Effects of Temperature on Emergence and Seasonality of West Nile Virus in California

Temperature has played a critical role in the spatiotemporal dynamics of West Nile virus transmission throughout California from its introduction in 2003 through establishment by 2009. We compared two novel mechanistic measures of transmission risk, the temperature-dependent ratio of virus extrinsic incubation period to the mosquito gonotrophic period (BT), and the fundamental reproductive ratio (R0) based on a mathematical model, to analyze spatiotemporal patterns of receptivity to viral amplification. Maps of BT and R0 were created at 20-km scale and compared throughout California to seroconversions in sentinel chicken flocks at half-month intervals. Overall, estimates of BT and R0 agreed with intensity of transmission measured by the frequency of sentinel chicken seroconversions. Mechanistic measures such as these are important for understanding how temperature affects the spatiotemporal dynamics of West Nile virus transmission and for delineating risk estimates useful to inform vector control agency intervention decisions and communicate outbreak potential

Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

Background:The recent West Nile virus (WNV) outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-)emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical sympt

Epidemics

Ivermectin (IVM)-treated birds provide the potential for targeted control of Culex mosquitoes to reduce West Nile virus (WNV) transmission. Ingestion of IVM increases mosquito mortality, which could reduce WNV transmission from birds to humans and in enzootic maintenance cycles affecting predominantly bird-feeding mosquitoes and from birds to humans. This strategy might also provide an alternative method for WNV control that is less hampered by insecticide resistance and the logistics of large-scale pesticide applications. Through a combination of field studies and modeling, we assessed the feasibility and impact of deploying IVM-treated birdfeed in residential neighborhoods to reduce WNV transmission. We first tracked 105 birds using radio telemetry and radio frequency identification to monitor their feeder usage and locations of nocturnal roosts in relation to five feeder sites in a neighborhood in Fort Collins, Colorado. Using these results, we then modified a compartmental model of WNV transmission to account for the impact of IVM on mosquito mortality and spatial movement of birds and mosquitoes on the neighborhood level. We found that, while the number of treated lots in a neighborhood strongly influenced the total transmission potential, the arrangement of treated lots in a neighborhood had little effect. Increasing the proportion of treated birds, regardless of the WNV competency status, resulted in a larger reduction in infection dynamics than only treating competent birds. Taken together, model results indicate that deployment of IVM-treated feeders could reduce local transmission throughout the WNV season, including reducing the enzootic transmission prior to the onset of human infections, with high spatial coverage and rates of IVM-induced mortality in mosquitoes. To improve predictions, more work is needed to refine estimates of daily mosquito movement in urban areas and rates of IVM-induced mortality. Our results can guide future field trials of this control strategy.U01CK000516/ACL/ACL HHSUnited States/UL1 TR001860/TR/NCATS NIH HHSUnited States/R01 AI148633/AI/NIAID NIH HHSUnited States/TL1 TR001861/TR/NCATS NIH HHSUnited States/U01 CK000516/CK/NCEZID CDC HHSUnited States

Mechanistic Elucidation of Protease–Substrate and Protein–Protein Interactions for Targeting Viral Infections

Viral infections represent an old threat to global health, with multiple epidemics and pandemics in the history of mankind. Despite several advances in the development of antiviral substances and vaccines, many viral species are still not targeted. Additionally, new viral species emerge, posing a menace without precedent to humans and animals and causing fatalities, disabilities, environmental harm, and economic losses. In this thesis, we present rational modeling approaches for targeting specific protease-substrate and protein-protein interactions pivotal for the viral replication cycle. Over the course of this work, antiviral research is supported beginning with the development of small molecular antiviral substances, going through the modeling of a potential immunogenic epitope for vaccine development, towards the establishment of descriptors for susceptibility of animals to a viral infection. Notably, all the research was done under scarce data availability, highlighting the predictive power of computational methods and complementarity between in-silico and in-vitro or in-vivo methods

Kinetic analysis of mouse brain proteome alterations following chikungunya virus infection before and after appearance of clinical symptoms

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV in

Investigation des interactions chez les Flavivirus dans le but de découvrir des nouvelles cibles thérapeutiques

Abstract : Mosquito-borne Flaviviruses include important human pathogens such as dengue virus, Zika virus, Yellow Fever virus, and West Nile virus. Although most infections are asymptomatic and most symptomatic cases are associated with a flu-like illness, a small fraction of Flavivirus infections can lead to severe illness, including encephalitis and hemorrhagic fever which may be fatal. Despite several decades of research efforts, no specific antiviral drugs are currently available to treat Flavivirus infections. Therefore, new potential targets for the discovery and/or design of antiviral therapies are needed. Identifying and understanding virus-host interactions has been proposed to be a promising avenue for the development of novel strategies to treat or prevent infections. Here, Flavivirus-host interactions were investigated using RNA sequencing as well as SILAC/MS, and the coding transcriptomes and proteomes of cells infected with three different Flaviviruses, namely Kunjin virus, Zika virus and Yellow Fever virus, was compared to those of mock-infected cells. Approximately 300-500 genes and 550-700 proteins were found to be differentially expressed during Flavivirus infections, and roughly 600-900 modulations in alternative splicing of host transcripts were identified. Moreover, 51 genes, 39 alternative splicing events and 57 proteins were found to be affected in all three Flavivirus infections. Several of these genes/proteins would be interesting candidates to study further for their roles in viral infections and their potential to be targeted to suppress viral replication. Another attractive target for the development of therapeutic compounds are protein-protein interactions. Here, the West Nile virus NS3:NS5 interaction, which is essential for viral replication, has been characterized using site-directed mutagenesis based on an interaction model that was developed in silico. Seven residues on the surface of the NS3 protein, clustered in two ‘hotspots’, were found to be critical for viral replication, possibly by mediating the NS3:NS5 interaction. These two ‘hotspots’ could be interesting targets for future anti-flaviviral drug development. In conclusion, the work presented in this thesis has highlighted several human genes/proteins as well as two regions on the surface of the viral protein NS3 that deserve further study to determine their potential to be targeted for the development of antiviral therapies.Les flavivirus comprennent d'importants agents pathogènes humains tels que le virus Zika, le virus de la fièvre jaune et le virus du Nil occidental. Bien que la majorité des infections soient asymptomatiques et la plupart des cas symptomatiques soient associés à une maladie pseudo-grippale, un faible pourcentage des infections par les flavivirus peut entraîner des maladies graves qui peuvent être mortelles. Malgré plusieurs décennies d'efforts de recherche, aucun médicament antiviral n'est actuellement disponible pour traiter ces infections. Par conséquent, de nouvelles cibles potentielles pour la découverte et/ou la conception de thérapies antivirales sont nécessaires. L'identification et la compréhension des interactions virus-hôte ont été proposées comme une voie prometteuse pour le développement de nouvelles stratégies pour traiter ou prévenir les infections. Ici, les interactions entre trois flavivirus (Kunjin, Zika, fièvre jaune) et leurs cellules hôtes ont été étudiées à l'aide du séquençage de l'ARN ainsi que de la protéomique quantitative (SILAC). Plusieurs centaines de gènes et protéines se sont avérés être exprimés de manière différentielle lors de ces infections, et environ 600 à 900 modulations dans l'épissage alternatif des transcrits cellulaires ont été identifiées. Plusieurs de ces gènes/protéines seraient des candidats intéressants à étudier davantage pour leurs rôles dans les infections virales et leur potentiel à être ciblés pour supprimer la réplication virale. Une autre approche intéressante pour le développement de composés thérapeutiques est de cibler les interactions protéine-protéine. Ici, l'interaction entre les protéines NS3 et NS5 du virus du Nil occidental, qui est essentielle pour la réplication virale, a été caractérisée. Sept résidus à la surface de la protéine NS3, regroupés en deux petites régions, se sont révélés critiques pour la réplication virale, possiblement en médiant l'interaction NS3:NS5. Ces deux petites régions pourraient être des cibles intéressantes pour le développement futur de médicaments contre les flavivirus. En conclusion, les travaux présentés dans cette thèse ont mis en évidence plusieurs gènes/protéines humains ainsi que deux régions à la surface de la protéine virale NS3 qui mériteraient d’être étudiés davantage afin de déterminer leur potentiel à être ciblés pour le développement de thérapies antivirales

The role of genetic diversity in the replication, pathogenicity and virulence of Murray Valley encephalitis virus

Genetic and phenotypic variation of genotype 1 (G1) and genotype 2 (G2) Murray valley encephalitis virus (MVEV) were characterised. G2 viruses were a minority, had lower levels of genetic diversity and an attenuated phenotype in the mouse model of MVE. G1 isolates were abundant with higher levels of genetic diversity and a virulent phenotype. The restricted evolution of MVEV was due to multiplication in mosquito cells. An RT-qPCR assay detecting all MVEV genotypes was developed

Network and Algebraic Topology of Influenza Evolution

Evolution is a force that has molded human existence since its divergence from chimpanzees about 5.4 million years ago. In that same amount of time, an influenza virus, which replicates every six hours, would have undergone an equivalent number of generations over only a hundred years. The fast replication times of influenza, coupled with its high mutation rate, make the virus a perfect model to study real-time evolution at a mega-Darwin scale, more than a million times faster than human evolution. While recent developments in high-throughput sequencing provide an optimal opportunity to dissect their genetic evolution, a concurrent growth in computational tools is necessary to analyze the large influx of complex genomic data. In my thesis, I present novel computational methods to examine different aspects of influenza evolution. I first focus on seasonal influenza, particularly the problems that hamper public health initiatives to combat the virus. I introduce two new approaches: 1. The q2-coefficient, a method of quantifying pathogen surveillance, and 2. FluGraph, a technique that employs network topology to track the spread of seasonal influenza around the world. The second chapter of my thesis examines how mutations and reassortment combine to alter the course of influenza evolution towards pandemic formation. I highlight inherent deficiencies in the current phylogenetic paradigm for analyzing evolution and offer a novel methodology based on algebraic topology that comprehensively reconstructs both vertical and horizontal evolutionary events. I apply this method to viruses, with emphasis on influenza, but foresee broader application to cancer cells, bacteria, eukaryotes, and other taxa

Genetic studies on the mosquito vector Culex pipiens.

As duas espécies do complexo Culex pipiens com maior distribuição geográfica, Culex quinquefasciatus e Culex pipiens sensu stricto, são importantes vectores de filárias e arbovírus. Culex pipiens s.s. apresenta categorias intra-específicas definidas por características ecológicas e fisiológicas, das quais as formas pipiens e molestus têm sido implicadas na transmissão do vírus da Febre do Nilo Ocidental na Europa e América do Norte. Hibridação entre Cx. quinquefasciatus e Cx. pipiens s.s. foi documentada em algumas regiões geográficas onde ambas espécies coexistem simpatricamente. Este fenómeno também foi descrito entre as formas molestus e pipiens, em áreas de simpatria e quando existe contacto limitado em certas épocas do ano. No entanto, o impacto da hibridação na divergência genética entre as espécies ou formas está por clarificar. Além disso, a hibridação pode afectar características ecológicas/fisiológicas das espécies/formas, que podem influenciar a sua capacidade vectorial. Neste contexto, foram analisadas populações do complexo Cx. pipiens da Europa, EUA e da Macaronésia com objectivo de determinar níveis de diferenciação genética e taxas de hibridação entre os membros do complexo. As amostras de mosquitos foram obtidas por diferentes métodos de colheita no terreno e a partir de colónias laboratoriais, entre 2005 e 2011. As análises genéticas realizadas foram baseadas em microssatélites e por polimorfismos de comprimento de fragmentos amplificados. Foram efectuadas comparações abordando questões específicas a diferentes níveis taxonómicos, que estão descritas nos cinco capítulos de resultados da tese. A distribuição e níveis de hibridação entre Cx. quinquefasciatus e Cx. pipiens s.s. foram avaliados nas ilhas da Macaronésia, o que permitiu detectar híbridos (~40%) em duas ilhas do arquipélago de Cabo Verde. A distribuição destas espécies na região reflecte a biogeografia e aspectos históricos da colonização humana. A coexistência em habitats de superfície das formas molestus e pipiens na região da Comporta (Portugal), foi demostrada pela combinação de análises fenotípicas e genéticas. As análises moleculares também sugerem a existência de um padrão de introgressão assimétrica, de molestus para pipiens. Estudos adicionais, sugerem uma maior tendência da forma molestus para explorar habitats intradomiciliares/antropogénicos quando comparada com a forma pipiens. Em ambas as formas, mais de 90% das refeições sanguíneas foram realizadas em aves. Foi ainda efectuada a primeira análise genómica focada na divergência entre os genomas das formas molestus e pipiens. Esta análise indicou uma baixa divergência entre os dois genomas (1,4%–3,1%), o que é consistente com um processo de especiação simpátrica com fluxo génico. Finalmente, foram realizadas análises genéticas em amostras de Cx. pipiens s.s. colhidas na Grécia durante um surto de Febre do Nilo Ocidental, em 2010. Populações simpátricas de molestus e pipiens com introgressão assimétrica foram identificadas na região onde o surto ocorreu, enquanto uma população homogénea de molestus foi encontrada numa região sem transmissão do vírus. Estes resultados evidenciam a importância da caracterização da variação genética e das relações evolutivas entre os membros do complexo Cx. pipiens para entender o seu potencial como vectores de doenças. Também abrem novas perspectivas para a investigação da ecologia e evolução deste complexo de espécies com importância médica.The two widespread species of the Culex pipiens complex, Culex quinquefasciatus and Culex pipiens sensu stricto, are major vectors of filarial worms and arboviruses. Culex pipiens s.s. is also divided into intraspecific categories defined by ecological and physiological traits. Of these, two forms, denoted pipiens and molestus, have been implicated in West Nile virus transmission in Europe and North America. Inter-specific hybridisation between Cx. quinquefasciatus and Cx. pipiens s.s.has been documented in some geographic regions where both species occur sympatrically. Likewise, hybridisation between molestus and pipiens forms has been described in areas of sympatry or when the forms become in contact during certain times of the year. However, the impact of hybridisation on the extent of genetic divergence between species or forms remains uncertain. Moreover, hybridisation may affect ecological and physiological traits of the species/forms, which may influence their vectorial capacity. In this context, the degree of genetic differentiation and hybridisation between members of the Cx. pipiens complex was studied in populations from Europe, USA and Macaronesian islands. Mosquito samples were obtained from field collections or laboratory colonies between 2005 and 2011. Genetic analyses were based on microsatellite genotypes and amplified fragment length polymorphisms. Comparisons were made at different taxonomic levels, addressing specific questions. These are described in the five results chapters of this thesis. The distribution and hybridisation between Cx. quinquefasciatus and Cx. pipiens s.s. were assessed in Macaronesian islands. Hybrid rates ~40% were detected in two islands of the Cape Verde archipelago. The distribution of the species reflects both the islands' biogeography and historical aspects of human colonization. A combination of phenotypic and genetic analyses conducted in Comporta (Portugal) revealed the co-occurrence of molestus and pipiens forms of Cx. pipiens s.s. in aboveground habitats.Moreover, a pattern of asymmetric introgression from molestus into pipiens was found. Subsequent molecular and ecological analyses carried out in the same region suggested that the molestus form has a higher tendency to explore indoor/anthropogenic habitats, when compared with the sympatric pipiens form. In both forms, over 90% of blood meals were made on avian hosts. The first genomic scan addressing levels of genome divergence between molestus and pipiens forms was implemented. Low levels of inter-form genomic divergence (1.4%–3.1%) were detected, consistent with a process of sympatric speciation with gene flow. Finally, Cx. pipiens s.s. samples collected in Greece during a WNV outbreak in 2010 were genetically characterised. Sympatric molestus and pipiens populations with asymmetric introgression were detected in the region where the outbreak occurred, whereas a more genetically homogenous molestus population was found in a region with no WNV transmission. These results highlight the importance of characterizing patterns of genetic variation and evolutionary relations among members of the Cx. pipiens complex as a requirement for understanding the potential of these species to act as disease vectors. They also open new perspectives for further research on the ecology and evolution of this species complex of medical importance