Estudos tem salientado que a expansão urbana, fragmentação do habitat, desmatamento e sobreposição das áreas de vida das populações humanas e silvestres tem constribuido para o surgimento de doenças emergentes e reemergentes nas últimas décadas. A paisagem da floresta amazônica vem sofrendo constantemente a dominância antropica e como resultado suas populações silvestres encontram-se cada vez mais expostas ao contato com populações humanas e domesticas com um alto risco de transmissão de agentes infecciosos. A cidade de Manaus, localizada na Amazônia Brasileira, tem afrontado um crescimento desordenado e vertiginoso devido ao seu desenvolvimento industrial causando uma alteração constante em sua paisagem; representando um modelo potencialmente útil para entender os mecanismos de transmissão de doenças. Os primatas são as espécies evolutivamente mais próximas dos humanos e essa proximidade filogenética tem facilitado o compartilhamento de diversos agentes infecciosos. O presente estudo propõe utilizar subpopulações de sauim-de-coleira (Saguinus bicolor) que ocupam os fragmentos urbanos de Manaus como espécie sentinela, para avaliar a presença de agentes infecciosos na interface-humano primata. A pesquisa objetiva também determinar se a perturbação antrópica dos locais de estudo estaria favorecendo a transmissão desses agentes. Entre os anos 2011 e 2014 um total de 55 sauins de coleira foram capturados em 9 fragmentos florestais urbanos e 1 area controle. Análises moleculares foram realizados para detectar Rotavirus A, Hantavirus, Coronavirus, Flavivirus, Enterovirus, Influenza A, Adenovirus, Metapneumovirus, Virus Sincitial Respiratório Humano, Parainfluenza 1, 2, 3, 4, Virus do Oeste de Nilo e Plasmodium spp. Os resultados indicaram uma prevalência para Hantavírus de (4/48), Rotavírus (9/48). Pela primeira vez é detectada a presença de hantavírus em primatas neotropicais. Nossos dados indicaram que a presença de infeção estaria associada com a existência de algum tipo de impacto antrópico nos locais pesquisados. Nenhum indivíduo resultou infectado na area controle. / In past decades, numerous studies have highlighted how urban expansion, habitat fragmentation, deforestation, and superposition of human and wildlife population areas contribute to surges in emergent and reemergent diseases. As a result of continuing anthropogenic disturbance in the Amazon, wildlife populations find themselves increasingly exposed to human populations and their domestic animals, bringing higher risks of transmission of a variety of infectious agents. Manaus, located in the Brazilian Amazon, represents a potentially useful model to understand the mechanisms of disease transmission. The city has undergone a disorganized and precipitous growth with ongoing industrial development, causing constant landscape alteration. Because non-human primates are closely evolutionary related to humans they share a diversity of infectious agents. The present study proposes to use subpopulations of Pied tamarins (Saguinus bicolor) occupying urban forest fragments in Manaus as a flagship species to evaluate the presence of infectious agents at the human-nonhuman primate interface. It will also assess whether anthropogenic perturbation at sites favors transmission of agents within this human dominated matrix. During the period of 2011-2014 a total of 55 pied tamarins in 9 urban forest fragments and 1 control area. Molecular analyses were performed for the detection of Rotavirus, Hantavirus, Coronavirus, Flavivirus, Enterovirus, Influenza A, Adenovirus, Metapneumovirus, Sincytial Human Respiratory virus, Parainfluenza 1, 2, 3, 4, West Nile Virus and Plasmodium spp. The results indicate prevalence for Hantavirus (n =4/48) and Rotavirus (n =9/48). This is the first record of Hantavirus in neotropical primates. Data indicate that the presence of infection in the study sites could be associated with anthropogenic impact. The control area resulted uninfected.

Submitted by biblioteca unifenas (biblioteca@unifenas.br) on 2018-03-02T18:56:27Z No. of bitstreams: 1 Rodrigo Felippin dissertação.pdf: 1035260 bytes, checksum: 2e8140a60a20265593d1c98c27337277 (MD5) / Made available in DSpace on 2018-03-02T18:56:27Z (GMT). No. of bitstreams: 1 Rodrigo Felippin dissertação.pdf: 1035260 bytes, checksum: 2e8140a60a20265593d1c98c27337277 (MD5) Previous issue date: 2016-12-08 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG / The experiment was conducted with the objective of evaluating the behavior of endemic diseases, which affect the sanity of dairy herds, directly influencing the productive and reproductive results of the animals. Blood samples of 216 animals were evaluated for BoHV-1 Ac, BVD Ac, BVD Ag and Neospora Caninum Ac, from 3 properties of the southern Minas Gerais, which belonged to the Dutch, Jersey and Girolando breeds. The animals were separated and distributed into groups according to the category, age, open days and test titration result. In order to identify the prevalence of these pathogens in the studied properties, the activity of these agents was monitored by the individual serology of the animals. For the BVD virus, 38.4% of the animals were positive, showing a significant effect (P <0.0001), with variation in viral activity ranging from 58% and 44.8% to 9.7%. Searching for viral fragments (antigen), which demonstrates the presence of the BVD virus, only 1 animal from the property with a higher incidence of positive serologies was diagnosed positive. This animal was later confirmed as permanently infected (PI), a condition in which the immune system identifies the virus as a particle of the organism itself, serving as a source of transmission for the herd. For the protozoan Neospora Caninum, 27.3% of the animals were seropositive, and again the property effect (P <0.01) showed higher prevalences (35% and 40%) in the same properties with high BVD indexes. The interpretation of the results obtained for IBR, however, should consider the behavior of a herpes virus, where, once infected, the animal remains infected for life. The indexes found (P <0.001) of positive prevalence ranged from 85.0% to 51.6%. We conclude that these infectious agents act on the reproduction of each property, varying according to the flow of the animals, animal concentration and sanitary aspects. And that the implementation of a health program based on the serological diagnosis is feasible and possible to be incorporated into the routine of the properties. For the BVD to identify the presence of PI animals is fundamental to maintain the health of the herd. / O experimento foi conduzido com objetivo de avaliar o comportamento de doenças endêmicas, que afetam a sanidade dos rebanhos leiteiros, influenciando diretamente nos resultados produtivos e reprodutivos dos animais. Foram avaliadas amostras sanguíneas de 216 animais para BoHV-1 Ac, BVD Ac, BVD Ag e Neospora Caninum Ac, oriundos de 3 propriedades do Sul de Minas Gerais, que pertenciam as raças: Holandês, Jersey e Girolando. Os animais foram separados e distribuídos em grupos de acordo com a categoria, idade, dias em aberto e resultado da titulação dos testes. Com intuito de identificar a prevalência desses patógenos nas propriedades estudadas, a atividade destes agentes foi monitorada pela sorologia individual dos animais. Para o vírus da BVD 38,4% dos animais foram positivos, demonstrando efeito significativo (P<0,0001) de propriedade, com variação da atividade viral desde valores altos como 58% e 44,8% até 9,7%. Pesquisando por fragmentos virais (antígeno), o que demonstra a presença do vírus da BVD, apenas um animal proveniente da propriedade com maior incidência de sorologias positivas foi diagnosticado positivo. Este foi posteriormente confirmado como permanentemente infectado (PI), condição em que o sistema imunológico identifica o vírus como partícula do próprio organismo, servindo de fonte de transmissão para o rebanho. Para o protozoário Neospora Caninum, 27,3% dos animais foram soropositivos e, novamente o efeito de propriedade (P<0,01) revelou maiores prevalências (35% e 40%) nas mesmas propriedades com altos índices para BVD. A interpretação dos resultados obtidos para IBR, no entanto, deve ser considerada a partir do comportamento de um herpes vírus, onde, uma vez infectado, o animal permanece assim por toda a vida. Os índices encontrados (P<0,001) de prevalências positivas variaram com resultados entre 85,0% a 51,6%. Concluímos que esses agentes infecciosos atuam na reprodução de cada propriedade, variando de acordo com o fluxo dos animais, concentração de animais e aspectos sanitários. E que a implantação de um programa sanitário baseado no diagnóstico sorológico, é viável e possível de ser incorporada na rotina das propriedades. Para a BVD identificar a presença de animais PI é fundamental para manter a saúde do rebanho.

No description available.

<p>The actin cytoskeleton is involved in many essential cellular events such as mitosis, cell migration, control of epithelial barrier function, and adherence of immune cells. Given the essential roles of the actin cytoskeleton, it is not surprising that it is a common target for bacterial virulence factors. <em>Legionella pneumophila</em>, the causative agent of Legionnaire’s pneumonia, establishes a replicative compartment using effectors secreted by its Dot/Icm secretion system. At least four Dot/Icm effectors, VipA, Ceg14, LegK2 and RavK have been shown to modulate the host actin cytoskeleton. Here, we identified RavJ (lpg0944) as an additional effector that interferes with the actin cytoskeleton in mammalian cells. We demonstrated that RavJ is a transglutaminase that functions to induce crosslink between actin and members of the Motin protein family, leading to inhibition of the binding between actin and ADF/cofilin. We also found that LegL1 (lpg0945) is a metaeffector of RavJ, which suppresses the transglutaminase activity of RavJ by blocking its enzymatic domain. </p>

<p dir="ltr"><i>Legionella pneumophila</i> strains harboring wild-type <i>rpsL</i> such as Lp02<i>rpsL</i>_WT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The mechanism of this unique infection-induced cell death remains unknown. Using a genome-wide CRISPR/Cas9 screening, we identified <i>Hmg20a </i>and <i>Nol9</i> as host factors important for restricting strain Lp02<i>rpsL</i>_WT in BMDMs. Depletion of <i>Hmg20a</i> protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by <i>Hmg20a</i> was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02<i>rpsL</i>_WT in BMDMs. Our results establish that <i>L. pneumophila</i> exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.</p>

<p dir="ltr"><i>Stenotrophomonas maltophilia</i> is an emerging multidrug-resistant, Gram-negative opportunistic pathogen. It causes many hospital-acquired infections such as sepsis, endocarditis, meningitis, and catheter-related urinary tract infections. It also affects individuals with cystic fibrosis, exacerbating their lung condition. <i>S. maltophilia</i> often causes pathogenesis through the formation of biofilms. However, the molecular mechanisms <i>S. maltophilia</i> uses to carry out these pathogenic steps are unclear. The SMF-1 chaperone/usher pilus has been thought to mediate <i>S. maltophilia</i> attachment. To confirm this role, we created an isogenic deletion of the <i>smf-1</i> pilin gene and observed a defect in biofilm compared to wild type. We also discovered 2 additional chaperone/usher pilus operons, mutation of which also caused attenuation in biofilm levels. Analysis of <i>S. maltophilia</i> clinical strains and <i>S. maltophili</i><i>a</i> complete genomes listed in NCBI showed that these three pili are prevalent and highly conserved, suggesting a vital role in infection. Intriguingly, through TEM studies, we found that the mutation of one pilus is not phenotypically compensated by another. Infection of <i>Galleria mellonella</i> larvae revealed increased virulence of the pilus mutants. Additionally, we also demonstrated a relationship between pilus and flagella contributing to the overall biofilm development of <i>S. maltophilia</i>. Understanding their activity may help identify therapeutic targets for this pathogen.</p>

L’empreinte écologique de l’homme sur les milieux naturels forestiers d’Afrique centrale n’a cessé de s’accroître au cours des dernières décennies, et rares sont les écosystèmes qui ne sont pas exploités. Ainsi, même des milieux a priori hostiles comme les grottes constituent des ressources répondant aux besoins primaires des populations environnantes (chasse, pêche, lieu de culte), mais permettant également le développement d’activités économiques (exploitation minière et écotourisme). Cette anthropisation est susceptible d’augmenter l’exposition des populations humaines à une multitude d'agents infectieux circulant au sein de la faune cavernicole. La présence d'animaux sauvages ou domestiques dans l'environnement immédiat des grottes contribue également à augmenter un tel risque. Parmi ces agents infectieux, certains peuvent être transmis par contact direct avec le réservoir animal, d’autres peuvent nécessiter l’intervention d’insectes hématophages. Le rôle de ces insectes dans l'épidémiologie de nombreux pathogènes est bien connu en Afrique en milieux épigés, mais demeure largement sous-investigué dans les environnements cavernicoles. Dans ce travail de thèse, nous nous sommes proposés de réponde à cette problématique. Dans six grottes du Gabon nous avons entrepris une étude de la diversité des diptères hématophages, en réalisant un inventaire taxonomique et l'étude de la dynamique temporelle des assemblages d'espèces. Dans un second temps, nous avons entrepris un criblage moléculaire d'agents infectieux parasitaires, bactériens et viraux en ayant recours à des techniques de détection par PCR nichée et séquençage à haut débit.Les travaux menés nous ont permis de réaliser un inventaire taxonomique de la faune diptérienne hématophage colonisant les grottes explorées. Notre étude a permis de découvrir une part importante de sa diversité de diptères hématophages cavernicoles, représentant plus de 60% des espèces de diptères (tout milieux confondus) actuellement au Gabon. Nos données ont montré que la composition des communautés de diptères était différente d'une grotte à une autre, et que ces dernières présentaient des variations au cours du temps en lien avec les variations microclimatiques des grottes. Le criblage d'agents infectieux chez les diptères collectés a permis 1) d'explorer la diversité parasitaire, virale et bactérienne qu'ils hébergent et 2) d'évaluer leur implication dans la transmission / The man's ecological mark on natural forest environments of central Africa has been increasing in recent decades, and only few natural areas remain non exploited yet. Thus, even supposedly hostile environments such as caves meet the primary needs of surrounding populations (hunting, fishing, shamanic practices), or economical needs (mining and ecotourism). That anthropization is likely to increase the exposure of human populations to multiple infectious agents carried by cave fauna. The presence of wild or domestic animals in the surroundings of caves also increase that risk. Among these infectious agents, some can be transmitted by direct contact with animal reservoirs, whereas others may require the involvement of blood-sucking insects. The role of these insects in the epidemiology of many pathogens is well known in Africa, particularly in epigeic environments, but remains largely investigated in caves. In this thesis work, we proposed to address that issue. In six caves of Gabon we firstly explored the diversity of blood-sucking Diptera, and we studied temporal dynamics of species assemblages. Secondly, we undertook a molecular screening of haemosporidia, bacteria and viruses using nested PCR and high-throughput sequencing.The work carried out has enable to make a taxonomic inventory of the Diptera fauna of the investigated caves. Our study revealed a significant diversity of the blood-sucking Diptera fauna, accounting for more than 60 percent of blood-sucking Diptera species currently known in Gabon, in all types of areas. Our data showed that the composition of Diptera communities was different from one cave to another, and that Diptera assemblages varied over time according to micro-climate fluctuations within caves. The screening of infectious agents in Diptera collected helped 1) to explore the parasitic, viral and bacterial diversity they host, and 2) to assess their involvement in transmission.

Made available in DSpace on 2016-01-26T18:55:36Z (GMT). No. of bitstreams: 1 Josaine.pdf: 238211 bytes, checksum: a26d1df7250354a0c0e8d5e909cc6db8 (MD5) Previous issue date: 2013-03-22 / Synanthropic flies are recognized as an important factor for the dissemination of infectious diseases to humans and animals. The aim of the present study was to determine the frequency of isolations and antimicrobial profile of Escherichia coli, Salmonella spp. and Staphylococcus spp. isolated from synanthropic flies captured with entomological traps, near of the in milk site of 30 dairy farms located in northern Paraná, Brazil. Surface washed and internal contents of 192 flies were submitted to microbiological analysis. In the 30 farms surveyed, the frequencies of caches of Muscidae (21/30 = 70%) and Calliphoridae (27/30 = 90%) were higher than Sarcophagidae (7/30 = 23.3%). E. coli was isolated only from Muscidae (14.3%) and Calliphoridae (33.3%). Salmonella spp. was isolated from 9.5% of Muscidae, 7.4% of Calliphoridae and 14.29% of Sarcophagidae. Staphylococcus spp. was only isolated from 28.5% of Muscidae and 29.6% of Calliphoridae. Isolates of E. coli were more common in flies captured on farms that kept domestic chickens near to the site milking (p = 0.031), and that did not use cane sugar for animal feed (p = 0.042). Two of the 27 strains (7.4%) of Staphylococcus spp. were coagulase-positive. Ceftriaxone, ciprofloxacin, enrofloxacin and gentamicin were the most effective antimicrobials against E. coli and Salmonella spp., and tetracycline was the less effective for the same microbial agents. None of the Staphylococcus spp. strains demonstrate resistance to oxacillin. It is concluded that the flies in the region are potential mechanical vectors of microbial agents that are able to cause enteritis in calves, mastitis in cows and contamination of dairy products produced on farms. / Moscas sinantrópicas são reconhecidas como fatores importantes na disseminação de várias doenças infecciosas para seres humanos e animais. O objetivo do presente estudo foi determinar a frequência de isolamentos e perfil de sensibilidade microbiano de Escherichia coli, Salmonella spp. e Staphylococcus spp. isolados de moscas sinantrópicas capturadas com armadilhas entomológicas próximas as salas de ordenha de 30 propriedades leiteiras localizadas no norte do Paraná, Brasil. Lavados superficiais e conteúdos internos de 192 moscas foram submetidos a análises microbiológicas. Nas 30 fazendas pesquisadas, as frequências de capturas de muscídeos (21/30 = 70%) e califorídeos (27/30 = 90%) foram superiores às capturas de sarcofagídeos (7/30 = 23,3%). E. coli foi isolada somente de muscídeos (14,3%) e califorídeos (33,3%). Salmonella spp. foi isolada de 9,5% dos muscídeos, 7,4% dos califorídeos e 14,29% dos sarcofagídeos. Staphylococcus spp. foi isolado de 28,5% dos muscídeos e 29,6% dos califorídeos. Isolamentos de E.coli foram mais comuns em moscas capturadas em fazendas que mantinham galinhas domésticas perto da sala de ordenha (p=0,031), e que não utilizavam cana de açúcar para alimentação animal (p=0,042). Duas de 27 (7,4%) linhagens de Staphylococcus spp. foram coagulase-positivas. Ceftriaxona, ciprofloxacina, enrofloxacina e gentamicina foram os antimicrobianos mais efetivos frente E. coli e Salmonella spp, enquanto tetraciclina foi o menos eficaz para os mesmos agentes microbianos. Nenhuma das estirpes de Staphylococcus spp. demonstraram resitência frente à oxacilina. Conclui-se que as moscas da região são potenciais vetores mecânicos de agentes microbianos capazes de causar enterite em bezerros, mastite em vacas e a contaminação de produtos lácteos.

Made available in DSpace on 2016-07-18T17:53:11Z (GMT). No. of bitstreams: 1 Josaine.pdf: 238211 bytes, checksum: a26d1df7250354a0c0e8d5e909cc6db8 (MD5) Previous issue date: 2013-03-22 / Synanthropic flies are recognized as an important factor for the dissemination of infectious diseases to humans and animals. The aim of the present study was to determine the frequency of isolations and antimicrobial profile of Escherichia coli, Salmonella spp. and Staphylococcus spp. isolated from synanthropic flies captured with entomological traps, near of the in milk site of 30 dairy farms located in northern Paraná, Brazil. Surface washed and internal contents of 192 flies were submitted to microbiological analysis. In the 30 farms surveyed, the frequencies of caches of Muscidae (21/30 = 70%) and Calliphoridae (27/30 = 90%) were higher than Sarcophagidae (7/30 = 23.3%). E. coli was isolated only from Muscidae (14.3%) and Calliphoridae (33.3%). Salmonella spp. was isolated from 9.5% of Muscidae, 7.4% of Calliphoridae and 14.29% of Sarcophagidae. Staphylococcus spp. was only isolated from 28.5% of Muscidae and 29.6% of Calliphoridae. Isolates of E. coli were more common in flies captured on farms that kept domestic chickens near to the site milking (p = 0.031), and that did not use cane sugar for animal feed (p = 0.042). Two of the 27 strains (7.4%) of Staphylococcus spp. were coagulase-positive. Ceftriaxone, ciprofloxacin, enrofloxacin and gentamicin were the most effective antimicrobials against E. coli and Salmonella spp., and tetracycline was the less effective for the same microbial agents. None of the Staphylococcus spp. strains demonstrate resistance to oxacillin. It is concluded that the flies in the region are potential mechanical vectors of microbial agents that are able to cause enteritis in calves, mastitis in cows and contamination of dairy products produced on farms. / Moscas sinantrópicas são reconhecidas como fatores importantes na disseminação de várias doenças infecciosas para seres humanos e animais. O objetivo do presente estudo foi determinar a frequência de isolamentos e perfil de sensibilidade microbiano de Escherichia coli, Salmonella spp. e Staphylococcus spp. isolados de moscas sinantrópicas capturadas com armadilhas entomológicas próximas as salas de ordenha de 30 propriedades leiteiras localizadas no norte do Paraná, Brasil. Lavados superficiais e conteúdos internos de 192 moscas foram submetidos a análises microbiológicas. Nas 30 fazendas pesquisadas, as frequências de capturas de muscídeos (21/30 = 70%) e califorídeos (27/30 = 90%) foram superiores às capturas de sarcofagídeos (7/30 = 23,3%). E. coli foi isolada somente de muscídeos (14,3%) e califorídeos (33,3%). Salmonella spp. foi isolada de 9,5% dos muscídeos, 7,4% dos califorídeos e 14,29% dos sarcofagídeos. Staphylococcus spp. foi isolado de 28,5% dos muscídeos e 29,6% dos califorídeos. Isolamentos de E.coli foram mais comuns em moscas capturadas em fazendas que mantinham galinhas domésticas perto da sala de ordenha (p=0,031), e que não utilizavam cana de açúcar para alimentação animal (p=0,042). Duas de 27 (7,4%) linhagens de Staphylococcus spp. foram coagulase-positivas. Ceftriaxona, ciprofloxacina, enrofloxacina e gentamicina foram os antimicrobianos mais efetivos frente E. coli e Salmonella spp, enquanto tetraciclina foi o menos eficaz para os mesmos agentes microbianos. Nenhuma das estirpes de Staphylococcus spp. demonstraram resitência frente à oxacilina. Conclui-se que as moscas da região são potenciais vetores mecânicos de agentes microbianos capazes de causar enterite em bezerros, mastite em vacas e a contaminação de produtos lácteos.

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