Au cours de ma thèse je me suis intéressée aux interactions protéine-protéine (PPI’s). Les PPI’s jouent un rôle majeur dans une grande diversité de processus cellulaires et sont maintenant considérées comme une cible majeure dans le but de développer de nouveaux médicaments. Cependant, cibler ce type d’interactions requiert le développement de chimiothèques dédiées, permettant d’accélérer la découverte de molécules « touches ». Pour surmonter ce problème, une chimiothèque orientée PPI (2P2I3D) a été conçu au laboratoire. Dans un premier temps, j’ai donc évalué cette chimiothèque sur différents complexes possédant des interfaces variées. Les résultats obtenus ont révélé des taux de touches supérieurs à ceux obtenus avec des chimiothèques non orientées, de 0,2 à 1,6% contre 0,01 à 0,1%, respectivement. Cette étude a permis d’établir une preuve de concept de la faisabilité de créer une chimiothèque orientée PPI, permettant ainsi une accélération de la découverte de composés biologiquement actifs.Dans un deuxième temps, je me suis intéressée à l’interaction entre deux protéines majeures du virus de la dengue : les protéines NS3 et NS5. J’ai tout d’abord identifié et caractérisé un nouveau site d’interaction, ce qui m’a permis de mettre en évidence que cette interaction avait pour conséquence d’augmenter l’activité enzymatique du domaine hélicase. J’ai par la suite recherché et identifié des petites molécules chimiques capable d’inhiber cette interaction. Les différentes caractérisations effectuées ont permis de mettre en évidence un effet antiviral. Ces inhibiteurs constituent un excellent point de départ afin d’étudier plus en détail le rôle biologique de ce complexe. / In my thesis I became interested in protein-protein interactions (PPI's). PPI's play a major role in a variety of cellular processes and are now considered a major target in order to develop new drugs. However, targeting such interactions requires the development of dedicated libraries, to accelerate the discovery of “hits”molecules .To overcome this issue, a focused chemical library PPI (2P2I3D) was designed in the laboratory.At first, I evaluated this chemical library on different complexes with diverse interfaces. The results showed higher hit rate to those obtained with non-oriented libraries, from 0.2 to 1.6% against 0.01 to 0.1%, respectively. This study has established a proof of concept of the feasibility of creating a focused chemical library PPI, thus accelerating the discovery of biologically active compounds.Secondly, I am interested in the interaction between two major proteins of dengue virus: the NS3 and NS5 proteins. I initially identified and characterized a novel interaction site, which allowed me to demonstrate that this interaction had the effect of increasing the enzymatic activity of the helicase domain. I searched and identified small molecules able to inhibit this interaction. The different characterizations helped to highlight an antiviral effect. These inhibitors are an excellent starting point to further explore the biological role of this complex.

A imunidade inata que o mosquito apresenta tenta combater os patógenos dentro do organismo do mosquito impedindo que este seja transmitido para outros hospedeiros. A resposta celular apresenta três diferentes processos: fagocitose, encapsulamento e formação nodular, todos estes processos buscam eliminar os patógenos. Peptídeos antimicrobianos fazem parte da resposta humoral do mosquito, sendo codificados por genes e secretados por diversos tipos celulares. Um peptídeo novo pouco conhecido descoberto em Anopheles gambiae, a gambicina, demonstrou bons resultados no combate de parasitas. Na infecção por Plasmodium galleceum, não há diferença significativa na expressão deste peptídeo entre o grupo controle e infectado nos intervalos analisados. Na infecção por vírus dengue, sorotipo 2, a gambicina não apresenta diferença significativa no intervalo de 24 horas após a infecção, quando comparamos grupo controle e infectado, nos intervalos de 7 dias e 14 dias após a infecção, a expressão da gambicina é maior no grupo controle quando comparemos com o grupo infectado. / Innate immunity presents a mosquito tries to combat the pathogens inside the body of the mosquito preventing it from being transmitted to other hosts. The cellular response has three different processes: phagocytosis, encapsulation and nodule formation, all these processes seek to eliminate pathogens. Antimicrobial peptides are part of the humoral response mosquito being encoded by genes and secreted by several cell types. A little known new peptide discovered in Anopheles gambiae, the gambicina showed good results in controlling pests. In Plasmodium infection galleceum, there is no significant difference in the expression of this peptide between the control group and infected in the analyzed intervals. In infection with dengue virus serotype 2, the gambicina no significant difference within 24 hours after infection when comparing the control group and infected at intervals of 7 days and 14 days after infection, the expression is higher in gambicina control group when compare to the infected group.

<p dir="ltr">It is with great pleasure that I present the culmination of my exploration into the process of flavivirus assembly, with particular emphasis on the envelope glycoproteins and C protein of ZIKV and DENV2, within the subsequent four chapters of this dissertation.</p><p dir="ltr">Beginning in Chapter 2, we describe findings from a structure-function study of the ZIKV prM and E transmembrane helices (TMHs) and their role in virus assembly. Using a mutagenesis approach in a ZIKV reporter virus particle (RVP) system to increase throughput and discovery, substantial information was obtained showcasing a novel function for specific residues located within a short (4 residue) connecting region between the two TMHs of prM protein – denoted as the prM TMH “turn” residues. During translation of the prM and E proteins, these TMH “turn” residues face towards the cytosolic side of the ER membrane. This orientation has been hypothesized to possibly play a role during viral assembly interactions between the envelope glycoproteins and the nucleocapsid core of flaviviruses. However, no information to date has supported or refuted this theory. Overall, a single amino acid change within the prM TMH “turn” residues was found to be highly detrimental to viral assembly, ultimately leading to the loss of capsid integration into released sub viral particles and the alteration of the lipid membrane architecture. We surmised that lipid interactions around the region of the mutation were perturbed, leading to a loss of assembly capabilities but interestingly maintaining the budding mechanisms. The work of Chapter 2 will be submitted for publication to a peer reviewed journal shortly after the submission of this dissertation.</p><p dir="ltr">Chapter 3 expands on the ZIKV RVP results described in Chapter 2 by detailing a series of mutagenesis experiments into the role of the prM and E TMHs in the fully infectious ZIKV and DENV2 systems. Mutations within the prM TMH “turn” residues of DENV2 were found to also perturb virus infectivity, with two mutations within prM completely eliminating infectivity. The two mutants were found to be capable of producing NS5 and intracellular E protein that had been glycosylated, indicating that translation was intact and that E protein trafficking into the trans-Golgi network still occurred. However, unlike the results discussed in Chapter 2, the DENV2 mutants did not release any detectable E protein into their supernatants. This suggested that while the mutants could generate viral proteins and somehow undergo protein trafficking into the Golgi (signifying potential particle maturation), no particles were released. The DENV2 results were supported by reciprocal mutations in the prM proteins of ZIKV using fully infectious cDNA clones. The ZIKV prM mutants also eliminated virus infectivity and prevented the release of the E protein into the supernatant, indicating no release of viral particles, infectious or otherwise. Overall, the mutations in the fully infectious DNEV2 and ZIKV systems add further support for a novel role of the prM TMHs in flavivirus assembly.</p><p dir="ltr">Chapter 4 describes our efforts to reconstitute the flavivirus envelope glycoproteins into natively derived lipid nanoparticles for in vitro assembly analysis. Styrene-maleic acid copolymers (SMAs) were utilized for this study due to their ability to self-polymerize into highly hydrophobic chains in aqueous solutions. These hydrophobic chains can imbed themselves into lipid membranes to escape the aqueous environment, and in doing so “cut out” ~10nm diameter “patches” of native lipid membranes, along with any integrated membrane proteins. This “lipid/protein patch” is referred to as a styrene-maleic acid lipid nanoparticle (SMALP). Initially, attempts were made to generate SMALPs using purified Kunjin virus (KUNV) particles as the source of membrane lipids and glycoproteins due to their rapid growth rate and homogenous particle population. Unfortunately, attempts to generate SMALPs using purified KUNV were unsuccessful. It is hypothesized that the membrane curvature of purified KUNV particles generated a sterically and energetically unfavorable environment for SMALP generation, leading to the complete destruction of the particles during SMA mixing. To circumvent this issue, cells transfected with either WT or mutant ZIKV RVP cDNA were fractionated and purified ER membrane samples were mixed with SMAs to generate SMALPs. Western blot analysis suggested that the SMALP generation was successful. However, further experimentation is warranted to confirm this outcome and the structural integrity of the envelope glycoproteins within the SMALP.</p><p dir="ltr">Chapter 5 describes collaborative work on the identification of a novel compound inhibitor against flavivirus assembly, specifically targeting C protein’s interactions with RNA. This work was done in conjunction with a visiting scholar from the Indian Institute of Technology Mandi – Dr. Prateek Kumar – during his time at Purdue University from August 2022-May 2023. Much of the foundational computation work was done by Prateek prior to his arrival at Purdue University. As such, while the full context and results for the entirety of the study will be discussed, this chapter will primarily focus on the in vitro experimental results that were gathered directly by me, or results that were produced by Prateek and myself equally. This chapter demonstrates that a novel small molecule inhibitor against ZIKV C protein can, in fact, diminish ZIKV assembly by impeding C protein’s binding to RNA, prevent efficient RNA replication through binding and disruption of NS2B/3 protease, and perturb virus binding and entry prior to infection by also binding to E protein. Moreover, the novel molecule was also found to disrupt DENV2 infection as well, albeit to a lesser degree than ZIKV. This multifaceted molecule was recommended for further study in animal systems to continue testing its safety and efficacy for treatment of ZIKV and DENV2 in humans. A co-authorship manuscript has been completed on the work from this chapter and is currently awaiting submission to a peer reviewed journal.</p><p dir="ltr">Finally, Chapter 6 will combine the conclusions from the above chapters and discuss, in detail, aspects pertaining to the future of studies aiming to better understand the assembly of flaviviruses. This chapter will focus on how the link between viral assembly and membrane lipid architecture fits with previously established literature and what future directions could be employed to answer the questions proposed within.</p>

Arbovirus infections are widespread, and their disease burden has increased in the past decade. In Africa, arbovirus infections and fever with unknown etiology are common. Due to the lack of well-established epidemiologic surveillance systems and accurate differential diagnosis in most African countries, little is known about the prevalence of human arbovirus infections in Africa. The aim of this review is to summarize the available epidemiological data and diagnostic laboratory tools of infections with dengue, yellow fever, Zika, and chikungunya viruses, all transmitted by Aedes mosquitoes. Studies indicate that these arboviral infections are endemic in most of Africa. Surveillance of the incidence and prevalence of the infections would enable medical doctors to improve the diagnostic accuracy in patients with typical symptoms. If possible, arboviral diagnostic tests should be added to the routine healthcare systems. Healthcare providers should be informed about the prevalent arboviral diseases to identify possible cases.

As principais matérias primas necessárias para a preparação de testes diagnósticos são as proteínas dos patógenos que necessariamente apresentem as estruturas nativas. O objetivo do presente estudo foi a obtenção das proteínas não estruturais 1 (NS1) dos vírus da dengue (DENV) e da zika (ZIKV) a partir dos corpúsculos de inclusão (CI) produzidos em bactérias Escherichia coli. Mostramos que a combinação de alta pressão hidrostática (APH) e pH alcalino é eficiente para a solubilização de NS1-CI. A incubação em 2,4 kbar das suspensões de NS1-CI em pH alcalino mostrou-se eficiente para a solubilização da NS1. A presença de Arg promove a dissociação de oligômeros. A aplicação de 2,4 kbar às suspensões de NS1-CI em pH de 10,5 (DENV) e de 11,5 (ZIKV) na presença de Arg e um par redox, seguida de diálise em tampão em pH 8,5, foram as condições escolhidas para o reenovelamento de NS1. Obtivemos ambas NS1 com rendimentos entre 75% e 90% em relação às quantidades totais das proteínas presente nos correspondentes CI de NS1. As NS1 reenoveladas apresentaram reatividade comparável às proteínas obtidas utilizando um protocolo convencional estabelecido, com rendimentos mais de 25 vezes superiores. Foi obtido um processo altamente eficiente para o reenovelamento de NS1 apresentando características biológicas preservadas em relação a reatividade com anticorpos específicos de antígeno, incluindo soro de paciente infectado com zikv e que, portanto, podem ser usados como antígeno para o desenvolvimento de vacinas ou testes de diagnóstico. Além disso, este estudo descreve a criação de um processo inovador, que é a utilização concomitante de APH e pH alcalino, para solubilização e posterior reenovelamento de NS1-CI que podem ser utilizados para outras proteínas relevantes. / The main products for the preparation of diagnostic tests are as proteins of the pathogens that necessarily present as the native structures. The objective of the present study was to obtain non-structural proteins 1 (NS1) from dengue virus (DENV) and zika virus (ZIKV) from the inclusion bodies (IBs) produced in Escherichia coli bacteria. We show that it is a combination of high hydrostatic pressure (HHP) and alkaline pH is efficient for a solubilization of NS1-IB. A 2.4 kbar incubation of NS1-IB suspensions at alkaline pH proved to be efficient for NS1 solubilization. The presence of Arg promotes the dissociation of oligomers. The application of 2.4 kbar to the suspensions of NS1-IB at pH 10.5 (DENV) and 11.5 (ZIKV) in the presence of Arg and a redox pair, dialysis in pH 8.5 buffer were as conditions chosen for the refolding of NS1. We obtained both NS1 at yields between 75% and 90% relative to the total amounts of the proteins present in the corresponding NS1 IB. Refolded NS1 showed similar to proteins obtained using an established standard protocol, with yields more than 25 times higher. A highly efficient process for the refolding of NS1 was obtained with preserved biological features regarding reactivity with antigen-specific antibodies, including sera of zikv-infected patients and that can be used as antigen for the development of vaccines or diagnostic tests. In addition, this study describes the creation of an innovative process, which is a concomitant use of HHP and alkaline pH, for solubilization and subsequent refolding of NS1-IB that can be used for other relevant proteins.

As principais matérias primas necessárias para a preparação de testes diagnósticos são as proteínas dos patógenos que necessariamente apresentem as estruturas nativas. O objetivo do presente estudo foi a obtenção das proteínas não estruturais 1 (NS1) dos vírus da dengue (DENV) e da zika (ZIKV) a partir dos corpúsculos de inclusão (CI) produzidos em bactérias Escherichia coli. Mostramos que a combinação de alta pressão hidrostática (APH) e pH alcalino é eficiente para a solubilização de NS1-CI. A incubação em 2,4 kbar das suspensões de NS1-CI em pH alcalino mostrou-se eficiente para a solubilização da NS1. A presença de Arg promove a dissociação de oligômeros. A aplicação de 2,4 kbar às suspensões de NS1-CI em pH de 10,5 (DENV) e de 11,5 (ZIKV) na presença de Arg e um par redox, seguida de diálise em tampão em pH 8,5, foram as condições escolhidas para o reenovelamento de NS1. Obtivemos ambas NS1 com rendimentos entre 75% e 90% em relação às quantidades totais das proteínas presente nos correspondentes CI de NS1. As NS1 reenoveladas apresentaram reatividade comparável às proteínas obtidas utilizando um protocolo convencional estabelecido, com rendimentos mais de 25 vezes superiores. Foi obtido um processo altamente eficiente para o reenovelamento de NS1 apresentando características biológicas preservadas em relação a reatividade com anticorpos específicos de antígeno, incluindo soro de paciente infectado com zikv e que, portanto, podem ser usados como antígeno para o desenvolvimento de vacinas ou testes de diagnóstico. Além disso, este estudo descreve a criação de um processo inovador, que é a utilização concomitante de APH e pH alcalino, para solubilização e posterior reenovelamento de NS1-CI que podem ser utilizados para outras proteínas relevantes. / The main products for the preparation of diagnostic tests are as proteins of the pathogens that necessarily present as the native structures. The objective of the present study was to obtain non-structural proteins 1 (NS1) from dengue virus (DENV) and zika virus (ZIKV) from the inclusion bodies (IBs) produced in Escherichia coli bacteria. We show that it is a combination of high hydrostatic pressure (HHP) and alkaline pH is efficient for a solubilization of NS1-IB. A 2.4 kbar incubation of NS1-IB suspensions at alkaline pH proved to be efficient for NS1 solubilization. The presence of Arg promotes the dissociation of oligomers. The application of 2.4 kbar to the suspensions of NS1-IB at pH 10.5 (DENV) and 11.5 (ZIKV) in the presence of Arg and a redox pair, dialysis in pH 8.5 buffer were as conditions chosen for the refolding of NS1. We obtained both NS1 at yields between 75% and 90% relative to the total amounts of the proteins present in the corresponding NS1 IB. Refolded NS1 showed similar to proteins obtained using an established standard protocol, with yields more than 25 times higher. A highly efficient process for the refolding of NS1 was obtained with preserved biological features regarding reactivity with antigen-specific antibodies, including sera of zikv-infected patients and that can be used as antigen for the development of vaccines or diagnostic tests. In addition, this study describes the creation of an innovative process, which is a concomitant use of HHP and alkaline pH, for solubilization and subsequent refolding of NS1-IB that can be used for other relevant proteins.

Por representar a mais importante arbovirose em nível mundial, as infecções causadas pelos vírus da dengue são de grande importância em nosso país, apresentando uma ampla variedade de sintomas clínicos que vão desde infecção assintomática até formas mais graves da doença. O título de anticorpos neutralizantes produzidos frente à infecção por dengue parece ser determinante na forma de apresentação da doença no paciente. Atualmente, a forma com que o teste de neutralização é realizado demanda tempo para sua realização. O objetivo deste trabalho foi padronizar um ensaio de neutralização viral por RT-PCR em tempo real em cepas virais dos quatro sorotipos dengue para, posteriormente, ser empregada na detecção rápida e em grande escala de anticorpos em soro de pacientes e de candidatos vacinais. Para isso, foram construídas curvas padrão, para cada sorotipo viral, por meio da transcrição in vitro do RNA viral. O ensaio de neutralização padronizado nesse estudo reduziu o número de dias de detecção da neutralização em 48 horas quando comparada com a técnica de neutralização tradicional (PRNT), além de utilizar técnicas moleculares sensíveis e específicas para detecção como a RT-PCR em tempo real que garantem maior aplicabilidade do teste. / Because Dengue virus is the most important arboviral disease worldwide, infections caused by this pathogen are of great importance in Brazil, producing a wide variety of clinical symptoms ranging from asymptomatic infection to more serious forms of the disease. The title of neutralizing antibodies produced against the dengue infection appears to be determinant in the outcome of the disease. Currently, neutralization tests that have been performed take time for its execution. The aim of this study was to standardize a viral neutralization assay by real-time RT-PCR of viral strains of the four Dengue serotypes to subsequently be used for rapid detection and large-scale using antibodies of patients and for vaccine candidates. For this matter, standard curves were constructed for each Dengue serotype, by in vitro transcription of viral RNA. The neutralization assay in this study reduced the period of neutralization to 48 hours, compared to traditional neutralization test (PRNT), and it uses a more sensitive and specific molecular technique for detection of neutralizing antibodies, such as real time RT-PCR, to ensure greater applicability of the test

Les maladies virales à transmission vectorielle émergentes et ré-émergentes comme la dengue, le chikungunya ou le zika sont responsables de nombreuses épidémies sévères à travers le monde. Récemment, la propagation rapide et très étendue du virus zika (ZIKV) ainsi que les complications neurologiques graves liées à l’infection par ZIKV ont incité l’Organisation Mondiale de la Santé (OMS) à déclarer le ZIKV comme une urgence de santé publique mondiale. Aujourd’hui, les mesures préventives ou curatives de l’infection par ZIKV sont quasiment inexistantes. D’autre part, la flore endémique de La Réunion est connue comme une source riche, renouvelable et prometteuse en produits naturels anti-infectieux. L’inscription à la pharmacopée française de 19 plantes médicinales réunionnaises souligne le potentiel thérapeutique des substances naturelles issues de la biodiversité locale. Les travaux ont été consacrés à l’identification de substances naturelles anti-ZIKV issues d’une sélection de sept plantes médicinales réunionnaises inscrites à la pharmacopée française. Dans une première étape, une extraction sans solvant assistée par micro-ondes a été appliquée sur les sept plantes médicinales sélectionnées afin d’obtenir des éco-extraits. Dans une deuxième étape, le criblage de l’activité antivirale, en utilisant un clone moléculaire du ZIKV avec un gène rapporteur, a permis d’identifier deux éco-extraits actifs provenant de Aphloia theiformis et de Psiloxylon mauritianum. Après avoir vérifié l’absence de cytotoxicité et de génotoxicité des extraits actifs, l’activité antivirale a été aussi démontrée sur d’autres types d’extraits réalisés via des méthodes d’extractions traditionnelles (infusion, décoction et macération). L’activité antivirale a été validée sur différentes souches de ZIKV (historique et épidémique) ainsi que sur les 4 sérotypes du virus de la dengue. Enfin, le mode d’action des deux extraits actifs a été étudié. Il a été montré que les éco-extraits d’A. theiformis et de P. mauritianum ciblent les phases précoces du cycle viral en inhibant l’attachement du virus à la cellule hôte. À l’aide de la microscopie électronique, il a été montré que l’éco-extrait d’A. theiformis déforme la particule virale empêchant cette dernière de s’attacher à la membrane de la cellule hôte. Ces résultats démontrent l’importance des plantes médicinales réunionnaises comme source de substances naturelles anti-infectieuses. / Emerging and re-emerging vector-borne viral diseases such as dengue, chikungunya or zika are responsible for many severe epidemics worldwide. Recently, the rapid spread of zika virus (ZIKV) worldwide and the serious neurological complications associated with ZIKV infection have prompted the World Health Organization (WHO) to declare ZIKV a public health emergency. Today, preventive or curative measures against ZIKV infection are almost non-existing. On the other hand, the endemic flora of Reunion Island is known as a rich, renewable and promising source of natural anti-infective products. The registration of 19 medicinal plants from Reunion Island in the French pharmacopoeia highlights the therapeutic potential of natural substances derived from local biodiversity. The work was dedicated to the identification of natural anti-ZIKV substances from a selection of seven medicinal plants from La Reunion registered in the French pharmacopoeia. In a first step, in collaboration with a local start-up (Bourbon Extracts), a solvent-free microwave-assisted extraction was applied to the seven selected plants in order to obtain eco-extracts. In a second step, the screening for antiviral activity, using a ZIKV molecular clone with a reporter gene, allowed the identification of two candidate plants: Aphloia theiformis and Psiloxylon mauritianum. After verifying the absence of cytotoxicity and genotoxicity of the active extracts, the antiviral activity was also demonstrated on other types of extracts using traditional extraction methods (infusion, decoction and maceration). The antiviral activity has been validated on different strains of ZIKV (historical and epidemic) as well as on the four serotypes of the dengue virus. Finally, the mode of action of the two active extracts has been studied. It has been shown that the eco-extracts from A. theiformis and P. mauritianum target the early steps of the viral cycle by inhibiting the attachment of the virus to the host cell. Using electron microscopy, it has been shown that the eco-extract of A. theiformis deforms the viral particle preventing its attachment to the membrane of the host cell. These results demonstrate the importance of medicinal plants from Reunion Island as a source of natural anti-infectious substances.

Les formes sévères de l’infection par les virus de la fièvre jaune (YFV) et de la dengue (DENV) sont caractérisées par une atteinte du foie, plus sévère lors d’une infection YFV. L’objectif de cette thèse est de comparer les infections de YFV et DENV dans un modèle d’hépatocytes humains dérivés de cellules souches (iHeps) afin d’identifier des facteurs à l’origine de cette différence de pathogenèse. Dans un premier temps, nous avons comparé le tropisme de YFV aux 4 sérotypes de DENV dans notre modèle hépatique établi en monocouche cellulaire. Nous avons observé une faible propagation de DENV dans les iHeps par rapport YFV. Les mêmes observations ont été faites dans des hépatocytes primaires. L’utilisation de souches chimériques 17D/DENV a permis de mettre en évidence que cette faible propagation serait liée à une faible efficacité d’entrée de DENV dans les hépatocytes. Nous avons également étudié l’infection dans des sphéroïdes iHeps, métaboliquement plus actifs que les iHeps 2D. Une infection productive a été observée uniquement avec YFV. Ce résultat pourrait s’expliquer par la faible accessibilité des cellules à l’intérieur des sphéroïdes. Dans un 2ème temps, nous avons étudié les réponses cellulaires induites dans les iHeps 2D après infection par les différents virus en utilisant une approche RNAseq. Les résultats préliminaires suggèrent un lien entre le taux de réplication et le nombre de gènes activés. La réponse interféron est plus précocement détectée dans le cas de YFV, mais l’infection par DENV induit un plus grand nombre de gènes. De plus, DENV-1 et DENV-4 induisent une augmentation d’expression de certains gènes impliqués dans la présentation d’antigène comme HLA-E et TAP-2, alors que YFV diminue l’expression de certains gènes de chimiokines et molécules d’adhésion. L’analyse préliminaire des voies liées au métabolisme hépatique révèle une inhibition de la voie de la coagulation dans le cas de l’infection par YFV, qui n’est pas observée lors de l’infection par DENV. Des observations similaires ont été décrites in vivo, au niveau protéique, confirmant la pertinence du modèle iHeps / Severe forms of infection with yellow fever virus (YFV) and dengue virus (DENV) are characterized by liver damage, with more severe symptoms observed during YFV infection. The aim of this thesis is to compare YFV and DENV infections in a model of human hepatocytes derived from stem cells (iHeps) in order to identify factors that could explain their difference in pathogenesis.First, we compared YFV tropism to the four DENV serotypes in 2D iHeps. We observed a low spread of DENV compared to YFV in both iHeps and primary hepatocytes. By using chimeric 17D/DENV strains, we demonstrate that this low propagation is linked to a low DENV entry efficiency in hepatocytes. We also studied infection in iHeps spheroids, metabolically closer to primary cells than 2D iHeps. A productive infection was observed with YFV only. The low accessibility of cells inside the spheroids could explained this result. Second, we studied cellular responses induced following infection by different viruses in 2D iHeps using an RNAseq approach. Preliminary results suggest a link between replication rate and the number of activated genes. The interferon response is detected earlier following YFV infection, but DENV induces a greater number of genes implicated in this pathway. Moreover, DENV-1 and DENV-4 up-regulate some genes involved in antigen presentation such as HLA-E and TAP-2, while YFV down-regulates genes encoding chemokines and adhesion molecules. Preliminary analysis of hepatic metabolism pathways reveals inhibition of the coagulation pathway induced by YFV infection, which is not observed during DENV infection. Similar observations have been described in vivo, at the protein level, confirming the relevance of the iHeps model

Source: Masters Abstracts International, Volume: 47-06, page: 3395. Advisers: Kristy O. Murray; George L. Delclos. Includes bibliographical references.