<div> <div> <div> <p>Flaviviruses is a genus within the family Flaviviridae. The genus consists of more than 70 viruses, including important threatening human pathogens such as dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). These viruses are causative agents for a range of mild to lethal diseases and there are currently no US- licensed therapeutic treatments for infection. The virus genome is a positive-sense, single-stranded RNA, encoding ten viral proteins. Of the ten flavivirus proteins, Non- Structural protein 1 (NS1) remains the most elusive in terms of its functions. To date NS1 has been linked to disease pathology and progression and plays roles in virus replication and assembly. However, little is understood how NS1 orchestrates these functions and how NS1 from different viruses function distinctively from one another. Moreover, flavivirus NS1 has a peculiar ability to associate with lipid membranes. During the life cycle of NS1, the protein travels through the classical secretory path- way, similar to infectious virus particles, and is secreted into the extracellular space as mostly hexameric oligomers containing a lipid core. How the protein binds to lipids and whether such lipid binding is important for NS1 functions and overall flavivirus pathology remain unknown. Using structure-based mutagenesis, we found a group of mutants on WNV NS1, which particularly altered the viral specific infectivity but maintained wild-type level of virus replication. Purified mutated virus particles revealed that the specific infectivity alteration was not because of the particle but interaction of the virus particles and NS1 mutated proteins. Here we demonstrated that specific residues on NS1 were responsible for distinctly roles in NS1 functions and the virus specific infectivity was regulated by NS1 protein. In other structure-base study, we focused on the membrane association ability of NS1. All structure-predicted regions on NS1 were examined for its contribution for the membrane/lipid binding function. This interaction was required for NS1 biology activities including intracel- lular trafficking, oligomerization, and endocytosis. The lipidomes from deletion of each membrane association region revealed differences in lipid classes binding to each region and the composition flexiblity of the lipid cargo of NS1 hexamer. </p> </div> </div> </div>

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Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:48:40Z No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:48:50Z (GMT) No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) / Made available in DSpace on 2015-05-12T13:48:50Z (GMT). No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A dengue é atualmente um dos principais problemas de saúde pública do mundo e segundo a Organização Mundial de Saúde (OMS) é a doença que mais acomete o homem na atualidade. Sua incidência vem aumentando e estima-se que 50-100 milhões de pessoas desenvolvam a doença a cada ano no mundo. O diagnóstico laboratorial da dengue é realizado por diferentes tipos de testes, entre eles estão o isolamento viral, o RT-PCR, e a detecção por ELISA ou por meio de testes rápidos do antígeno viral NS1 e de anticorpos IgM específicos contra o vírus. A fim de contribuir para um melhor entendimento sobre a validade destes testes em diferentes circunstâncias, o objetivo principal deste trabalho foi avaliar a validade dos diferentes métodos laboratoriais no diagnóstico da dengue. A sensibilidade dos testes diagnósticos, ELISA IgM, ELISA NS1 e RT-PCR, foi avaliada de forma individual e de forma combinada utilizando amostras de soro de 623 pacientes incluídos em um estudo prospectivo de vigilância de base populacional entre fevereiro e julho de 2010. A sensibilidade destes testes também foi avaliada de acordo com a duração dos sintomas, com o tipo de infecção (primária vs secundária) e, por sorotipo infectante. A especificidade de cada método foi avaliada em um grupo de amostras de pacientes com diagnóstico laboratorial de leptospirose, hepatite, doadores de sangue e indivíduos sadios. Os resultados encontrados mostraram que 240 (38%) dos pacientes com doença febril aguda apresentaram dengue no período do estudo sendo que 194 (81%) dos pacientes com dengue representavam pacientes com infecções secundárias, o sorotipo predominante foi o DENV-2 (70%). As sensibilidades do RT-PCR, do ELISA NS1 e do ELISA IgM na amostra de fase aguda foram de 83,3%, 31,7% e 30%, respectivamente. O uso combinado do teste RT-PCR e do teste ELISA IgM em uma amostra de fase convalescente foi capaz de identificar 100% dos casos confirmados de dengue. As especificidades encontradas variaram de 97% a 100% para o ELISA NS1 e de 55% a 85% para o ELISA IgM. Os resultados indicam que na fase aguda da doença o RT-PCR é mais sensível a detecção de anticorpos IgM e do antígeno NS1 por ELISA, entretanto, o uso de métodos diagnósticos adicionais pode ser necessário em pacientes com uma suspeita da doença e resultado negativo do RT-PCR. / Dengue is currently one of the main problems of public health and the world according to the World Health Organization (WHO) is the disease that affects more men today. Its incidence is increasing and it is estimated 50-100 million people develop the disease each year worldwide. Laboratory diagnosis of dengue is done by testing different types, which include viral isolation, RT-PCR, and detection by ELISA or by rapid viral tests NS1 antigen and specific IgM antibodies against the virus. In order to contribute to a better understanding of the validity of these tests in different circumstances, the aim of this study was to evaluate the validity of different laboratory methods for diagnosis of dengue. The sensitivity of diagnostic tests, IgM ELISA, ELISA NS1 and RT-PCR was evaluated individually and in combination form using serum samples from 623 patients enrolled in a prospective population-based study of surveillance between February and July 2010. The sensitivity of these tests was also evaluated according to the duration of symptoms of infection with the type (primary versus secondary), and the infecting serotype. The specificity of each method was evaluated in a group of samples from patients with laboratory diagnosis of leptospirosis, hepatitis, blood donors and healthy individuals. The results showed that 240 (38%) of patients with acute febrile disease had dengue during the study period of which 194 (81%) of patients with dengue represented patients with secondary infections, the predominant serotype was DENV-2 (70% ). The sensitivity of the RT-PCR of NS1 and IgM ELISA ELISA in the acute phase of the sample were 83.3%, 31.7% and 30%, respectively. The combined use of RT-PCR and ELISA IgM in a sample convalescent phase was able to identify 100% of confirmed cases of dengue fever. The specificities found varied from 97% to 100% for ELISA NS1 and 55% to 85% for the IgM ELISA. The results indicate that the acute phase of the disease the RT-PCR is more sensitive detection of IgM antibodies and NS1 antigen by ELISA, however, the use of additional diagnostic methods may be necessary in patients with a suspicion of disease and negative outcome of RT-PCR.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-12-06T14:22:57Z No. of bitstreams: 2 Dissertação - Ricardo Lemes Gonçalves - 2017.pdf: 6297486 bytes, checksum: 3393a7cb0ffe26eafd0acd04f1198ab0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-12-07T10:18:10Z (GMT) No. of bitstreams: 2 Dissertação - Ricardo Lemes Gonçalves - 2017.pdf: 6297486 bytes, checksum: 3393a7cb0ffe26eafd0acd04f1198ab0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-07T10:18:10Z (GMT). No. of bitstreams: 2 Dissertação - Ricardo Lemes Gonçalves - 2017.pdf: 6297486 bytes, checksum: 3393a7cb0ffe26eafd0acd04f1198ab0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Flavivirus-induced pathogens are considered a serious public health problem in the world, because they affect primarily the poor populations of underdeveloped tropical countries. This family of viruses has an enveloped positive RNA genome encoding tree structural proteins and seven non-structural proteins. In particular, non-structural protein 1 (NS1) is associated mainly with the viral replication process and immune system leakage, and can be found in different glycosylated oligomeric forms, but only "mature" after its dimerization, or when in the hexamer form. In this context, inhibition of the dimerization process of NS1 has been pointed as a great target for the rational design of drugs. However, for this type of rationalized approach to be successful, it is necessary to understand the molecular mechanisms involved in the process of dimerization, as well as characterize its functional regions. In this study, through Molecular Dynamics (DM) simulations, the structural behavior of the NS1's of the Dengue and Zika were evaluated and compared in the monomeric and dimeric forms, from structures crystallized with glycosylations. For the structure of the Dengue (PDB: 5k6k), there was a need to previously model the regions of missing residues, then making necessary to replace the sugars in the structures. All of the N-glycosylations and intercysteine bonds of each monomer and dimer were performed in the Amber14 software. Specific histidine protonation was predicted by the H++ assay before proceeding with DM. All structures were submitted to DM by 100ns using the GROMACS 5.1.2 software. The stability and flexibility of the structures observed in the trajectories were used to elucidate the mechanisms responsible for the monomer-monomer interaction, as well as to understand the singularities between the regions that stood out. In the monomeric form of the two species, large conformational fluctuations were observed in their N-terminal loops involved in the β-roll composition, which in the dimeric form were stabilized by the triad Ile/Val19 - Phe20 - Ile21 for creating a framework favorable to the interlacing of the N-terminal loops between the A and B chains. Concomitantly, Lysines-189 of the β-ladder domain acted as a key residue (staple) to stabilize the central portion of β-roll. The stability mechanisms of NS1 revealed in this work for the Zika and Dengue species, contribute to make the possibility of pharmacological intervention more accessible, in a rationalized way, whose approach could be directed at specific structural points responsible for its biological role. / As patogenias causadas pelos Flavivírus são consideradas um sério problema de saúde pública no mundo, pois afetam principalmente populações pobres de países tropicais subdesenvolvidos. Esta família de vírus apresenta um genoma de RNA positivo envelopado que codifica três proteínas estruturais e sete proteínas não estruturais. Em particular, a proteína não estrutural 1 (NS1) está associada principalmente aos processos de replicação viral e escape do sistema imune, sendo encontrada em diferentes formas oligoméricas glicosiladas, mas na sua forma “madura” somente após sua dimerização, ou quando na forma de hexâmero. Neste contexto, a inibição do processo de dimerização da NS1 tem sido apontada como um ótimo alvo para o design racional de fármacos. Contudo, para que este tipo de abordagem racional seja bem sucedida, é necessário compreender os mecanismos moleculares que estão envolvidos no seu processo de dimerização, bem como caracterizar suas regiões funcionais. Neste estudo, através de simulações de Dinâmica Molecular (DM), o comportamento estrutural das NS1’s do Zika e Dengue foram analisados e comparados nas formas monoméricas e diméricas, partindo de estruturas cristalizadas nas formas glicosiladas. No caso da estrutura do Dengue (PDB: 5k6k) houve a necessidade de modelar previamente as regiões dos resíduos faltantes, tornando em seguida necessária a recolocação dos açucares nas estruturas. Todas as N-glicosilações e ligações entre cisteínas de cada monômero e dímero, foram efetuadas a partir do software Amber 14. A protonação especifica das histidinas foi predita pelo servirdor H++ antes de proceder com a DM. Todas as estruturas foram submetidas à DM por 100ns usando o software GROMACS 5.1.2. A estabilidade e flexibilidade das estruturas observadas nas trajetórias foram usadas para elucidar os mecanismos responsáveis pela interação monômero-monômero, bem como compreender as singularidades entre as regiões que se destacaram. Na forma monomérica das duas espécies, observou-se grandes flutuações conformacionais nas suas alças N-terminais envolvidas na composição do β-roll, que na forma dimérica foram estabilizadas pela tríade Ile/Val19 - Phe20 - Ile21 por criar um arcabouço favorável ao entrelaço das alças N-terminais entre as cadeias A e B. Concomitantemente, as Lisinas-189 do domínio β-leadder atuaram como um resíduo chave (grampo) para estabilizar a porção central do β-roll. Os mecanismos de estabilidade da NS1 revelados neste trabalho para as espécies Zika e Dengue, contribuem para tornar mais acessível a possibilidade de uma intervenção farmacológica de forma racionalizada, cuja abordagem poderia ser dirigida em pontos estruturais específicos responsáveis por seu papel biológico.

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.</p>

All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.