such as CD36, ICAM or CSA the parasite prevents the cell from being cleared by the spleen and therefore prolongs its own survival. A key protein involved in this process is the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) which is a parasite derived protein transported to the RBC surface to mediate cytoadherence. With this process exposes the parasite itself to the host immune system leading to the production of specific antibodies. In order to evade this host immune response the parasite undergoes antigenic variation by switching to another member of the same protein family. PfEMP1 is encoded by approximately 60 var genes per haploid genome and is expressed at the surface in a mutually exclusive manner, i.e. only 1 of the 60 proteins is expressed and exposed at any one time whilst the others remain silenced. Protection against severe malaria is thought to be mediated to a large degree by the piecemeal acquisition of anti-PfEMP1 antibodies during early childhood, since adults still get infected but rarely develop severe malaria symptoms. Recent observations suggest that not all PfEMP1 proteins expressed by a parasite are equally virulent, but only a subset of distinct var genes might render a parasite more pathogenic than parasites expressing different var gene variants. To generate potential anti-severe disease interventions members of this particular subset need to be identified. To date, only 6 studies have been published investigating the repertoire of expressed var genes in vivo. We have further used samples collected in Papua New Guinea from a case control study and analyzed var transcripts by RT-PCR followed by cloning and sequencing. We determined the 3 main groups of 5’UTR and analysed the data with respect to the clinical presentation of the children they were collected from. The detected number of different var group B and C transcipts was not significantly different between asymptomatic, mild or severe malaria cases, whereas an increase of group A var genes was observed in symptomatic cases when compared to children without any malaria symptoms. We identified an amino acid substitution mainly occurring in asymptomatic children with high parasitemia that might influence the binding affinity of parasites expressing these variants. However, using phylogenetic analyses we were not able to identify other distinct var genes or subsets associated with severe malaria. Blasting DBL1α domains against the 3D7 genome to obtain information on the upstream region was found to be suitable for group A var genes only, whereas 28% of group B and 62% of group C sequences were assigned to the wrong subgroup using this method. Even though we observed a 7% sequence overlap, bioinformatic analyses estimated the var gene repertoire in this region of PNG to be unlimited. It has previously been shown, that isolates causing severe disease are recognized more frequently than those causing mild malaria. In the second part of this thesis, we wanted to obtain information on the importance of distinct PfEMP1 domains in the recognition by the host immune system. For that purpose, fragments of 2 representative var genes shown to be associated with severe malaria were recombinantly expressed in E.coli and analyzed for their recognition by naturally exposed sera of different origin. Analysis of synthetic peptides using the same sera served to complement the results of ELISAs using recombinant proteins if expression of distinct domains was not possible. ELISA and Western blot analysis determined that 3 recombinant fragments and 2 synthetic peptides harbor epitopes that might play a role in the generation of protective antibodies. However, since sample size was small further investigations are required to confirm these findings. In the third part of this thesis, we tested the usefulness of the GeneMapper® analysis software to genotype var genes. It has been successfully established for genotyping the polymorphic marker gene msp2 and since var genes also show some length polymorphism it was investigated whether this technique could replace tedious cloning and sequencing approaches, used so far to dissect var gene diversity. Therefore, purified PCR products of UTR-DBL domains generated during the sequence analysis were reamplified with fluorescently labeled DBL-specific primers and analyzed by GeneMapper®. The results were then compared to the sequencing data. GeneMapper® sizing was highly accurate with a mean deviation of 1bp and showed a high consistency with sequencing data. Furthermore, GeneMapper® detected 141 sequences which were not identified with the sequencing approach, whereas vice verca, this was only the case for 16 sequences. However, a significant proportion of var genes could not be distinguished because the analyzed DBL domains were identical in size. Despite this shortcoming, we belive that GeneMapper® would greatly facilitate the analysis of expressed var genes and their dynamics

Genetic variation associated with infection and the environment in the accidental pathogen Burkholderia pseudomallei

Funder: Thailand National Science and Technology Development Agency (FDA-CO-2562-8764-TH) and Thailand Science Research and Innovation fund (MRG6280226)Funder: Food Standards Agency Fellow and is supported by the BBSRC Institute Strategic Programme Microbes in the Food Chain BB/R012504/1 and its constituent projects BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain) and BBS/E/F/000PR10351 (Theme 3, Microbial Communities in the Food Chain)Abstract: The environmental bacterium Burkholderia pseudomallei causes melioidosis, an important endemic human disease in tropical and sub-tropical countries. This bacterium occupies broad ecological niches including soil, contaminated water, single-cell microbes, plants and infection in a range of animal species. Here, we performed genome-wide association studies for genetic determinants of environmental and human adaptation using a combined dataset of 1,010 whole genome sequences of B. pseudomallei from Northeast Thailand and Australia, representing two major disease hotspots. With these data, we identified 47 genes from 26 distinct loci associated with clinical or environmental isolates from Thailand and replicated 12 genes in an independent Australian cohort. We next outlined the selective pressures on the genetic loci (dN/dS) and the frequency at which they had been gained or lost throughout their evolutionary history, reflecting the bacterial adaptability to a wide range of ecological niches. Finally, we highlighted loci likely implicated in human disease

Molecular evolution of the proopiomelanocortin system in Barn owl species.

Examination of genetic polymorphisms in outbred wild-living species provides insights into the evolution of complex systems. In higher vertebrates, the proopiomelanocortin (POMC) precursor gives rise to α-, β-, and γ-melanocyte-stimulating hormones (MSH), which are involved in numerous physiological aspects. Genetic defects in POMC are linked to metabolic disorders in humans and animals. In the present study, we undertook an evolutionary genetic approach complemented with biochemistry to investigate the functional consequences of genetic polymorphisms in the POMC system of free-living outbred barn owl species (family Tytonidae) at the molecular level. Our phylogenetic studies revealed a striking correlation between a loss-of-function H9P mutation in the β-MSH receptor-binding motif and an extension of a poly-serine stretch in γ3-MSH to ≥7 residues that arose in the barn owl group 6-8 MYA ago. We found that extension of the poly-serine stretches in the γ-MSH locus affects POMC precursor processing, increasing γ3-MSH production at the expense of γ2-MSH and resulting in an overall reduction of γ-MSH signaling, which may be part of a negative feedback mechanism. Extension of the γ3-MSH poly-serine stretches ≥7 further markedly increases peptide hormone stability in plasma, which is conserved in humans, and is likely relevant to its endocrine function. In sum, our phylogenetic analysis of POMC in wild living owls uncovered a H9P β-MSH mutation subsequent to serine extension in γ3-MSH to 7 residues, which was then followed by further serine extension. The linked MSH mutations highlight the genetic plasticity enabled by the modular design of the POMC gene

Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease

The source of the severe acute respiratory syndrome (SARS) epidemic was traced to wildlife market civets and ultimately to bats. Subsequent hunting for novel coronaviruses (CoVs) led to the discovery of two additional human and over 40 animal CoVs, including the prototype lineage C betacoronaviruses, Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5; these are phylogenetically closely related to the Middle East respiratory syndrome (MERS) CoV, which has affected more than 1,000 patients with over 35% fatality since its emergence in 2012. All primary cases of MERS are epidemiologically linked to the Middle East. Some of these patients had contacted camels which shed virus and/or had positive serology. Most secondary cases are related to health care-associated clusters. The disease is especially severe in elderly men with comorbidities. Clinical severity may be related to MERS-CoV's ability to infect a broad range of cells with DPP4 expression, evade the host innate immune response, and induce cytokine dysregulation. Reverse transcription-PCR on respiratory and/or extrapulmonary specimens rapidly establishes diagnosis. Supportive treatment with extracorporeal membrane oxygenation and dialysis is often required in patients with organ failure. Antivirals with potent in vitro activities include neutralizing monoclonal antibodies, antiviral peptides, interferons, mycophenolic acid, and lopinavir. They should be evaluated in suitable animal models before clinical trials. Developing an effective camel MERS-CoV vaccine and implementing appropriate infection control measures may control the continuing epidemic. © 2015, American Society for Microbiology. All Rights Reserved.postprin