Les Alphavirus sont des virus à ARN enveloppés, d’un diamètre de 70 nm, à structure icosaédrique à symétrie de type T=4 (Choi et al. 1991; Cheng et al. 1995; Garoff et al. 2004). Ces virus, dont la répartition est mondiale, sont capables d’infecter une grande variétés d’animaux vertébrés (mammifères, oiseaux, poissons). Ces virus sont des arbovirus, c’est-à_dire des virus transmis par des arthropodes. Dans le cas des Alphavirus, la vectorisation est faite par des moustiques appartenant à plusieurs espèces. A ce jour, 29 espèces d’Alphavirus ont été identifiés, dont au moins 6 sont pathogènes pour l’Homme. Chez l’Homme, certains Alphavirus sont responsables d'encéphalites, d'arthrites, de fièvres, d'éruptions cutanées et peuvent être fatals (Thiruvengadam et al. 1965; Pialoux et al. 2006).Le premier Alphavirus isolé fut l'Encephalite Equine de l'Ouest (WEEV), en 1930 (Meyer et al. 1931). Les virus de l'encéphalite de l'Est (VEEV) et le virus de l'Encephalite Equine du Vénézuéla (VEEV) furent isolés respectivement en 1933 et 1938 (Gibbs EP. 1976 ; Beck et al. 1938 ; Kubes et al. 1939). Le Virus Sindbis isolé en Egypte en 1952 (Taylor et al. 1955), fut le premier Alphavirus responsables d’arthrites à être isolé. La mise en évidence de l'existence du CHIKV se fera 1952 en Tanzanie (Robinson 1955) (Lumsden 1955). Suivent alors les découvertes de l'ensemble des autres Alphavirus. Le South Elephent Seal virus (SESV), identifié en 2000 sur l'île australienne de Macquarie, est à l’heure actuelle le dernier Alphavirus découvert. La phylogénétique des souches de Chikungunya permet d’identifier des clades différents pour les souches d’Afrique de l’Est, de l’Ouest ou d’Asie, et l’analyse phylogénétique est très proche du O’Nyong-Nyong (Powers et al. 2000), Le séquençage de différents isolats de l’épidémie de 2005, a permis de mettre en évidence chez certains d’entre eux une mutation dans la glycoprotéine de l'enveloppe plus précisément dans E1, (Schuffenecker et al. 2006). Cette mutation entraine la substitution d'une arginine en position 226 au lieu de la valine (A226V), est un élément clé pour déterminer le choix d'un nouveau vecteur pour la transmission ou Aedes albopictus (qui le transmet sur l'île de La Réunion) par rapport au vecteur Aedes aegypti (Tsetsarkin et al. 2007). Cette mutation a ensuite été également trouvée en Inde en 2007. (Arankalle et al. 2007; Kumar et al. 2008; Santhosh et al. 2008).Le tableau clinique classique débute souvent par l’apparition brutale d’une forte fièvre (40°C) pendant 3 / 10 jours accompagnée de frissons intermittents (Deller et al.1967). La fièvre est, dans certains cas, bi-phasique, c’est-à-dire qu’elle diminue durant un ou deux jours, avant de remonter brutalement. Elle est généralement suivie d’érythèmes, de courbatures douloureuses ou myalgies et douleurs musculaires (Ozden et al. 2007) en particulier celles impliquant la douleur au niveau des extrémités (poignés, phalanges et chevilles) (Robinson 1955; Jadhav et al. 1965; Thiruvengadam et al. 1965). Egalement migraine, éruptions cutanées maculo-papuleuses parfois prurigineuses. L'éruption touchant le thorax et le visage les mains et les pieds, chez les enfants ont été observées des éruptions de type bulleux accompagné par un détachement cutané (Talarmin et al. 2007). L'évolution de la maladie régresse progressivement. Il n’y a aucun traitement antiviral efficace contre le CHIKV. Le traitement est donc essentiellement symptomatique et composé d'antalgiques non salicylés, de paracétamol et d'anti-inflammatoires non stéroïdiens. Ce travail se compose de deux parties : 1 partie sur l’étude phylogénétique du CHIKV et 1 partie sur l'étude des molécules antivirales. [...] / The Alphavirus RNA viruses are enveloped with a diameter of 70 nm, icosahedral structure with symmetry of type T = 4 (Choi et al. 1991; Cheng et al. 1995; Garoff et al. 2004). These viruses, whose distribution is worldwide, can infect a wide variety of vertebrates (mammals, birds, fish). These viruses are arboviruses, is à_dire viruses transmitted by arthropods. In the case of Alphavirus, the vectorization is done by mosquitoes from several species.To date, 29 species of Alphavirus have been identified, including at least six are pathogenic for humans. In humans, some are responsible for Alphavirus encephalitis, arthritis, fever, rash and can be fatal (Thiruvengadam et al. 1965; Pialoux et al. 2006).The first was isolated Alphavirus Equine Encephalitis West (Weeve) in 1930 (Meyer et al. 1931). The encephalitis virus Eastern (VEEV) and virus Venezuelan equine encephalitis (VEEV) were isolated respectively in 1933 and 1938 (Gibbs EP. 1976; Beck et al. 1938; Kubes et al. 1939 ). Sindbis virus isolated in Egypt in 1952 (Taylor et al. 1955), was the first Alphavirus responsible for arthritis to be isolated. The demonstration of the existence of CHIKV in Tanzania will be 1952 (Robinson 1955) (Lumsden 1955). Then follow the discoveries of all other Alphavirus. The South Elephent Seal virus (SESV), identified in 2000 on the Australian island of Macquarie is now the last Alphavirus discovered. The phylogenetic strains of Chikungunya can identify different clades for strains of East African, Western or Asian, and phylogenetic analysis is very close O'Nyong-Nyong (Powers and al. 2000), The sequencing of different isolates of the epidemic of 2005, helped to highlight some of them a mutation in the envelope glycoprotein more specifically in E1 (Schuffenecker et al. 2006). This mutation causes the substitution of an arginine at position 226 instead of valine (A226V), is a key element in determining the choice of a new vector for the transmission or Aedes albopictus (which transmits on the island of La meeting) with respect to the vector Aedes aegypti (Tsetsarkin et al. 2007). This mutation was later also found in India in 2007. (Arankalle et al. 2007; Kumar et al. 2008; Santhosh et al. 2008).The classic presentation often begins with sudden onset of high fever (40°C) for 3 / 10 days intermittent chills (Deller and al.1967). Fever is, in some cases, bi-phasic, that is to say, it decreases during a day or two before rising sharply. It is usually followed by erythema, pain or stiffness of muscle pain and muscle aches (Ozden et al. 2007) especially those involving pain in the extremities (wrists, ankles and knuckles) (Robinson 1955; Jadhav et al. 1965; Thiruvengadam et al. 1965). Also headache, rash maculopapular itchy sometimes. The rash affecting the chest and face hands and feet, children were seen eruptions like bullous skin accompanied by a detachment (Talarmin et al. 2007). The evolution of the disease regresses gradually. There is no antiviral therapy effective against CHIKV. Treatment is essentially symptomatic and consists of non-analgesic salicylates, paracetamol and anti-inflammatory drugs. This work consists of two parts: one part on the phylogenetic study of CHIKV and one part of the study of antiviral molecules. [...]

Orientador: Adriano Mondini / Resumo: Arbovírus são vírus transmitidos por artrópodes. Apresentam grande importância no âmbito da saúde pública devido à morbidade e mortalidade de suas infecções. Nos últimos quatro anos, muitos casos de infecção pelos vírus Zika (ZIKV) e Chikungunya (CHIKV) foram notificados no Brasil. O controle de seu principal vetor, o mosquito Aedes aegypti, é uma das formas de combate a esses vírus. Não há, até o momento, terapia antiviral específica contra infecções causadas por esses vírus, e os meios de controle do vetor, disponíveis, geram resistência e danos ambientais. Assim, compostos naturais, com ação antiviral ou que controlem o vetor, tornam-se uma via importante e atrativa. O extrato de inhame (Colocasia esculenta) possui entre suas substâncias a tarina, uma lectina que demonstrou atividade antiviral contra o DENV e HCV, além de atividade larvicida contra mosquitos Diaphania nitidalis. O objetivo do trabalho foi avaliar a atividade do extrato de Colocasia esculenta, obtidos por dois métodos de extração, quanto a sua atividade antiviral contra o CHIKV e ZIKV, bem como quanto a sua ação larvicida contra o mosquito Aedes aegypti. Os extratos foram obtidos a partir das folhas e do caule do inhame, e extraídos com a utilização de água e metanol. Os extratos aquosos e metanólico foram obtidos por técnica de maceração. Para a avaliação da ação antiviral, os extratos foram utilizados em ensaios de citotoxidade e de redução de placas em cultura celular, bem como PCR em tempo real (qPCR) p... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Le ré-encodage aléatoire des codons à grande échelle est une nouvelle méthode d'atténuation virale qui consiste en l'insertion d'un grand nombre de mutations synonymes, individuellement peu délétères, de façon aléatoire dans une ou plusieurs régions codantes d'un virus. Cette approche permet de diminuer de façon significative et modulable le fitness réplicatif des virus in cellulo et in vivo, ainsi que la pathogénicité du virus chez la souris, tout en induisant une protection immunitaire spécifique et efficace lors d'une nouvelle infection par le virus sauvage. Les virus ré-encodés présentent également une grande stabilité et une absence de réversion ce qui en font des candidats vaccins très prometteurs en termes d'efficacité et de fiabilité pour la conception de candidats vaccins vivants atténués contre une grande variété de virus à ARN. La combinaison du ré-encodage aléatoire et d'une nouvelle méthode de génétique inverse permettant de générer de nouveaux virus en quelques jours: ISA (Amplicon Subgenomique Infectieux), est une approche prometteuse qui pourrait aider au développement de vaccins vivants atténués de nouvelle génération en un temps record. / Large-scale random codon re-encoding is a new method of viral attenuation consisting in the insertion of a high number of slightly deleterious synonymous mutations, randomly, in one or several coding regions of a virus. This approach significantly reduces the replicative fitness of re-encoded viruses in cellulo and in vivo, as viral pathogenicity, while inducing a specific and effective immune response in mice against a new infection with wild-type viruses. Re-encoded viruses also present a high stability and an absence of reversion, making them promising vaccine candidates in term of reliability and efficiency for the conception of new vaccine candidates against a wide variety of RNA viruses. Combination of random re-encoding with a new method of revers genetics allowing to generate new viruses in days : ISA (Infectious Subgenomic Amplicons) would be very helpful to develop new-generation vaccine candidates.

The world is grappling with an escalating risk of viral outbreaks of pandemic proportion, with zoonotic RNA viruses such as chikungunya virus (CHIKV) and SARS-CoV-2 posing significant threats to global health. Several environmental and evolutionary factors have fueled the emergence and spread of infection, creating a constant arms race against emerging pathogens. Current prevention and mitigation strategies are inadequate, necessitating tools to prevent and control viral infections; innovative strategies are needed in the pipeline to address significant challenges. CHIKV is a mosquito-borne virus that has caused millions of disease cases worldwide and is a reemerging threat with increasing potential to become endemic in the US. Currently, there are no licensed treatments available to protect against CHIK disease, making the development of a vaccine crucial. Live-attenuated vaccines (LAVs) have traditionally been a promising strategy due to their high immunogenicity and cost-effectiveness. However, concerns regarding adverse side effects and the potential for viral replication leading to pathogenic reversions or transmission into mosquitoes have limited their use. To that end, we have developed a new generation of safer vaccines by modifying the standard LAV platform through innovative attenuating strategies. Our dual-attenuated platform utilizes a previously developed chimera of CHIKV and the closely related Semliki Forest virus (SFV) as a vaccine backbone which expresses antiviral mouse cytokines IFN-γ or IL-21, as an additional mechanism to control infection. In several mouse models, both cytokine-expressing candidates showed reduced footpad swelling and minimal to no systemic replication or dissemination capacity compared to the parental vaccine post-vaccination. Importantly, these candidates conferred full protection from wildtype CHIK disease. Our IFNγ-expressing vaccine showed the most significant attenuation of viral replication. To understand the underlying mechanism, we identified three IFNγ-regulated antiviral genes (Gbp1/2 and Ido1) that were highly upregulated in 3T3 mouse fibroblasts post-infection with the IFN-γ-expressing candidate but not the parental backbone. To further investigate the role of these genes in restricting viral replication and enhance the clinical relevance of our vaccine platform, we redesigned our vaccine to express human IFNγ (hIFNγ) and performed viral growth kinetics in MRC5 human lung fibroblasts. Our vaccine showed reduced viral replication compared to controls and high expression of human GBP1/2/3 was observed post-infection. Overexpression of these genes demonstrated a direct impact on viral replication against wildtype CHIKV. These findings shed light on the mechanism of action of our vaccine and highlight the potential of targeting IFNγ-regulated antiviral genes for developing effective vaccines against CHIKV. Our results provided a foundation for investigating the broad-use application of IFN-γ against other alphaviruses for vaccine or therapeutic design. We evaluated the effects of increasing levels of exogenous hIFNγ on Mayaro virus (MAYV), Ross River virus (RRV), and Venezuelan Equine Encephalitis virus (VEEV). We observed a positive dose-dependent relationship between hIFNγ and decreasing viral titers for all three viruses. Interestingly, we also observed similar patterns of GBP upregulation with MAYV and RRV, both Old World alphaviruses, but not with VEEV, a New World alphavirus. This finding may indicate an alternative IFNγ-stimulated pathway responsible for controlling different alphaviruses. Overall, these studies establish a fundamental role of IFNγ in controlling viral infection and highlight its potential use in both vaccine and therapeutic intervention. While LAVs are a gold standard for developing immunity against a virus, the urgency of responding to an active and deadly pandemic has promoted the use of faster strategies such as mRNA vaccines. Once the viral sequence was known, these vaccines were comparatively quick to produce for SARS-CoV-2 and prevented millions of disease cases at the height of their introduction. However, the emergence of variants of concerns bypassing previous immunization efforts has demonstrated the need for complementary treatments such as antivirals to control disease. To that end, we evaluated several rhodium organometallic complexes as potential antivirals against SARS-CoV-2. We show that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(ICy)Cl2 and Cp*Rh(dpvm)Cl are non-toxic in Vero E6 and Calu3 cells and reduce SARS-CoV-2 plaque formation up to 99%. These complexes have previously demonstrated high antimicrobial activity against multiple antibiotic-resistance bacteria and with our results, support their potential application as pharmaceuticals, warranting further investigation into their activity. / Doctor of Philosophy / The global response to the COVID-19 pandemic, and its far-reaching impact, revealed significant shortcomings in public health preparedness for emerging viruses. Despite efforts to develop vaccines and antivirals to prevent and treat disease, current mitigation strategies have proven insufficient to eradicate the pathogen. The emergence of viral outbreaks caused by viruses such as chikungunya (CHIKV) and SARS-CoV-2 underscores the ongoing threat posed by emerging infectious diseases. Improved countermeasures are urgently needed to address gaps in vaccine and antiviral development. CHIKV is a mosquito-borne virus that has caused millions of infections across hundreds of countries with the emergent potential to become endemic in the US. Currently, there are no vaccines available to the public; therefore, it is important to generate and administer an effective vaccine before further spread of the virus. To this end, we developed innovative live-attenuated vaccines (LAVs) against CHIKV using a weakened chimeric backbone of CHIKV and its close relative, Semliki Forest virus (SFV), along with vaccine-driven expression of antiviral cytokines to control viral replication. Vaccination of highly susceptible mice with these cytokine-expressing vaccines produced significantly decreased side-effects compared to the parental virus not expressing the cytokines. Additionally, these viruses had significantly restricted viral replication capabilities while robustly protecting mice from a semi-lethal CHIKV infection. Our interferon-gamma (IFNγ) expressing vaccine had the greatest impact on viral replication, and we investigated the mechanism leading to this attenuation. To assess the clinical relevance of our vaccine platform, we redesigned the virus to express human IFNγ and identified a specific pattern of IFNγ-stimulated genes that are potentially responsible for limiting CHIKV replication. Furthermore, we demonstrated the broad therapeutic use of IFNγ against other medically relevant alphaviruses. Overall, these studies establish an improved mechanism to create safer vaccines without compromising efficacy and highlight the therapeutic potential of IFNγ against alphaviruses. Lastly, in a collaborative effort to respond to the COVID-19 pandemic, we also explored and characterized the use of a new class of antiviral drugs. With the advent of increasing drug resistance, it is essential to develop novel and resilient therapeutics. We demonstrated the first antiviral potential of rhodium organometallics, which was previously shown to be effective against multiple antibiotic-resistant bacteria. Two complexes demonstrated high virucidal activity against SARS-CoV-2 and low toxicity in mammalian cell lines. Moreover, these complexes can be further derivatized to improve efficacy, making them a promising new antiviral strategy.

Zusammenfassung Yauhen Karliuk Einfluss der 3' nichttranslatierten Region von Chikungunya-Virus auf die Replikation in verschiedenen Stechmückenarten Institut für Tierhygiene und Öffentliches Veterinärwesen der Veterinärmedizinischen Fakultät, Universität Leipzig Eingereicht im Februar 2022 52 Seiten, 7 Abbildungen, 116 Literaturangaben, 1 Publikation Schlüsselwörter: Chikungunya-Virus; 3′ UTR; direct repeats (DRs); CHIKV 3́ UTR-Deletionsmutante; Vektorkompetenz; Aedes vexans; Culex pipiens Einleitung: Arthropoden-übertragene Viren (Arboviren) spielen weltweit eine große Rolle für die Gesundheit von Menschen und Tieren. Das Chikungunya-Virus (CHIKV) wird v.a. durch Stechmücken der Gattung Aedes übertragen. Die Hauptvektoren sind Aedes aegypti (Ae. aegypti) und Aedes albopictus (Ae. albopictus), wobei letzterer sich zunehmend auch in gemäßigten Breiten etabliert. Ziele der Untersuchungen: Zum einen sollte untersucht werden, ob auch die Stechmückenarten Aedes vexans (Ae. vexans) und Culex pipiens molestus (Cx. pipiens), die in gemäßigten Klimazonen vorkommen, als CHIKV-Vektoren fungieren können. Zum anderen sollte der Einfluss von Deletionen der Sequenzwiederholungen (DR) in der 3‘ nichttranslatierten Region (3‘ UTR) auf die virale Replikation in Zellkultur und in Stechmücken untersucht werden. Tiere, Material und Methoden: Zunächst wurde eine CHIKV 3́ UTR-Deletionsmutante mit einer Deletion von DR1a und DR2a in der 3́ UTR (CHIKV-ΔDR) hergestellt und diese bezüglich der Wachstumskinetik mit Chikungunya-Wildtyp-Virus (CHIKV-WT) in C6/36- und Aag2-Stechmückenzellen sowie in BHK-21/J- Wirbeltier-Zellen verglichen. Um die Vektorkompetenz von beiden Viren in Stechmücken zu untersuchen, wurden Ae. aegypti, Ae. albopictus, Ae. vexans und Cx. pipiens in einem Insektarium gezüchtet. Bei den Infektionsexperimenten im S3-Labor wurden insgesamt 27 Ae. aegypti, 20 Ae. albopictus, 78 Ae. vexans und 62 Cx. pipiens Stechmücken verwendet. In diesen Experimenten wurden diese mit CHIKV-ΔDR und CHIKV-WT sowohl oral mit je 1x 10^6 PFU/ml über eine Fütterungsmembran als auch intrathorakal mit je 200 PFU (zur Umgehung der Mitteldarmbarriere) infiziert und an verschiedenen Tagen nach der Infektion und in verschiedenen Körperteilen sowie im Speichel auf virale RNA mittels Real-Time Reverser Transkription-Polymerase Kettenreaktion (RT-PCR) untersucht. Unterschiede in der Virusreplikation wurden entweder mit Mann-Whitney- oder Fisher’s Exakt-Test überprüft. Das Signifikanzniveau lag bei p < 0,05. Ergebnisse: Beide Viren, das CHIKV-WT und das CHIKV-ΔDR, zeigten ein vergleichbares Wachstum in Wirbeltier-Zellen (BHK-21/J) und erreichten einen Titer von 5x 10^8 PFU/ml. Das Wachstum beider Viren war auch in von Ae. albopictus abgeleiteten C6/36- Stechmückenzellen effizient, wobei CHIKV-WT ein um knapp eine Log-Stufe höheres Wachstum zeigte als CHIKV-ΔDR. In unseren Experimenten zeigte CHIKV-WT ein weniger effizientes Wachstum in von Ae. aegypti abgeleiteten Aag2-Stechmückenzellen, als in Ae. albopictus abgeleiteten C6/36-Stechmückenzellen, obwohl Ae. aegypti als Hauptvektor für CHIKV-WT gilt. In einer intrathorakalen und oralen Infektion konnten sowohl die bekannten CHIKV-Vektoren Ae. aegypti und Ae. albopictus als auch die einheimische Stechmückenarten Ae. vexans und Cx. pipiens erfolgreich infiziert werden. Bei einer intrathorakalen Infektion mit Umgehung der Mitteldarmbarriere wurde bei Ae. vexans oder Cx. pipiens eine effizientere Virusreplikation beobachtet als bei einer oralen Infektion. CHIKV-WT zeigte eine signifikant höhere Replikation in Ae. vexans im Vergleich zu CHIKV-ΔDR am Tag 7 und am Tag 14 nach der Infektion. Bei Cx. pipiens wurden signifikante Unterschiede für CHIKV-WT im Vergleich zu CHIKV-ΔDR nur am Tag 7 beobachtet. Schlussfolgerungen: Das beeinträchtigte Wachstum in C6/36- und Aag2-Zellen von CHIKV-ΔDR deutet darauf hin, dass die deletierten Sequenzwiederholungen spezifisch mit noch unbekannten Faktoren in Stechmückenzellen interagieren. Dennoch konnte CHIKV-ΔDR die bekannten CHIKV-Vektoren Ae. aegypti und Ae. albopictus problemlos nach intrathorakaler und oraler Infektion infizieren. Die Mitteldarm-Entweichungsbarriere scheint also nicht der einzige Faktor zu sein, der die Vektorkompetenz von Stechmücken beeinflusst. Auch die Replikationskinetik des Virus in den Sekundärgeweben scheint bei den verschiedenen Stechmückenarten unterschiedlich zu sein. Zwar umfassten unsere Studien zur oralen Infektion mit CHIKV nur einige einheimische Ae. vexans und Cx. pipiens Stechmücken, jedoch deuten die Ergebnisse darauf hin, dass diese Stechmücken potenziell als Vektoren für CHIKV dienen können.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 Einleitung 1 2 Literaturübersicht 2 2.1 Alphaviren 2 2.1.1 Klassifikation 2 2.1.2 Virusmorphologie und Genomaufbau 2 2.1.3 Virusreplikation 4 2.2 Chikungunya-Virus 5 2.2.1 Übertragungszyklus 5 2.2.2 Epidemiologie 7 2.2.3 Genotypen und die 3′ UTR Region 9 2.2.4 Chikungunya-Fieber 12 2.3 Stechmücken 13 2.3.1 Taxonomie und Stechmückenarten in Deutschland 13 2.3.2 Allgemeine Morphologie, Biologie und Ökologie 14 2.3.2.1 Eiablage und Schlüpfen der Larven 15 2.3.2.2 Aquatische Entwicklungsstadien 16 2.3.2.3 Adulte 17 2.3.2.4 Flugverhalten und Überwinterungsstrategien 19 2.3.3 Arboviren in Deutschland 20 3 Publikation 26 3.1 Stellungnahme zum Eigenanteil an den Arbeiten zur Publikation 26 3.2 Publikation 27 4 Diskussion 42 5 Zusammenfassung 49 6 Summary 51 7 Literaturverzeichnis 53 8 Danksagung 66