Rozprawy doktorskie na temat „Flavivirus Infection”

The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.

Resistance to flavivirus-induced disease is conferred by a single gene that encodes oligoadenylate synthetase (Oas) 1b (Oas1b). Oas1b is not a functional synthetase suggesting its anti-flavivirus mechanism is RNase L-independent and that it may be mediated by interactions with other host cell protein(s). A yeast two-hybrid screen was used to identify host cell binding partners of Oas1b. Candidate partners were confirmed by yeast co-transformation and co-immunoprecipitation analyses. Oxysterol binding protein-related 1L (ORP1L) and ATP binding cassette subfamily F 3 (ABCF3) were found to interact with Oas1b. RNAi knockdown studies suggested that ORP1L and ABCF3 form a tripartite complex with Oas1b that is critical for the flavivirus-induced disease resistance mechanism. Stresses including oxidation, nutrient starvation, and viral infections often induce the formation of stress granules (SGs) in eukaryotic cells. In response to stress, eIF2α kinases phosphorylate eIF2α leading to stalled 48S pre-initiation complexes and SG formation. West Nile virus (WNV) Eg101 infections were previously shown not to induce the formation of SGs. Infections with viruses of other natural WNV strains, as well as a WNV lineage 1/2-based infectious clone (W956IC) were analyzed and only W956IC infections were found to induce SGs. eIF2α kinase knockout MEFs were used to show that the W956IC-induced SGs were PKR-dependent. WNV chimeras were made by inserting Eg101 genes into the W956IC backbone. Chimeras replacing NS5 or NS1 and NS5 or NS1 and NS3 and NS4a reduced SG formation as well as early viral RNA synthesis similar to Eg101 infections. W956IC infections but not Eg101 infections were shown to produce exposed viral dsRNA at early times after infection. The data suggest that natural WNV infections evade the cell SG response by suppressing the amplification of viral RNA until cytoplasmic membranes have been remodeled to protect replication complexes from detection. It was previously reported that WNV Eg101 infections inhibited the formation of arsenite-induced SGs. The ability of other natural WNV strain infections to inhibit SG formation by arsenite (HRI), DTT (PERK), W956IC co-infection (PKR), and heat shock treatments was assessed. WNV infections only inhibited arsenite-induced SG formation suggesting that WNV infections specifically suppress the response to oxidative intermediates.

Background: A characteristic feature of many infections is that only a portion of exposed individuals develop clinical disease. These include mosquito-borne flaviviruses such as West Nile virus (WNV), Zika virus (ZIKV) and Usutu virus (USUV) infections, which generally cause mild illness or asymptomatic infections in humans. Nonetheless, WNV can cause serious neuroinvasive diseases in less than 1% of infected patients, mainly elderly and immunocompromised subjects; ZIKV may cause fetal microcephaly in about 5% of infections acquired during pregnancy and 1 in 10,000 infected adults develop Guillain-Barré syndrome; USUV seems less pathogenic than WNV and most human infections described so far were asymptomatic, with rare cases of encephalitis or meningitis. Aim of the study: The different infection outcomes or progression to severe disease can be partly explained by host genetic variations, but the genetic traits associated with susceptibly to severe infection remain poorly understood. Aim of this study was to develop a patient-specific in vitro platform, based on human induced pluripotent stem cells (hiPSCs), to investigate the mechanisms of variations in human susceptibility to severe flavivirus infection. Methods: iPSCs were generated from erythroblasts of two blood donors with asymptomatic WNV infection (controls) and from two patients who developed WNV encephalitis but had no co-morbidity or other risk factors (cases). Patient-specific iPSCs were differentiated into neural stem cells (NSCs) and infected with WNV lineage 1 (GU011992), ZIKV Asian lineage (KU853013), and USUV lineage Europe 1 (AY453411) at different MOIs. Time course experiments were performed to evaluate viral replication kinetics in infected NSCs, cell viability and cell death following infection, and expression of genes involved in antiviral innate immunity. Next-generation sequencing of 2,600 genes related to immune system in iPSCs of cases and controls was performed to detects mutations potentially associated with increased susceptibility to neuroinvasive disease. Results: USUV and WNV replicated more efficiently, yielding 10 and 100-fold higher viral load and inducing 40% and 70% higher cell mortality, respectively, in NSCs derived from cases than in NSCs derived from controls. WNV induced 3-fold higher caspase 3 activity in infected NSC derived from encephalitis patients than in NSCs derived from asymptomatic donors. Several genes involved in the antiviral IFN pathway were significantly upregulated after USUV, ZIKV and WNV infection (in particular, type 3 IFNs genes), but the general trend indicated an attenuated response in NSCs derived from WNV encephalitis cases, which showed significantly lower mRNA levels of IFN pathway regulators such as TLR3, MAVS and IRF7. Exome sequencing analysis identified heterozygous inactivating mutations in the PSIP1 and DDX58 genes of cases, but not in controls, as polymorphism in other genes that could play a role in disease susceptibility. Conclusions: Patient-specific iPSCs are useful tools to model individual susceptibility to viral infectious diseases and allowed to demonstrate that WNV and USUV and, to a lesser extent, ZIKV, replicated more efficiently and induced more cell death and apoptosis in NSCs derived from patients with WNV encephalitis than in cells derived from blood donors with asymptomatic infection. This increased susceptibility to neurotropic flaviviruses was associated with a significantly attenuated innate antiviral response. Exome sequencing revealed inactivating mutations in genes that represent good candidates for further investigation.

Vector-borne diseases are an increasing global threat to humans due to climate changes, elevating the risk of infections transmitted by mosquitos, ticks, and other arthropod vectors. Ixodes ricinus, a common tick in Europe, transmits dangerous tick-borne pathogens to humans. Tick-borne encephalitis (TBE) is a vector-borne disease caused by TBE virus (TBEV). Climate change has contributed to increased tick abundance and incidence of tick-borne diseases, and between 10,000 and 15,000 human TBE cases are reported annually in Europe and Asia. TBEV shows a patchy geographical distribution pattern where each patch represents a natural focus. In nature, TBEV is maintained within the tick-rodent enzootic cycle. Co-feeding is the main route for TBEV transmission from infected to uninfected ticks and for maintenance within the natural foci. The increasing number of TBE cases in Scandinavia highlights the importance of characterizing additional TBEV sequences and of identifying novel natural foci, and in this work we sequenced and phylogenetically characterized four TBEV strains: Saringe-2009 (from a blood-fed nymph), JP-296 (from a questing adult male), JP-554 (from a questing adult male), and Mandal-2009 (from a pool of questing nymphs, n = 10). Mandal-2009 represents a TBEV genome from a natural focus in southern Norway. Saringe-2009 is from a natural endemic focus in northern Stockholm, Sweden, and JP-296 and JP-554 originate from a natural focus “Torö” in southern Stockholm. In addition, we have studied the effect of different biotic and abiotic factors on population dynamics of I. ricinus in southern Stockholm and observed significant spatiotemporal variations in tick activity patterns. Seasonal synchrony of immature stages and total tick abundance are important factors for the probability of horizontal transmission of TBEV among co-feeding ticks. We found that the probability of co-occurrence of larvae, nymphs, and female adults was highest during early summer whereas increasing vegetation height and increasing amounts of forest and open water around the study sites had a significant negative effect on co-occurrence of larvae, nymphs, and female adults. The proximal part of the 3 ́non-coding region (3 ́NCR) of TBEV contains an internal poly(A) tract, and genomic analysis of Saringe-2009 revealed variability in the poly(A) tract indicating the existence of different variants within the TBEV pool of Saringe-2009. Like other RNA viruses, TBEV exists as swarms of unique variants called quasispecies. Because Saringe-2009 came from an engorged nymph that had been feeding on blood for &gt;60 h, we propose that Saringe-2009 represents a putative shift in the TBEV pool when the virus switches from ectothermic/tick to endothermic/mammalian environments. We investigated the role of poly(A) tract variability in replication and virulence of TBEV by generating two infectious clones of the TBEV strain Toro-2003, one with a short/wild-type (A)3C(A)6 poly(A) tract and one with a long (A)3C(A)38 poly(A) tract. The infectious clone with the long poly(A) tract showed poor replication in cell culture but was more virulent in C57BL/6 mice than the wild-type clone. RNA folding predictions of the TBEV genomes suggested that insertion of a long poly(A) tract abolishes a stem loop structure at the beginning of the 3 ́NCR. Next generation sequencing (NGS) analysis of the TBEV genomes after passaging in cell culture and/or mouse brain revealed molecular determinants and quasispecies structure that might contribute to the observed differences in virulence. Our findings suggest that the long poly(A) tract imparts instability to the TBEV genome resulting in higher quasispecies diversity that in turn contributes to TBEV virulence. Phylogenetic analysis of Saringe-2009, JP-296, JP-554, and Mandal-2009 predicted a strong evolutionary relationship among the four strains. They clustered with Toro-2003, the first TBEV strain from Torö, demonstrating a Scandinavian clade. Except for the proximal part of the 3 ́NCR, TBEV is highly conserved in its genomic structure. Genomic analysis revealed that Mandal-2009 contains a truncated 3 ́NCR similar to the highly virulent strain Hypr, whereas JP-296 and JP-554 have a genomic organization identical to Toro-2003, the prototypic TBEV strain from the same natural focus. NGS revealed significantly higher quasispecies diversity for JP-296 and JP-554 compared to Mandal-2009. In addition, single nucleotide polymerphism (SNP) analysis showed that 40% of the SNPs were common between quasispecies populations of JP-296 and JP-554, indicating the persistence and maintenance of TBEV quasispecies within the natural focus. Taken together, these findings indicate the importance of environmental factors for the occurrence pattern of the different life-stages of the tick vector, which are important for the persistence of TBEV in nature. Our findings also show that the selection pressure exerted by specific host also affects the population structure of the TBEV quasispecies. In addition, our results further demonstrate that the evolution of quasispecies has effect on TBEV virulence in mice.<br>Vektorburna sjukdomar är ett växande globalt hot mot både människor och djur. De pågående klimatförändringarna kan leda till förhöjda risker för infektioner överförda av myggor, fästingar och andra leddjursvektorer. Ixodes ricinus är en vanlig fästing i Europa som överför fästingburna patogener som är farliga för människor. Fästingburen encefalit (TBE) är en vektorburen sjukdom som orsakas av TBE-virus (TBEV). De pågående klimatförändringarna har bidragit till en ökning både av vektorn och sjukdomsfrekvensen. Mellan 10 000 och 15 000 mänskliga TBE-fall rapporteras årligen i Europa och Asien. Den geografiska fördelningen av TBEV visar ett ojämnt fördelningsmönster där viruset är koncentrerat till vissa fokusområden. TBEV återfinns i naturen i en livscykel där viruset hela tiden överförs mellan fästingar och däggdjur. Spridningen sker dels från en infekterad fästing till ett ryggradsdjur när fästingen äter på värddjuret. Spridning mellan fästingar sker troligen främst genom så kallad “co-feeding”, det vill säga att flera fästingar suger blod samtidigt från samma värddjur. Viruset kan då passera från en infekterad fästing, genom värddjuret till oinfekterade fästingar. Virus kan identifieras och studeras med genetiska metoder. Det ökande antalet TBE-fall i Skandinavien styrker vikten av att hitta och karakterisera ytterligare TBEV-stammar och identifiera nya naturliga fokusområden. Vi har sekvenserat och fylogenetiskt beskrivit fyra TBEV-stammar: Saringe-2009 (blodfylld nymf), JP-296 (födosökande vuxen hane), JP-554 (födosökande vuxen hane) och Mandal-2009 (födosökande nymfer, n = 10). Mandal-2009 är ett TBEV från ett naturligt fokusområde i södra Norge. Saringe-2009 kommer från ett naturligt fokusområde i norra Stockholms län, Sverige. JP-296 och JP-554 härstammar från Torö som är ett naturligt fokusområde i södra Stockholms län, Sverige. Förutom den genetiska sekvenseringen av TBEV har vi också studerat effekten av olika biotiska och abiotiska faktorer på populationsdynamik av I. ricinus i södra Stockholm och observerade variation i fästingsaktivitetsmönster både temporalt och spatialt. Förekomstmönster av fästinglarver, nymfer och vuxna honor, och det totala antalet fästingar är viktiga faktorer för sannolikheten för horisontell överföring av TBEV mellan fästingar. Vi fann att sannolikheten för synkron förekomst av larver, nymfer och honor var högst under försommaren. Vegetationshöjd, mängden skog och mängd öppet vatten runt undersökningsområden hade signifikanta negativa effekter på sannolikheten för att larver, nymfer och honor skulle förekomma samtidigt. Den variabla delen av den icke-kodande 3 ́regionen (3'NCR) av TBEV-genomet innehåller ofta en intern poly(A)-sekvens. Liksom andra RNA-virus, förekommer TBEV som så kallade ”quasispecies” vilka definieras som grupper av olika genetiska varianter av virus. Genom analysen av TBEV-stam Saringe-2009 avslöjades variation i poly(A)-sekvensen vilket indikerar förekomst av ”quasispecies”. Eftersom Saringe-2009 kom från en blodfylld nymf som hade sugit blod i &gt; 60 timmar, föreslår vi att Saringe-2009 visar en förändring i ”quasispecies”-poolen när viruset överförs från exoterm fästingmiljö till endoterm däggdjursmiljö. Vi undersökte poly(A)-ekvensens variabilitet och dess roll vid replikering och för virulens hos TBEV, genom att skapa två infektiösa kloner av Torö-2003 stammen; en med en kort/vild-typ (A)3C(A)6 poly(A)-sekkvens, och en med en lång (A)3C(A)38 poly(A)-sekvens. Den infektiösa klonen med lång poly(A)-sekvens replikerade sämre än vildtypklonen i cellkultur, men (A)3C(A)38 poly(A) var mer virulent i C57BL/6-möss än (A)3C(A)6 poly(A). Datasimulering av TBEV-genomets sekundär-RNA-struktur visade att de längre poly(A)-sekvenserna påverkar veckningen av en specifik sekundärstruktur (SL14) i början av 3 ́NCR. Djupsekvenseringsanalys av TBEV-gnomen avslöjade skillnader för specifika gener och ”quasispecies”-strukturen efter passering i cellkultur och/eller mushjärna. Dessa förändringar föreslås bidra till de observerade skillnaderna i virulens. Våra resultat indikerar att den långa poly(A)-sekvensen ger instabilitet i TBEV-genomet, vilket resulterar i ökad mångfald av ”quasispecies”-populationen som i sin tur kan bidra till TBEV-virulens. Fylogenetisk analys av Saringe-2009, JP-296, JP-554 och Mandal-2009 visade på ett nära släktskap mellan de fyra skandinaviska TBEV-stammarna. De nya stammarna formerade ett kluster med en tidigare TBEV-stam identifierad på Torö (Toro-2003), vilket skapade ett skandinaviskt klad. Genetisk analys visade att Mandal-2009 innehåller en trunkerad 3 ́NCR som liknar den högvirulenta stammen HYPR. JP-296 och JP-554 hade däremot samma genetiska struktur som den längre Torö-2003 stammen från samma fokusområde. Djupsekvensering visade höge mångfald av ”quasispecies”-populationen för JP-296 och JP- 554 jämfört med Mandal-2009. Analys av enkel nukleotid polymorfism (SNP) visade att 40 % av alla SNP var gemensamma mellan ”quasispecies”-populationen för JP-296 och JP-554. Detta indikerar att TBEV-”quasispecies”-strukturen kan vara konserverad för närbesläktade virus vilken kan leda till att den bevaras inom specifika fokusområden. Sammantaget så visar dessa studier att miljöfaktorer påverkar förekomsten av fästingvektorn och dess olika livsstadier, vilket är en bakomliggande faktor för utbredning av TBEV i naturliga fokusområden. Det visar även på att värdmiljön påverkar strukturen för ”quasispecies”-populationen. Dessutom visar våra studier att evolution och utveckling av ”quasispecies”-strukturen kan påverka virulensen för TBEV i möss.

Dengue Fever virus (DENV) is a very old mosquito-borne flavivirus that has made a modern worldwide re-emergence as a result of population movement and growth, urbanisation and lapse of vector control. The World Health Organisation estimates that 2.5 billion people, or two-fifths of the world’s population are at risk from DENV, which can cause serious illness and in its severe forms, death. Despite the humanitarian and economic burden that DENV and its flaviviral relatives create, there are no chemotherapeutic drugs available and vaccine development is challenging. Mammalian host cell infection by DENV is mediated by the Envelope glycoprotein (EGP), which covers the entire exposed surface of the mature virus particle and is comprised of three exposed protein domains (DI, DII and DIII) and a transmembrane anchor. While significant effort has been invested to better understand how DIII of EGP participates in receptor mediated endocytosis of DENV into host cells, including the site and structure of the receptor binding site, or carbohydrate recognition domain (CRD), and the structure of ligands involved remain undefined. A recent study of mammalian cell surface glycans involved in DENV infection by Dr Kazuya Hidari and co-workers identified DENV inhibition by the glycolipid Paragloboside, which includes the tetrasaccharide Lacto-N-neotetraose (nLc4)1. This thesis reports an investigation of DENV-2 EGP DIII ligand specificity and characterisation of the DIII CRD involved in mammalian cell infection. To achieve this, soluble and high level expression of DENV-2 ThNH-7/93 EGP DIII was established from Pichia pastoris (P. pastoris) yeast and the recombinant DIII was successfully purified to near homogeneity by single step affinity chromatography. The biological activity of DIII was assessed by DENV permissible cell based assays and the recombinant protein was shown to have retained its wildtype host cell receptor binding activity. Recombinant DIII protein was utilised to successfully establish glycan microarray and saturation transfer difference nuclear magnetic resonance (STD NMR) methodologies, Confidential – not to be copied ii which are useful in the study of EGP ligand specificity. Investigation of nLc4 ligand binding to EGP confirmed that this tetrasaccharide binds to the CRD DIII, involving each of its carbohydrate moieties. Epitope mapping by STD NMR spectroscopy also revealed that the H-1 proton of the N-acetyl-D-glucosamine (GlcNAc) makes closest contact with DIII via its N-acetyl group. Screening of carbohydrate libraries with DENV-2 and a multivalent DIII complex identified additional EGP specificity to several novel binding ligands that share a GlcNAc moiety at the first or second non-reducing cytoplasmexposed positions.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Institute for Glycomics<br>Full Text

Les flavivirus sont des virus responsables d'une considérable morbidité et mortalité dans le monde en causant des encéphalites sévères, des fièvres hémorragiques, ainsi que des symptômes fébriles chez l'homme. L'absence de symptômes cliniques spécifiques pour une infection liée à un virus donné, et la présence simultanée de différents arbovirus dans une même région impose la nécessité de disposer d'un diagnostic de genre par PCR. Parmi les amorces proposées dans la littérature, une seule paire a permis la détection des différents flavivirus testés avec une sensibilité limite de 105 doses infectieuses. ML-1. La PCR semi-nichée développée au laboratoire utilise 2 nouvelles amorces et permet la détection des différents flavivirus avec une sensibilité de moins de 200 doses infectieuses. ML-1. Après le séquençage des produits amplifiés, la construction d'un dendrogramme permet d'orienter le diagnostic vers les principales espèces de flavivirus. Actuellement, il n'existe pas de traitement spécifique des infections à flavivirus. La protéase virale est une cible privilégiée pour ce type de thérapie. La protéase NS3 du virus Langat, virus utilisé comme modèle du virus de l'encéphalite à tiques, a été exprimée, purifiée et son activité enzymatique, sous sa forme recombinante, a été caractérisée. L'activité protéasique a été réalisée par hydrolyse de différents substrats peptidiques chromogéniques. Cette protéase virale clive in vitro les substrats comportant en position P1 un acide aminé basique tel l'arginine ou la lysine. Ces observations sont confirmées par l'étude comparative des sites de clivage naturels au sein de la polyprotéine des flavivirus. Cette protéase recombinante devrait pouvoir être utilisée pour le criblage de molécules antiprotéases utilisables dans le traitement des infections à flavivirus<br>Flaviviruses are responsible for considerable morbidity and mortality and may cause severe encephalitis, hemorrhagic fever, hepatitis, and febrile symptoms in vertebrates, including humans (. . . ). Different p-nitroanilide substrates, defined on canonic sequences for their susceptibility to Ser-protease, were applied to the proteolytic assays of the protein. The highest values were obtained from substrates containing an Arg or Lys (amino acid) residue at the P1 position. This purification method will facilitate the future development of reliable testing procedures for anti-proteases directed to NS3 proteins

L'encéphalite japonaise est une maladie croissante en Asie du Sud-Est et dans les pays du Pacifique occidental puisqu'elle s'étend aujourd'hui à l'Australie et à la Russie. Elle est actuellement la première cause d'encéphalite virale en Asie et se rencontre surtout en zone rurale. Cette maladie est provoquée par un virus de la famille des Flavivirus. Elle est véhiculée par une piqûre de moustique de la famille des Culex. Les porcs et les oiseaux aquatiques ont un rôle de réservoirs dans la transmission de la maladie et seule leur augmentation explique la transmission à l'homme, puisqu'e celui-ci est un hôte accidentel. L'encéphalite japonaise est une maladie souvent bénigne mais peut cependant être fatale et laisser d'importantes séquelles chez les survivants. Les personnes les plus touchées sont les enfants et les personnes âgées ou immunodéficientes. Actuellement, aucun médicament efficace n'existe, mais la vaccination et une protection mécanique permettenet d'empêcher la maladie. De nouveaux vaccins sont en cours de recherche et la terminologie du "Chimerivax" va apporter dans peu de temps une vaccination à immunisation rapide et aussi efficace que celle utilisée contre le virus de la fièvre jaune. De plus, un accord entre professionnels pharmaceutiques et instituts de recherches donne l'espoir de la fabrication d'un traitement curatif.

Dans le but d'explorer la capacité des moustiques à répliquer le VHC, nous avons réalisé une série d'infections expérimentales sur Aede caspius, Aedes vexans et genre Cu/ex pipiens sauvage et d'élevage. Après collecte des moustiques, les femelles ont été nourries par un repas sanguin virémique (VHC 1 b positif). Les moustiques ont ensuite été maintenus en élevage pendant plusieurs jours. Après mise au point des conditions de détection, nous avons réussi à apporter la preuve de la réplication du VHC dans les moustiques du genre Aedesvexans. Dans la première infection expérimentale réalisée sur Ae. Vexans , nous avons détecté de l'ARN du VHC dans -50% des moustiques du 21,ème jours post-infection (JPI), par PCR en point final. La seconde expérience d'infection, réalisée sur les deux genres dE moustiques: Aedes Sp, et Cu/ex pipiens, nous a permis de : (i) confirmer par qRT-PCR le résultat de la première expérience avec un tau: d'infection de -33% ; (ii) démontrer que seuls les moustiques du genre Aedes contenaient de l'ARN du VHC, les moustiques du genre Cu/ex étaient tous négatif; (iii) montrer que l'ARN détecté était bien issu de la réplication, dans la mesure où des mutations d'adaptation ont été identifiées dans la séquence de la RdRp (ARN Polymérase ARN dépendante), avec une spécificité pour le genre Aedes, puisque tous les moustiques Cu/ex à 21 JPI étaient ARN VHC-négatifs. Un autre moyen de distinguer l'ARN issu de la réplication de l'A RN résiduel a été de montrer l'existence d'une dissémination au sein du moustique. Pour ce faire, nous avons réalisé une troisième expérience d'infections (genres Ae. Caspius et Ae. Vexans), sur une durée de 15 jours et avons analysé la présence de l'ARN du VHC distinctement dans les têtes et les corps. En effectuant des prélèvements à différentes dates après infection (0, 4, 8 et 15 jours), nous avons réussi à obtenir une cinétique d'infection, séparément, dans les têtes et dans les corps, détection réalisée parWTA (Whole Transcriptome Amplification) suivie de qPCR. Au jour de l'infection (JO), le taux d'infection dans les têtes et les corps des moustiques éta de 9,1 % et 100% respectivement, ce taux est passé à 57,1 % et 85,7% respectivement à J15 après une négativation huit jours après infection. D'autre part, nous avons identifié des mutations d'adaptation dans la séquence de l'IRES des moustiques à J15 comparés à ceux à JO. Il est intéressant de noter que les moustiques ARN VHC-positifs appartenaient tous à l'espèce vexans, montrant ainsi que la réplication est spécifique d'espèce. Différentes régions du génome du VHC ont, ensuite, étaient amplifiées. Enfin, nous avons eu recoun à des moustiques d'élevage. L'infection de cette souche a également montré, en utilisant différentes approches de détection (qRT-PCR, WTA-qPCR, HCV-GA notamment), qu'à 30 JPI, l'ARN du VHC était présent dans -33% des têtes et -66% des corps, alors que les taux d'infection à JO étaient de -28% et -71 % dans les têtes et les corps respectivement. Les résultats se sont appuyés sur des séquençage effectués sur les régions amplifiées du génome du VHC. L'ensemble de ces résultats montrent que le VHC est capable de se répliquer dans les moustiques du genre Ae. Vexans, ce qui suggère que le VHC, à l'image d'autres F/avivirus, puisse alterner entre deux hôtes rimates et moustiQues) et Qu'il ait perdu son potentiel de réplication chez le moustiQue au fil du temps<br>Ln order to explore the ability of mosquitoes to replicate HCV, we conducted a series of experimental infections caspius on Aedes, Aedes vexans and Culex pipiens wild and farmed. After collection of mosquitoes, the females were fed a blood meal viremic (HCV 1b positive). The mosquitoes were th en maintained in breeding for several days. After development of detection conditions, we were able to demonstrate HCV repli cation in the mosquito Aedes vexans. Ln the first experimental infection performed on Ae. Vexans, we detected HC' RNA in - 50% of mosquitoes 21st days post-infection (JPI), PCR end point. The second infection experiment, conducted on Iwo types of mosquitoes: Aedes SP, and Culex pipiens, has allowed us to: (i) confirmation by qRT-PCR results of the first experience with an infection rate of - 33%, (ii) demonstrate that only the mosquito Aedes contained RNA HCV, the Culex mosquitoes were ail negative, (iii) show that the RNA detected was good of replication, the extent of adaptive mutations were identified in the sequence of the RdRp (RNA polymerasi RNA dependent), with specificity for the genus Aedes, since ail Culex mosquitoes at 21 YPI were HCV RNA-negative. Another way to distinguish RNA from the replication of RNA remaining was to show the existence of a release within the mosquito. To do this, we conducted a third experiment infections (gender Ae. Caspius and Ae. Vexans), over a'period of 15 days and analyzed the presence of HCV RNA in distinct heads and bodies. Ln conducting samples at different times after infection (0, 4, 8 and 15 days), we managed to obtain a kinetic infection separately in heads and bodies, detection performed by WTA (Whole transcriptomics Amplification) followed by qPCR. On the day of infection (DO), the infection rate in the heads and bodies of mosquitoes was 9. 1 % and 100% respectively, the rate rose to 57. 1% and 85. 7% respectively J15 negativity after eight days after infection. On the other hand, we have identified mutations in th adaptation of the IRES sequence of mosquito D15 compared to those on day O. It is interesting to note that mosquito-positive HCV RNA ail belonged to the species vexans, showing that replication is species specific. Different regions of the HCV genome, then, were amplified. Finally, we made use of mosquito breeding. Infection of this strain has also shown, using different approaches to detection (qRT-PCR, qPCR-WTA, including HCV-GA), only 30 JPI, HCV RNA was present in - 33% heads and - 66% of the body, while infection rates were JO - 28% - 71 % in the head and body respectively. The results were based on sequencing performed on the amplified region! of the HCV genome. Ali these results show that HCV is able to replicate in mosquitoes of the genus Ae. Vexans, suggesting that HCV, likt other flaviviruses, can alternate belween Iwo hosts (primates and mosquitoes) and has lost its potential for replication in the mosquito ove time

La dengue est une maladie ré-émergente qui menace le tiers de la population mondiale, et pour laquelle aucun vaccin n'est disponible. Dans ce travail, nous avons évalué une nouvelle stratégie de vaccination basée sur l'expression d'un antigène combiné du virus de la dengue par un vecteur dérivé du vaccin vivant atténué pédiatrique contre la rougeole. La compréhension des mécanismes de neutralisation des flavivirus par les anticorps est essentielle pour la conception d’approches vaccinales innovantes contre la dengue. C’est pourquoi, dans un premier temps, nous avons étudié le mécanisme de l’inhibition de fusion par des anticorps neutralisants dirigés contre les trois domaines de la protéine d’enveloppe (E). Le virus de l’encéphalite à tiques (TBEV) a été utilisé comme modèle dans des tests de fusion, de neutralisation et d’association aux membranes lipidiques. Les résultats montrent, que les anticorps peuvent interférer avec les étapes précoces ou tardives du processus de fusion. Cette étude montre aussi que les anticorps dirigés contre le domaine III de la protéine E peuvent bloquer à la fois l’entrée et la fusion virale. Pour faire la preuve de concept de notre stratégie de vaccination, nous avons donc inséré dans le vecteur rougeole le domaine III de la glycoprotéine E (EDIII) qui contient le site de liaison au récepteur ainsi que des épitopes neutralisants sérotype-spécifiques. Pour renforcer son immunogénicité, le EDIII a été fusionné à l'ectodomaine pro-apoptotique de la protéine de membrane (ectoM) du VDEN-1. Testé chez des souris sensibles à la rougeole, ce candidat vaccin s’est montré immunogène et capable d'induire des anticorps neutralisants sérotype-spécifiques à long terme. La présence de l'ectoM a été déterminante pour l'immunogénicité du EDIII. Sa capacité adjuvante a été corrélée avec sa capacité à induire la maturation des cellules dendritiques et à favoriser la sécrétion de cytokines pro inflammatoires et antivirales ainsi que des chimiokines impliquées dans l'établissement de l'immunité adaptative. Un candidat rougeole-dengue tétravalent a été ensuite construit dans le but d’induire la même immunité contre les 4 sérotypes du virus de la dengue. Cette stratégie de vaccination combinée rougeole-dengue permettrait d’offrir des vaccins pédiatriques accessibles particulièrement attrayants pour immuniser les enfants simultanément contre la rougeole et la dengue dans des régions du monde où les deux maladies coexistent<br>Dengue fever is a reemerging disease that threatens one third of the world's population, for which no vaccine is available. In this work, we evaluated a new vaccination strategy based on the expression of a combined dengue antigen by a vector derived from the pediatric live attenuated measles vaccine. Understanding the mechanisms of flavivirus neutralization by antibodies is essential for the design of innovative vaccine approaches against dengue. Therefore, as a first step, we studied the mechanism of inhibition of fusion by neutralizing antibodies directed against the three domains of the envelope protein (E). The Tick-borne encephalitis virus (TBEV) was used as a model in in vitro fusion tests, and coflottation assays with lipid membranes. The results showed that the neutralizing antibodies can interfere with the early or late stages of the fusion process. This study also shows that antibodies against domain III of the E protein can block both the viral entry and fusion. As a proof-of-concept of our vaccination strategy, we inserted into measles vector the domain III of the glycoprotein E of dengue virus (EDIII), which contains the putative receptor binding site and serotype-specific neutralizing epitopes. To strengthen its immunogenicity, EDIII was fused with the pro-apoptotic ectodomain of the membrane protein (ectoM) of VDEN-1. Tested in mice susceptible to measles, this vaccine candidate was immunogenic and able to induce long term serotype-specific neutralizing antibodies. The presence of the ectoM proved crucial for the immunogenicity of EDIII. Its adjuvant capacity correlated with its ability to mature dendritic cells and to enhance the secretion of proinflammatory and antiviral cytokines, as well as chemokines involved in the development of adaptive immunity. A tetravalent measles-dengue candidate was then generated in order to induce the same immunity against the 4 serotypes of dengue virus. This vaccination strategy combining measles and dengue might offer an affordable pediatric vaccine particularly attractive to immunize children both against measles and dengue fever in areas of the world where the two diseases co-exist

Une étude de la séroprévalence des arboviroses à flaviviridae a été entreprise sur l'île de la Réunion. Deux mille cinq cent sept sérums humains, prélevés en 1987 sur un échantillon randomisé de la population réunionnaise, ont été testés à l'aide de la réaction d'inhibition de l'hémagglutination vis-à-vis des 5 antigènes suivants: fièvre jaune, dengue de type 1, dengue de type 2, West Nile et Wesselsbron. Cent treize sérums provenant de diverses espèces animales, récoltés en 1989, ont été étudiés avec la même technique et la même batterie d'antigènes. Des commémoratifs individuels, recueillis lors de l'enquête de 1987, et les résultats d'une autre enquête à la réunion datant de 1971 (F. Rodhain) ont été exploités en corrélation avec nos résultats sérologiques. La prévalence globale trouvée en 1987 (42,68%) est significativement plus élevée que celle constatée en 1971 (16,5%). Les réactions multivalentes représentent pratiquement les trois quarts des réponses positives. Aucune focalisation précise n'est relevée. Deux phénomènes semblent à l'origine de ces positivités: d'une part une épidémie sévère de dengue sur l'île en 1977-1978, d'autre part la circulation probable d'un (voire de plusieurs) flavivirus dans cette région. Des éléments d'ordre épidémiologique sont en faveur d'un virus de type dengue ou West Nile. Parmi les facteurs de risque étudiés, le sexe, la profession et le contact avec les espèces animales recensées semblent sans effet sur le taux de positivité. En revanche, une étude précise de l'âge des sujets infectés, de leur type d'habitat (rural ou urbain) et de la répartition géographique des augmentations de séroprévalence entre 1971 et 1987 confirment l'intervention de la dernière épidémie du dengue enregistrée sur le taux de positivité enregistré, ainsi que la circulation d'un (ou de plusieurs) flavivirus parmi cette population. Une nouvelle enquête entomologique concernant toute l'île de la Réunion apparaît nécessaire.

L'émergence des zoonoses est à relier directement avec les perturbations générées par l'Homme sur son environnement naturel à plus ou moins grande échelle. Sur l'ensemble des zoonoses émergentes chez l'Homme, la majorité provient d'animaux sauvages. L'étude du rôle de la faune sauvage dans la circulation des agents pathogènes est donc cruciale en particulier quand les interfaces faune sauvage/Homme sont fortes. L'objectif de cette thèse a été de comprendre par des approches éco-épidémiologiques à large échelle, la circulation d'agents pathogènes dans les populations d'un oiseau sauvage en contact étroit avec l'Homme, le goéland leucophée (Larus michahellis). Le premier chapitre présente les différentes méthodes utilisées dans la surveillance d'agents pathogènes ainsi que leurs limites lorsqu'elles sont menées en populations naturelles. Ce chapitre met en évidence au travers d'une étude portant sur les virus influenza aviaires, que la quantification des anticorps maternels dans les œufs est un outil efficace. Le second chapitre consiste à élargir l'échelle spatiale de l'étude afin de mettre en évidence plus finement les facteurs éco-épidémiologiques influençant la transmission des virus influenza aviaires dans et entre les populations de goéland leucophée de l'ouest méditerranéen. Enfin, le dernier chapitre repose sur la comparaison des patrons d'expositions obtenus pour des agents pathogènes au mode de transmission vectoriel : les flavivirus. Cette thèse permet de mettre en évidence les patrons d'exposition de certains agents pathogènes (virus influenza aviaire et flavivirus) et d'appréhender les facteurs éco-épidémiologiques potentiellement impliqués dans leurs circulations. Les résultats permettent d'envisager de futurs axes de recherches, nécessaires pour évaluer plus précisément la dispersion de ces virus en Méditerranée<br>The emergence of zoonotic diseases is directly linked to the noise generated by humans on the natural environment to a greater or lesser extent. Of all the emerging zoonoses in humans, the majority comes from wild animals. The study of the role of wildlife in the circulation of pathogens is crucial, especially when wildlife/human interfaces are prominent. The aim of this thesis is to understand, using large scale eco-epidemiological approaches, the circulation of pathogens in a close to human population of wild bird, the yellow-legged gull (Larus michahellis). The first chapter presents the different methods used in the monitoring of pathogens and their limitations when they are conducted in natural populations. This chapter further highlights, through a study of avian influenza viruses, that the quantification of maternal antibodies in eggs is an effective tool. The second chapter expands the spatial scale of the study to highlight the finer eco-epidemiological factors influencing the transmission of avian influenza viruses within and between populations of Western Mediterranean yellow-legged gull. Finally, the last chapter is based on the comparison of exposition patterns obtained for vectorial transmission mode pathogens: flaviviruses. This thesis highlights patterns of exposure for certain pathogens (avian influenza virus and flaviviruses) and enables the understanding of the eco-epidemiological factors potentially involved in their circulations. The results allow to consider future areas of research needed to more accurately assess the dispersion of these viruses over the Mediterranean

Les virus de la dengue (DENV) et du Zika (ZIKV) sont des virus transmis par les moustiques qui causent de graves problèmes de santé publique à travers le monde. Ces virus transmis par des arthropodes (arbovirus) sont des virus à ARN du genre Flavivirus principalement transmis à l'homme par le moustique Aedes aegypti. De plus, les moustiques Ae. aegypti sont naturellement infectés par le cell-fusing agent virus (CFAV), le premier flavivirus spécifique d'insecte (FSI) ayant été décrit. L'évolution du CFAV, ses interactions avec les arbovirus chez les moustiques co-infectés et les réponses immunitaires des moustiques vis-à-vis du CFAV, sont mal connus. Ce travail doctoral a abordé plusieurs de ces aspects peu étudiés de la biologie du CFAV. Premièrement, de nouvelles séquences du génome complet du CFAV ont permis une analyse phylogénétique globale. Un manque de congruence phylogénétique entre le CFAV et Ae. aegypti indique que d’autres facteurs que la structure de la population hôte façonnent la diversité génétique du CFAV. Deuxièmement, dans des expériences de co-infection, le CFAV a inhibé la réplication du DENV et du ZIKV in vitro et leur dissémination in vivo. Ces résultats étayent l'hypothèse que les FSIs pourraient réduire la transmission des arbovirus dans la nature. Troisièmement, il a été montré qu’un élément viral endogène (EVE) dérivé du CFAV dans le génome d’Ae. aegypti régule la réplication du CFAV dans les ovaires via la voie des piARNs. Ce résultat suggère que les EVEs pourraient minimiser des coûts de reproduction associés à l'infection par des virus apparentés. Cette thèse a éclairé les interactions complexes entre le CFAV, Ae. aegypti et les arbovirus<br>Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause major public health problems worldwide. These arthropod-borne viruses (arboviruses) are RNA viruses of the Flavivirus genus that are primarily transmitted among humans by the mosquito Aedes aegypti. In addition, Ae. aegypti mosquitoes are naturally infected with cell-fusing agent virus (CFAV), the first-described insect-specific flavivirus (ISF). Little is known about CFAV evolution, interactions with arboviruses in coinfected mosquitoes, and mosquito immune responses to CFAV. This PhD work addressed such understudied aspects of CFAV biology. First, novel full-genome sequences of CFAV allowed a global phylogenetic analysis. A lack of phylogenetic congruence between CFAV and Ae. aegypti indicates that other factors than host population structure shape CFAV genetic diversity. Second, in coinfection experiments, CFAV inhibited DENV and ZIKV replication in vitro and their dissemination in vivo. These results support the hypothesis that ISFs may reduce arbovirus transmission in nature. Third, a CFAV-derived endogenous viral element (EVE) in the Ae. aegypti genome was found to regulate CFAV replication in the ovaries through the piRNA pathway. This finding suggests that EVEs may help to minimize reproductive costs associated with infection by cognate viruses. Overall, this PhD thesis shed light on the complex interactions between CFAV, Ae. aegypti, and arboviruses

Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most relevant arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. In our recent work we demonstrated that TBEV is able to trigger the stress response of infected cells leading to the formation of stress granules (SG) (Albornoz et al. 2014). We also found that the formation of SG in TBEV infected cells is delayed, following the same delayed kinetic of the IFN response (Miorin et al. 2012). Indeed, while TBEV replication is evident at early time points post infection, SG and IFN-β mRNA become detectable only after 16 hours. Transcriptome analysis of TBEV infected cells showed that, in addition to interferon and interferon stimulated genes, also genes of the unfolded protein response (UPR) were activated. Interestingly, the spliced form of XBP1 and phosphorylation of PERK occurred early during infection (<12h) indicating that the UPR occurs before induction of interferon. We then investigated the role of the UPR as an early cellular response to the infection and as a possible trigger of the interferon response. Interestingly, when cells were infected following treatment with Tunicamycin, a known inducer of the UPR, the IFN response was already active at 8 hours post-infection and the virus titres were significantly decreased. In this condition formation of stress granules was also anticipated with the same kinetic. These data suggest that TBEV is able to evade both the stress and interferon responses and that the UPR may play a critical and unexpected role in the delayed activation of both.

Les flavivirus ayant un impact en médecine vétérinaire sont largement distribués dans le monde (à l’exemple de la fièvre West Nile (WNF) présente sur les cinq continents ou de l’Encéphalite japonaise (EJ) en Asie du Sud-Est) et sont responsables de maladies à dominante neurologique chez l’homme et/ou le cheval.La virémie étant généralement brève lors de ces infections virales, les méthodes de diagnostic utilisées sont essentiellement sérologiques. Or le chevauchement fréquent des aires de répartition des flavivirus complique le diagnostic sérologique. En effet, des réactions sérologiques croisées entre flavivirus sont observées lors de l’utilisation de méthodes de diagnostic usuelles telles que l’ELISA et l’immunofluorescence (IF). Les résultats sérologiques doivent donc être confirmés par la méthode fastidieuse de séroneutralisation (SNT) virale avec les différents flavivirus existants dans la région. De plus, le risque d’émergence sur un territoire donné de nouveaux flavivirus comme le virus Zika au Brésil ou en Amérique du Nord ne peut être exclu.Nous avons donc développé dans la première partie de ce travail une nouvelle stratégie de diagnostic sérologique multiplexe à l’aide de la méthode d’immuno-essai (MIA) sur billes. Sachant que la glycoprotéine d’enveloppe soluble (sE) des flavivirus est divisée en 3 domaines (D) structuraux, DI, DII et DIII et que les épitopes du DIII sont spécifiques de chaque flavivirus, des protéines EDIII recombinantes de différents flavivirus d’intérêt ont été synthétisées en système d’expression Drosophila S2 et leur antigénicité a été testée sur sérums équins et ovins. Nous avons obtenu des résultats très encourageants en démontrant que l’utilisation des EDIII associée avec la capacité de multiplexage de la méthode MIA apparaît comme une réponse adaptée au défi du diagnostic sérologique des infections à flavivirus.Nous avons en outre utilisé la même méthode de multiplexage mais avec des antigènes NS1 du WNV pour mettre en place un test DIVA (Differentiating Infected from Vaccinated Animals) permettant de différencier les chevaux infectés par le WNV des chevaux vaccinés avec un vaccin recombinant WNV.Un autre écueil en médecine vétérinaire est le traitement des affections à flavivirus. En effet l’arsenal thérapeutique est limité et le traitement est avant tout symptomatique. Nous avons dans la seconde partie de ce travail testé in vitro une molécule antivirale à large spectre, le sr1057 sur nos virus d’intérêt (WNV et JEV). Cette molécule qui provient d’une stratégie de criblage développée par l’Institut Pasteur a probablement pour cible la cellule de l’hôte car elle est capable d’inhiber des virus très différents, à génome à ARN de polarité positive ou négative et ADN.Les résultats que nous avons obtenus avec ce composé sont en demi-teinte pour les flavivirus testés avec une efficacité démontrée pour le JEV mais plus modeste pour le WNV. Ils n’excluent cependant pas une possible utilisation de cet antiviral en médecine vétérinaire équine car une activité inhibitrice in vitro sur les virus de l’Herpes équin de type I et de l’artérite virale a été confirmée par d’autres collaborateurs<br>Flaviviruses with a major impact in veterinary medicine are widely distributed (e.g. West Nile fever (WNF) has spread across the five continents and Japanese Encephalitis (JE) is reported in South-East Asia) and are mainly responsible for neurological diseases in humans and/or horses.After flavivirus infection, viremia in mammal hosts is generally short and consequently indirect methods are mostly used to diagnose flavivirus infections. However, frequent spatial overlapping in their circulation areas renders the interpretation of serological assays difficult. Indeed, cross-reactions between flaviviruses are observed in rapid serological tests such as in ELISA and immunofluorescence assays (IFA). Serological assay results should thus be confirmed by the tedious comparative virus neutralization test (VNT) using a panel of viruses known to circulate in the area. Moreover, the risk of emergence of new flaviviruses such as reported recently with the Zika virus in Brazil or in North America should be considered when studying flavivirus epidemiology.In the first section, a new strategy aiming at improving the serological diagnosis of flavivirus infections was developed using the multiplexing capacity of microsphere immunoassays (MIA). The flavivirus soluble envelope (sE) glycoprotein ectodomain is composed of three domains (D), e.g. DI, DII and DIII, with EDIII containing virus-specific epitopes. Recombinant EDIIIs of different flaviviruses were synthesized in the Drosophila S2 expression system. The microspheres coupled with rEDIIIs were assayed with equine and ovine sera from natural and experimental flavivirus infections or non-immune samples. Very encouraging results have been obtained and this innovative multiplex immunoassay based on flavivirus rEDIIIs appears to be a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.MIA with WNV nonstructural 1 protein were also implemented to differentiate Infected from Vaccinated Animals (DIVA). Such a DIVA approach was only successful when horses had been immunized with a recombinant canarypox vaccine, while horses receiving inactivated WNV vaccine developed immune responses close to the ones induced after natural infection.Another pitfall in veterinary medicine is the lack of therapeutics for viral diseases and specifically for flaviviruses. The therapeutic arsenal is indeed rather limited and animals are generally administered supportive treatments only. In the second part, the results of the in vitro testing of a broad spectrum antiviral named sr1057 on WNV and JEV replication are presented. This chemical, identified from a unique screening strategy developed by Pasteur Institute, is probably targeting the host cell and was found to inhibit the replication of varied RNA and DNA viruses belonging to different virus families. The sr1057 compound was not as efficient at inhibiting the replication of flaviviruses as for other RNA+ viruses, with a modest antiviral effect demonstrated for WNV and a higher efficacy on JEV. This antiviral presents however potentials for applications in equine veterinary medicine because it efficiently inhibited equine herpes virus-1 and equine arteritis virus in vitro, as clearly shown by other collaborators

Mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) are major threats to global public health resulting in millions of infections and hundreds of thousands of deaths annually. The presence of these viruses and their increasing emergence/spread continues to escalate. Notably, Usutu virus (USUV; Genus: Flavivirus; Family: Flaviviridae) is one such pathogen currently causing mass die-offs of avian hosts throughout Europe. USUV is categorized in the Japanese Encephalitis virus (JEV) antigenic complex and thus shares many antigenic and pathologic characteristics with fellow members, such as JEV and WNV. Respective to human infections, USUV cases are generally asymptomatic; nonetheless, acute cases have been reported. These acute cases typically cause mild symptoms, such as fevers and rashes; however, more severe cases can result in neurologic diseases, such as encephalitis and/or meningoencephalitis. In addition to these pathologic similarities, USUV shares several ecological and geographical traits with WNV, a pathogen responsible for several outbreaks during its spread from Africa, to Europe, and eventually the United States. Currently, WNV is considered endemic in areas across the United States due to its transmission via Culex spp.; mosquitoes that are ubiquitous in the United States. These parallels suggest the possible emergence of USUV into the United States and therefore, it is imperative to broaden our knowledge of USUV and assess its potential to become a major global health concern. The overall goal of this thesis was to characterize USUV and evaluate its emergence potential in the United States by: (1) developing infectious clones of recent European and African USUV isolates as tools for characterization and analysis of USUV and (2) assessing the transmission potential of several species of North American mosquitoes. In Aim 1, we show that the aforementioned infectious clones infect and replicate similarly to their parental strains in vitro in both vertebrate and invertebrate models, as well as in transiently immunocompromised CD-1 and IFNAR-/- murine models, and thus serve as useful tools for future molecular studies focusing on USUV. Furthermore, in Aim 2, we describe the ability of field-caught (Southwest Virginia, USA) Culex spp. and Aedes spp. mosquitoes to become infected with a recent European isolate of USUV; although, we report an overall limited potential for these species to transmit this virus. Altogether, these studies form a foundation for understanding the potential emergence of USUV in the United States as well as provide necessary tools needed to aid future research on USUV emergence, transmission, and pathogenesis.<br>Master of Science<br>Usutu virus (USUV) is an emerging mosquito-borne virus that was first isolated from a mosquito in 1959 in South Africa, and since then, has become a major problem throughout Africa and Europe causing acute to severe infection in dozens of patients. Additionally, this virus is causing massive die-offs in Eurasian blackbird populations. This is particularly problematic because birds play a critical role in ecosystems as they act as forms of pest control, pollinators, and seed dispersers. Depletion of these species could lead to an imbalance and, eventually, collapse of our natural ecosystem. Additionally, there is a growing concern of USUV making its way into the United States, following a similar track of emergence to WNV's introduction in New York in 1999 and its subsequent spread throughout the states. WNV's introduction to the United States was detrimental to native bird populations and humans, and has caused tens of thousands of infections and thousands of deaths since this introduction. Research has shown USUV causes similar disease symptoms to WNV. The self-limiting illness from these viruses typically includes fever and rashes but some infections can result in more severe cases causing inflammation of the brain and surrounding areas. Like many other prominent mosquito-borne viruses, there is no specific treatment or vaccine for WNV or USUV. Because USUV is so closely related to WNV, and their similar characteristics may point towards similar emergence in the United States, it is essential to garner more information on USUV. The overall goal of this thesis was to establish a reliable tool(s) for further characterization of USUV and demonstrate the potential for USUV emergence in the United States. We first developed molecular tools, known as viral clones, that are valuable to the scientific community which allows the manipulation of USUV genetic material to perform further downstream studies. Our objective for this initial study was to create a molecular tool that would behave similarly to their natural, or "parental", virus. The results from this study suggest we have successfully produced these tools. Furthermore, we sought to determine the potential for field-caught mosquitoes from Southwest Virginia, USA to transmit a recently isolated strain of USUV. These data suggest that while these mosquitoes do have the ability to become infected with USUV, they have a limited potential to transmit this virus to animal hosts. Altogether, these studies have allowed us to expand our knowledge on USUV's potential emergence in the United States and develop powerful tools to continue this essential research.

Zika virus (ZIKV) has emerged in many regions of the world, with infection outcomes spanning from no apparent illness to crippling nervous system disease. ZIKV and its close relatives, West Nile virus, Japanese encephalitis virus, dengue virus, and yellow fever virus are primarily transmitted by mosquitoes. Three ZIKVs were selected: MR-766 (Uganda, 1947), P6-740 (Malaysia, 1966), and PRVABC-59 (Puerto Rico, 2015), whose place of origin and time of isolation differ substantially. Stable, complementary DNA (cDNA) copies of the three ZIKV RNA genomes were cloned to examine the significance of viral and host genetic variations in directing ZIKV infection outcomes. Using a new toolbox for ZIKV genome engineering and protein analysis, combined with various cell culture and mouse infection model systems, the following were determined: (1) Genome-wide landscape of viral gene products and their related species, with several immuno-reactive gene products identified in the case of all three cloned ZIKVs. (2) Viral replicability in cultured cells, varied significantly depending on the virus strain and host cell type, with one cow cell line being resistant to ZIKV infection. (3) Virus induced neurological disease in mice, differed dramatically depending on the virus dose and strain, mouse age and strain, route of infection, and presence or absence of immune system components. Overall, the findings demonstrate the impact of the viral and host genetic backgrounds on the ability of ZIKV to replicate and cause disease. The ZIKV strain-specific characterizations and molecular instruments described will provide multiple avenues for developing and testing medical countermeasures.

West Nile Virus (WNV) forms part of the Japanese encephalitis serocomplex in the genus Flavivirus, family Flaviviridae. This enveloped positive single-stranded RNA (+ssRNA ) virus is the etiological agent of West Nile fever, and in more severe cases WNV neuroinvasive disease, in both humans and animals. WNV is distributed worldwide and is phylogenetically classified into five distinct lineages. The WNV genome is ~11 Kb in length and encodes a single open reading frame (ORF) that is post-translationally cleaved into three structural proteins and seven non-structural proteins. In this study, two contemporary and two historic South African WNV strains were genetically characterised as lineage 2 strains based on complete genome sequences. Genetic change as a result of passage number and propagation system was quantified on both the consensus genome- and quasispecies level. A lack of variation was observed amongst the consensus genome sequences of WNV strains subject to changes in propagation system from BHK-21 cell culture to mouse brain and vice versa. In contrast, variation amongst the latter was observed on the quasispecies level. Genome-wide single nucleotide polymorphism (SNP) profiles as well as full-length haplotype sequences reconstructed from ultra deep sequence data indicated that high levels of quasispecies diversity persists, particularly in the capsid gene region, during changes in propagation environment. The changes in frequency of variants were consistent throughout isolates propagated in different systems. The increased variation in the capsid gene region may result from selective pressures brought about by differences in host cell type between propagation systems. This study is the first to demonstrate quasispecies dynamics resulting from changes in propagation system of a lineage 2 WNV based on the reconstruction of full-length haplotype sequences from ultra deep sequence data. The approach demonstrates a cost-effective alternative to the estimation of viral population structure in light of viral evolutionary dynamics, which may in turn be assessed by the single plasmid reverse genetic system designed in this study. Although early attempts at rescuing an infectious WNV clone were unsuccessful, the system shows promise in the application of future studies concerning vaccine and diagnostic development, virulence studies and disease control.<br>Dissertation (MSc)--University of Pretoria, 2013.<br>gm2013<br>Zoology and Entomology<br>Unrestricted

La protéine d'enveloppe des Flavivirus comprend 3 ectodomaines et un segment membranaire. J'ai caractérisé les propriétés de son domaine III (ED3) in vitro. J'ai mesuré la stabilité thermodynamiques d'ED3 par des expériences d'équilibres de dépliement, induits chimiquement ou thermiquement, et suivis par spectrofluorimétrie; augmenté la stabilité d'ED3 par des changements de résidus dans son coeur hydrophobe sans affecter son antigénicité; et montré qu'un domaine ED3 consensus des virus de la dengue (DENV) était hautement stable. La Protéine Ribosomale SA (RPSA) humaine comprend un domaine N-terminal replié et un domaine C-terminal désordonné. Nous avons quantifié leurs interactions avec les domaines ED3 de Flavivirus pathogènes, la laminine, l'héparine et un anticancéreux (EGCG) par des méthodes immunochimiques et spectrofluorimétriques in vitro. J'ai montré que les domaines-N ,et -C avaient à la fois des fonctions idiosyncratiques et des fonctions partagées, et que le domaine-C mimait l'héparine. J'ai déterminé la spécificité de serotype des IgMs dirigées contre le domaine ED3 d'un des 4 sérotypes de DENV en utilisant des sérums humains infectés ou non-infectes par DENV et une analyse par courbe ROC (Receiving Operator Characteristic). Ces sérums ont été testées en MAC-ELISA au moyen des 4 sérotypes d'un hybride dimérique entre ED3 et phosphatase alcaline d'E. Coli. La discrimination par ED3 entre sérums infectés par le DENV homotypique et sérums non-infectes variait avec le serotype; elle était maximale pour ED3 de DENV1 quel que soit le serotype infectant. Certains domaines ED3 discriminaient entre sérotypes de DENV. Des applications potentielles sont considérées<br>The envelope protein of Flaviviruses includes 3 ectodomains and a membrane segment. I have characterized the properties of its domain III (ED3) in vitro. I measured the thermodynamic stability of ED3 by experiments of unfolding equilibria, chemically or thermically induced and monitored by spectrofluorimetry; increased the stability of ED3 by changes of residues in its hydrophobic core without affecting its antigenicity; and showed that an ED3 domain, consensus for all the dengue viruses (DENV), was highly stable. The human Ribosomal Protein SA (RPSA) includes a folded N-terminal domain and a disordered C-terminal domain. We quantified their interactions with the ED3 domains of pathogenic Flaviviruses, laminin, heparin and an anticarcinogen (EGCG) by immunochemical and spectrofluorimétrie methods in vitro. I showed that the N- and C-domains had both idiosyncratic and shared fonctions, and that the C-domain mimicked heparin. We determined the serotype specificity of IgMs that were directed to the ED3 domain of each serotype of DENV by using DENV-infected or -uninfected human serums and a statistical analysis by receiving operator characteristic (ROC) curves. These serums were tested in MAC-ELISA with the 4 serotypes of a dimeric hybrid between ED3 and E, coli alkaline phosphatase. The discrimination by ED3 between serums infected by the homotypic DENV and uninfected serums varied with the serotype; it was maximal with the ED3 from DENV1 whatever the infecting serotype. Some ED3 domains discriminated between sérotypes of DENV. Potential applications are described

Le genre Flavivirus regroupe plus de 70 espèces dont plusieurs sont des pathogènes humains de première importance responsables dans les formes les plus graves de manifestations hémorragiques ou d’encéphalites parfois mortelles. L’absence de traitements antiviraux spécifiques et l’augmentation croissante des flaviviroses, surtout dans les régions tropicales, justifient un effort de recherche et développement pour lutter contre ces maladies.Ce travail a abordé deux aspects de l’infection à flavivirus : un aspect épidémiologique et un aspect plus fondamental sur l’immunité innée et les contremesures flavivirales. L’étude épidémiologique a été menée en collaboration avec le CENETROP (Centro national de enfermedades tropicales) de Bolivie grâce à la contribution de l’IRD. De par l’analyse des différentes souches circulantes dans ce pays, elle a permis une meilleure compréhension de l’épidémiologie de la dengue et de la fièvre jaune et nous a fait prendre conscience de la variabilité génétique de ces virus. Devant le peu de données répertoriées en Bolivie, nos travaux serviront de référence pour comprendre les épidémies futures, peut-être améliorer les techniques de diagnostic et permettre le développement de stratégies de prévention adaptées et l’amélioration des politiques de lutte contre la fièvre jaune en Amérique du Sud. La cohabitation entre le virus et l’hôte immunocompétent est le résultat d’un équilibre subtil entre le taux de réplication virale et la clairance du système immunitaire pour garantir la survie des deux espèces. Chacun a évolué en développant des mécanismes de défenses contre l’autre. Notre second travail visait à analyser l’influence de la protéine flavivirale non structurale NS1 sur la réponse interféron de l’hôte. L’identification de stratégies virales d’évasion face à l’immunité de l’hôte et l’analyse de leurs fonctions dans l’infection virale permettrait de mieux comprendre le système immunitaire ainsi que l’interaction virus–hôte. Ceci aiderait au développement de nouvelles stratégies antivirales afin de traiter les pathologies associées à ces arbovirus<br>The Flavivirus genus consists of sevevral human pathogens responsible for hemorragic syndrome or encephalitis. The absence of specific antiviral treatment and an increase in Flavivirus incidence has led to a greater research effort in fighting these diseases. The study takes an epidemiological and a fundamental approach in its analysis of the innate immune response to flavivirus infection as well as flaviviral adaptation to evade this response. The analysis of circulating strains in Bolivia has led to a better understanding of dengue and yellow fever and also an awareness of their genetic variability. Given the limited information available in Bolivia, our studies could be used as a reference to understand future epidemics, improve diagnostic methods and allow the development of prevention strategies to fight against yellow fever in south Africa. The relationship between virus and host results from a subtle balance between viral replication and immunity clearance allowing the survival of both species. Each one as developed defence mechanisms against the other. We also examined the role of the non structural protein NS1 in the interferon respons to Flaviviral infection. Knowledge on viral escape strategies from host immunity could help to develop antiviral treatment for these arbovirus diseases

As Neotropical migratory birds, Prothonotary Warblers are exposed to parasites in both tropical and temperate regions and may act as dispersal agents between geographic areas. This study identifies the prevalence of Haemosporidia, West Nile Virus (WNV), and St. Louis Encephalitis virus (SLEV) in this species. A total of 71.6% of captured Prothonotary Warblers were infected with Haemosporidia during the 2008 breeding season, and infection prevalence increased throughout the season. This temporal change in prevalence is likely due to infection relapse and transmission of new infections. No correlations between reproductive effort and infection status were observed, nor were any associations between infection prevalence and nest box location identified. WNV and SLEV were present in 37.5% and 6.3% of sampled Prothonotary Warblers, respectively. These results warrant more detailed analyses of pathogen transmission dynamics in this population, physiological mechanisms that affect infection susceptibility, and spatial and temporal trends in infection that may exist.

Le virus de l’encéphalite à tiques (TBEV), membre de la famille des Flaviviridae et du genre Flavivirus, est d’un point de vue médical, l’arbovirus plus important en Europe et en Asie du Nord-Est. Il est responsable de symptômes fébriles et de manifestations neurologiques allant de la méningite légère à l'’encéphalomyélite sévère pouvant être fatale. En dépit de son importance médicale, la neuropathogenèse induite par TBEV reste peu caractérisée. Ici, nous avons utilisé des cellules neurales humaines différenciées à partir de progéniteurs neuraux fœtaux pour modéliser l’infection in vitro et élucider les mécanismes par lesquels le virus endommage le cerveau humain. Nos résultats ont montré que les neurones et les cellules gliales (astrocytes et oligodendrocytes) étaient permissifs au TBEV. Les neurones étaient massivement infectés et la cible d’un effet cytopathique important (perte de 60 % des neurones 7 jours après l’infection). Les astrocytes étaient également infectés, bien qu’à des niveaux inférieurs, et l’infection avait un effet modéré sur leur survie (perte de 30 % des astrocytes 7 jours après l’infection), induisant une hypertrophie caractéristique d’une astrogliose. Ainsi, deux événements majeurs décrits dans les cerveaux de patients infectés par TBEV (perte neuronale et astrogliose) étaient reproduits dans ce modèle cellulaire in vitro, démontrant ainsi sa pertinence pour des études de neuropathogenèse. Nous l’avons donc utilisé pour étudier la réponse antivirale induite par TBEV. En utilisant des PCR arrays, nous avons d’abord montré que le virus induisait une forte réponse antivirale caractérisée par une surexpression de senseurs viraux, de cytokines et de gènes stimulés par l’interféron. Puis, en établissant des cultures enrichies en neurones humains et astrocytes humains, nous avons montré que ces deux types cellulaires participaient à la réponse antivirale globale. Cependant, les astrocytes élaboraient une réponse antivirale plus forte que les neurones. Ces résultats, en démontrant que les neurones humains et les astrocytes humains élaborent chacun une réponse antivirale unique suite à l’infection, suggèrent que leur susceptibilité particulière à TBEV serait due à leur différente capacité à établir une réponse antivirale protectrice<br>Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, genus Flavivirus, is, from a medical point of view, the most important arbovirus in Europe and North-East Asia. It is responsible for febrile illness and, in some cases, for neurological manifestations ranging from mild meningitis to severe encephalomyelitis that can be fatal. Despite its medical importance, TBEV-induced neuropathogenesis remains poorly understood. Here, we used human neural cells differentiated from fetal neural progenitor cells (hNPCs) to model the infection in vitro and to decipher the mechanisms by which the virus damages the human brain. Our results showed that neurons and glial cells, namely astrocytes and oligodendrocytes, were permissive to TBEV. Neurons were massively infected and subjected to a dramatic cytopathic effect (60% loss 7 days post-infection). Astrocytes were also infected, although at lower levels, and the infection had a moderate effect on their survival (30% loss 7 days post infection), inducing a hypertrophied morphology characteristic of astrogliosis. Thus, two major cellular events described in TBEV-infected human brain (i.e. neuronal loss and astrogliosis) were reproduced in this in vitro cellular model, showing its relevance to study TBEV-induced neuropathogenesis. We therefore used it to tackle TBEV induced antiviral response. Using PCR arrays, we first showed that TBEV induced a strong antiviral response characterized by the overexpression of viral sensors, cytokines and interferon-stimulated genes (ISGs). Then, setting up enriched cultures of human neurons and human astrocytes, we further showed that the two cellular types were participating in the global antiviral response. However, astrocytes developed a stronger antiviral response than neurons. These results, by demonstrating that human neurons and human astrocytes have unique antiviral potential, suggest that their particular susceptibility to TBEV infection is due to their different capacity to mount a protective antiviral response

The recent emergence of a number of new viral diseases as well as the re-emergence of tuberculosis (TB), indicate an urgent need for new drugs against viral and bacterial infections. Coronavirus nsp1 has been shown to induce suppression of host gene expression and interfere with host immune response. However, the mechanism behind this is currently unknown. Here we present the first nsp1 structure from an alphacoronavirus, Transmissible gastroenteritis virus (TGEV) nsp1. Contrary to previous speculation, the TGEV nsp1 structure clearly shows that alpha- and betacoronavirus nsp1s have a common evolutionary origin. However, differences in conservation, shape and surface electrostatics indicate that the mechanism for nsp1-induced suppression of host mRNA translation is likely to be different in the alpha- and betacoronavirus genera. The Modoc virus is a neuroinvasive rodent virus with similar pathology as flavivirus encephalitis in humans. The flaviviral methyltransferase catalyses the two methylations required to complete 5´ mRNA capping, essential for mRNA stability and translation. The structure of the Modoc NS5 methyltransferase domain was determined in complex with its cofactor S-adenosyl-L-methionine. The observed methyltransferase conservation between Modoc and other flaviviral branches, indicates that it may be possible to identify drugs that target a range of flaviviruses and supports the use of Modoc virus as a model for general flaviviral studies. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is part of the methylerythritol phosphate (MEP) pathway that produces essential precursors for isoprenoid biosynthesis. This pathway is used by a number of pathogens, including Mycobacterium tuberculosis and Plasmodium falciparum, but it is not present in humans. Using a structure-based approach, we designed a number of MtDXR inhibitors, including a novel fosmidomycin-analogue that exhibited improved activity against P.falciparum in an in vitro blood cell growth assay. The approach also allowed the first design of an inhibitor that bridge both DXR substrate and co-factor binding sites, providing a stepping-stone for further optimization.

Aedes albopictus est un vecteur de transmission d’arbovirus, et ceci fait de lui une sérieuse menace. Durant la dernière décennie, NIRVS (séquences intégrées de virus à ARN non-rétroviraux) issues de Flavivirus spécifiques d’insectes (FSIs) ont été découvert intégrées dans le génome du moustique Aedes albopictus il y a très longtemps. De plus, il a été montré que ces éléments pourraient avoir un rôle antiviral chez le vecteur par la production de piARNs qui réduirait la réplication virale. Ici nous avons caractérisé 8 NIRVS chez 12 populations naturelles d’Aedes albopictus. Nous montrons qu’il y a une forte diversité inter- et intrapopulation, suggérant une évolution complexe de ces NIRVS dans le génome. De plus, les analyses microsatellites basées sur les mêmes populations ont révélé que ces NIRVS ont évolué différemment de gènes neutres, suggérant une potentielle fonction de ces éléments. Nous montrons que cette fonction pourrait être liée à la compétence vectorielle d’Aedes albopictus pour les arbovirus, mais que celle-ci reste pourtant incertaine et nécessite de plus amples investigations. Enfin, nous montrons par l’utilisation des lignées cellulaires infectées de manière persistante que la formation de NIRVS est un évènement qui se produit rarement dans le génome des moustiques Aedes, et que les arbovirus, avec les FSIs, sont également capables d’endogénisation chez les moustiques<br>Aedes albopictus is a vector for transmitting arboviruses and this makes it a serious threat. In the last decade, NIRVS (non-retroviral integrated RNA virus sequences) from insect-specific flaviviruses (ISFs) have been found integrated in Aedes albopictus mosquito genome a long time ago. Moreover, it has been shown that these elements may have an antiviral role in vectors by producing piRNAs that would reduce viral replication. Here we characterized 7 NIRVS in 12 Aedes albopictus wild populations. We show that there is a high diversity inter- and intra-population, suggesting a complex evolution of these NIRVS in Aedes albopictus genome. Moreover, microsatellite analysis based on those same populations revealed that those NIRVS evolved differently from neutral genes, suggesting a potential function of these elements. We show that this function may be linked to the vector competence of Aedes albopictus to arboviruses, but remains unclear and require more investigations. Finally, we show by establishing persistent infected cells that NIRVS formation is an event that occurs rarely in the Aedes mosquito genome, and that arboviruses, along with ISFs, are also capable of endogenization in mosquitoes

In under 6 decades dengue has emerged from South East Asia to become the most widespread arbovirus affecting human populations. Recent dramatic increases in epidemic dengue fever have mainly been attributed to factors such as vector expansion and ongoing ecological, climate and socio-demographic changes. The failure to control the virus in endemic regions and prevent global spread of its mosquito vectors and genetic variants, underlines the urgency to reassess previous research methods, hypotheses and empirical observations. This thesis comprises a set of studies that integrate currently neglected and emerging epidemiological, ecological and evolutionary factors into unified mathematical frameworks, in order to better understand the contemporary population biology of the dengue virus. The observed epidemiological dynamics of dengue are believed to be driven by selective forces emerging from within-host cross-immune reactions during sequential, heterologous infections. However, this hypothesis is mainly supported by modelling approaches that presume all hosts to contribute equally and significantly to the selective effects of cross-immunity both in time and space. In the research presented in this thesis it is shown that the previously proposed effects of cross-immunological reactions are weakened in agent-based modelling approaches, which relax the common deterministic and homogeneous mixing assumptions in host-host and host-pathogen interactions. Crucially, it is shown that within these more detailed models, previously reported universal signatures of dengue's epidemiology and population genetics can be reproduced by demographic and natural stochastic processes alone. While this contrasts with the proposed role of cross-immunity, it presents demographic stochasticity as a parsimonious mechanism that integrates, for the first time, multi-scale features of dengue's population biology. The implications of this research are applicable to many other pathogens, involving challenging new ways of determining the underlying causes of the complex phylodynamics of antigenically diverse pathogens.

De nombreux virus transmis par les arthropodes (arbovirus), tels que ceux de la dengue (DENV) et de la fièvre jaune (YFV), circulaient à l’origine dans des cycles selvatiques et ont émergé chez l’Homme via des moustiques « bridge vectors » qui connectent les cycles de transmission selvatiques et humains. Ces « bridge vectors » peuvent aussi par transfert inverse établir de nouveaux cycles selvatiques. Cette thèse a évalué le potentiel de vecteur d’arbovirus d’un moustique répandu en Asie du sud-est, Aedes malayensis. Nous avons identifié Ae. malayensis pour la première fois au Laos lors de captures de moustiques dans une forêt de la réserve de Nakai Nam Theun. En utilisant des pièges à appâts humains sur le terrain, nous avons observé qu’Ae. malayensis pouvait piquer l’Homme et donc potentiellement agir comme « bridge vector ». En laboratoire, cette population selvatique d’Ae. malayensis a montré une faible compétence vectorielle relative pour DENV et YFV, et une absence d’attraction détectable pour l’odeur humaine. Cependant, des tests de compétence vectorielle et de pièges à appâts humains ont révélé qu’une population péri-domestique d’Ae. malayensis à Singapour était compétente pour YFV et entrait en contact avec l’Homme. Au final, ce travail de doctorat a souligné l’importance de ne pas négliger les vecteurs secondaires dans l’évaluation du risque d’émergence des arbovirus<br>Many emerging arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and yellow fever virus (YFV) originated in sylvatic cycles and have emerged among humans through spillover transmission by mosquito species that ‘bridge’ sylvatic and human transmission cycles. These bridge vectors can also mediate ‘spillback’ transmission of arboviruses from humans into novel sylvatic cycles. This PhD focused on Aedes malayensis, a mosquito species widely distributed in South East Asia, to assess its potential as an arbovirus vector. We identified Ae. malayensis for the first time in Laos during mosquito surveys conducted in a forested area of the Nakai Nam Theun National Protected Area (NNT NPA). Using field-based human-baited traps, we found that Ae. malayensis engaged in human-biting behavior and therefore could act as bridge vector in the NNT NPA. In laboratory conditions, this sylvatic population of Ae. malayensis displayed a relatively low vector competence for DENV and YFV and a lack of detectable attraction to human odor. However, vector competence assays and a human-baited trap survey showed that a peridomestic Ae. malayensis population in Singapore was competent for YFV and engaged in contact with humans. Overall, this PhD work highlighted that ancillary vectors should not be overlooked to fully assess the risk of arbovirus emergence

碩士<br>國防醫學院<br>微生物及免疫學研究所<br>102<br>Japanese encephalitis virus (JEV) and dengue virus (Den) are flaviviruses whose genome is a single-stranded, positive-sense RNA with 5’ cap but without 3’ poly-A tail. After viral entry and uncoating, translation of flaviviral RNA is initiated by the host mRNA translation machineries. Cellular cap-dependent mRNA translation is tightly controlled by phosphorylation of eukaryotic initiation factor 2 α subunit (eIF2α) at residue Ser51. In response to certain stress condition, eIF2α can be phosphorylated by at least four different cellular kinases, for example PKR activated by dsRNA and PERK activated by endoplasmic reticulum stress, to temporally shut down cellular protein synthesis. Our previous studies indicate that JEV NS2A can block PKR-mediated eIF2α phosphorylation and eIF2α phosphorylation was only noted at late stage of JEV infection. To directly address the role of eIF2α phosphorylation in flavivirus infection, we manipulated the cellular eIF2α phosphorylation level by overexpressing eIF2α-S51D or -S51A mutants, mimicking the phosphorylated and unphosphorylated forms of eIF2α, respectively, and by treatment with an inhibitor of eIF2α de-phosphorylation, Salubrinal, which sustains endogenous eIF2α in phosphorylated status. JEV and Den-2 replication were suppressed in cells with eIF2α-S51D overexpression and in cells treated with Salubrinal. However, cells with eIF2α-S51A overexpression did not affect JEV and Den-2 replication. Thus, eIF2a phosphorylation play an antiviral role in flavivirus infection.

博士<br>國防醫學院<br>生命科學研究所<br>93<br>In this study, we found that infection with flaviviruses, such as Japanese encephalitis virus (JEV) and Dengue virus serotype 2 (DEN-2), leads to interferon-beta (IFN-beta) gene expression in a virus-replication- and de novo protein-synthesis-dependent manner. NF-kappa B activation is essential for IFN-beta induction in JEV- and DEN-2-infected cells. However, these two viruses seem to preferentially target different members of the interferon regulatory factor (IRF) family. The activation of constitutively expressed IRF-3, characterized by slower gel mobility, dimer formation, and nuclear translocation, is more evident in JEV-infected cells. Other members of the IRF family, such as IRF-1 and IRF-7 are also induced by DEN-2, but not by JEV infection. The upstream molecules responsible for IRF-3 and NF-kappa B activation were further studied. Evidently, a cellular RNA helicase, retinoic acid-inducible gene I (RIG-I), and a cellular kinase, phosphatidylinositol-3 kinase (PI3K), are required for flavivirus-induced IRF-3 and NF-kappa B activation, respectively. Therefore, we suggest that JEV and DEN-2 initiate the host innate immune response through a molecular mechanism involving RIG-I/IRF-3 and PI3K/NF-kappa B signaling pathways.

博士<br>國防醫學院<br>生命科學研究所<br>100<br>The untranslated regions (UTRs) located at the 5and 3 ends of the Japanese encephalitis virus (JEV) genome, a positive-sense RNA, are involved in viral translation, the initiation of RNA synthesis, and the packaging of nascent virions. The cellular and viral proteins that participate in these processes are expected to interact with the UTRs. In this study, we used biotinylated RNA-protein pulldown and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses to identify that the far upstream element (FUSE) binding protein 1 (FBP1) binds with JEV 5 and 3 UTRs. The impact of FBP1 on JEV infection was determined in cells with altered FBP1 expression. JEV replication was enhanced by knockdown and reduced by the overexpression of FBP1, indicating a negative role for FBP1 in JEV infection. FBP1, a nuclear protein, was redistributed to the perinuclear region and appeared as cytoplasmic foci that partially colocalized with JEV RNA in the early stage of JEV infection. By using a JEV replicon reporter assay, FBP1 appeared to suppress JEV protein expression mediated by the 5 and 3UTRs. Thus, we suggest that FBP1 binds with the JEV UTR RNA and functions as a host anti-JEV defense molecule by repressing viral protein expression. FBP1 was found to partially colocalize with stress granules (SGs) in JEV-infected cells and some viruses have been found to interact with the SG pathway. We thus study the role of SG in JEV infection. We found that JEV infection induced SGs formation and did not interfere with SG induction triggered by sodium arsenite stimulation. JEV- and Dengue virus serotype 2 (DEN-2)-induced SGs formation can not be blocked by specific inhibitors Y-27632 and Toxin B that inactive signaling of RhoA and its downstream kinase ROCK1, respectively, suggesting that RhoA/ROCK1 pathway is not involved in SGs induction during JEV and DEN-2 infection. In addition, monocyte chemotactic protein-induced protein 1 (MCPIP1), also known as Zc3h12a, could block the assembly of SGs in cells infected with JEV and DEN-2. Furthermore, several viral proteins such as core, prM, E, NS1, NS2B, NS4A, and NS4B, but not NS2B3 and NS5 are able to trigger SG formation . Overall, we demonstrate that SGs formation was induced in DEN-2- and JEV-infected cells. The mechanism and consequence of SGs formation during DEN-2 and JEV infection are of interest to be further studied.

archives@tulane.edu<br>Zika virus (ZIKV), a member of the Flaviviridae family, was the cause of a large viral outbreak reaching across the Americas during 2015 and 2016. Discovered in 1947, it has historically been a neglected disease, due to its emergence in humans on a large scale being recent. At the time of the outbreak, no FDA approved ZIKV diagnostics existed, and those that were able to detect the virus were unable to distinguish it from related viruses such as Dengue virus (DENV), and at this time, no approved therapeutics or vaccines are available. We investigated the ability of diagnostics targeted toward both anti-NS1 antibodies and NS1 antigen circulating during infection to detect current or past ZIKV disease, as well as the capability of NS1 to produce a protective response. Our studies suggest anti-NS1diagnostics are feasible, though some populations may display an immune response reminiscent of a prior infection. Levels of circulating NS1 were lower than those produced during DENV infection, though were still detectable with our assay. Additionally, intraperitoneal immunization with NS1 produced an anti-ZIKV NS1 response that coincided with a decrease in viremia, though further work was needed to discern life-prolonging effects. Together, this work furthers the development of the tools necessary to combat future outbreaks of ZIKV in vulnerable populations.<br>1<br>Brandon Beddingfield

abstract: Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches.<br>Dissertation/Thesis<br>Doctoral Dissertation Microbiology 2016

碩士<br>國防醫學院<br>微生物及免疫學研究所<br>98<br>MicroRNAs (miRNAs) are small noncoding RNAs of approximately 21~23 nucleotides in length. They are derived from cellular or viral transcripts and bind to their target mRNAs in a sequence-specific manner, resulting in either mRNA degradation or translational repression. Recent studies suggest that cellular miRNAs are among the key determinants controlling virus infection in mammalian hosts, for example cellular miRNAs can serve as restriction factors to limit infection by several viruses, including human immunodeficiency virus and vesicular stomatitis virus. However, our understanding on the role played by cellular miRNA on flaviviruses is limited.

博士<br>國防醫學院<br>生命科學研究所<br>93<br>Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. As JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, as only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3 or -7 alleviated the levels of PARP cleavage following virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, as overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an antiapoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death. Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells via receptor-mediated endocytosis and low-pH-triggered membrane fusion, then replicate at intracellular membrane structures. Lipid rafts, the cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion of chelating with methyl--cyclodextrin or filipin III reduced JEV and DEN-2 infections mainly at the intracellular replication steps, and to a lesser extent on viral entry. By membrane flotation assay, we further demonstrated that several flaviviral nonstructural proteins were associated with detergent-resistant membrane structures, indicating the replication complex of JEV and DEN-2 are localized at the membranes possessing lipid raft property. Interestingly, we also found that extra amount of cholesterol readily blocked flaviviral infection, in great contrast to an alphavirus, sindbis virus whose infection was enhanced by cholesterol. Cholesterol might reduce the levels of viral RNA uncoating, but not at the initial infection steps of viral binding and entry. The anti-flaviviral effects of cholesterol might also take place intracellularly in the steps post viral adsorption. Our results thus suggest a stringent requirement of membrane components, especially the amounts of cholesterol for various steps of flavivirus life cycle.

碩士<br>國立臺灣海洋大學<br>食品科學系<br>95<br>Viral infectious diseases caused by arthropod-borne flaviviruses have been gradually increased worldwide in recent years, especially including dengue virus (DEN) and Japanese encephalitis virus (JEV) prevalent in Taiwan and other South Asia countries. Dengue viruses cause dengue fever and dengue related diseases that represent a global public health problem. Seaweeds have known to possess an ingredient of sulfated polysaccharides that have been widely used as curative agents for anti-virus, anti-tumor, anti-oxidation, anti-coagulant, and control of normal cholesterol levels. The main purpose of this thesis is to study, by using cell cultures, the anti-DEN effect of the water-soluble extracts from Ulva lactuca, which belongs to green seaweed. Three homogeneous extracts of Ulva lactuca leaves, A1, A4 and A8, were used in this study and we found them had no cytotoxicity to the cultured cells at as high as 100 µg/ml. By indirect immunofluorescence assay, we observed that 100 µg/ml of A4 or A8 could suppress DEN infection to BHK-21 cells by 90%, and similarly we found they could also inhibit DEN infection to human hepatocyte HepG2 cells. We further noticed that these extracts were active against DEN infection only during the early stage of virus adsorption and penetration. Moreover, another flavivirus JEV was also found to be blocked to certain degrees by Ulva lactuca extracts when infected to BHK-21 cells. However, Sindbis virus, a member of Alphaviruses, appeared to be not suppressed by these extracts even their concentrations were used as high as 500 µg/ml. Together, our results demonstrate that the extracts from green seaweed Ulva lactuca, comprising abundant sulfated polysaccharides, have a specific anti-flavivirus effect in cultured system and the medical values of these extracts are worth further studying.