Os norovírus (NV) são uma importante causa de hospitalização infantil. Crianças internadas por gastroenterite por NV (GENV) são consideradas portadoras de diarreia grave. O objetivo desse estudo, realizado na cidade do Rio de Janeiro, Brasil, é descrever as características clínicas e a frequência da diarreia por NV em crianças hospitalizadas, comparando as taxas de detecção de NV em crianças vacinadas e não vacinadas contra rotavírus (Rotarix). Foram coletadas 659 amostras de fezes de igual número de crianças e encaminhadas para análise pela reação em cadeia pela polimerase, precedida de transcrição reversa no período de janeiro de 2004 a dezembro de 2009. O percentual de amostras positivas para os NV foi de 27,3% nesse período. Das 180 amostras positivas para NV, 55% tiveram origem na comunidade (aqCo) e 45% foram de aquisição nosocomial (aqNo). O percentual de GENV nos dois anos anteriores (2004 e 2005) à introdução da vacina Rotarix foi de 28,3%, sendo 11,3% o percentual de amostras aqCo. Nos dois anos posteriores (2008 e 2009), a GENV significou 24,4%, e as amostras aqCo foram 14,9% (p<0,05). Em 647 crianças, 494 não receberam a vacina Rotarix, enquanto 151 crianças receberam, pelo menos, uma dose. O percentual de GENV foi de 23,8% e 39,7%, respectivamente (p<0,05). Apesar do comportamento sazonal dos casos de GENV aqCo, esse fato não teve significância estatística. Das 180 crianças, 61,6% tinham peso &#8804; p10 do NCHS, 82,2% tinham idade &#8804; 5anos. As crianças com idade &#8804; 2 anos foram mais acometidas nos casos de aqCo do que àquelas de aqNo (p<0,05). Foram observados em 82 crianças: vômitos (73,2%), febre (54,9%), tosse (20,7%), coriza (2,2%), sangue nas fezes (8,5%), erupção cutânea (4,9%) e broncoespasmo (7,3%). Houve significância estatística com relação à frequência maior de febre, coriza, tosse e broncoespasmo nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). De 69 crianças, 73,9% apresentaram desidratação e, dessas, 76,5% necessitaram de hidratação venosa. Esses dados tiveram significância estatística, representada por maiores percentuais nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). Esse estudo demonstra que os NV foram um importante agente etiológico nos casos de gastroenterites em crianças hospitalizadas e responsável por altas taxas de infecções nosocomiais. Estatisticamente, não foi comprovada uma tendência de aumento dos casos de GENV no período do estudo, como também do aumento da frequência de GENV nos anos posteriores em relação aos anos anteriores à introdução da vacina Rotarix no Brasil em 2006. No entanto, houve significância estatística quando foi avaliado o percentual de GENV em crianças hospitalizadas vacinadas e não vacinadas contra RV. Um aumento dos casos de GENV em crianças poderá vir a acontecer nos próximos anos, quando é esperado que um número maior de crianças será vacinado contra RV. Tosse, coriza e broncoespasmo são sintomas que devem ser mais detalhadamente investigados. Estratégias de prevenção contra a disseminação dos NV são condutas importantes em unidades de internação. Uma vacina eficaz contra norovírus pode ser um benefício significativo para reduzir o percentual de crianças hospitalizadas por diarreia. / Noroviruses (NV) are a major cause of infant hospitalization. Children hospitalized for gastroenteritis NV (NVGE) are considered to have severe diarrhea. The purpose of this study, conducted in the city of Rio de Janeiro, Brazil, is to describe the clinical characteristics and frequency of norovirus diarrhea in hospitalized children, comparing the rates of detection of NV in vaccinated and unvaccinated children against rotavirus (RV). We collected 659 fecal samples from equal numbers of children and sent for analysis by polymerase chain reaction, reverse transcription from January 2004 to December 2009. The percentage of samples positive for NV was 27.3% in this period. Of the 180 samples positive for NV, 55% meant source community (Coaq) and 45% of nosocomial acquisition (Noaq). The percentage of NVGE the previous two years (2004 and 2005) the introduction of the vaccine against RV was 28.3% and 11.3% represented the percentage of samples Coaq. In the two subsequent years (2008 and 2009), NVGE meant to 24.4%, and the samples were Coaq 14.9% (p <0.05). In 647 children, 494 received no vaccine against RV while 151 children received at least one dose. The percentage of NVGE was 23.8% and 39.7%, respectively (p <0.05). Despite the seasonal behavior of NVGE aqCo cases, this did not reach statistical significance. Of the 180 children, 61.6% had weight &#8804; p10 NCHS, 82.2% were aged &#8804; 5 years old. Children aged &#8804; 2 years were most affected in cases of Coaq than those of Noaq (p <0.05). Were observed in 82 children: vomiting (73.2%), fever (54.9%), cough (20.7%), coryza (2.2%), blood in stools (8.5%), rash (4.9%) and bronchospasm (7.3%). There was statistical significance with respect to the higher frequency of fever, coryza, coughing and wheezing in children with NVGE of Coaq than those of Noaq (p <0.05). Of 69 children, 73.9% had dehydration and of these, 76.5% required intravenous hydration. These data were statistically significant, represented by the highest percentage of children with NVGE of Coaq than those of Noaq (p <0.05). This study demonstrates that the NV were an important etiologic agent in cases of gastroenteritis in hospitalized children and responsible for high rates of nosocomial infections. Statistically, , it was not shown a tendency to increase in cases of NVGE during the study period, as well as the increased frequency NVGE in later years relative to years prior to the introduction of RV vaccine in Brazil. However, statistical significance was assessed as the percentage of NVGE in hospitalized children vaccinated and unvaccinated against RV. An increase in cases of children in NVGE could happen in the next few years, when it is expected that a greater number of children will be vaccinated against RV. Cough, coryza and wheezing are symptoms that should be further investigated. Strategies for preventing the spread of NV are important conduits in inpatient units. An effective vaccine against norovirus can be a significant benefit to reduce the percentage of children hospitalized for diarrhea.

Os norovírus (NV) são uma importante causa de hospitalização infantil. Crianças internadas por gastroenterite por NV (GENV) são consideradas portadoras de diarreia grave. O objetivo desse estudo, realizado na cidade do Rio de Janeiro, Brasil, é descrever as características clínicas e a frequência da diarreia por NV em crianças hospitalizadas, comparando as taxas de detecção de NV em crianças vacinadas e não vacinadas contra rotavírus (Rotarix). Foram coletadas 659 amostras de fezes de igual número de crianças e encaminhadas para análise pela reação em cadeia pela polimerase, precedida de transcrição reversa no período de janeiro de 2004 a dezembro de 2009. O percentual de amostras positivas para os NV foi de 27,3% nesse período. Das 180 amostras positivas para NV, 55% tiveram origem na comunidade (aqCo) e 45% foram de aquisição nosocomial (aqNo). O percentual de GENV nos dois anos anteriores (2004 e 2005) à introdução da vacina Rotarix foi de 28,3%, sendo 11,3% o percentual de amostras aqCo. Nos dois anos posteriores (2008 e 2009), a GENV significou 24,4%, e as amostras aqCo foram 14,9% (p<0,05). Em 647 crianças, 494 não receberam a vacina Rotarix, enquanto 151 crianças receberam, pelo menos, uma dose. O percentual de GENV foi de 23,8% e 39,7%, respectivamente (p<0,05). Apesar do comportamento sazonal dos casos de GENV aqCo, esse fato não teve significância estatística. Das 180 crianças, 61,6% tinham peso &#8804; p10 do NCHS, 82,2% tinham idade &#8804; 5anos. As crianças com idade &#8804; 2 anos foram mais acometidas nos casos de aqCo do que àquelas de aqNo (p<0,05). Foram observados em 82 crianças: vômitos (73,2%), febre (54,9%), tosse (20,7%), coriza (2,2%), sangue nas fezes (8,5%), erupção cutânea (4,9%) e broncoespasmo (7,3%). Houve significância estatística com relação à frequência maior de febre, coriza, tosse e broncoespasmo nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). De 69 crianças, 73,9% apresentaram desidratação e, dessas, 76,5% necessitaram de hidratação venosa. Esses dados tiveram significância estatística, representada por maiores percentuais nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). Esse estudo demonstra que os NV foram um importante agente etiológico nos casos de gastroenterites em crianças hospitalizadas e responsável por altas taxas de infecções nosocomiais. Estatisticamente, não foi comprovada uma tendência de aumento dos casos de GENV no período do estudo, como também do aumento da frequência de GENV nos anos posteriores em relação aos anos anteriores à introdução da vacina Rotarix no Brasil em 2006. No entanto, houve significância estatística quando foi avaliado o percentual de GENV em crianças hospitalizadas vacinadas e não vacinadas contra RV. Um aumento dos casos de GENV em crianças poderá vir a acontecer nos próximos anos, quando é esperado que um número maior de crianças será vacinado contra RV. Tosse, coriza e broncoespasmo são sintomas que devem ser mais detalhadamente investigados. Estratégias de prevenção contra a disseminação dos NV são condutas importantes em unidades de internação. Uma vacina eficaz contra norovírus pode ser um benefício significativo para reduzir o percentual de crianças hospitalizadas por diarreia. / Noroviruses (NV) are a major cause of infant hospitalization. Children hospitalized for gastroenteritis NV (NVGE) are considered to have severe diarrhea. The purpose of this study, conducted in the city of Rio de Janeiro, Brazil, is to describe the clinical characteristics and frequency of norovirus diarrhea in hospitalized children, comparing the rates of detection of NV in vaccinated and unvaccinated children against rotavirus (RV). We collected 659 fecal samples from equal numbers of children and sent for analysis by polymerase chain reaction, reverse transcription from January 2004 to December 2009. The percentage of samples positive for NV was 27.3% in this period. Of the 180 samples positive for NV, 55% meant source community (Coaq) and 45% of nosocomial acquisition (Noaq). The percentage of NVGE the previous two years (2004 and 2005) the introduction of the vaccine against RV was 28.3% and 11.3% represented the percentage of samples Coaq. In the two subsequent years (2008 and 2009), NVGE meant to 24.4%, and the samples were Coaq 14.9% (p <0.05). In 647 children, 494 received no vaccine against RV while 151 children received at least one dose. The percentage of NVGE was 23.8% and 39.7%, respectively (p <0.05). Despite the seasonal behavior of NVGE aqCo cases, this did not reach statistical significance. Of the 180 children, 61.6% had weight &#8804; p10 NCHS, 82.2% were aged &#8804; 5 years old. Children aged &#8804; 2 years were most affected in cases of Coaq than those of Noaq (p <0.05). Were observed in 82 children: vomiting (73.2%), fever (54.9%), cough (20.7%), coryza (2.2%), blood in stools (8.5%), rash (4.9%) and bronchospasm (7.3%). There was statistical significance with respect to the higher frequency of fever, coryza, coughing and wheezing in children with NVGE of Coaq than those of Noaq (p <0.05). Of 69 children, 73.9% had dehydration and of these, 76.5% required intravenous hydration. These data were statistically significant, represented by the highest percentage of children with NVGE of Coaq than those of Noaq (p <0.05). This study demonstrates that the NV were an important etiologic agent in cases of gastroenteritis in hospitalized children and responsible for high rates of nosocomial infections. Statistically, , it was not shown a tendency to increase in cases of NVGE during the study period, as well as the increased frequency NVGE in later years relative to years prior to the introduction of RV vaccine in Brazil. However, statistical significance was assessed as the percentage of NVGE in hospitalized children vaccinated and unvaccinated against RV. An increase in cases of children in NVGE could happen in the next few years, when it is expected that a greater number of children will be vaccinated against RV. Cough, coryza and wheezing are symptoms that should be further investigated. Strategies for preventing the spread of NV are important conduits in inpatient units. An effective vaccine against norovirus can be a significant benefit to reduce the percentage of children hospitalized for diarrhea.

Les Rotavirus appartiennent à la famille des Reoviridae, famille du groupe III des virus à ARN double brin. Identifiés en 1973 par Ruth Bishop, ces virus non enveloppés sont la première cause de diarrhée aiguë sévère du jeune enfant dans le monde. La capside virale icosaédrique est constituée de 3 couches protéiques de structure : la couche externe formée par la glycoprotéine VP7 d’où émergent les spicules de protéine VP4, la couche intermédiaire constituée par la protéine VP6 représentant près de 50 % du poids du virus et enfin, la couche interne appelée core, résultant de l’assemblage des protéines VP2, d’où émergent vers l’intérieur les protéines VP1 et VP3. Cette capside renferme un génome divisé en 11 segments d’ARN bicaténaires. A ces 6 protéines structurales s’ajoutent les protéines non structurales qui interviennent lors de la réplication du virus. Les deux protéines structurales, VP4 et VP7 sont essentielles pour la fixation de la particule triple couche (TLP) aux membranes des cellules hôtes, par interaction aux récepteurs intégrines, elle sont également impliqués dans la déstabilisation des membranes endosomales, indispensable à la libération de la particule double couche (DLP) infectieuse dans le cytoplasme. Actuellement, contrairement au mécanisme d’action de la protéine VP5*, celui de la glycoprotéine VP7 est inconnu. L’objectif de cette thèse, a été de comprendre le mécanisme moléculaire de déstabilisation des membranes par les peptides dérivés de VP7. Dans un premier temps nous avons montré, par des études in silico, l’existence d’un domaine prédit en hélice membranaire bordé de résidus arginine et lysine hautement conservés, situé à l’extrémité C-terminale de la glycoprotéine VP7. Ces résultats ont conduit à la synthèse de quatre peptides avec lesquels des tests de perméabilisation de membranes modèles de larges vésicules unilamellaires (LUVs) ont été menés. Ceux-ci ont permis d’identifier le domaine minimum le plus actif, VP723, parmi les peptides sélectionnés. Dans un second temps nous avons déterminé la structure de ces peptides par RMN, dans des conditions mimant l’environnement hydrophobe de la membrane. Le peptide minimal VP723 s’organise en hélice α-amphipathique, structure souvent impliquée dans la déstabilisation des membranes cellulaires. La comparaison de sa structure obtenue par RMN à celle du domaine correspondant dans la structure cristallographique de la protéine native montre le réarrangement conformationnel de ce segment après maturation par la trypsine. Ces résultats ont été confirmés par deux mutants de synthèse, dont l’un est inactif pour la perméabilisation des membranes modèles. Ces travaux ont été complétés par des expériences de Résonance Plasmonique aux Ondes guidée (PWR). Des études par RMN du solide sont en cours afin de déterminer l’orientation du peptide dans les membranes modèles. En conclusion, nos résultats mettent en évidence l’importance du domaine C-terminal VP723 de la protéine VP7 dans la déstabilisation des membranes, permettant d’assurer la translocation de la particule virale infectieuse (DLP) de l’endosome vers le cytoplasme. Un modèle du mécanisme d’entrée du virus, médié par les peptides dérivés de la maturation par la trypsine de la glycoprotéine VP7 est proposé. / Rotaviruses belong to the Reoviridae family, belonging to the group III of dsRNA viruses. Identified in 1973 by Ruth Bishop, these non-enveloped viruses are the leading cause of severe diarrhea in young children worldwide. The icosahedral capsid is composed of three structural protein layers: the outer one, formed by the glycoprotein VP7, emerges spicules protein VP4, the intermediate one consists of VP6 protein representing nearly 50% of the weight of the virus and finally, the inner one called core, results from the assembly of proteins VP2, emerges towards the inside of proteins VP1 and VP3. The capsid contains a genome divided into 11 segments of dsRNA. To these six structural proteins are added nonstructural proteins involved in virus replication. The two structural proteins, VP4 and VP7, are involved in the interaction of the triple layer particle (TLP) to integrin receptors, necessary for the release of the infectious double layer particle (DLP) into the cytoplasm following the permeabilization of the membrane of the endosome compartments. Currently, unlike the mechanism of action of the protein VP5*, the glycoprotein VP7 remains unknown. The objective of this work was to understand the molecular mechanism involved in the destabilization of membranes by peptides derived from VP7. In a first step, we have shown, by in silico studies, the existence of a helical trans-membrane domain predicted containing a highly conserved arginine and lysine residues, located at the C-terminus of the VP7 glycoprotein. These results led to the synthesis of four peptides with which permeabilizing tests of model membranes were conducted. We have identified the minimum of the most active domain, named VP723, among the selected peptides. In a second step, we determined the structure of these peptides by NMR under conditions mimicking the hydrophobic environment of the membrane. The VP723 peptide is organized like an α-helical amphipathic structure often involved in the destabilization of cell membranes. The comparison of the structure obtained by NMR to that of the corresponding domain in the crystallographic structure of the native protein shows a conformational rearrangement of the segment after trypsin maturation. These results were confirmed by two synthetic mutants, one of which is inactive for the permeabilization of model membranes. These studies were complemented by experiments Plasmon Resonance guided the Waves (PWR). Studies by solid state NMR are in progress to determine the orientation of the peptide in model of membranes. In conclusion, our results highlight the importance of the C-terminal domain of the VP7 protein, named VP723, in the destabilization of membranes, to ensure the translocation of the infectious viral particle (DLP) from the endosome into the cytoplasm compartments. A mechanism of virus entry mediated by peptides derived from trypsin maturation of the VP7 glycoprotein is proposed in this study.

Structuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66. Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds. Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property.

Rotavirus is one of the leading etiological agents of gastroenteritis in young of many species including humans worldwide and is responsible for about 600,000 infant deaths per annum. Rotavirus belongs to the Reoviridae family, and its genome is composed of 11 double-stranded RNA segments that encode six structural proteins and six nonstructural proteins. Rotavirus replication is fully cytoplasmic and occurs within highly specialized regions called viroplasms. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm¬like structures. A recent study suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins, containing four structural (VP1, VP2, VP3 and VP6) and two nonstructural (NSP2 and NSP5) proteins. NSP5, the translation product of gene segment 11 has a predicted molecular eight of 21 kDa. NSP5 has been reported to exist in multiple isoforms ranging in size from 28-and 32-35 kDa from a 26-kDa precursor has been attributed to O-glycosylation and hyperphosphorylation. To study different properties of the protein, recombinant NSP5 containing an N-terminal hisidine tag was expressed in bacteria and purified by affinity chromatography. A significant observation was the similarity in phosphorylation property of the bacterially expressed and that expressed in mammalian cells. While the untagged recombinant protein failed to undergo phosphorylation in vitro, addition of His tag or deletions at the N-terminus promoted phosphorylation of the protein in vitro, which is very similar to the reported properties exhibited by the corresponding proteins expressed in mammalian cells. Phosphorylation of NSP5 in vitro is independent of the cell type from which the extract is derived suggesting that the kinases that phosphorylate NSP5 are distributed in all cell types. Among the C-terminal deletion mutants studied, NH-∆C5 and NH-∆C10 were phosphorylated better than full-length NSP5, but NH-∆C25 and NH¬∆C35 showed substantial reduction in the level of phosphorylation compared to full-length NSP5. These results indicate that the C-terminal 30 residues spanning the predicted α-helical domain of NSP5 are critical for its phosphorylation in vitro which is in correspondence with previous findings that C-terminal 21 amino acids of NSP5 direct its insolubility, hyperphosphorylation, and VLS formation. The results revealed that though the tagged full-length and some of the mutants could be phosphorylated in vitro, they are not suitable substrates for hyperphosphorylation unlike the similar proteins expressed in mammalian cells or infected cells. Analysis by western blot and mass spectrometry revealed that the bacterially expressed NH-NSP5 is indeed phosphorylated. It appears that prior phosphorylation in bacteria renders the protein conformationally not amendable for hyperphosphorylation by cellular kinases in vitro. Mutation of the highly conserved proline marginally enhanced its phosphorylation in vitro but the stability of protein is affected. Notably, mutation of S67A, identified as a critical residue for the putative caesin kinase-I and-II pathways of NSP5 phosphorylation, affected neither the phosphorylation nor the ATPase activity of NSP5. These results suggest that bacterially expressed NSP5 by itself has undectable auto-kinase activity and it is hypophosphorylated. Purified recombinant NSP5 has been reported to possess an Mg¬ 2+-dependent ATP-specific triphosphatase activity. The results indicated that deletion of either C-terminal 48 amino acids or N-terminal 33 residues severely affected the ATPase activity of recombinant NSP5, underlying the importance of both N-and C-terminal domains for NSP5 ATP hydrolysis function. NSP5 expressed in rotavirus infected cells exists as inter-molecular disulfide-linked dimeric forms and it appears that the 46 kDa isoforms, that are phosphorylated, corresponds to dimer as revealed by western blotting. Analytical gel filtration analysis of NH-NSP5, NH-ΔN43 and NH-ΔN33-ΔC25 showed most of the proteins in void volume, but an additional peak corresponding to the mass of dimeric species further supports that NSP5 is basically a dimer that undergoes oligomerization. Analysis by sucrose-gradient fractionation revealed that NH-NSP5 and NH-ΔN43 proteins were mainly distributed in the lower fraction of the gradient suggesting the existence of high molecular weight complexes or higher oligomers. The multimeric nature of NSP5 and its mutants was further confirmed by dynamic light scattering which suggests that high molecular weight complexes are of homogenous species. The correlation curves showed a low polydispersity distribution and a globular nature of recombinant NH-NSP5 proteins. The present results clearly demonstrate that dimer is the basic structural unit of NSP5 which undergoes oligomerization to form a complex consisting of about 20-21 dimers. The nonstructural protein 5 is hyperphosphorylated in infected cells and cellular kinases have been implicated to be involved in its phosphorylation. NSP5 contains multiple consensus sites for phosphorylation by several kinases, but the cellular kinases that specifically phosphorylate NSP5 in infected cells are yet to be identified. Previous studies from our laboratory using signaling pathway inhibitors revealed that recombinant NH¬NSP5 and its deletion mutants can be phosphorylated in vitro by purified cellular kinases and by mammalian cell extracts. These studies also showed the involvement of PI3K-Akt and MAPK signaling pathways in NSP5 phosphorylation and a negative role for GSK3β in the phosphorylation of bacterially expressed recombinant NSP5 in vitro. In the present work, using phospho-specific anti-Ser9 GSK3β antibody, we observed that GSK3β is inactivated in a rotavirus infected MA104 cells in a strain-independent manner. GSK3β¬specific small interfering RNA (siRNA-GSK3β) reduced GSK3β levels leading to increased level of synthesis of the structural rotavirus protein VP6 and NSP5 hyperphosphorylation compared to control siRNA. The pharmacological kinase inhibitors (LY294002, Genistein, PD98059, and Rapamycin) studies at the concentrations tested did not significantly affect rotavirus infection as seen from the number foci, while U0126 severely affected rotavirus replication. The results clearly demonstrated the importance of the MEK1/2 signaling pathway in the successful replication of rotavirus and NSP5 hyperphosphorylation in rotavirus-infected cells. In contrast inhibition of GSK3β activity by LiCl, increased in general, the number of foci by greater than 2-fold for all viral strains studied. These results suggest that MEK1/2 pathway majorly contributes to GSK3β inactivation in rotavirus infected cells. Thus, our results reveal that rotavirus activates both the PI3K/Akt and FAK/ERK1/2 MAPK pathways and appears to utilize them as a strategy to activate mTOR, and inhibit GSK3β through phosphorylation on serine 9, the negative regulator of rotavirus NSP5 phosphorylation, and thus facilitate translational competence of rotaviral mRNAs during virus replication cycle. It was shown previously in the laboratory by co-immunoprecipitation assay that Hsp70 interacts with rotaviral proteins VP1 and VP4 in rotavirus-infected mammalian cells. In this study, the interactions between Hsp70 with VP1 and VP4 were further evaluated in vitro by GST-pull down assay. It was observed that the N-terminal ATPase and C-terminal peptide-binding domain of Hsp70 is necessary for its direct interaction with VP1 and VP4. The presence of Hsp70 in purified double-and triple-layered virus particles further supported the observed interactions of rotaviral proteins VP1 and VP4 with Hsp70. However, the specific interaction observed between Hsp70 and rotaviral capsid proteins, VP1 and VP4 in viral particles suggests that Hsp70 has an important role during rotavirus assembly which requires further investigation.

No description available.

Um total de 8.961 amostras fecais coletadas de pacientes com diarréia aguda, no período de 1996 a 2006, no Estado de São Paulo foi testado para rotavírus por EIE. Destas, 20,0% foram positivas e posteriormente realizadas a caracterização dos rotavírus em genótipos G e P por nested RT-PCR. O genótipo G1 de rotavírus foi o mais freqüente, detectado em 35,2% das amostras, seguidos dos tipos G9, G2, G3, G4, infecção mista e G12. A associação mais freqüente foi a P[8]G1 e P[8]G9. Foi realizada a sequencia nucleotídica do gene 9 (VP7) de 38 rotavirus genótipo G1. Duas cepas foram analisadas dos anos de 1997, 1998, 2001 e 2002, três cepas dos anos 1996, 1999 e 2003, quatro cepas em 2000, sete cepas em 2004 e 2005, e cinco em 2006. Os 38 rotavírus G1 foram classificados em duas linhagens distintas, linhagem G1-I e linhagem G1-II. A linhagem G1-I foi detectada durante seis anos, 1996-1997, 2001-2002 e 2004-2006, e a linhagem G1-II foi detectada durante os anos de 1998-2001, e 2003-2005. Análises preliminares mostraram que Rotarix ® foi eficiente contra estas linhagens G1. / Rotavirus (RV) infections are recognized as a major cause of severe gastroenteritis in children worldwide. In March 2006, a monovalente P[8]G1, human RV vaccine (Rotarix® GlaxoSmithKline Biologicals) was introduced in Brazil into the routine childhood immunization schedule. Therefore, the study of genetic diversity among rotavirus strains before and after the introduction of this vaccine may be important for the development of vaccination strategies. A total of 8,961 fecal samples collected from patients with acute diarrhea, during the 11-year period surveillance in São Paulo State (1996 to 2006) were tested for rotavirus by ELISA. One thousand seven hundred eighty- four (1,784, 20.0%) were positive, and the characterization of the G and P genotypes was performed on 1,300 rotavirus samples by nested RT-PCR. The G1 type was the most prevalent rotavirus strain (35.2%). The second most prevalente was the G9 type (31.2%), followed by G2 (4.0%), G3 (3.5%), G4 (2.2%), mix infection (1.8%) and G12 (0.5%). The more frequent association was P[8]G1 and P[8]G9. We performed a sequence analysis of 38 P[8]G1 rotavirus strains, selected from a total of 341 P[8]G1.Two strains from 1997, 1998, 2001, and 2002 were analyzed; three strains from 1996, 1999, and 2003; four strains from 2000; seven strains from 2004, and 2005; and five strains from 2006. All 38 rotavirus G1 sequence in this study were found to be classified into two distinct lineages, lineage I with 44.7% (17/38) and lineage II with 55.3% (21/38). The G1I lineages were detected during six rotavirus seasons 1996-1997, 2001- 2002, and 2004-2006 whereas and lineage G1- II was detected during 1998-2001, and 2003-2005. Preliminary analyses 4 demonstrated that Rotarix® has been efficacious against these G1 lineages.

INTRODUÇÃO: A doença diarreica aguda é a segunda causa de morte em crianças abaixo de 5 anos de idade. No Brasil, entre 2003 e 2009, a diarreia aguda foi responsável por cerca de 100.000 internações por ano, e por 4% das mortes em crianças abaixo de 5 anos de idade. O rotavírus é a principal etiologia de diarreia aguda grave no mundo todo, sendo responsável por 40% das internações por diarreia aguda, e 29% de todas as mortes por diarreia aguda. A vacina monovalente (RV1) contra o rotavírus foi introduzida no Programa Nacional de Imunizações em 2006. OBJETIVOS: Verificar o impacto da vacina monovalente contra rotavírus nas consultas de pronto-socorro e internações por doença diarreica aguda em crianças menores de 5 anos de idade, verificar a positividade do exame \"pesquisa de rotavírus nas fezes\", e verificar a presença ao ausência de imunidade de rebanho. METODOLOGIA: Foi realizado um estudo ecológico retrospectivo no Hospital Universitário da Universidade de São Paulo. O período foi dividido em pré-vacina (2003 a 2005) e pós-vacina (2007 a 2009). Foram incluídas todas as crianças abaixo de 5 anos que passaram em consulta no pronto-socorro e verificado o diagnóstico do atendimento e internação através de registro eletrônico. Foram obtidas as taxas de consultas no pronto-socorro e internações por doença diarreica aguda, foram selecionadas as crianças não vacinadas para cálculo da imunidade de rebanho, e verificado se houve coleta do exame pesquisa de rotavírus nas fezes. A redução nas taxas foi obtida através da fórmula: redução (%) = (1 - odds ratio) x 100. RESULTADOS: No período pré-vacina a taxa de consultas por diarreia aguda foi de 85,8 consultas por 1.000 consultas gerais, no período pós vacina a taxa de consultas por diarreia aguda foi 80,9 por 1.000, e a redução foi 6% (IC 95%, 4% a 9%, p < 0,001), chegando a 40% (IC 95%, 36% a 44%, p<0,001) nos meses de maio e junho. A taxa de internação por diarreia aguda era 40,8 internações por 1.000 e caiu para 24,9 por 1.000, redução de 40% (IC 95%, 22% a 54%, p < 0,001), chegando a 82% (IC 95%, 62% a 92%, p < 0,001) nos meses de maio e junho. Nas crianças não vacinadas não houve redução na taxa de consultas de pronto-socorro (IC 95%, -4% a 5%, p=0,903), e não se pode afirmar se houve redução ou aumento das internações por diarreia aguda (IC 95%, -212% a 35%, p=0,381). Houve queda da positividade do exame pesquisa de rotavírus em 2009 (redução de 70%, IC 95%, 26% a 88%, p=0,007). CONCLUSÕES: Após a introdução da vacina contra rotavírus (RV1) houve uma redução de 6% nas consultas por diarreia aguda no pronto-socorro, de 40% nas internações por diarreia aguda e de 70% na positividade do exame pesquisa de rotavírus nas fezes. Não foi detectada imunidade de rebanho / INTRODUCTION: Acute diarrheal disease is the second cause of death in children under five years. In Brazil, from 2003 to 2009, acute diarrhea was responsible for nearly 100,000 deaths per year and 4% of the deaths in children under five years. Rotavirus is the leading cause of severe acute diarrhea worldwide, accounting for 40% of hospital admissions and 29% of all the acute diarrhea-related deaths. In 2006, the rotavirus monovalent vaccine (RV1) was added to the Brazilian National Immunization Program. OBJECTIVES: To analyze the impact of the RV1 on Emergency Department (ED) visits and hospital admissions for acute diarrhea, the rates of positivity of the stool rotavirus tests and the presence or absence of herd immunity. METHODS: A retrospective ecologic study at the University Hospital, University of Sao Paulo. The study analyzed two periods: the pre-vaccine (2003-2005) and the post-vaccine (2007-2009) periods. We screened the main diagnosis of all ED attendances and hospital admissions of children under five years in an electronic registry system database and calculated the rates of ED visits, hospital admissions and positivity of the stool rotavirus test in patients with acute diarrhea. Herd immunization was evaluated in non-vaccinated children. The reduction rate was analyzed according to the formula: reduction (%) = (1- odds ratio) x 100. RESULTS: The rates of ED visits for acute diarrhea was 85.8 and and 80.9 per 1,000 total ED visits in the pre and post vaccination periods, respectively, resulting in 6% reduction (95% CI, 4% to 9%, p< 0.001) in the overall period and reaching 40% reduction on May and June. The rates of hospital admissions for acute diarrhea was 40.8 per 1,000 in the pre-vaccine period and dropped to 24.9 per 1,000 hospitalizations, resulting in 40% reduction (95% CI, 22% to 54%, p < 0.001) in the overall period and 82% (95% CI, 62% to 92%, p < 0.001) on May and June. Among non-vaccinated children, no reduction on ED visits was observed (95% CI, -4% to 5%, p=0.903), while an increase or reduction in the hospitalization rates could not be determined (95% CI, -212% to 35%, p=0.381). There was a decrease in the positivity of the stool rotavirus tests in 2009 (70% reduction, 95% CI, 26% to 88%, p=0.007). CONCLUSIONS: The introduction of the RV1 vaccine resulted in 6% reduction in the ED visits and 40% reduction in hospital admissions for acute diarrhea, and 70% reduction in the rates of positive stool rotavirus tests. No herd immunity was observed

Os rotavírus são um dos principais agentes causadores de doenças entéricas em várias espécies animais, com ocorrência generalizada na suinocultura do Brasil. O seu diagnóstico é um componente essencial para estudos epidemiológicos e delimitação de medidas profiláticas visando o controle da doença. Apesar da relevância, não existem testes, tais como PCR em tempo real desenvolvido para detectar a diversidade genética de rotavírus suíno do grupo A (RVA). Este trabalho descreve o desenvolvimento de uma PCR em tempo real com SYBR® Green, para a detecção de rotavírus suíno e os seus resultados comparados com a PCR convencional e ELISA. Foram desenhados primers visando o segmento codificador da proteína NSP5 (137pb) e também foram utilizados primers visando o mRNA do gene mitocondrial bovino NADH5 (191pb) para o controle interno exógeno. Amostras de fezes de suínos de até 60 dias de idade de suínos do estado de São Paulo foram usadas para compor um painel de teste. Foram utilizadas como amostras de referencia o isolado 32/00 (controle positivo de rotavírus suíno do grupo A), concentrado de células MDBK (controle positivo do controle interno exógeno) e água tratada com DEPC (controle negativo). A extração do RNA total foi realizado com Trizol a partir de suspensões fecais contendo MDBK e o cDNA foi sintetizado utilizando primers aleatórios e M-MLV. Para as reações de PCR em tempo real utilizou-se o reagente MaximaTM SYBR Green qPCR Master Mix (Fermentas Life Science). Durante a padronização da PCR convencional, a temperatura de 54°C foi definida como a Tm ótima para a reação. O desempenho do ensaio foi validado em sete amostras positivas inicialmente testadas pelos métodos ELISA e PAGE. O isolado viral RV8209 foi utilizado para determinar o limiar de detecção da PCR em tempo real através de diluições seriadas sendo defino como ponto de corte Ct=33,5 (10-12,28TCID50%). O segmento codificador da NSP5 foi clonado no vetor pTZ57R/T submetido a restrição enzimática e usado como alvo para gerar uma curva padrão, onde obteve-se uma eficiência de 93,39%, slope de -3,49 e R2 de 0,993. A detecção do controle interno exógeno mostrou 82,9% de positividade para PCR convencional e 76,31% para a PCR em tempo real, com correlação significativa (0,718). O ensaio de ELISA para RVA apresentou 10,5% (8/78) de positividade, enquanto que as taxas de detecção da PCR em tempo real e PCR convencional foram de 50% (29/58) e 30,1% (24/63), respectivamente. Foi encontrada uma correlação moderada (0,546) entre PCR convencional e PCR em tempo real; baixa (0,056) entre a PCR convencional e ELISA; ausente (0,0) entre a PCR em tempo real e ELISA. Os resultados obtidos sugerem que a detecção por PCR em tempo real para a detecção do rotavírus suíno do grupo A em amostras fecais possa ser utilizada como diagnostico rápido e eficiente aumentando o repertório dos testes já estabelecidos, de modo a proporcionar uma maior sensibilidade para o diagnóstico clínico e epidemiológico. / Rotavirus is one of the main causative agents of enteric diseases in several animal species with widespread occurrence in Brazilian pig farm. Diagnosis is an essential component for epidemiological studies and delineation of prophylactic measures aiming disease control. Despite the relevance, there are no assays such as real-time PCR developed to detect the genetic diversity of porcine rotavirus from group A (RVA). This work describes the development of SYBR Green real-time PCR assay for the detection of porcine rotavirus and the results were compared with conventional PCR and ELISA. Primers were designed targeting the coding segment of the protein NSP5 (137pb) and also primers targeting the bovine mitochondrial gene mRNA NAD5 (191pb) were used for the exogenous internal control. Fecal samples from pigs up to 60 days of age from São Paulo state were used to compose a panel test. The reference sample was the isolate 32/00 (positive control for porcine rotavirus group A), concentrated MDBK cells (Exogenous Internal Positive Control) and DEPC-treated water (negative control). Total RNA extraction from supernatants of fecal samples containing MDBK cells were carried out with TRIzol reagent and cDNA was synthesized using random primers and M-MLV Reverse Transcriptase. For real-time PCR reactions were used MaximaTM SYBR Green qPCR Master Mix (Fermentas Life Science). For conventional PCR optimization, 54oC was defined as the optimum reaction temperature (Tm). The performance of the assay was validated on seven samples initially tested positive by ELISA and PAGE methods. The limit of detection of the developed real-time-PCR assay was determined using serial dilutions of the isolated RV8209 with Ct=33,5 (10-12,28TCID50%). The NSP5 gene segment was cloned into vector pTZ57R/T submitted to enzymatic restriction and used as template to generate a standard curve which Efficiency of 93.39%, slope of -3.49 and R2 &#9633; 0.993. The Exogenous internal control showed 82.9% positivity for conventional PCR and 76.31% for real-time PCR with substantial correlation (0.718). The ELISA assay detected porcine RVA in 10,5% (8/78) of fecal samples, whereas the detection rates of both SYBR Green real-time PCR and conventional PCR assays were 50% (29 of 58) and 30.1% (24 of 63), respectively. A moderate correlation (0.546) was found between conventional PCR and real-time PCR; low (0.056) between the conventional PCR and ELISA; absent (0.0) between the real-time PCR and ELISA. Our findings suggest that detection of Group A porcine rotavirus in fecal samples by use of the real-time PCR assay may be fast and efficient increasing the repertoire of tests established to improve sensitivity for epidemiology and clinical diagnosis.

Made available in DSpace on 2014-07-29T15:26:22Z (GMT). No. of bitstreams: 1 Tese Talissa de Morais Tavares.pdf: 2094396 bytes, checksum: cca589203914a2c676b55bcf55e62bbc (MD5) Previous issue date: 2008-04-28 / Group A rotaviruses are the major cause of gastroenteritis in children throughout the world. Epidemiological surveys and molecular analysis of rotavirus strains are required for gastroenteritis control and prevention. Studies using VP6, an important immunogenic structural protein, and NSP4, a transmembrane nonstructural glycoprotein which is critical to rotavirus morphogenesis and pathogenesis, have been performed. In this study, 330 rotavirus-positive fecal samples previously obtained from children with or without diarrhea, between 1987 and 2003, in three cities of Central West Region of Brazil (Goiânia, Brasília and Campo Grande), were characterized for VP6- and NSP4-encoding genes. The VP6 and NSP4 genes were amplified by reverse transcription- polymerase chain reaction followed by sequencing and phylogenetic analysis. Detection rates of 84.8% and 78.5% were observed for VP6 and NSP4 genes, respectively. Two distinct genotypes could be recognized for NSP4 (A and B). It was observed that the G9P[6] samples were associated with genotype A, whereas the G1P[6], G1P[8], G2P[8], G3P[8], G4P[8] and G9P[8] samples were associated with genotype B. The analysis of VP6 gene allowed genogrouping of samples in two clusters, genogroups I and II. The G2P[4], G3P[4] and G9P[6] samples were identified as genogroup I, whereas the G1P[6], G1P[8], G2P[8], G4P[6], G4P[8] and G9P[8] samples were identified as genogroup II. In addition, it was showed that samples identified as VP6 genogroup I were associated with NSP4 genotype A and samples identified as VP6 genogroup II were associated with NSP4 genotype B. This investigation described different genetic groups representing diversity of group A rotavirus samples circulating in the Central West Region of Brazil. / Os rotavírus do grupo A são considerados como os principais agentes de gastroenterite em crianças em todo o mundo. Investigações de vigilância epidemiológica e molecular são importantes para o controle e prevenção da doença. Nesse sentido, destacam-se os estudos utilizando VP6, proteína estrutural que se tem mostrado como a mais imunogênica, e NSP4, uma glicoproteína não estrutural transmembrana envolvida na morfogênese e patogênese viral. No presente estudo, 330 amostras de rotavírus coletadas de 1987 a 2003, provenientes de espécimes fecais de crianças com ou sem diarréia, em três cidades da Região Centro-Oeste do Brasil (Goiânia, Brasília e Campo Grande), foram caracterizadas em relação aos genes que codificam as proteínas VP6 e NSP4. Inicialmente, foi feita a amplificação dos genes de VP6 e NSP4 pela reação em cadeia pela polimerase pós-transcrição reversa, seguida do seqüenciamento genômico e de análise filogenética. Os genes de VP6 e NSP4 foram detectados em 84,8% e 78,5% das amostras, respectivamente. Dois genótipos de NSP4 foram identificados (A e B). Foi observado que as amostras G9P[6] associaram-se ao genótipo A e as amostras G1P[6], G1P[8], G2P[8], G3P[8], G4P[8] e G9P[8] associaram-se ao genótipo B. A análise do gene VP6 permitiu a identificação de dois genogrupos distintos (I e II). As amostras G2P[4], G3P[4] e G9P[6] foram caracterizadas como genogrupo I, enquanto as amostras G1P[6], G1P[8], G2P[8], G4P[6], G4P[8] e G9P[8] foram caracterizadas como genogrupo II. Ainda foi evidenciado que as amostras genogrupo I de VP6 estavam associadas com genótipo A de NSP4, e as amostras genogrupo II de VP6 estavam associadas com genótipo B de NSP4. A presente investigação identificou diferentes variantes genéticas, mostrando a diversidade dos rotavírus do grupo A circulantes na Região Centro-Oeste do Brasil.