Orientadores: Clarice Weis Arns, Márcia Bianchi dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T11:13:27Z (GMT). No. of bitstreams: 1 Carvalho_RicardoDuraesde_D.pdf: 3518273 bytes, checksum: 7b6f8b159eb057429823e23f6852c29b (MD5) Previous issue date: 2015 / Resumo: A evolução e a dinâmica populacional dos Coronavírus (CoVs) ainda permanecem pouco exploradas. No presente estudo, análises filogenéticas e de filogeografia foram conduzidas para investigar a dinâmica evolutiva dos CoVs detectados em aves silvestres e sinantrópicas. Um total de 500 amostras, que inclui os suabes traqueais e cloacais coletados de 312 aves silvestres pertencentes a 42 espécies, foram analisadas através da RT-qPCR. Sessenta e cinco amostras (13%) provenientes de 23 espécies foram positivas para o Coronavírus aviário (AvCoV). Trezentos e duas amostras foram investigadas para a pesquisa do Pan-Coronavírus (Pan-CoV) através do nPCR, destas, 17 (5,6%) foram positivas, sendo que 11 foram detectadas em espécies diferentes. Análises filogenéticas dos AvCoVs revelaram que as sequências de DNA das amostras coletadas no Brasil não agruparam com nenhuma das sequências do gene Spike (S1) dos AvCoVs depositados no banco de dados GenBank. Análise Bayesiana estimou uma variante do AvCoV proveniente da Suécia (1999) como o ancestral comum mais recente dos AvCoVs detectados neste estudo. Além disso, as análises realizadas através do "Bayesian Skyline Plot" (BSP) inferiram um aumento na dinâmica da população demográfica do AvCoV em diferentes espécies de aves silvestres e sinantrópicas. As análises filogenéticas do Pan-CoV mostrou que a maioria das amostras se agruparam com o Vírus da Hepatite Murina A59 (MHV A59), CoV pertencente ao grupo dos Beta-CoVs. Uma amostra [CoV detectado em Amazona vinacea(Papagaio-de-peito-roxo)] se agrupou com um CoV de Suínos, o PCoV HKU15, que pertence ao gênero Delta-CoV, ainda não relatado na América do Sul. Nossos achados sugerem que as aves podem ser novos potenciais hospedeiros responsáveis pela propagação e disseminação de diferentes CoVs para diferentes espécies de animais / Abstract: The evolution and population dynamics of Coronaviruses (CoVs) still remain underexplored. In the present study, phylogenetic and phylogeographic analyseswere conducted to investigate the evolutionary dynamics of CoV detected in wild and synanthropic birds. A total of 500 samples, including tracheal and cloacal swabs collected from 312 wild birds belonging to 42species, were analysed by RT-qPCR. A total of 65 samples from 23bird species were positive for Avian Coronaviruses (AvCoVs).Three hundred and two samples were screened for the Pan-Coronavirus (Pan-CoV) through the nPCR, 17 (5.6%) were positive, being that 11 were detected in different species. AvCoVs phylogenetic analyses revealed that the DNA sequences from samples collected in Brazil did not cluster with any of the AvCoV S1 gene sequences deposited in the GenBank database. Bayesian framework analysis estimated an AvCoV strain from Sweden (1999) as the most recent common ancestor of the AvCoVs detected in this study. Furthermore, Bayesian Skyline Plot (BSP) analysis inferred an increase in the AvCoV dynamic demographic population in different wild and synanthropic bird species. Phylogenetic analysis of the Pan-CoV showed that most of the samples clustered with the Murine Hepatitis Virus A59 strain (MHV A59) belong to the BetaCoV group. Besides, one of our samples [CoV detected in Amazona vinacea (parrot-breasted-purple)] clustered with a CoV isolated from pigs, PCoV HKU15, belonging to the DeltaCoV genus, still not reported in South America. Our findings suggest that birds may be potential new hosts responsible for spreading of different CoVs for different species of animals / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

No description available.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Chuck Chi-pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 138-160). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiii / Abbreviations --- p.xiv / Acknowledgement --- p.xviii / Introduction / Chapter 1. --- Background / Chapter 1.1 --- SARS / Chapter 1.1.1 --- Outbreak and Influence --- p.1 / Chapter 1.1.2 --- Clinical Features --- p.4 / Chapter 1.2 --- SARS-CoV / Chapter 1.2.1 --- Genomic Organization --- p.5 / Chapter 1.2.2 --- Morphology --- p.7 / Chapter 1.2.3 --- Phylogenetic Analysis --- p.9 / Chapter 1.3 --- S Glycoprotein / Chapter 1.3.1 --- Functional Roles --- p.11 / Chapter 1.3.2 --- Structure and Functional Domains --- p.12 / Chapter 1.3.3 --- Interacting Partners --- p.15 / Chapter 1.3.4 --- Viral Entry Mechanism --- p.17 / Chapter 1.4 --- Aim of Study / Chapter 1.4.1 --- Mismatch of SARS-CoV Tissue Tropism and Tissue Distribution of ACE2 --- p.20 / Chapter 1.4.2 --- Presence of Other Interacting Partner(s) --- p.22 / Chapter 1.4.3 --- Significance of the Study Materials and Methods --- p.22 / Chapter 2. --- Plasmid Construction / Chapter 2.1 --- Fragment Design / Chapter 2.1.1 --- Functional Domain Analysis --- p.23 / Chapter 2.1.2 --- Secondary Structure and Burial Region Predictions --- p.24 / Chapter 2.2 --- Vector Amplification / Chapter 2.2.1 --- E. coli Strain DH5a Competent Cell Preparation --- p.30 / Chapter 2.2.2 --- Transformation of E. coli --- p.30 / Chapter 2.2.3 --- Small-scale Vector Amplification --- p.31 / Chapter 2.3 --- Cloning of DNA Fragments into Various Vectors / Chapter 2.3.1 --- Primer Design --- p.32 / Chapter 2.3.2 --- DNA Amplification --- p.35 / Chapter 2.3.3 --- DNA Purification --- p.35 / Chapter 2.3.4 --- "Restriction Enzyme Digestion, Ligation and Transformation" --- p.36 / Chapter 2.3.5 --- Colony PCR --- p.37 / Chapter 2.4 --- DNA Sequence Analysis / Chapter 2.4.1 --- Primer Design --- p.35 / Chapter 2.4.2 --- DNA Amplification and Purification for DNA Sequence Analysis --- p.39 / Chapter 2.4.3 --- Sequence Detection and Result Analysis --- p.40 / Chapter 3. --- "Protein Expression, Purification and Analysis" / Chapter 3.1 --- Protein Expression in E. coli / Chapter 3.1.1 --- Molecular Weight and pI Predictions --- p.41 / Chapter 3.1.2 --- Glycerol Stock Preparation --- p.41 / Chapter 3.1.3 --- Protein Expression Induction --- p.41 / Chapter 3.1.4 --- Protein Extraction --- p.42 / Chapter 3.1.5 --- Affinity Chromatography --- p.42 / Chapter 3.1.6 --- Removal of GroEL --- p.43 / Chapter 3.1.7 --- Protein Solubilization and Refolding --- p.44 / Chapter 3.2 --- Protein Expression in P. pastoris / Chapter 3.2.1 --- Large-scale Plasmid Amplification --- p.46 / Chapter 3.2.2 --- Restriction Enzyme Digestion and Ethanol Precipitation --- p.47 / Chapter 3.2.3 --- Preparation of KM71H Competent Cells --- p.47 / Chapter 3.2.4 --- Electroporation --- p.48 / Chapter 3.2.5 --- Colony PCR --- p.48 / Chapter 3.2.6 --- Protein Expression Induction and Time Course Study --- p.49 / Chapter 3.2.7 --- Deglycosylation --- p.49 / Chapter 3.3 --- Protein Analysis / Chapter 3.3.1 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.50 / Chapter 3.3.2 --- Western Blotting --- p.50 / Chapter 3.3.3 --- Mass Spectrometry --- p.51 / Chapter 3.3.4 --- N-terminal Sequencing --- p.52 / Chapter 3.3.5 --- Size Exclusion Chromatography --- p.52 / Chapter 4. --- Identification of Interacting Partner(s) / Chapter 4.1 --- VeroE6 Preparation / Chapter 4.1.1 --- Cell Culture --- p.53 / Chapter 4.1.2 --- Protein Extraction and Western Blotting --- p.53 / Chapter 4.2 --- Pull-down Assay --- p.54 / Chapter 4.3 --- Two-dimensional Gel Electrophores --- p.is / Chapter 4.3.1 --- Isoelectric Focusing --- p.56 / Chapter 4.3.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.56 / Chapter 4.3.3 --- Silver Staining --- p.57 / Chapter 4.4 --- Mass Spectrometry / Chapter 4.4.1 --- Destaining --- p.58 / Chapter 4.4.2 --- In-gel Digestion --- p.58 / Chapter 4.4.3 --- Desalting by Zip-tip --- p.59 / Chapter 4.4.4 --- Loading Sample --- p.59 / Chapter 4.4.5 --- Peptide Mass Detection and Data Analysis --- p.59 / Results / Chapter 5. --- S Protein Expression / Chapter 5.1 --- Plasmid Construction --- p.61 / Chapter 5.2 --- Molecular Weight and pi Predictions --- p.63 / Chapter 5.3 --- Protein Expression and Optimization in E. coli / Chapter 5.3.1 --- "Comparison of Expression Levels, Solubility and Purities of S Protein Fragments" --- p.64 / Chapter 5.3.2 --- "Alteration of the Solubility in Various Cell Strains, Expression Conditions and Lysis Buffers" --- p.68 / Chapter 5.3.3 --- Identification and Remove of the non-target proteins --- p.72 / Chapter 5.3.4 --- Unfolding and Refolding --- p.79 / Chapter 5.4 --- Protein Expression and Optimization in P. pastoris / Chapter 5.4.1 --- "Expression Levels, Solubility and Purities of Various S Protein Fragments" --- p.85 / Chapter 5.4.2 --- Characterization of De-N-glycosylated Recombinant Proteins --- p.89 / Chapter 6. --- Identification of Interacting partners / Chapter 6.1 --- Practicability of Pull-down Assay / Chapter 6.1.1 --- ACE2 Extraction --- p.95 / Chapter 6.1.2 --- Pull-down of ACE2 by the P. pastoris-expressed recombinant RBD --- p.96 / Chapter 6.2 --- Pull-down Assay and Two-dimensional Gel Electrophoresis --- p.97 / Chapter 6.3 --- Identification of Putative Interacting Partners by MALDI-TOF-TOF --- p.107 / Chapter 7. --- Discussion / Chapter 7.1 --- S Protein Expression in E. coli / Chapter 7.1.1 --- Improving Recombinant Protein Expression Level and Solubility --- p.114 / Chapter 7.1.2 --- S Recombinant Protein Bound by GroEL --- p.117 / Chapter 7.2 --- S Protein Expression in P. pastoris / Chapter 7.2.1 --- Advantages of Using P. pastoris --- p.119 / Chapter 7.2.2 --- Variation of S Fragment Expression Levels --- p.120 / Chapter 7.2.3 --- Sizes of S Protein Fragments --- p.123 / Chapter 7.3 --- Identification of Interacting Partners / Chapter 7.3.1 --- Relationship between S Protein and Putative Interacting Partners --- p.124 / Chapter 7.3.2 --- Failure of Finding ACE2 --- p.125 / Chapter 7.3.2 --- Difficulty in the Identification of Protein Spots --- p.126 / Chapter 7.4 --- Conclusion --- p.131 / Chapter 7.5 --- Future Perspective --- p.132 / Chapter 8. --- Appendix --- p.133 / Chapter 9. --- References --- p.138

Algumas espécies de morcegos têm sido reconhecidas como reservatórios naturais de várias famílias virais, desempenhando um importante papel na trasmissão e manutenção desses micro organismos. Devido à descaracterização e fragmentação de habitats naturais, esses mamíferos buscam alternativas de abrigo e alimento, e assim, ficam cada vez mais expostos aos meios antrópicos e em contato com humanos e animais domésticos. Com exceção do vírus rábico, existem poucos trabalhos realizados na detecção de vírus em morcegos no Brasil. Em virtude disso, o presente estudo objetivou a detecção de vírus (circovírus, astrovírus, coronavírus e lyssavírus relacionados ao vírus da raiva) em amostras de órgãos de morcegos do estado do Rio Grande do Sul. Os ácidos nucléicos foram extraídos das amostras de órgãos de morcegos e submetidos à detecão por PCR e RT-PCR. Após a detecção, os fragmentos obtidos foram sequenciados para realizar análise filogenética dos vírus encontrados. Ao total foram analisadas 108 amostras de diferentes espécies e localidades, das quais dez foram positivas para circovírus, seis para coronavírus e 25 para astrovírus, este último sendo o primeiro registro do vírus em morcegos para o Brasil. Todas as amostras foram negativas para lyssavírus relacionados ao vírus da raiva. Análises filogenéticas revelaram que as sequências de circovírus agruparam em ambos os gêneros Circovirus e Cyclovirus, coronavírus no gênero Alphacoronavirus em dois clados diferentes e astrovírus no gênero Mamastrovirus junto com outros astrovírus de morcegos, o qual formam um clado separado dos outros mamíferos. Os resultados demonstram uma diversidade genética entre os vírus encontrados em diferentes espécies de morcegos, que possuem dietas alimentares e habitats distintos. / Some bat species have been recognized as natural reservoirs of several viral families, playing an important role in the transmission and maintaining of these micoorganism. Due to mischaracterization and fragmentation of natural habitats, these mammals seek shelter alternatives and food, and thus are increasingly exposed to anthropism, which make the contact with humans and domestic animals closer. With the exception of the rabies virus, there are few studies on the detection of viruses in bats in Brazil. Therefore, the present study aimed the detection of viruses (circovirus, astrovirus, coronavirus and rabies-related virus) in bats organs samples from Rio Grande do Sul state. Nucleic acids were extracted from bat organs samples and submitted to detection by PCR and RT-PCR. After detection, the obtained fragments were sequenced to perform phylogenetic analysis of the viruses found. From a total of 108 samples analyzed of different species and locations, ten were positive for circoviruses, six for coronaviruse and 25 for astrovirus, which was the first report of this virus in bats in Brazil. All samples were negative for rabies-related virus. Phylogenetic analyzes revealed that the sequences of circoviruses grouped in both Circovirus and Cyclovirus genus, coronaviruses in Alphacoronavirus genus in two different clades and astroviruses in Mamastrovirus genus along with other bats astrovirus, which form a separate clade from other mammals. Results demonstrate a genetic diversity among viruses found in different species of bats, which have different diets and habitats.

Choy Wai Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 98-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / Table of contents --- p.xi / List of figures --- p.xv / List of tables --- p.xviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe acute respiratory syndrome (SARS) - An overview --- p.1 / Chapter 1.1.1 --- Epidemiology of SARS --- p.1 / Chapter 1.1.2 --- Clinical presentation of SARS --- p.2 / Chapter 1.1.3 --- Diagnostic tests of SARS --- p.5 / Chapter 1.1.4 --- Treatment of SARS --- p.7 / Chapter 1.2 --- Severe acute respiratory syndrome coronavirus (SARS- CoV) --- p.8 / Chapter 1.2.1 --- The etiological agent of SARS --- p.8 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- Genome of SARS-CoV --- p.11 / Chapter 1.3 --- Spike (S) glycoprotein of SARS-CoV --- p.14 / Chapter 1.3.1 --- Functions of SARS-CoV S glycoprotein --- p.15 / Chapter 1.3.2 --- Receptors for S glycoprotein of SARS-CoV --- p.17 / Chapter 1.4 --- 3C-like protease (3CLPro) of SARS-CoV --- p.20 / Chapter 1.4.1 --- Extensive proteolytic processing of SARS-CoV replicase polyproteins --- p.20 / Chapter 1.4.2 --- SARS-CoV 3CLPro --- p.21 / Chapter 1.4.3 --- Substrate specificity of SARS-CoV 3CLPro --- p.22 / Chapter 1.5 --- Combating SARS - Vaccine and drug development --- p.24 / Chapter 1.5.1 --- Vaccine development against SARS --- p.24 / Chapter 1.5.2 --- Drug development against SARS --- p.25 / Chapter 1.6 --- Project objectives of this thesis --- p.27 / Chapter 1.6.1 --- Synthetic Peptide Studies on SARS-CoV S glycoprotein --- p.27 / Chapter 1.6.2 --- Synthetic Peptide Studies on SARS-CoV 3CLPro --- p.28 / Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.30 / Chapter 2.1.1 --- Bioinformatics analyses of SARS-CoV S gly- coprotein --- p.30 / Chapter 2.1.2 --- Peptide design and molecular modeling --- p.32 / Chapter 2.1.3 --- Solid phase peptide synthesis (SPPS) --- p.33 / Chapter 2.1.4 --- Peptide conjugation --- p.35 / Chapter 2.1.5 --- Immunization in rabbits and monkeys --- p.36 / Chapter 2.1.6 --- ELISA analysis --- p.37 / Chapter 2.1.7 --- Immunofluorescent confocal microscopy --- p.39 / Chapter 2.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.40 / Chapter 2.2.1 --- Protein expression and purification --- p.40 / Chapter 2.2.2 --- Solid phase peptide synthesis (SPPS) --- p.41 / Chapter 2.2.3 --- Peptide cleavage assay --- p.44 / Chapter 2.2.4 --- Molecular docking --- p.46 / Chapter 3 --- Results --- p.48 / Chapter 3.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.48 / Chapter 3.1.1 --- General features and structural analyses of the S glycoprotein --- p.48 / Chapter 3.1.2 --- Peptides design and synthesis --- p.53 / Chapter 3.1.3 --- ELISA analysis and immunofluorescent con- focal microscopy --- p.55 / Chapter 3.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.62 / Chapter 3.2.1 --- Substrate specificity of SARS-CoV 3CLPro . . --- p.62 / Chapter 3.2.2 --- Molecular docking of SARS-CoV 3CLPro and peptide substrates --- p.74 / Chapter 4 --- Discussion --- p.78 / Chapter 4.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.78 / Chapter 4.1.1 --- Synthetic peptides elicited SARS-CoV specific antibodies --- p.78 / Chapter 4.1.2 --- Factors affecting the specificity and antigenic- ity of synthetic peptides --- p.80 / Chapter 4.1.3 --- Next step towards vaccine development --- p.83 / Chapter 4.1.4 --- A synthetic peptide-based approach --- p.84 / Chapter 4.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.86 / Chapter 4.2.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.87 / Chapter 4.2.2 --- Sequence comparison between SARS-CoV 3CLpro cleavage sites --- p.90 / Chapter 4.2.3 --- A rapid and high throughput approach to screen protease substrate specificity --- p.94 / Bibliography --- p.98

Les virus à ARN sont à l’origine de nombreuses épidémies depuis ces dernières décennies. Malgré des avancées thérapeutiques majeures, une majorité d’infection est orpheline de traitement. Le développement d’antiviraux à spectre large est une alternative thérapeutique pour maximiser le nombre de virus ciblés, minimiser les coûts de production et améliorer la prise pour les patients. Afin de trouver de nouvelles cibles cellulaires, la compréhension des mécanismes moléculaires utilisés par les virus pour infecter l’hôte est essentielle.Les virus utilisent des facteurs cellulaires pour survivre et se propager. Parmi ceux-ci, on trouve les microARNs (miARNs) et les longs ARNs non-codants (lncARNs) qui peuvent participer à la réponse antivirale mais peuvent également être détournés par les virus pour favoriser l’infection. Ces d’ARN non-codants peuvent interagir avec des protéines cellulaires (« RNA-binding protein » (RBP)) telles que la protéine KSRP. Cette RBP est impliquée dans le contrôle de l’expression des ARNs en participant à l’épissage de certains pré-ARNm, à la dégradation des ARNs contenant des séquences riches en AU et à la maturation de certains miARNs. Ses fonctions et sa localisation sont dépendantes de la phosphorylation de certains résidus par les kinases cellulaires Akt, ATM et p38/MAPK.Le but de ma thèse a été d’étudier la modulation de l’expression de ces deux classes d’ARN non-codants au cours de l’infection par des virus à ARN tels que le virus de l’Hépatite C (VHC) et la souche HCoV-229E des Coronavirus. Plus particulièrement nous avons cherché à étudier l’implication de KSRP dans la régulation d’ARN non-codants essentiels pour ces infections.Mes recherches ont commencé par l’étude de la maturation du microARN-122 (miR-122), un facteur proviral de l’infection par le VHC. Nous avons montré que KSRP phosphorylée sur le résidu S193 par Akt interagissait avec le complexe nucléaire DROSHA/DGCR8 et ainsi était essentielle à la maturation du pri-miR-122 en miR-122 favorisant la réplication virale. Notre avons ensuite étudié le rôle des phosphorylations de KSRP par ATM et p38/MAPK sur la réplication et sur la maturation du miR-122. La phosphorylation par ATM ne semble pas jouer un rôle majeur sur ces deux paramètres. En revanche, la phosphorylation de KSRP sur le résidu T692 par la kinase p38/MAPK semble jouer un rôle positif sur la réplication VHC.Dans un second temps, par homologie avec les résultats obtenus dans le cas du VHC, nous avons étudié le rôle de KSRP lors de l’infection par la souche HCoV-229E des Coronavirus. En transfectant un siKSRP ou un plasmide exprimant la protéine KSRP, nous avons pu démontrer que KSRP était un facteur proviral pour la réplication virale.Afin d’identifier les ARN non-codants modulés au cours de l’infection HcoV-229E et dont l’expression pouvait être régulée par KSRP, nous avons effectué deux analyses de séquençage à haut débit (« NGS »). L’analyse réalisée sur des cellules infectées vs non-infectées nous a permis d’identifier l’ensemble des miARNs et lncARNs dérégulés par le virus. Nous avons croisé ces résultats avec un second « NGS » fait sur des cellules infectées, inhibées pour KSRP et nous avons trouvé que l’expression du LinC00473 était modulée dans les deux conditions expérimentales. En étudiant ce facteur cellulaire au cours de l’infection nous avons observé une forte induction KSRP-dépendante du LinC00473 à 24 h post-infection, puis une diminution à 48 h post-infection. L’inhibition de ce facteur entraîne une diminution de la réplication virale suggérant que le LinC00473 est un facteur proviral au début de l’infection.Nos résultats ont permis de montrer le rôle proviral de la protéine KSRP lors de deux infections virales (VHC et HCoV-229E des Coronavirus). Son implication dans la régulation de l’expression des ARNs fait de cette protéine un outil efficace pour découvrir de nouvelles cibles thérapeutiques ARN non-codants au cours d’autres infections virales. / Résumé en anglaisRNA viruses have been the cause of many epidemics in recent decades. Despite major therapeutic advances, a majority of infection is currently orphan for treatment. The development of new broad spectrum antivirals is a therapeutic alternative to maximize the number of targeted viruses, minimize production costs and improve access to population. In order to find new cellular targets for this type of therapeutic approach, understanding the molecular mechanisms used by RNA viruses to infect the host is essential.Viruses exploit cellular factors to survive and to disseminate. Among those factors, microRNA (miRNA) and long non-coding RNA (lnCRNA) can participate to cellular antiviral response but can also be hijacked by the virus to improve the infection. These two families of non-coding RNA could interact with cellular RNA-binding protein (RBP) such as KSRP. This ubiquitous protein is involved in RNA expression control via its participation to pre-mRNA splicing, decay of AU-rich element mRNA and maturation of microRNAs. The functions and localization of KSRP are dependent of post- modification by the cellular kinases Akt, ATM and p38/MAPK.The aim of my thesis was to study the modulation of the expression of these two classes of non-coding RNA during infection by RNA viruses such as the hepatitis C virus (HCV) and the HCoV-229E strain of the Coronaviruses. More specifically, we evaluated the involvement of KSRP in the regulation of non-coding RNAs essential for these infections.My research project began with the study of microRNA-122 (miR-122) the maturation. This miRNA is a proviral factor for HCV infection. We have shown that the Akt-dependent phosphorylation of S193-KSRP promoted the interaction of pri-miR-122 with the DROSHA / DGCR8 nuclear complex and thus was essential for the maturation of miR-122, finally promoting viral replication. We then investigated the role of KSRP phosphorylation by ATM and p38 / MAPK on viral replication and on miR-122 maturation. ATM phosphorylation does not seem to play a major role in these two parameters. In contrast, phosphorylation of KSRP on the T692 residue by p38 / MAPK kinase appears to play a positive role on viral replication.In a second step, by homology with the results obtained in the case of the HCV infection, we studied the role of KSRP during the infection with the HCoV-229E strain of Coronaviruses. After siKSRP transfection or exogenous expression of the KSRP protein, we were able to demonstrate that KSRP was a proviral cellular factor for HCoV-229E replication.In order to characterize the modulation of non-coding RNAs expression during HcoV-229E infection and to identify the non-coding RNAs whose expression could be regulated by KSRP, we performed two high-throughput sequencing ("NGS") assays. The analysis performed on infected and non-infected cells allowed us to identify all the miRNAs and lncRNAs whose expression was altered by the virus. We cross-examined these results with a second "NGS" performed on HCoV-229E infected cells inhibited for KSRP. We found that the expression of an InCARN (LinC00473) was modulated under both experimental conditions. We demonstrated a strong KSRP-dependent induction of LinC00473 expression at 24 h post-infection, then a decrease at 48 h post-infection. Inhibition of this factor results in decreased viral replication suggesting that LinC00473 is a proviral cell factor at the onset of infection.Our results have shown the proviral role of the KSRP protein during two viral infections (HCV and HCoV-229E of the coronaviruses). Its involvement in the regulation of RNA expression makes of KSRP an effective tool for discovering new non-coding RNA therapeutic targets for other viral infections

Algumas espécies de morcegos têm sido reconhecidas como reservatórios naturais de várias famílias virais, desempenhando um importante papel na trasmissão e manutenção desses micro organismos. Devido à descaracterização e fragmentação de habitats naturais, esses mamíferos buscam alternativas de abrigo e alimento, e assim, ficam cada vez mais expostos aos meios antrópicos e em contato com humanos e animais domésticos. Com exceção do vírus rábico, existem poucos trabalhos realizados na detecção de vírus em morcegos no Brasil. Em virtude disso, o presente estudo objetivou a detecção de vírus (circovírus, astrovírus, coronavírus e lyssavírus relacionados ao vírus da raiva) em amostras de órgãos de morcegos do estado do Rio Grande do Sul. Os ácidos nucléicos foram extraídos das amostras de órgãos de morcegos e submetidos à detecão por PCR e RT-PCR. Após a detecção, os fragmentos obtidos foram sequenciados para realizar análise filogenética dos vírus encontrados. Ao total foram analisadas 108 amostras de diferentes espécies e localidades, das quais dez foram positivas para circovírus, seis para coronavírus e 25 para astrovírus, este último sendo o primeiro registro do vírus em morcegos para o Brasil. Todas as amostras foram negativas para lyssavírus relacionados ao vírus da raiva. Análises filogenéticas revelaram que as sequências de circovírus agruparam em ambos os gêneros Circovirus e Cyclovirus, coronavírus no gênero Alphacoronavirus em dois clados diferentes e astrovírus no gênero Mamastrovirus junto com outros astrovírus de morcegos, o qual formam um clado separado dos outros mamíferos. Os resultados demonstram uma diversidade genética entre os vírus encontrados em diferentes espécies de morcegos, que possuem dietas alimentares e habitats distintos. / Some bat species have been recognized as natural reservoirs of several viral families, playing an important role in the transmission and maintaining of these micoorganism. Due to mischaracterization and fragmentation of natural habitats, these mammals seek shelter alternatives and food, and thus are increasingly exposed to anthropism, which make the contact with humans and domestic animals closer. With the exception of the rabies virus, there are few studies on the detection of viruses in bats in Brazil. Therefore, the present study aimed the detection of viruses (circovirus, astrovirus, coronavirus and rabies-related virus) in bats organs samples from Rio Grande do Sul state. Nucleic acids were extracted from bat organs samples and submitted to detection by PCR and RT-PCR. After detection, the obtained fragments were sequenced to perform phylogenetic analysis of the viruses found. From a total of 108 samples analyzed of different species and locations, ten were positive for circoviruses, six for coronaviruse and 25 for astrovirus, which was the first report of this virus in bats in Brazil. All samples were negative for rabies-related virus. Phylogenetic analyzes revealed that the sequences of circoviruses grouped in both Circovirus and Cyclovirus genus, coronaviruses in Alphacoronavirus genus in two different clades and astroviruses in Mamastrovirus genus along with other bats astrovirus, which form a separate clade from other mammals. Results demonstrate a genetic diversity among viruses found in different species of bats, which have different diets and habitats.

Algumas espécies de morcegos têm sido reconhecidas como reservatórios naturais de várias famílias virais, desempenhando um importante papel na trasmissão e manutenção desses micro organismos. Devido à descaracterização e fragmentação de habitats naturais, esses mamíferos buscam alternativas de abrigo e alimento, e assim, ficam cada vez mais expostos aos meios antrópicos e em contato com humanos e animais domésticos. Com exceção do vírus rábico, existem poucos trabalhos realizados na detecção de vírus em morcegos no Brasil. Em virtude disso, o presente estudo objetivou a detecção de vírus (circovírus, astrovírus, coronavírus e lyssavírus relacionados ao vírus da raiva) em amostras de órgãos de morcegos do estado do Rio Grande do Sul. Os ácidos nucléicos foram extraídos das amostras de órgãos de morcegos e submetidos à detecão por PCR e RT-PCR. Após a detecção, os fragmentos obtidos foram sequenciados para realizar análise filogenética dos vírus encontrados. Ao total foram analisadas 108 amostras de diferentes espécies e localidades, das quais dez foram positivas para circovírus, seis para coronavírus e 25 para astrovírus, este último sendo o primeiro registro do vírus em morcegos para o Brasil. Todas as amostras foram negativas para lyssavírus relacionados ao vírus da raiva. Análises filogenéticas revelaram que as sequências de circovírus agruparam em ambos os gêneros Circovirus e Cyclovirus, coronavírus no gênero Alphacoronavirus em dois clados diferentes e astrovírus no gênero Mamastrovirus junto com outros astrovírus de morcegos, o qual formam um clado separado dos outros mamíferos. Os resultados demonstram uma diversidade genética entre os vírus encontrados em diferentes espécies de morcegos, que possuem dietas alimentares e habitats distintos. / Some bat species have been recognized as natural reservoirs of several viral families, playing an important role in the transmission and maintaining of these micoorganism. Due to mischaracterization and fragmentation of natural habitats, these mammals seek shelter alternatives and food, and thus are increasingly exposed to anthropism, which make the contact with humans and domestic animals closer. With the exception of the rabies virus, there are few studies on the detection of viruses in bats in Brazil. Therefore, the present study aimed the detection of viruses (circovirus, astrovirus, coronavirus and rabies-related virus) in bats organs samples from Rio Grande do Sul state. Nucleic acids were extracted from bat organs samples and submitted to detection by PCR and RT-PCR. After detection, the obtained fragments were sequenced to perform phylogenetic analysis of the viruses found. From a total of 108 samples analyzed of different species and locations, ten were positive for circoviruses, six for coronaviruse and 25 for astrovirus, which was the first report of this virus in bats in Brazil. All samples were negative for rabies-related virus. Phylogenetic analyzes revealed that the sequences of circoviruses grouped in both Circovirus and Cyclovirus genus, coronaviruses in Alphacoronavirus genus in two different clades and astroviruses in Mamastrovirus genus along with other bats astrovirus, which form a separate clade from other mammals. Results demonstrate a genetic diversity among viruses found in different species of bats, which have different diets and habitats.

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