Interactions of host miRNAs in the flavivirus 3´UTR genome:From bioinformatics predictions to practical approaches

The genus Flavivirus of the Flaviviridae family includes important viruses, such as Dengue, Zika, West Nile, Japanese encephalitis, Murray Valley encephalitis, tick-borne encephalitis, Yellow fever, Saint Louis encephalitis, and Usutu viruses. They are transmitted by mosquitoes or ticks, and they can infect humans, causing fever, encephalitis, or haemorrhagic fever. The treatment resources for these diseases and the number of vaccines available are limited. It has been discovered that eukaryotic cells synthesize small RNA molecules that can bind specifically to sequences present in messenger RNAs to inhibit the translation process, thus regulating gene expression. These small RNAs have been named microRNAs, and they have an important impact on viral infections. In this review, we compiled the available information on miRNAs that can interact with the 3’ untranslated region (3’UTR) of the flavivirus genome, a conserved region that is important for viral replication and translation

Bioinformatic analysis of genome-scale data reveals insights into host-pathogen interactions in farm animals

This thesis documents the contribution of my bioinformatics research activities, including novel software development, to a range of research projects aimed at investigating the interactions between bacterial and viral pathogens and their hosts. The focus is largely on farm animal species and their pathogens, although some of the research has a wider scientific impact. RNA interference (RNAi) refers to a variety of related regulatory pathways present in animals, plants and insects. The major pathways are microRNAs (miRNAs), small-interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). Marek’s disease virus is an important pathogen of poultry, causing T-cell lymphoma. We identified the presence and expression patterns of several MDV-encoded microRNAs, including the identification of 5 novel microRNAs. We also showed that not only do virus-encoded microRNAs dominate the mirNome within chicken cells, but also that specific host-microRNAs are down-regulated. We also identify novel virus-encoded microRNAs in other Herpesviridae and provide the first evidence of miRNA evolution by duplication in viruses. In related work, we present a novel microRNA generated by the canonical miRNA biogenesis pathway in Avian Leukosis Virus, another avian oncogenic virus, and publish data showing the expression pattern of known chicken microRNAs across a range of important avian cells. Two of the other RNAi pathways (siRNA and piRNA) form an important part of the antiviral response in arthropods. We have published work demonstrating an siRNA antiviral response to bluetongue virus and Schmallenberg virus in cells from the Culicoides midge, an important insect vector, as well as work demonstrating the importance of the piRNA pathway in the antiviral response to Semliki forest virus (SFV). Further work on flaviviruses in ticks demonstrates the active suppression of the siRNA response by Langat Virus, as well as a key difference between the siRNA responses in Mosquitos compared to ticks. Salmonella is one of the most important zoonoses, with an estimated 1.4 million cases of human salmonellosis per annum in the USA alone. Salmonella infections of farm animals are an important route into the human food chain. This thesis presents work on the comparative structure and function of 13 fimbrial operons within Salmonella enterica serovar Enteritidis as well as a genomic comparison of that serovar with Salmonella enterica serovar Gallinarum, a chicken-specific serovar. We characterised the global expression profile of Salmonella enterica serovar Typhimurium during colonization of the chicken intestine, and we have published the genomes of four strains of Salmonella eneterica serovars of well-defined virulence in food-producing animals. Our work in this area led to us publishing an important and comprehensive review of the automatic annotation of bacterial genomes. Finally, I present work on novel software development. ProGenExpress, a software tool that allows the easy and accurate integration and visualisation of quantitative data with the genome annotation of bacteria; Meta4 is a web application that allows data sharing of bacterial genome annotations from metagenomes; CORNA, a software tool that allows scientists to link together microRNA targets, gene expression and functional annotation; viRome, a software tool for the analysis of siRNA and piRNA responses in virus-infection studies; DetectiV, a software tool for the analysis of pathogen-detection microarray data; and poRe, a software tool that enables users to organise and analyse nanopore sequencing dat

Human Cytomegalovirus Reprograms the Expression of Host Micro-RNAs whose Target Networks are Required for Viral Replication: A Dissertation

The parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection. The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs

Identification and characterization of microRNAs and their putative target genes in Anopheles funestus s.s

Philosophiae Doctor - PhDThe discovery of microRNAs (miRNAs) is one of the most exciting scientific breakthroughs in the last decade. miRNAs are short RNA molecules that do not encode proteins but instead, regulate gene expression. Over the past several years, thousands of miRNAs have been identified in various insect genomes through cloning and sequencing, and even by computational prediction. However, information concerning possible roles of miRNAs in mosquitoes is limited. Within this context, we report here the first systematic analysis of these tiny RNAs and their target mRNAs in one of the principal African malaria vectors, Anopheles funestus s.s. Firstly, to extend the known repertoire of miRNAs expressed in this insect, the small RNAs from the four developmental stages (egg, larvae, pupae and the adult females), were sequenced using next generation sequencing technology. A total of 98 miRNAs were identified, which included 65 known Anopheles miRNAs, 25 miRNAs conserved in other insects and 8 novel miRNAs that had not been reported in any species. We further characterized new variants for miR-2 and miR-927 and stem-loop precursors for miR-286 and miR-2944. The analysis showed that many miRNAs have stage-specific expression, and co-transcribed and co-regulated during development. Secondly, for a better understanding of the molecular details of the miRNAs function, we identified the target genes for the Anopheles miRNAs using a novel approach that identifies overlap genes among three target prediction tools followed by filtering genes based on functional enrichment of GO terms and KEGG pathways. We found that most of the miRNAs are metabolic regulators. Moreover, the results suggest implication of some miRNAs not only in the development but also in insect-parasite interaction. Finally, we developed the InsecTar database (http://insectar.sanbi.ac.za) for miRNA targets in the three mosquito species; Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus, which incorporates prediction and the functional analysis of these target genes. The proposed database will undoubtedly assist to explore the roles of these regulatory molecules in insects. This type of analysis is a key step towards improving our understanding of the complexity and regulationmode of miRNAs in mosquitoes. Moreover, this study opens the door for exploration of miRNA in regulation of critical physiological functions specific to vector arthropods which may lead to novel approaches to combat mosquito-borne infectious diseases

Characterisation of small RNAs in response to human vaccination and infection

Background Heterogeneity in host responses plays a role in vaccine failure, vaccine-related adverse events and infection outcomes and undermines efforts to produce universally effective vaccines and treatments. Gene expression analyses have provided insights into the immune response, but a deeper understanding requires a broader view encompassing gene regulation. MicroRNAs (miRNAs) are small RNAs (sRNAs) that post-transcriptionally regulate protein-coding genes. Small RNA sequencing (sRNA-seq) has revealed the presence of other small RNA molecules ~20 to 30 nucleotides long, which arise from RNAs classes, including tRNAs, small nucleolar RNAs and yRNAs. The action of these so-called sRNA-derived fragments (sRNA-dfs) is poorly understood; they may play a role in gene regulation. Their role in the immune system is not known. Research questions: 1. Does vaccination and infection alter miRNA expression? 2. Can miRNA expression predict or shape the response to vaccination and/or infection? 3. What is the role of miRNAs in the immune response to infection/vaccination? Methods sRNA-seq was run on samples from three studies: an infant vaccine study involving the reactogenic 4CMenB vaccine, a randomised controlled trial of two typhoid vaccines set within a typhoid vaccine-challenge study, and a ChAdOx1 nCoV-19 vaccine study that followed participants through COVID-19 infection. Results This thesis found vaccination and infection strongly influence miRNAs and sRNA-df expression in plasma, whole blood and PBMCs. In total, 1846 sRNAs were DE after vaccination or infection in at least one study. These features comprised miRNAs and sRNA-dfs derived from tRNAs, snoRNAs, lncRNAs and protein-coding genes. Associations were found between aspects of the immune response and sRNA expression. The infant vaccine study revealed an association between vaccine fever and post-vaccination expression of hsa-miR-122-5p at 4 months of age. Integration of miRNA, mRNA expression, and proteomics revealed a possible link between liver-derived miRNAs and the acute phase response to vaccination via the action of IL6 on hepatocytes. An in-vitro model supported this. The typhoid vaccine-challenge study revealed a divergent pattern of miRNA expression 10 days after vaccination is associated with vaccine-mediated protection from typhoid, and that a hypo-responsiveness to S. Typhi challenge is associated with susceptibility to typhoid fever. Finally, the COVID-19 study showed downregulation of miRNAs at COVID-19 onset was associated with wide-scale upregulation of pro-inflammatory genes and pathways. Amelioration of COVID-19 disease severity after ChAdOx1 nCoV-19 vaccination was reflected at the sRNA level, with less perturbation occurring compared with COVID-19 infection in unvaccinated individuals. Conclusion This thesis reports for the first time that vaccination and infection alter the expression of sRNA fragments derived from snoRNA, lncRNAs and protein-coding genes. This thesis opens up exciting avenues for investigating the role of miRNAs and sRNA-dfs as purveyors of the acute phase response and correlates of vaccine-mediated protection, whose dysregulation may contribute to disease pathogenesis, aspects hitherto overlooked

Computational Methods for the Analysis of Genomic Data and Biological Processes

In recent decades, new technologies have made remarkable progress in helping to understand biological systems. Rapid advances in genomic profiling techniques such as microarrays or high-performance sequencing have brought new opportunities and challenges in the fields of computational biology and bioinformatics. Such genetic sequencing techniques allow large amounts of data to be produced, whose analysis and cross-integration could provide a complete view of organisms. As a result, it is necessary to develop new techniques and algorithms that carry out an analysis of these data with reliability and efficiency. This Special Issue collected the latest advances in the field of computational methods for the analysis of gene expression data, and, in particular, the modeling of biological processes. Here we present eleven works selected to be published in this Special Issue due to their interest, quality, and originality

A Meeting of Minds: In Recognition of the Contributions of Randall J. Cohrs

A Special Issue in memory of Randall J. Cohrs, Ph.D. Topics include original research reports on a variety of viruses as well as reviews and commentaries on Randy’s contributions to many investigations

Influence and regulation of PCBP2 and YTHDF2 RNA-binding proteins during self-renewal and differentiation of human induced pluripotent stem cells

2019 Fall.Includes bibliographical references.Embryonic stem cells (ESCs) are able to self-renew or differentiate into any cell type in the body, a property known as pluripotency that enables them to initiate early growth and development. However, the ethical implications of harvesting and manipulating ESCs hinders their use in basic research and the clinical applications. Thus, the discovery that somatic cells can be exogenously reprogrammed into induced pluripotent stem cells (iPSCs) offers new and exciting possibilities for gene therapy, personalized medicine and basic research. However, more research is needed into the mechanisms involved in regulating pluripotency in order for iPSCs to reach their full potential in the research lab and clinic. To maintain a state of self-renewal, yet also be able to rapidly differentiate in response to external signals, pluripotent stem cells need to exert tight control over gene expression through transcriptional and post-transcriptional mechanisms. There are several notable transcriptional networks that regulate pluripotency, but the post-transcriptional mechanisms remain poorly characterized. mRNA decay is one form of post-transcriptional regulation that can help to both maintain the steady-state of a transcriptome or facilitate its rapid remodeling. To this end, degradation rates are influenced by the elements contained in an mRNA and the RNA-binding proteins (RBPs) they associate with. Previous reports have indicated the RNA modification N6-methyladenosine (m6A) and C-rich sequence elements (CREs) can affect mRNA decay in pluripotent stem cells. Therefore, we sought to further understand the roles of m6A and CREs in mRNA decay in stem cells by characterizing the expression and mRNA targets of two RBPs that recognize these elements, YTHDF2 and PCBP2, respectively. In this thesis, I report YTHDF2 is differentially regulated in pluripotent and differentiated cells and that YTHDF2 contributes to pluripotency by targeting a group of mRNAs encoding factors important for neural development. The down-regulation of YTHDF2 during neural differentiation is consistent with increased expression of neural factors during this time. Moreover, YTHDF2 expression is regulated at the level of translation via elements located in the first 300 nucleotides of the 3' untranslated region of YTHDF2 mRNA. Based on these results, I propose that stem cells are primed for rapid differentiation by transcribing low levels of mRNAs encoding neural factors that are subsequently targeted for degradation, in part by YTHDF2, until differentiation is induced. On the other hand, PCBP2 is up-regulated upon differentiation of pluripotent stem cells and regulates several mRNAs associated with pluripotency and development, including LIN28B. Notably, expression of long non-coding RNAs (lncRNAs) that contain human endogenous retrovirus element H (HERV-H) is influenced by PCBP2. HERV-H lncRNAs are almost exclusively expressed in stem cells and play a role in maintaining a pluripotent state, although their functions are not fully understood. Intriguingly, some HERV-lncRNAs can also regulate PCBP2 expression, as altering the expression of LINC01356 or LINC00458 effects PCBP2 protein levels. Based on these results, I propose the reciprocal regulation of PCBP2 and HERV-H lncRNAs influences whether stem cells maintain a state of self-renewal or differentiate. Taken together, these findings demonstrate that YTHDF2 and PCBP2 post-transcriptionally regulate gene expression in stem cells and influence pluripotency

Mapping transcriptional regulation of cell types and states using systems genetics in mouse

Complex traits are intricately intertwined with an organism's genome, a relationship underscored by the dynamic landscape of its transcriptome. Selective gene expression regulates cell type specialization and fluctuation of cell states. The development of RNA sequencing has facilitated the capture of the whole transcriptome of a given sample. However, a bulk approach obscures cell type heterogeneity, impeding the precise dissection of cell-specific effects, including those modulated by genotype, developmental stage, and disease state. In contrast, single-cell and single-nucleus RNA-seq preserves cellular identity, enabling a comprehensive mapping of gene expression across various cell types and states.Here, I describe my work in single-cell transcriptomics to characterize cell types and cell states in mouse. First, I present our long-read single-cell RNA-seq method, benchmarked in the C2C12 mouse myogenic system, which revealed cell type-specific isoform switching in key genes during myogenesis. Next, I characterize 5 mouse tissues at single-nucleus resolution during postnatal development using the ENCODE4 mouse dataset, where I used topic modeling to reveal cell type- and state-specific cellular programs. Lastly, I investigate the impact of genetic variation on gene expression across 8 diverse tissues from 8 mouse genotypes, pinpointing genotype- driven variation in specific celltypes in both wild-derived and classical lab strains. Together, these projects lay the groundwork for cohesive cell type and cell state annotation and comparative analyses, contributing to future characterization of these tissues in other contexts such as human diseases and hybrid mouse genotypes

Vector-pathogen interactomics: connecting the dots

As carraças e doenças associadas a carraças têm um impacto negativo considerável na saúde humana e animal. Rhipicephalus bursa é uma carraça multihospedeiro hematófaga e é o principal vetor de Babesia ovis, um hemoparasita altamente patogénico em pequenos ruminantes, que pode levar a uma taxa de mortalidade de 30- 50% em animais suscetíveis e, indiretamente contribuir para um impacto socioeconómico negativo na sociedade humana. O controlo de carraças e doenças associadas depende principalmente do uso de fármacos, que apresentam grandes desvantagens, como a contaminação de alimentos e ambiente e o aumento da resistência, reforçando assim a necessidade de medidas alternativas, como a vacinação. Com base na premissa de que as glândulas salivares da carraça têm um papel crucial no comportamento hematófago e na transmissão de agentes patogénicos, o objetivo principal deste trabalho é aumentar o conhecimento sobre a interação R. bursa-B. ovis neste tecido, de forma a identificar novos candidatos a antigénios protetores para o desenvolvimento de vacinas. Assim sendo, os sialotranscritos e as sialoproteínas de R. bursa foram analisados em diferentes condições, para compreender melhor os processos de alimentação e infeção e contribuir para o desenvolvimento de novas vacinas anti-carraça e doenças associadas. A análise comparativa dos transcriptomas e proteomas revelou que a alimentação por sangue induz a produção de moléculas por parte da carraça, o que se traduziu no aumento da expressão genética e da síntese proteica. Além disso, os dados mostram que a combinação de estímulos (alimentação e infeção) influenciou positivamente a expressão genética, mas negativamente a tradução, podendo sugerir a manipulação de B. ovis no sialoma de R. bursa. Estes resultados aliados a diferentes metodologias como RNA de interferência (in vitro e in vivo) e vacinologia reversa, permitem explorar a maquinaria celular da carraça e identificar vários alvos como potenciais antigénios para vacinas. Os ensaios de silenciamento revelaram o impacto direto de algumas moléculas na sobrevivência da carraça e a sua fixação ao hospedeiro (como a putativa “Vitelogenin-3” e uma proteína do “cement”), enquanto que outros demonstraram um efeito duplo divergente na sobrevivência do vetor e do agente patogénico (como a “lachesin” e a “UB2N”). A análise imunoinformática dos dados anteriores de sequenciação permitiu a identificação de proteínas/peptídeos capazes de induzir, no hospedeiro vertebrado, uma resposta imunológica forte e robusta contra o vetor e o agente patogénico. Nesta análise, uma proteína membranar (proteína contendo domínios “Marvel”) e duas secretórias (uma “Evasin” e uma proteína contendo domínios de “Ricin”) foram selecionadas e promissores "immunological kernels" foram encontrados, contendo características ideais de uma vacina baseada em peptídeos, sem causar alergia e toxicidade. Além disso, a integração de diferentes análises ómicas de diferentes espécies de carraças foi usada como uma estratégia para pesquisar e caracterizar vias biológicas conservadas, a fim de selecionar novos alvos capazes de impactar uma ampla gama de espécies de carraças e bloquear a transmissão de vários agentes patogénicos transmitidos por estas. Deste estudo, destacou-se a via de biossíntese de folato, ao observar que durante a infeção da carraça, quer por bactéria quer por protozoário, a expressão de genes relacionados com esta via era aumentada. No entanto, ensaios de silenciamento numa linha celular de carraça mostraram que, a curto prazo, a redução da expressão de um gene relacionado ao folato (gch-I), não exorta alterações significativas nas células de carraça ou no comportamento do agente patogénico em termos de invasão ou multiplicação. Estudos aplicados e ensaios de vacinação precisam ser conduzidos para validar o potencial desses alvos promissores para o desenvolvimento de abordagens anti-carraça e de bloqueio de transmissão de doenças.health. Rhipicephalus bursa is a hematophagous multi-host tick and the main vector of Babesia ovis, a highly pathogenic hemoparasite in small ruminants, which leads to a 30-50 % of mortality rate in susceptible animals and, indirectly, to a negative socioeconomic impact in human society. Tick and disease control rely mainly in the use of chemotherapy and acaracides, which has major drawbacks including food and environment contamination and the increase of resistance, reinforcing the need for alternatives measures, such as vaccination. Based on the premise that tick salivary glands have a crucial role on hematophagous behaviour and on pathogen transmission, the main objective of this research was to increase the understanding on the Rhipicephalus bursa- Babesia ovis interaction in this organ, in order to find new protective antigen candidates for vaccine design. Thus, the R. bursa sialotranscripts and sialoproteins were screened under different conditions, to better understand the feeding and infection processes and contribute for the development of new anti-tick and tick-borne diseases. The comparative analyses of the transcriptomes and proteomes revealed that blood feeding induces the production of tick molecules, which was translated by the increased gene expression and protein synthesis. Moreover, the data unveiled that the combination of stimuli (feeding and infection) influenced positively gene expression but negatively translation, suggesting that B. ovis might manipulate R. bursa sialome. These results allied to interference RNA (in vitro and in vivo) and reverse vaccinology, allowed to explore the tick cellular machinery and pinpointed several targets as potential vaccine antigens. The silencing assays revealed the direct impact of some molecules in tick survival and attachment to the host (such as putative Vitellogenin-3 and a Cement protein), while others demonstrated a divergent dual-effect on both vector and parasite survival (such as Lachesin and UB2N). Immunoinformatic analysis of the previous sequencing data allowed the identification of proteins/peptides capable of elicit, in the vertebrate host, a strong and robust immune response against both vector and pathogen. In this experiment, one membrane-related (Marvel-containing protein) and two secreted (a Evasin and a ricin-containing protein) proteins were selected and promising “immunological kernels” were found to have ideal characteristics for an anti-tick peptide-based vaccine, without causing allergy and toxicity. Furthermore, the integration of different omics analyses from different tick species was used as a strategy to search and characterize conserved biological pathways in order to select new targets able to impact a wide range of tick vectors and block the transmission of several transmitted pathogens. From this study the folate biosynthesis pathway stood out by observing that during tick infection, by either bacteria or protozoan, the expression of genes related to this pathway were increased. However, silencing assays in a tick cell line demonstrate that, in a short term, the reduction of expression of a folate-related gene (gch-I), did not lead to significant changes in tick cells or pathogen behaviour of invasion or multiplication. Applied studies and vaccination trials need to be conducted to validate the potencial of these promising targets for the development of anti-tick and transmission blocking approaches