Estudos genômicos, filogenéticos e proteômicos de alfavírus (CHIKV e MAYV)

Dissertação (mestrado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2019.Os arbovírus são vírus que necessitam de um vetor artrópode para serem transmitidos. Entre eles, estão o Chikungunya virus (CHIKV) e o Mayaro virus (MAYV), dois integrantes do gênero Alphavirus, família Togaviridae. Ambos são arbovírus artritogênicos que apresentam genoma de RNA fita simples senso positivo de aproximados 12kb. O CHIKV é amplamente distribuído e, apesar de já ter causado graves epidemias no Brasil, há poucos isolados brasileiros com genoma completo sequenciado. Por outro lado, o MAYV possui distribuição quase restrita à América do Sul, sendo um vírus historicamente negligenciado. Apesar de ser transmitido principalmente por Haemagogus spp., o MAYV também pode ser transmitido por mosquitos de outros gêneros, como o Aedes aegypti. Assim, no Capítulo 2 do presente trabalho, foram realizados o sequenciamento de alto desempenho do genoma completo e um estudo de filogenia de seis isolados de CHIKV do Centro-Oeste brasileiro. Esses isolados, os primeiros a serem publicados de tal região, apresentaram sequências de 11.811 nucleotídeos. Nenhum deles possui as mutações A226V (E1) ou L210Q (E2), presentes em isolados transmitidos por Aedes albopictus, sendo provavelmente transmitidos pelo vetor urbano principal A. aegypti. A análise filogenética mostrou que os isolados de CHIKV do Centro-Oeste pertencem à linhagem East-Central-South Africa. Já o Capítulo 3 focou na análise proteômica qualitativa de cultura de células Aag-2 de A. aegypti durante infecção por MAYV, visto que as proteínas são as principais mediadoras da comunicação intracelular. Após espectrometria de massas, foram identificadas 191 proteínas estatisticamente significativas. Ao longo das 48h de infecção, as proteínas da célula hospedeira, em geral, tiveram sua abundância reduzida. Já as proteínas virais foram super expressas. As proteínas de A. aegypti que foram super expressas mostraram-se importantes para a replicação viral, como a proibitina, a enolase fosfatase e1, chaperonas, uma sinaptobrevina e o fator de transcrição HCFC1. Tais proteínas já foram também identificadas como super expressas em estudos envolvendo outros arbovírus. Além do mais, vias de secreção clássicas de proteínas foram menos utilizadas durante a infecção, enquanto vias não convencionais foram ativadas. Juntos, esses dados auxiliam na compreensão da evolução e da dispersão de CHIKV, assim como elucidam componentes que são essenciais para a infecção por MAYV, sendo possivelmente importantes para a relação arbovírus-vetor em geral.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Arboviruses are viruses that require an arthropod vector to be transmitted. Among them are Chikungunya virus (CHIKV) and Mayaro virus (MAYV), two members of the genus Alphavirus, family Togaviridae. Both of them are arthritogenic arboviruses that have a single-strand positive sense RNA genome of approximately 12kb. CHIKV is widely distributed and, although it has already caused serious epidemics in Brazil, there are few Brazilian isolates with sequenced complete genome. On the other hand, the MAYV is distributed almost exclusively to South America, being a virus historically neglected. Although being transmitted mainly by Haemagogus spp., MAYV can also be transmitted by mosquitoes of other genera, such as Aedes aegypti. Thus, in Chapter 2 of the present work, high-throughput sequencing of the complete genome and a phylogeny study of six CHIKV isolates from the Brazilian Midwest were performed. These isolates, the first ones to be published from this region, had sequences of 11,811 nucleotides. None of them has the mutations A226V (E1) or L210Q (E2), which were detected in isolates transmitted by Aedes albopictus, being probably transmitted by the main urban vector A. aegypti. Phylogenetic analysis showed that the Midwest CHIKV isolates belong to the East-Central-South Africa lineage. Chapter 3 focused on the qualitative proteomic analysis of A. aegypti Aag-2 cell culture during MAYV infection, given proteins are the main mediators of intracellular communication. After mass spectrometry, we identified 191 statistically significant proteins. Over the 48 hours of infection, the host cell proteins had their abundance reduced in general. Viral proteins, in contrary, were upregulated. Upregulated A. aegypti proteins have proven to be important for viral replication, such as prohibitin, enolase phosphatase e1, chaperones, synaptobrevins and the transcription factor HCFC1. Such proteins have also been identified as upregulated in studies involving other arboviruses. In addition, classical protein secretion pathways were less used during infection, while non-conventional pathways were activated. Together, these data assist in understanding the evolution and dispersion of CHIKV, as well as elucidating components that are essential for MAYV infection, possibly being important for the arbovirus-vector relationship in general

IMPACTOS DA PANDEMIA DO CORONAVÍRUS NOS CONTRATOS ADMINISTRATIVOS:: UM ESTUDO DE CASOS

This article sought, from a bibliographic study involving both doctrinal research and case-by-case analyses, to verify the existence and possible extent of impacts of the coronavirus pandemic on signed administrative contracts. The relevance of the analysis is evident, above all, due to the need for debate about the effects of a pandemic crisis that is still prevalent and without comparable precedents in recent history. Therefore, contractual cases were used, detailed from a conceptual and legislative approach of contemporary Brazilian law. During the research, the following situations were identified: (i) the incidence of greater State action; (ii) the adoption of the concepts of force majeure and fact of the prince as a guide; and (iii) the possibility of breach of contractual obligations by the individual and the right of the contractor to readjust the economic-financial balance of the contract.  O presente artigo buscou, a partir de um estudo bibliográfico envolvendo tanto pesquisa doutrinária quanto análises casuísticas, verificar a existência e a possível extensão de impactos da pandemia do coronavírus sobre os contratos administrativos celebrados. A relevância da análise evidencia-se, sobretudo, em função da necessidade de debate acerca dos efeitos de uma crise pandêmica ainda prevalente e sem precedentes equiparáveis na história recente. Assim, foram utilizados causos contratuais, esmiuçados a partir de uma abordagem conceitual e legislativa do Direito brasileiro contemporâneo. Ao longo da pesquisa, foram identificadas as seguintes conjunturas: (i) a incidência de maior atuação estatal; (ii) a adoção dos conceitos de força maior e fato do príncipe como norte; e (iii) a possibilidade de descumprimento das obrigações contratuais por parte do particular e o direito desse contratado à readequação do equilíbrio econômico-financeiro do contrato

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus

Background Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. Methods After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. Results After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. Conclusions To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation

Análises proteômica e fosfoproteômica de células Aag-2 de Aedes aegypti infectadas com os arbovírus MAYV e CHIKV

Tese (doutorado) — Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2024.Mayaro virus (MAYV) e Chikungunya virus (CHIKV) são dois arbovírus pertencentes ao gênero Alphavirus e que são transmitidos pelo mosquito Aedes aegypti. Enquanto o MAYV é endêmico da América do Sul, o CHIKV já foi responsável por epidemias em todo o globo. Os elevados números de casos de arboviroses pelo mundo e o fato de muitas populações de mosquitos serem resistentes a inseticidas levaram à necessidade de desenvolvimento de estratégias alternativas de controle desses vírus. Houve um aumento expressivo na quantidade de estudos focados na interação vírus-vetor, muitos deles analisando aspectos moleculares, como as proteínas envolvidas nessa interação. Neste sentido, o presente estudo abordou a análise proteômica de células Aag-2 de Ae. aegypti infectadas com CHIKV em multiplicidade de infecção (MOI) 0,1 e tempos de coleta t = 0 h, 12 horas pós-infecção (h p.i.) e 48 h p.i. As amostras, em triplicatas biológicas, foram identificadas por espectrometria de massas após redução, alquilação e digestão. Entre outros resultados, foi observada uma maior abundância de proteínas relacionadas à tradução em 12 h p.i., enquanto proteínas relacionadas à produção de energia, em 48 h p.i. As imagens de microscopia eletrônica de transmissão revelaram, além de muitas partículas virais, mitocôndrias com formato alongado em 48 h p.i. Tal morfologia pode ser consequência de um provável desbalanço da rede mitocondrial decorrente da infecção viral, gerando uma maior demanda energética. Ademais, sabese que a fosforilação de proteínas está altamente relacionada à regulação espaçotemporal de diversos processos essenciais ao funcionamento dos organismos. Como os vírus sequestram a maquinaria celular do hospedeiro para sua própria replicação, espera-se encontrar uma modulação diferencial no padrão de fosforilação nas células infectadas. Assim, objetivou-se realizar uma análise fosfoproteômica de células Aag-2 infectadas com MAYV e CHIKV no presente trabalho. Foi realizada uma predição in silico de fosfoproteínas de Ae. aegypti e as 433 proteínas resultantes foram enriquecidas para processos como autofagia e via de sinalização MAPK. Para as análises experimentais, foram utilizados o MOI 1 e os tempos de coleta t = 0 h, 2 h p.i., 4 h p.i., 8 h p.i. e 12 h p.i., em quadruplicatas biológicas. As proteínas extraídas a partir das células Aag-2 foram utilizadas para Western Blot e espectrometria de massas. Os ensaios de Western Blot contra anticorpos anti-fosfoserina, fosfotirosina e fosfotreonina apresentaram diferentes padrões de marcação na comparação por anticorpo. A partir do processamento do extrato total de proteínas para a espectrometria de massas e dessalinização, foi realizado o enriquecimento de fosfopeptídeos por colunas de dióxido de titânio (TiO2). Após a obtenção dos dados da análise por espectrometria de massas, a análise de bioinformática nos permitirá verificar a influência da infecção por MAYV e CHIKV sobre a homeostase das células hospedeiras de Ae. aegypti nas vias metabólicas que são reguladas por eventos de fosforilação.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).Mayaro virus (MAYV) and Chikungunya virus (CHIKV) are two arboviruses belonging to the genus Alphavirus and transmitted by the Aedes aegypti mosquito. While MAYV is endemic to South America, CHIKV has been responsible for epidemics across the globe. The high number of arbovirus cases around the world and the fact that many mosquito populations are resistant to insecticides have led to the need to develop alternative strategies to control these viruses. There has been a significant increase in the number of studies focused on the virus-vector interaction, many of them analyzing molecular aspects, such as the proteins involved in this interaction. In this sense, the present study addressed the proteomic analysis of Aag-2 cells from Ae. aegypti infected with CHIKV at multiplicity of infection (MOI) 0.1 and collection times t = 0 h, 12 hours post-infection (h p.i.) and 48 h p.i. The samples, in biological triplicates, were identified by mass spectrometry after reduction, alkylation, and digestion. Among other results, a greater abundance of proteins related to translation was observed at 12 h p.i., while proteins related to energy production were observed at 48 h p.i. Transmission electron microscopy images revealed, in addition to many viral particles, mitochondria with an elongated shape at 48 h p.i. This morphology may be a consequence of a probable imbalance in the mitochondrial network resulting from the viral infection, generating a greater energy demand. Furthermore, it is known that protein phosphorylation is highly related to the spatiotemporal regulation of several processes essential to the functioning of organisms. As viruses hijack the host's cellular machinery for their own replication, we expect to find a differential modulation in the phosphorylation pattern in infected cells. Thus, the aim was to perform a phosphoproteomic analysis of Aag-2 cells infected with MAYV and CHIKV in the present work. An in silico prediction of Ae. aegypti phosphoproteins was performed and the resulting 433 proteins were enriched for processes such as autophagy and the MAPK signaling pathway. For experimental analyses, MOI 1 and collection times t = 0 h, 2 h p.i., 4 h p.i., 8 h p.i. were used. and 12 h p.i., in biological quadruplicates. Proteins extracted from Aag-2 cells were used for Western blot and mass spectrometry. Western blot assays against anti-phosphoserine, phosphotyrosine, and phosphothreonine antibodies showed different staining patterns when compared by antibody. From the processing of the total protein extract for mass spectrometry and desalination, the enrichment of phosphopeptides was carried out using titanium dioxide (TiO2) columns. After obtaining data from mass spectrometry analysis, bioinformatics analysis will allow us to verify the influence of MAYV and CHIKV infection on the homeostasis of Ae. aegypti host cells in metabolic pathways that are regulated by phosphorylation events.Faculdade de Medicina (FM)Programa de Pós-Graduação em Patologia Molecula

Chikungunya virus produced by a persistently infected mosquito cell line comprises a shorter genome and is non-infectious to mammalian cells

Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work, Aedes aegypti Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in Ae. albopictus C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the nsP3 gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.</jats:p

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus

Abstract Background Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. Methods After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. Results After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. Conclusions To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation. </jats:sec

Aedes aegypti Aag-2 Cell Proteome Modulation in Response to Chikungunya Virus Infection

Chikungunya virus (CHIKV) is a single-stranded positive RNA virus that belongs to the genus Alphavirus and is transmitted to humans by infected Aedes aegypti and Aedes albopictus bites. In humans, CHIKV usually causes painful symptoms during acute and chronic stages of infection. Conversely, virus–vector interaction does not disturb the mosquito’s fitness, allowing a persistent infection. Herein, we studied CHIKV infection of Ae. aegypti Aag-2 cells (multiplicity of infection (MOI) of 0.1) for 48 h through label-free quantitative proteomic analysis and transmission electron microscopy (TEM). TEM images showed a high load of intracellular viral cargo at 48 h postinfection (hpi), as well as an unusual elongated mitochondria morphology that might indicate a mitochondrial imbalance. Proteome analysis revealed 196 regulated protein groups upon infection, which are related to protein synthesis, energy metabolism, signaling pathways, and apoptosis. These Aag-2 proteins regulated during CHIKV infection might have roles in antiviral and/or proviral mechanisms and the balance between viral propagation and the survival of host cells, possibly leading to the persistent infection

Aedes aegypti Aag-2 Cell Proteome Modulation in Response to Chikungunya Virus Infection

Chikungunya virus (CHIKV) is a single-stranded positive RNA virus that belongs to the genus Alphavirus and is transmitted to humans by infected Aedes aegypti and Aedes albopictus bites. In humans, CHIKV usually causes painful symptoms during acute and chronic stages of infection. Conversely, virus–vector interaction does not disturb the mosquito’s fitness, allowing a persistent infection. Herein, we studied CHIKV infection of Ae. aegypti Aag-2 cells (multiplicity of infection (MOI) of 0.1) for 48 h through label-free quantitative proteomic analysis and transmission electron microscopy (TEM). TEM images showed a high load of intracellular viral cargo at 48 h postinfection (hpi), as well as an unusual elongated mitochondria morphology that might indicate a mitochondrial imbalance. Proteome analysis revealed 196 regulated protein groups upon infection, which are related to protein synthesis, energy metabolism, signaling pathways, and apoptosis. These Aag-2 proteins regulated during CHIKV infection might have roles in antiviral and/or proviral mechanisms and the balance between viral propagation and the survival of host cells, possibly leading to the persistent infection.</jats:p

Characterization of diagnostic delay in oral and oropharyngeal cancer at two referral centers

Abstract Objectives To analyze the chronology of diagnosis and determine whether clinical and epidemiological variables have an influence on diagnostic delay at two referral centers. Methods The medical records of all patients older than 18 years diagnosed with oral/oropharyngeal cancer from June 2005 to June 2013 were analyzed using SPSS® 20. The association between epidemiological and clinical variables with patient and professional delay was performed using ANOVA, Student’s t-test, and Kruskal-Wallis test. Results In total, 121 medical records were included in the study. Patients were predominantly brown, male, illiterate, living in country towns, smokers, and heavy drinkers (mean age 64.3 years, SD=12.94). The majority (85.1%) of patients were diagnosed at advanced stages of their disease. The greatest delay was patient-related, mean 197.8 days (SD=323.9). Delay in establishing the medical diagnosis averaged 20 days (SD=25.9), and health care system-related delay was 71.1 days (SD=71.7). There was no association of clinical and epidemiological variables with delayed diagnosis (patient and professional). Conclusion Data from the present study suggest that clinical and epidemiological variables do not influence diagnostic delay.</p