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

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3′ untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions.

Alphaviruses are a group of medically relevant arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that are maintained by mosquito vectors. These zoonotic viruses are clustered into two groups: New World and Old World, depending on their geographical origin/distribution and clinical manifestations. Both of these groups cause disease symptoms of an acute febrile illness; however, each group has a distinct, hallmark disease symptom; New World alphaviruses, such as Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), present with severe encephalitis while Old World alphaviruses, such as Sindbis, chikungunya, and Mayaro viruses (SINV, CHIKV, and MAYV, respectively) present with an incapacitating polyarthralgia that can persist for years following initial infection. To date, the most effective means of controlling these arboviral infections is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. Given the close phylogenetic and antigenic relationship between MAYV and CHIKV, we hypothesized that prior CHIKV immunity may affect the outcome of MAYV disease and/or limit its emergence in humans. Our work has shown that anti-CHIKV neutralizing antibodies can provide cross-protective immunity against MAYV disease. Alongside these studies, we have characterized the potency of a camelid-derived single-domain antibody (sdAb) that neutralizes a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express two anti-CHIKV sdAbs to target infection, dissemination, and transmission of MAYV and CHIKV within this deadly vector. These findings are particularly significant because they highlight the ability to co-target two emerging alphaviruses that are crippling public health and obliterating quality of life around the globe within a single defense strategy. / Doctor of Philosophy / Alphaviruses are arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that infect millions of people annually via the bite of female mosquitoes. These viruses are major public health threats due to their ability to infect humans and animals and infections resulting in a range of debilitating diseases. Viruses within this genus are clustered into two groups: Old World and New World, based on geographical origin and distribution. While New World alphaviruses are known for inducing severe encephalitis (i.e., swelling in the brain), a hallmark symptom of the Old World alphaviruses is the development of incapacitating polyarthralgia (i.e., widespread joint pain) that can persist for years following initial infection. To date, the most effective means of combatting these viruses is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens. Given the close genetic relationship between chikungunya virus (CHIKV) and Mayaro virus (MAYV), our research has focused on harnessing cross-reactive immunity between these emerging alphaviruses. We discovered this cross-reactivity provides protective immunity to both viruses (i.e., CHIKV and MAYV) after exposure to only one (i.e., CHIKV) of the viruses. Next, we characterized the potency of a small, single-domain antibody (sdAb) to neutralize a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express this sdAb to target both CHIKV and MAYV within this deadly mosquito vector. These findings are particularly significant because they provide the foundation for a novel approach to controlling and preventing outbreaks of these emerging alphavirus pathogens that obliterate quality of life in public health settings around the globe.

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

[Truncated abstract] In March 2005, the Indian Ocean island of Reunion, a former colony and now overseas department of France, saw the first cases of what was to become a massive epidemic of the mosquito borne viral infection Chikungunya. More than 250,000 people, one third of the Island's population, were subject to high fevers, rash, and joint and muscle pains over the next 18 months, yet the public health authorities in metropolitan France were arguably slow to take the epidemic seriously. The research presented here explores attitudes underlying the management of the epidemic by examining both metropolitan and local representations of mosquito borne disease from historical, epidemiological and media perspectives. The research seeks to answer the general question Does colonial history continue to influence the representation and management of mosquito borne disease in Reunion? Three parallel approaches are taken to answering this question, using a common framework of tropicality (a Western discourse that exalts the temperate world over its tropical counterpart, and overlaps with colonialism and orientalism). ... Several factors are likely to have contributed to the persistence of tropicality in public health practice in Reunion: Othering as a universal phenomenon; the cost of administering interventions to combat tropical diseases in the remote environments of French overseas departments and territories; the denial of a serious public health risk as a cultural trait in Reunion; and the significant role of the colonies in forming and maintaining the French national identity. It has to be acknowledged that historically, tropicalism does appear to have played one positive role in the management of mosquito borne disease:

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-04-03T14:48:30Z No. of bitstreams: 1 HannalyWanaBezerraPereira_DISSERT.pdf: 2518932 bytes, checksum: 2d0f7247523a10a3475dd41f16e8f017 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-04-10T20:00:29Z (GMT) No. of bitstreams: 1 HannalyWanaBezerraPereira_DISSERT.pdf: 2518932 bytes, checksum: 2d0f7247523a10a3475dd41f16e8f017 (MD5) / Made available in DSpace on 2018-04-10T20:00:29Z (GMT). No. of bitstreams: 1 HannalyWanaBezerraPereira_DISSERT.pdf: 2518932 bytes, checksum: 2d0f7247523a10a3475dd41f16e8f017 (MD5) Previous issue date: 2018-02-27 / A febre Chikungunya ? uma s?ndrome febril com grave artralgia debilitante, podendo evoluir para casos at?picos, como manifesta??es neurol?gicas e mucocut?neas. Geralmente ? transmitida por mosquitos do g?nero Aedes. O agente etiol?gico ? o v?rus Chikungunya (CHIKV) que pertence ? fam?lia Togaviridae e ao g?nero Alphavirus. At? pouco tempo essa doen?a era negligenciada no Brasil, por?m com o surto epid?mico que houve no ano de 2016, essa arbovirose se tornou um desafio para a sa?de p?blica. O objetivo do presente estudo foi realizar a caracteriza??o gen?tica do CHIKV identificado no Estado do Rio Grande do Norte (RN), no bi?nio 2016-2017. Um total de 10 amostras de soro, l?quor ou conte?do de ves?culas-bolhosas foram analisadas pela metodologia de qRT-PCR para detec??o do CHIKV e todas se apresentaram positivas. Foi realizada a filogenia e a caracteriza??o gen?tica do v?rus, por meio do sequenciamento da regi?o codificadora da poliprote?na estrutural, com posterior an?lise da estrutura da prote?na por meio da modelagem. A an?lise filogen?tica indicou que o gen?tipo circulante no Estado do Rio Grande do Norte ? o Leste-Centro-Sul Africano II (ECSA II). A compara??o entre as sequencias dos CHIKV deste estudo com aquelas de seu ancestral da linhagem ECSA II identificado em Uganda em 1982 (GenBank: HM045812) revelou a presen?a de 21 muta??es n?o sin?nimas. O sequenciamento dos CHIKV do soro e do l?quor de um mesmo paciente revelou duas muta??es n?o sin?nimas potencialmente associadas a neurovirul?ncia viral: N606K e P677L. Adicionalmente, a an?lise da modelagem de prote?nas mostrou que o v?rus circulante no Rio Grande do Norte n?o apresenta a muta??o na posi??o A226V, que determina uma maior infectividade do v?rus para o Aedes albopictus. Em conclus?o, esse estudo revela a origem do CHIKV circulante no Estado do Rio Grande do Norte e poss?veis marcadores de neurovirul?ncia viral, informa??es ?teis para compreender a evolu??o viral e a patog?nese da doen?a. / Chikungunya fever is a febrile syndrome with severe debilitating arthralgia, which may progress to atypical cases, such as neurological and mucocutaneous manifestations. It is usually transmitted by mosquitoes of the genus Aedes. The etiological agent is the Chikungunya virus (CHIKV) that belongs to the family Togaviridae and to the genus Alphavirus. Until recently this disease was neglected in Brazil, but with the epidemic outbreak that occurred in the year 2016, this arbovirose has become a challenge for public health. The objective of the present study was to perform the genetic characterization of the CHIKV identified in the State of Rio Grande do Norte (RN), in the biennium 2016-2017. A total of 10 serum samples, cerebrospinal fluid (CSF) or blister samples were analyzed by the qRT-PCR methodology for CHIKV detection and all were positive. Phylogeny and genetic characterization of the virus were performed by sequencing the coding region of the structural polyprotein, with subsequent analysis of the protein structure through modeling. Phylogenetic analysis indicated that the circulating genotype in the State of Rio Grande do Norte is East-Central-South-African II (ECSA II). The comparison between CHIKV sequenced in this study and its ancestor from ECSA II lineage identified in Uganda in 1982 (GenBank: HM045812) revealed the presence of 21 non-synonymous mutations. Sequencing of serum and CSF CHIKV from the same patient revealed two non-synonymous mutations potentially associated with viral neurovirulence: N606K and P677L. In addition, the analysis of the protein model showed the circulating non-RN virus has no mutation at position A226V which is the major part of virus infection for Aedes albopictus. In conclusion, this study reveals the origin of CHIKV circulating in the State of Rio Grande do Norte and possible markers of viral neurovirulence, useful information to understand the viral evolution and the pathogenesis of the disease.

Dans différents domaines de recherche, la modélisation est devenue un outil efficace pour étudier et prédire l’évolution possible d’un système, en particulier en épidémiologie. En raison de la mondialisation et de la mutation génétique de certaines maladies ou vecteurs de transmission, plusieurs épidémies sont apparues dans des régions non encore concernées ces dernières années. Dans cette thèse, un modèle décrivant la transmission de l’épidémie de chikungunya à la population humaine est étudié. Ce modèle prend en compte la mobilité spatiale des humains, ce qui est nouveau. En effet, c’est un facteur intéressant qui a influencé la réapparition de plusieurs maladies épidémiques. Le déplacement des moustiques est omis puisqu’il est limité à quelques mètres. Le modèle complet (modèle EDOs-EDPs) est alors composé d’un système à réaction-diffusion (prenant la forme d’équations différentielles partielles (EDPs) paraboliques semi-linéaires) couplé à des équations différentielles ordinaires (EDOs). Nous démontrons pour ce modèle, d’abord l’existence et l’unicité de la solution globale, sa positivité et sa bornitude, puis nous donnons quelques simulations numériques. Dans ce modèle, certains paramètres ne sont pas directement accessibles à partir des expériences et doivent être estimés numériquement. Cependant, avant de rechercher leurs valeurs, il est essentiel de vérifier l’identifiabilité des paramètres pour déterminer si l’ensemble des paramètres inconnus peut être déterminé de manière unique à partir des données. Cette étude permettra de s’assurer que les procédures numériques peuvent être couronnées de succès. Si l’identifiabilité n’est pas assurée, certaines données supplémentaires doivent être ajoutées. En fait, une première étude d’identifiabilité a été effectuée pour le modèle EDOs en considérant que le nombre d’œufs peut être facilement compté. Toutefois, après avoir discuté avec les chercheurs épidémiologistes, il apparaît que c’est le nombre de larves qui peut être estimé semaines par semaines. Ainsi, nous ferons une étude d’identifiabilité pour le nouveau modèle EDOs-EDPs avec cette hypothèse. Grâce à l’intégration de l’une des équations du modèle, on obtient des équations plus faciles reliant les entrées, les sorties et les paramètres, ce qui simplifie vraiment l’étude d’identifiabilité. A partir de l’étude d’identifiabilité, une méthode et une procédure numérique sont proposés pour estimer les paramètres sans en avoir connaissance. / In different fields of research, modeling has become an effective tool for studying and predicting the possible evolution of a system, particularly in epidemiology. Due to the globalization and the genetic mutation of certain diseases or transmission vectors, several epidemics have appeared in regions not yet concerned in the last years. In this thesis, a model describing the transmission of the chikungunya epidemic to the human population is studied. As a novelty, this model incorporates the spatial mobility of humans. Indeed, it is an interesting factor that has influenced the re-emergence of several epidemic diseases. The displacement of mosquitoes is omitted since it is limited to a few meters. The complete model (ODEs-PDEs model) is then composed of a reaction-diffusion system (taken the form of semi-linear parabolic partial differential equations (PDEs)) coupled with ordinary differential equations (ODEs). We prove the existence, uniqueness, positivity and boundedness of a global solution of this model at first and then give some numerical simulations. In such a model, some parameters are not directly accessible from experiments and have to be estimated numerically. However, before searching for their values, it is essential to verify the identifiability of parameters in order to assess whether the set of unknown parameters can be uniquely determined from the data. This study will insure that numerical procedures can be successful. If the identifiability is not ensured, some supplementary data have to be added. In fact, a first identifiability study had been done for the ODEs model by considering that the number of eggs can be easily counted. However, after discussing with epidemiologist searchers, it appears that it is the number of larvae which can be estimated weeks by weeks. Thus, we will do an identifiability study for the novel ODEs-PDEs model with this assumption. Thanks to an integration of one of the model equations, some easier equations linking the inputs, outputs and parameters are obtained which really simplify the study of identifiability. From the identifiability study, a method and numerical procedure are proposed for estimating the parameters without any knowledge of them.

Dans le but de rechercher de nouveaux inhibiteurs de l’ARN polymérase NS5 du virus de la dengue (DENV), un criblage a été mené sur 650 plantes néo-calédoniennes. A la suite de ce criblage, deux espèces (Cryptocarya chartacea Kostermans et Trigonostemon cherrieri Veillon) ont été sélectionnées. L’extrait AcOEt des écorces de Cryptocarya chartacea (Lauraceae) a montré une forte inhibition de la NS5 polymérase (99 % à 10 µg/ml). L’étude phytochimique de l’extrait a mis en évidence une série de nouvelles flavanones 6-mono et 6,8-dialkylées, nommées chartacéones. Celles-ci sont présentes sous forme de mélanges racémiques au sein de C. chartacea. La chartacéone A a été purifiée sur colonne chirale conduisant à l’isolement de quatre diastéréoisomères optiquement purs. Une étude configurationnelle basée sur le calcul théorique de spectres de dichroïsme circulaire a permis la détermination de leur configuration absolue. Les chartacéones inhibent de façon sélective la NS5 polymérase du DENV. L’étude des extraits AcOEt des écorces et du bois de Trigonostemon cherrieri (Euphorbiaceae) a mis en évidence une série de métabolites secondaires originaux de type Diterpènes Daphnane Orthoester (DDO) chlorés : les trigocherrines (non-macrocycliques) et les trigocherriolides (macrocycliques). Ces composés ont montré une inhibition de l’activité enzymatique de la NS5 polymérase du DENV et une activité antivirale sur le virus du chikungunya in cellulo. / In order to identify new inhibitors of the dengue virus (DENV) NS5 RNA polymerase, a screening was led on 650 new-caledonian plants. Two species, Cryptocarya chartacea Kostermans and Trigonostemon cherrieri Veillon were selected. The EtOAc bark extract of Cryptocarya chartacea (Lauraceae) showed a potent inhibition of the NS5 polymerase activity (99 % at 10 µg/ml). The phytochemical study of the extract led to the isolation of a series of new 6-mono and 6,8-dialkylated flavanones, called chartaceones. Chartaceones are present as racemic mixtures in the plant. Chartaceone A was purified on chiral column leading to the isolation of 4 optically pure diastereoisomers. A configurational study based on the theoretical calculation of circular dichroism spectra allowed the determination of their absolute configuration. Chartaceone are selective inhibitors of the DENV NS5 polymerase. The study of the EtOAc extract from the bark and wood of Trigonostemon cherrieri (Euphorbiaceae) led to the isolation of a series of unusual chlorinated daphnane diterpene orthoesters (DDO) : trigocherrins (non macrocylic) and trigocherriolides (macrocyclic). These compounds inhibit the DENV NS5 polymerase activity and present an antiviral activity on the chikungunya virus in cellulo.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes disease in humans and equines eliciting both an agricultural and public health threat. In humans, the disease typically presents as a febrile illness with common signs of fever and malaise. Four to fourteen percent of Venezuelan equine encephalitis (VEE) cases are associated with severe neurological complications due to encephalitis caused by VEEV's propensity to infect the brain. Public health concerns are exacerbated by VEEV's aerosolization capabilities, low infectious dose and affordability to mass produce. These qualities drove interest in the pathogen as a bioweapon by the US and the former Soviet Union during the cold war. As a precautionary response to VEEV's notoriety as a biothreat, the National Institute of Allergies and Infectious Diseases has classified VEEV as a category B priority pathogen, and the Human Health Services and United States Department of Agriculture list live virulent strains of VEEV as a select agent and require the pathogen to be manipulated in highly regulated biosafety level 3 (BSL3) facilities. There are currently no FDA approved vaccines or antivirals to target VEEV or other closely related alphaviruses associated with clinical disease in humans. The research performed in this dissertation aimed to elucidate new antiviral targets and treatments to help bridge gaps in current understanding of alphaviruses. The current market lacks available antibodies for E1 specific isolation. In response, a recombinant VEEV TC-83 was produced with a V5 tag at the C-terminal of the E1 sequence to enable VEEV E1 detection. Sequencing was used to verify V5 insertion in the plasmid and immunoprecipitation was used to verify V5 insertion within the E1 glycoprotein. Replication kinetics experiments verified the virus replicated similarly to the parental VEEV TC-83 strain, while passaging experiments verified the tag was highly stable for up to 10 passages. This research produced a cost-effective and highly efficient means to probe and isolate the E1 glycoprotein without modifying the viability of the virus. Knowledge of host protein interactions with VEEV E1 glycoprotein has been limited, with most E1 research focusing on its fusion capabilities. Utilizing 293-T cells infected with E1-V5 TC-83, co-immunoprecipitation was performed to isolate E1 and associated interactors. A total of 486 host and 5 viral protein interactors of E1 were discovered after normalization to the negative control. The top peptide spectrum matches (PSMs) revealed a number of chaperone proteins and ubiquitin proteins as top interactors of VEEV E1. These results effectively revealed a number of previously unknown alphavirus interactions that can be targeted by antivirals and explored further for implications in viral replication. LC-MS/MS results showed that protein disulfide isomerase family A member 6 (PDIA6) interacted with E1. High PSMs, presence in all 3 replicates, similar cellular localization to E1 and known associations between other viruses and protein disulfide isomerase (PDI) family members made this protein an optimum target for further analysis. Co-immunoprecipitation and co-localization experiments were used to validate the LC-MS/MS results. Involvement of PDIs in VEEV replication were explored utilizing two known PDI inhibitors, LOC14 and Nitazoxanide. LOC14, a non-FDA approved broad-spectrum PDI inhibitor, showed broad-spectrum alphavirus antiviral potential, decreasing titers of VEEV TC-83, VEEV Trinidad Donkey strain, eastern equine encephalitis virus (EEEV), chikungunya virus (CHIKV) and Sindbis (SINV) virus in a dose dependent manner. Nitazoxanide, an FDA approved drug known to inhibit PDIA3, was shown to have minimal toxicity and effectively reduced VEEV TC-83 and EEEV titers at concentrations with 100% cell viability. Time of addition assays, E1 expression time course studies, and early event assays showed PDI inhibition with these drugs effects early viral production events. RNA quantification, confocal microscopy and biotin switch assay experiments show that the drugs also prevented proper folding of the E1 glycoprotein and decreased expression of E1 on the peripheral membrane. With no current treatments for alphaviruses, these data provide an effective broad-spectrum target that affects viral replication at multiple stages in-vitro. Nitazoxanide also presents as a promising, non-toxic drug that could be repurposed to combat a number of clinically relevant alphaviruses. Valosin containing protein (VCP) was also shown to interact with the E1 glycoprotein. Exploration of VCP's interaction with alphavirus E1 has never been explored, yet it was previously shown to be involved in alphavirus replication. Co-localization and co-immunoprecipitation experiments were performed validating the interaction between VCP and E1. siRNA knockdown of VCP in 293-T cells and U87-MG cells showed a significant reduction in VEEV TC-83 titers. The allosteric VCP inhibitor, NMS-873, also reduced VEEV TC-83 titers, but was shown to be less effective against CHIKV, SINV and EEEV, suggesting the NMS-873 mechanism is more selective for VEEV. Mechanism experiments showed that reduction of VCP with NMS-873 inhibits early events of VEEV replication. These results elucidate VCP's association with E1 and show that VCP can be targeted to decrease VEEV viral replication. / Doctor of Philosophy / Venezuelan equine encephalitis virus (VEEV) causes disease in humans, as well as horses, donkeys and other closely related animals. In humans, the virus causes a flu-like disease and sometimes swelling of the brain. This can be associated with symptoms such as light sensitivity, confusion and sometimes coma. Prior to the Cold War, VEEV was researched by the US and previous Soviet Union's militaries in hopes to deploy the virus as a bioweapon. Current treaties prevent active production of such weapons, yet allows for defensive research to continue in preparation for a worst-case scenario. Currently no FDA approved medications or vaccines exist to combat the virus further exacerbating concerns. In order to protect laboratorians and prevent unintentional or intentional introduction of the virus into the community, the virus is only manipulated in highly secure facilities with barriers that separate the virus from personnel and the outside environment. A component of the virus called E1, allows for the virus to be released from a structure, called an endosome, that transports the virus into the cell. Currently, E1 is mostly known for this function, yet our research found that E1 interacts with 486 protein components of the host cell, suggesting a more elaborate role of E1 than previously understood. This list of interactors provides numerous new targets for potential medications to combat VEEV and other closely related viruses. Discovered E1 interactors, protein disulfide isomerase family A member 6 (PDIA6) and valosin containing protein (VCP), were validated through extensive experimentation and their function in viral replication was further explored. Protein disulfide isomerases (PDI), such as PDIA6, play an important role in folding proteins, which are cellular components made of organic building blocks called amino acids. PDIs do so by creating organic pillars, called disulfide bonds, between two cysteine amino acid residues. These disulfide bonds contribute to the 3D shape of the proteins they fold which are essential for the protein's function. E1 of VEEV has a total of eight disulfide bonds within its structure, highlighting that disulfide bonds are likely essential for the protein's structure, and therefore, function. We verified that E1 could not properly fold without PDI function by using two compounds that prevented PDI from forming or breaking disulfide bonds, specifically LOC14 and FDA approved drug nitazoxanide. Cells treated with one of either compound before and after infection with VEEV, were found to produce E1 protein with significantly less disulfide bonds therefore producing less viable virus. Further experiments also showed that the compounds also affected early stages in the virus production cycle. These two mechanisms explain the significant reduction in production of VEEV and related viruses when PDI is inhibited. These results provide a new VEEV drug target, PDIs, as well as two compounds that can potentially be used to combat VEEV and other related viruses that have no current treatment options. Another host interactor, VCP, functions throughout the cell and is known for unfolding of numerous substrates, including proteins. It is involved in numerous cellular functions thus making this interactor a promising target for drug treatment. Cells with reduced VCP function were shown to produce less progeny VEEV. Cells treated with NMS-873, a compound that reduces VCP function was also shown to reduce VEEV production. NMS-863 inhibition of VCP was shown to effect early events in VEEV replication. These results further emphasize the E1 interactors discovered are invaluable novel targets for VEEV drug treatment.

Submitted by Luiz Cesar Maranho (lc-maranho@bol.com.br) on 2018-10-11T20:16:50Z No. of bitstreams: 1 Dissertação Final.pdf: 1883342 bytes, checksum: 85a25606a3113b39d6d4354dcaa161d8 (MD5) / Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2018-10-15T12:39:20Z (GMT) No. of bitstreams: 1 maranho_lc_me_sjrp.pdf: 5069791 bytes, checksum: 2501e6acc67bdd7103eb807f326a4c0b (MD5) / Made available in DSpace on 2018-10-15T12:39:20Z (GMT). No. of bitstreams: 1 maranho_lc_me_sjrp.pdf: 5069791 bytes, checksum: 2501e6acc67bdd7103eb807f326a4c0b (MD5) Previous issue date: 2018-08-20 / O mosquito Aedes aegypti é o principal vetor responsável por diversas arboviroses como a dengue, a febre amarela, o vírus zika e a febre chikungunya. Devido a sua resistência, adaptabilidade e proximidade ao homem, o Aedes aegypti é atualmente um dos maiores problemas de saúde pública no Brasil e nas Américas. Mesmo com os avanços e investimentos em pesquisas com vacinas, monitoramento, campanhas educativas e diversos tipos de controle deste vetor, ainda não existe um método eficaz para controlar e erradicar o mosquito. Portanto, esse trabalho destina-se ao auxílio na criação de estratégias para controlar esse agente transmissor, mediante a análise do espaço de estados e a estabilidade assintótica de uma dinâmica não linear para controle populacional do Aedes aegypti via a técnica de linearização de Lyapunov, além de apresentação de formas de prevenção e combate aos criadouros do mosquito. A dinâmica não linear proposta é uma dinâmica simplificada obtida de um modelo não linear existente na literatura, proposto por Esteva e Yang em 2005 e se baseia no ciclo de vida do mosquito, que é dividido em duas fases: fase imatura ou aquática (ovos, larvas e pupas) e fase alada (mosquitos adultos). Na fase adulta, os mosquitos são divididos em machos, fêmeas imaturas e fêmeas fertilizadas, sendo que a dinâmica proposta nesta dissertação de mestrado é baseada nos estudos efetuados por Reis desde 2016, obtendo um modelo simplificado no qual a soma das densidades das populações de fêmeas imaturas e fêmeas fertilizadas serão consideradas como fêmeas adultas. / The mosquito Aedes aegypti is the main vector responsible for several arboviruses such as dengue fever, yellow fever, zika virus and chikungunya fever. Due to its resistance, adaptability and proximity to humans, Aedes aegypti is currently one of the major public health problems in Brazil and the Americas. Even with the advances and investments in research with vaccines, monitoring, educational campaigns and various types of control of this vector, there is still no effective method to control and eradicate the mosquito. Therefore, this work is intended to aid in the creation of strategies to control this transmitting agent by analyzing the state space and the asymptotic stability of a nonlinear dynamics for population control of Aedes aegypti via the Lyapunov linearization technique to present ways of preventing and combating mosquito breeding sites. The proposed nonlinear dynamics is a simplified dynamics obtained from a nonlinear model existing in the literature, proposed by Esteva and Yang in 2005 and based on the life cycle of the mosquito, which is divided into two phases: immature or aquatic phase (eggs, larvae and pupae) and winged phase (adult mosquitoes). In the adult phase, mosquitoes are divided into males, immature females and fertilized females, and the dynamics proposed in this dissertation is based on studies carried out by Reis since 2016, obtaining a simplified model in which the sum of the densities of the populations of females immature and fertilized females will be considered as adult females.