Thèse (Ph. D.)--Université Laval, 2008. / Bibliogr.: f. 117-136. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (MSc (Med)(Virology))--University of Limpopo (Medunsa Campus), 2009. / Background: The most important causes of viral gastroenteritis include rotaviruses, caliciviruses, adenoviruses and astroviruses. Globaly, rotavirus infection has been estimated to result in 527 000 deaths of young children annually. In South Africa rotaviruses were found to be responsible for 20 – 30% of diarrhoeal diseases in children younger than 5 years of age. Noroviruses are the leading cause of non-bacterial acute gastroenteritis outbreaks worldwide, with human noroviruses accounting for more than 86.0% of all outbreaks caused by viruses. Aim: To investigate the prevalence of rotaviruses and noroviruses and to determine the genotypes of rotaviruses circulating in children under 5 years of age attending the private health facilities in the Tshwane region of South Africa. Material and methods: A total of 1227 stool specimens and the corresponding patient‟s data were obtained from the Lancet Pathology Laboratory (Pretoria, South Africa) from children under the age of 5 years with gastroenteritis who submitted stools for testing. The MRC-DPRU detected rotaviruses using the commercially available enzyme immunoassay DAKO IDEIATM assay whereas the Lancet Pathology Laboratory detected rotaviruses using VIKIA assay. The rotavirus genome was extracted from EIA positive samples using phenol-chloroform and analyzed utilizing polyacrylamide gel electrophoresis (PAGE). Molecular characterization of the rotavirus-positive samples was done using reverse transcriptase polymerase chain reaction (RT-PCR) and genotyping assays. Samples were sequenced for further confirmation and to type the nontypeable samples. Due to lack of sufficient raw stool materials, only 673 of the specimens were screened for noroviruses using the RIDASCREEN® Norovirus testing assay. No molecular characterization of noroviruses isolates was performed. In addition, the Lancet Pathology Laboratory detected other diarrhoeal pathogens such as bacteria, parasites fungi and viruses, and the results were used to determine the relative significance of rotaviruses and noroviruses as the causes of diarrhoeal disease in comparison to other potential enteric pathogens in the Tshwane region. Results: The Lancet Laboratory reported 216 (17.6%) rotavirus positives with VIKIA assay and the MRC-DPRU reported 534 (43.5%) rotavirus positives with DAKO assay, clearly indicating that the DAKO assay has superior sensitivity. The use of a diagnostic assay which is not sensitive such as VIKIA assay could lead to misdiagnosis, under-reporting and x mismanagement of patients. Rotavirus infection was higher in children below 12 months of age (58.2% vs 41.7% for all other age groups). There was no significant difference between black and white children infected with rotaviruses [(249 or 46.6% for black versus 279 or 52.3% for white children)]. However, only 5 (0.9%) of rotavirus positives were Indians most probably due to small sample size. Rotaviruses were mostly predominant during the months of May (66.0%), June (71.0%) and July (57.4%). The prevalence of norovirus was relatively small compared to rotavirus for the same period. Only 53 (7.9%) samples were norovirus positive by ELISA. Noroviruses were detected in 11.1% of Indian children, followed by 7.9% of black children and 7.8% in white children. There was no significant difference amongst black and white children infected with noroviruses. A total of 74.2% of the EIA rotavirus positives were confirmed by PAGE. Nine PAGE patterns were obtained, displaying six long and three short electrophoretypes. A total of 319 stool specimens were genotyped for both VP4 and VP7. Of these, 289 (90.6%) were typeable for the VP4 [P] gene and 298 (93.4%) were typeable for the VP7 [G] gene. The most predominant single P genotypes were P[8] at 179 (56.1%), P[9] at 31 (9.7%) and P[4] at 25 (7.8%). Among the genotyped samples, the most predominant single G genotypes were G3 at 54 (16.9%), G8 at 48 (15.1%), G9 at 31 (9.7%) and G2 at 27 (8.5%). Mixed P and G rotavirus genotypes were detected in 12.5% and 29.5% for VP4 and VP7 respectively. Dual, triple and quadra rotavirus infections were detected for both VP4 and VP7 genotyping. The most predominant G and P genotypes found in this study were G8P[8] (8.9%), G3P[8] (8.3%) and G9P[8] (6.0%). Phylogenetic analysis indicated that of the 8 samples sequenced for VP4, all were P8 genotypes. Of these, five (DPRU1570, DPRU1754, DPRU7596, DPRU7310, and DPRU7335) clustered with Hun9-like lineage and 3 (DPRU7581, DPRU2030 and DPRU7173) clustered with Wa-like lineage on the P[8] phylogenetic tree. The only VP7 sample that was successfully sequenced (DPRU7381) clustered with lineage II of G3 on the phylogenetic tree. Children infected with rotavirus (83.0%) were also found to be co-infected with other diarrhoeal pathogens and these included Candida spp (33.2%), E. coli (19.8%), Cryptosporidium spp (8.9%), Adenoviruses types 40 and 41 (8.2%) and Giardia lamblia (7.7%). Viruses were the most predominant enteric pathogens detected in the samples at 63.0%, followed by fungi at 19.0%, bacteria at 15.0% and parasites at 3.0%. xi Conclusion: Compared to other enteric pathogens, rotaviruses are the leading cause of diarrhoea in children attending private health care facilities in Tshwane region. It was interesting to note that the most predominant G and P genotypes were G8P[8] (8.9%), G3P[8] (8.3%) and G9P[8] (6.0%). The predominance of these strains clearly indicate that the rotavirus vaccination programme that started in August 2009 in South Africa will be effective in protecting children against major circulating strains. However, more studies should be done to monitor the epidemiological trends of all enteric pathogens in South Africa, especially that rotavirus is now a vaccine-preventable disease.

The noroviruses are important pathogen of epidemic and sporadic gastroenteritis in all age group and show great genetic diversity. The aim of the present study was to describe the prevalence and genetic diversity of noroviruses among children hospitalized with acute sporadic gastroenteritis in Kaohsiung, Taiwan. Fecal samples were collected from hospitalized pediatric patients with sporadic gastroenteritis below age of 18 years during a 2-year period (2007 to 2008). Norovirus RNA was detected by reverse transcription-polymerase chain reaction and comfirmed by sequence analysis. Two different sets of primers were used. Region C primers target shell domain at 5¡¦ end of capsid gene and region D primers target highly variable P2 subdomain at 3¡¦ end of capsid gene. Noroviruses were identified in 5 of 39 (12%) rotavirus-negative specimens using region C primers. Using region D primers only one among these 5 samples could yield PCR product, which showed concordant noroviral genotype. 3 (10%, n=30) specimens from children below age of 5 years tested positive. All these 5 patients had symptoms of vomiting and 3 had fever. All PCR products were sequenced and showed 2 strains of genogroup 1 (G 1.4) and 3 strains of genogroup2 (G 2.4). To our knowledge, this is the first report that demostrated G1.4 genotype norovirus from Taiwan. Norovirus accounted for 10% of sporadic non-bacterial gastroenteritis cases among hospitalized children below 5 years of age in Kaohsiung, Taiwan.

Der Replikation des NV-Genoms geht die kotranslationale Spaltung des vom ORF1 kodierten Polyproteins durch die viruseigene Protease, voraus. Erst in cis und in trans Prozessierungen an definierten Spaltmotiven innerhalb des Polyproteins ermöglichten die Freisetzung der strukturellen und nicht-strukturellen Proteine, die für den Aufbau des Replikationskomplexes und für die Initiation der Replikation essentiell sind. Die Regulation der Polyproteinprozessierung war allerdings bislang unbekannt. In der Charakterisierung der sequentiellen und differentiellen Prozessierung des Polyproteins bestand demnach die Hauptaufgabe dieser Arbeit. Dafür wurde zunächst ein NV-Volle-Länge-cDNA-Klon aus sechs sich überlappenden cDNA-Einzelfragmenten, unter Verwendung der overlap-extension PCR, hergestellt. Dieser Volle-Länge cDNA-Klon diente als Template für die Generierung von Vorläuferkonstrukten, die für die Charakterisierung der in cis und in trans Prozessierung des Polyproteins verwendet wurden. Die cis-Spaltung des Polyproteins konnte sowohl in E.coli als auch in humanen 293-T Zellen anhand eines Vorläuferproteins, das die komplette Sequenz der 3CLpro enthält, die 5`-seitig von der Spaltstelle zum VPg Protein und 3`-seitig vom N-Termins der 3DLpol flankiert ist, verifiziert werden. Infolge der kotranslationalen cis-Spaltung dieses Vorläuferproteins kam es zur Freisetzung der 3CLpro, die anschließend aufgereinigt wurde. Zusätzlich wurde das Fusionsprotein 3CLproµE1189A3DLpol aufgereinigt, bei dem das Spaltmotiv E/G zwischen der 3CLpro und der 3DLpol so mutiert wurde, dass die kotranslationale Spaltung des Fusionsproteins verhindert wurde. Anhand der 3CLpro sowie dessen Vorläufers 3CLproµE1189A3DLpol sollte die differentielle und sequentielle Spaltung des Polyproteins in trans charakterisiert werden. Dafür wurden synthetisch hergestellte Peptide, die die authentischen Spaltmotive innerhalb des Polyproteins aufwiesen, sowohl mit der 3CLpro als auch mit der 3CLproµE1189A3DLpol in einem trans-Spaltungsassay inkubiert und anschließend die Spaltung mit Hilfe der reversed-phase Chromatographie analysiert. Im Rahmen dieser Versuche konnte gezeigt werden, dass nur die Spaltmotive zwischen dem N-terminalem Protein p37 und der 2CNTPase sowie zwischen der 2CNTPase und dem Protein p20 in trans von der 3CLpro gespalten wurden. Massenspektrometrische Analysen wiesen nach, dass die Spaltungen jeweils zwischen den Aminosäureresten Q/G stattgefunden hatte. Zusätzlich zu den Peptiden p37/2CNTPase und 2CNTPase/p20 konnte das Peptid VPg/3CLpro durch das Fusionsprotein 3CLproµE1189A3DLpol an der Schnittstelle E/G prozessiert werden. Anhand von Berechnungen zur relativen Spalteffizienz von 3CLpro und 3CLproµE1189A3DLpol wurde nachgewiesen, dass 3CLpro eine signifikant höhere Spezifität zu den Spaltmotiven der Peptide p37/2CNTPase und 2CNTPase/p20 aufwies als das Fusionsprotein 3CLproµE1189A3DLpol. Die Spaltspezifität der 3CLpro war dabei an dem Spaltmotiv zwischen der 2CNTPase und dem Protein p20 signifikant höher als an dem Spaltmotiv zwischen dem N-terminalem Protein p37 und der 2CNTPase. In vitro Translationsstudien des NV-ORF1 im zellfreien System bestätigten, dass die kotranslationale in trans-Spaltung des Polyproteins nur zwischen dem Protein p37 und der 2CNTPase und der 2CNTPase und dem Protein p20 stattfand. Darüber hinaus konnte anhand dieses Versuches gezeigt werden, dass die initiale Prozessierung des Polyproteins auf der trans-Aktivität der 3CLpro beruht, was in der Freisetzung der Proteine p37, 2CNTPase und des Fusionsproteins VPg3CLpro[delta]3DLpol resultiert. Eine weitere Spaltung von VPg3CLpro[delta]3DLpol konnte im Rahmen dieses Versuches nicht gezeigt werden. Mit Hilfe des humanen Zellkultursystems sollte anschließend untersucht werden, ob zelluläre Faktoren in die Prozessierung des p20VPg Vorläuferproteins involviert sind. Dafür wurden Koexpressionsstudien des Vorläuferproteins p20VPg mit dem 3CLpro Vorläuferprotein [delta]VPg3CLpro[delta]3DLpol durchgeführt. Doch auch in dieser Expressionsstudie konnte eine Spaltung des Vorläuferproteins nicht beobachtet werden. Interessanterweise wurde allerdings die cis-Spaltung des Vorläuferproteins [delta]VPg3CLpro[delta]3DLpol durch die Koexpression mit [delta]VPg3CLpro[delta]3DLpol verhindert. Unter Verwendung des trans-Spaltungsassays konnte daraufhin in vitro verifiziert werden, dass p20VPg die Aktivität der 3CLpro am Peptid p37/2CNTPase signifikant reduzierte. Der initiale Schritt der NV-Replikation basiert auf der Aktivität der 3CLpro. Eine Inaktivierung dieses Enzyms würde demnach den Replikationszyklus verhindern. Somit stellt dieses Enzym ein geeignetes Target für anti-NV Medikamente dar. Aus diesem Grund wurden in dieser Arbeit mögliche 3CLpro Inhibitoren getestet. Anhand dieser Testreihen konnte gezeigt werden, dass die Substanz CMK eine mögliche Grundlage, für die Entwicklung von NV-3CLpro spezifischen Chlormethylketon-Peptidanaloga als Inhibitoren, darstellen könnte.

Le norovirus est le virus de la gastroentérite virale aiguë chez les humains et les animaux. Chaque année, il est responsable de la mort de près de 220 000 personnes et un coût de près de 65 milliards d’euros.C’est un petit virus à ARN simple brin de polarité positive. Sa capside icosaédrique est composée de 90 dimères d’une seule protéine structurale (VP1) à deux domaines : le domaine de spicule (P) exposé à l’environnement biologique, et le domaine shell (S) qui est le module d’assemblage de la capside. Un intermédiaire d'assemblage serait le pentamère de dimères, mais expérimentalement c'est un intermédiaire de 10-11 dimères qui a été rapporté.En utilisant des approches computationnelles (modélisation par homologie, recuit simulé, simulation de dynamique moléculaire avec des systèmes tout atomes ou en gros grains) nous cherchons à déterminer les bases moléculaires de l’assemblage de la capside des norovirus.Nos résultats sur la brique d’assemblage de la capside (dimère de VP1) indiquent la présence d’une asymétrie pouvant avoir un rôle dans les premières étapes d’autoassemblage, que nous confirmons expérimentalement par SAXS.Nos résultats sur de plus gros assemblages (pentamère et hexamère de dimère) révèlent également une rupture de symétrie dans le pentamère de dimère, laissant une position privilégiée pour l’insertion d’un dimère supplémentaire et favorisant une croissance anisotropique.Nous complétons et précisons ainsi le modèle d’assemblage initialement publié par notre équipe en 2013. / Noroviruses are the leading cause of acute viral gastroenteritis in humans and animas. Each year, they are responsible for 220 000 deaths and cost close to 65 billion euros.It’s a small single-stranded positive sense RNA virus. The capsid is composed of 90 dimers of a single structural protein (VP1) which consists of two main domains: the protruding domain (P) exposed to the biological environment, and the shell domain (S) which is the assembly module of the capsid .Using computational approaches such as homology modelling, simulated annealing, molecular dynamic simulations in all atoms and coarse grains systems, we are looking to determine the molecular basis of norovirus capsid assembly.Our results on the capsid assembly brick (dimer of VP1) indicate the presence of an asymmetry that can have a role in the first stages of self-assembly, which we confirm experimentally by SAXS.Our results on larger VP1 assemblages (pentamer and hexamer of dimers) also reveal a disruption of symmetry in the pentamer of dimer, leaving a preferred position for the insertion of an additional dimer and promoting anisotropic growth.We complete and specify the assembly model originally published by our team in 2013.

Norovirus (NoV) is a major cause of infectious human viral gastroenteritis. Detection is important to understanding the epidemiology of NoV and the viral dynamics of NoV infection which is poorly understood. In 2006, a marked increase in gastroenteritis outbreaks occurred worldwide. During this period, a total of 231 stool samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. A total of 186 isolates of NoV were detected and sequenced to determine the genotype and relatedness to known epidemic NoV GII.4 variants. Two GII.4 variants, 2006a and 2006b, were identified in 61.8% and 11.3%, in these cases, respectively. Thus, the increase in NoV gastroenteritis in 2006 was linked to the emergence of two novel co-circulating GII.4 variants, 2006a and 2006b. During an outbreak in an aged-care facility, stool samples were collected from the onset of illness to cessation of viral excretion. Here, viral shedding peaked in the acute stage of illness and continued for an average of 28.7 days. The viral decay rate was 0.76 per day. Prolonged asymptomatic shedding of NoV was detected in the elderly. A quality control for the assessment of molecular based viral assays for NoV and other RNA viruses is necessary to meet current testing requirements. Available controls only monitor the RNA and DNA amplification steps. An MS2 bacteriophage BioBallTM with 100 pfu was evaluated and applied as a multi-purpose phage control. It was assessed as a quality control, in comparison to MS2 phage stock, to validate MS2 phage assays. Furthermore, MS2 BioBallTM was used as a process control for the molecular detection of RNA viruses. It validated every performed step, determined if the assay worked and its sensitivity. Thus, MS2 BioBallTM offered uniformity, stability and reproducibility across molecular based viral detection systems. Overall, this thesis provided valuable insight into the molecular epidemiology of NoV in the southern hemisphere and nature of NoV infections in the elderly. The MS2 BioBallTM provides standardisation and quality control of viral RNA assays. Understanding the genetic diversity and viral dynamics of NoV will be crucial to developing effective intervention and treatment strategies, and ultimately lead to reduced viral gastroenteritis worldwide.

Norovirus (NoV) is a major cause of infectious human viral gastroenteritis. Detection is important to understanding the epidemiology of NoV and the viral dynamics of NoV infection which is poorly understood. In 2006, a marked increase in gastroenteritis outbreaks occurred worldwide. During this period, a total of 231 stool samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. A total of 186 isolates of NoV were detected and sequenced to determine the genotype and relatedness to known epidemic NoV GII.4 variants. Two GII.4 variants, 2006a and 2006b, were identified in 61.8% and 11.3%, in these cases, respectively. Thus, the increase in NoV gastroenteritis in 2006 was linked to the emergence of two novel co-circulating GII.4 variants, 2006a and 2006b. During an outbreak in an aged-care facility, stool samples were collected from the onset of illness to cessation of viral excretion. Here, viral shedding peaked in the acute stage of illness and continued for an average of 28.7 days. The viral decay rate was 0.76 per day. Prolonged asymptomatic shedding of NoV was detected in the elderly. A quality control for the assessment of molecular based viral assays for NoV and other RNA viruses is necessary to meet current testing requirements. Available controls only monitor the RNA and DNA amplification steps. An MS2 bacteriophage BioBallTM with 100 pfu was evaluated and applied as a multi-purpose phage control. It was assessed as a quality control, in comparison to MS2 phage stock, to validate MS2 phage assays. Furthermore, MS2 BioBallTM was used as a process control for the molecular detection of RNA viruses. It validated every performed step, determined if the assay worked and its sensitivity. Thus, MS2 BioBallTM offered uniformity, stability and reproducibility across molecular based viral detection systems. Overall, this thesis provided valuable insight into the molecular epidemiology of NoV in the southern hemisphere and nature of NoV infections in the elderly. The MS2 BioBallTM provides standardisation and quality control of viral RNA assays. Understanding the genetic diversity and viral dynamics of NoV will be crucial to developing effective intervention and treatment strategies, and ultimately lead to reduced viral gastroenteritis worldwide.

Made available in DSpace on 2015-10-21T12:19:29Z (GMT). No. of bitstreams: 2 ana_sa_ ioc_mest_2012.pdf: 3171036 bytes, checksum: cd95297b24f95941e437fe4f73f068f1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-05-21 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A gastronterite aguda (GA) é uma causa importante de morbidade e mortalidade entre crianças com menos de cinco anos no mundo. Segundo a Organização Mundial de Saúde, a GA e as infecções respiratórias agudas são os mais importantes agravos à saúde das crianças \2264 5 anos, responsáveis por 17% das 10,4 milhões de mortes a cada ano. As GA causadas por rotavírus da espécie A (RVA) são responsáveis por aproximadamente 390.000 mortes ao ano, 80% dessas nos países em desenvolvimento, principalmente na Ásia e na África. Os rotavírus são classificados em cinco espécies (A-E), sendo os da espécie A os principais agentes etiológicos da diarreia aguda em crianças menores de 5 anos. Pertencem à família Reoviridae, gênero Rotavirus. Baseando-se nas proteínas de superfície, VP4 e VP7, os RVA são classificados em genótipos P e G, respectivamente. Estudos de epidemiologia molecular demonstraram que, mundialmente, cinco genótipos G são prevalentes: G1-4 e 9; em associação com os genótipos P[8], P[4] ou P[6]. Os norovírus (NoV), gênero Norovirus da família Caliciviridae, são amplamente reconhecidos como os principais agentes causadores de surtos de GA não bacteriana e como o segundo vírus mais prevalente em infecções esporádicas. Neste estudo, foi analisada a presença de RVA e NoV em 200 espécimes clínicos coletados de maio de 2008 a abril de 2009, em Fortaleza, Ceará. Os resultados revelaram 12% e 17% de prevalência para RVA e NoV, respectivamente. Todas as amostras positivas para RVA pertencem ao genótipo G2P[4], sugerindo a predominância deste genótipo Diferentes estudos realizados em vários Estados brasileiros revelaram que o genótipo G2P[4] é o mais prevalente desde 2005. Entretanto, foi demonstrado que existem flutuações, tanto temporais quanto geográficas, das combinações G-P de RVA circulantes. As análises filogenéticas permitiram identificar 3 variantes do gene que codifica para a proteína VP7, 2 variantes do gene que codifica a proteína VP4, 3 variantes do gene que codifica para a proteína NSP4, demonstrando a segregação independente dos genes de RV-A analisados. Em 2009, uma nova variante foi identificada. Dos mecanismos de geração de diversidade em RVA, foi possível evidenciar a ocorrência de: i) mutações pontuais; ii) reassortment entre amostras humanas; iii) reassortment entre amostras humanas e amostras bovinas para o gene que codifica para a proteína NSP4. Dentre os NoV, foram detectados os seguintes genótipos GII.4 (59%), GII.12 (17%), GII.6 (9%), GII.3 (6%) e GII.? (9%). O genótipo GII.4 foi predominante, seguido de GII.12, corroborando o fenômeno de emergência deste, descrito mundialmente a partir de 2008. Os resultados aqui apresentados mostram que os RVA e os NoV são importantes causas de GA no Estado do Ceará e o acompanhamento contínuo da epidemiologia destes vírus, em diferentes regiões do Brasil, será essencial para determinar o impacto real destas infecções no país / Acute gastroenteritis (AGE) is an important cause of morbidity and mortality of children <5 years old worldwide. According to WHO, AGE and the acute respiratory infections are the most important health problems in 5 - year - old children, being responsible of 17% of 10.4 million of death e very year. AGE caused by Rotavirus species A (RVA) are responsible for approximately 390,000 deaths annually, 80% of those in developing countries, mainly in Asia and Africa. Rotav iruses are classified into five species (A - E ) and species A constitute the m ost important etiological agent of acute diarrhea in children with less than 5 years old. Based on surface proteins, VP4 and VP7, R VA are classified as genotype P and G, respectively. Molecular epidemiology studies have shown that five G genotypes are prev alent worldwide: G1, G2, G3, G4 and G9, in association with the genotypes P[8], P[4] or P[6]. Noroviruses (NoV), gender Norovirus from the Caliciviridae family, are widely recognized as the most important causative agents of non - bacterial AGE outbreaks and the second most prevalent viruses in sporadic infections. In this study, we screened the presence of RVA and NoV in 200 fecal samples originated from clinical specimens in Fortaleza, Ceará collected from May 2008 to April 2009. The results obtained reveal ed prevalence of 12% and 17% for RVA and NoV, respectively. All positive samples for RVA belong to P[4]G2 genotype, suggesting the predominance of this genotype. Studies in various Brazilian states showed that the genotype P[4]G2 is the most prevalent sinc e 2005 . However, it was shown that there are fluctuations, both temporal and geographic, on the circulating RVA genotypes. Phylogenetic analyzes have identified 3 variants of the VP7, 2 variants of the VP4 gene, 3 variants of the NSP4 gene, demonstrating the independent assortment of the RVA genes analyzed. In 2009, a new variant was identified. Between the mechanisms of diversity generation in RVA, it was possible to demonstrate the occurrence of: i ) mutations ii ) reassortment between human samples iii ) r eassortment between human and bovine samples, regarding NSP4 gene analysis. Among NoV, t he following genotypes were detected: GII.4 (59%), GII.12 (17%), GII.6 (9%), GII.3 (6%) and GII.? (9%). The genotype GII.4 was predominant, followed by GII.12, corrobor ating its global emergency, described since 2008. The results presented here show that RVA and NoV are important causes of AGE in Ceará State and its continuous monitoring in different regions of Brazil will be essential to determine their real impact in A GE infections in Brazil .

Made available in DSpace on 2016-04-15T12:56:11Z (GMT). No. of bitstreams: 2 juliana_andrade_ioc_mest_2013.pdf: 2734146 bytes, checksum: 7430ee31960a9248ee0ab399f4659502 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Os vírus do gênero Norovirus pertencem à família Caliciviridae e são divididos em cinco genogrupos (G) e 35 genótipos, sendo GI, GII e GIV capazes de infectar humanos e o genótipo GII.4 com suas variantes o de maior impacto epidemiológico. Os norovirus (NoV) são os principais agentes de surtos de gastroenterite aguda (GA) em todo o mundo, infectando indivíduos de todos os grupos etários. No Brasil, a importância destes vírus em casos esporádicos e de surtos de GA tem sido demonstrada pela prevalência e diversidade dos vírus circulantes em alguns estados, embora ainda não exista um sistema de vigilância rotineiro que investigue a prevalência desses vírus em todo o país. A ausência de dados sobre a caracterização molecular dos NoV no estado do Rio Grande do Sul (RS) chama atenção pelo número de surtos de GA relatados nos últimos anos. O objetivo desta dissertação foi realizar um estudo de epidemiologia e caraterização molecular de NoV provenientes de casos de GA decorrentes de surtos ocorridos no RS no período de 2004 a 2011, contribuindo para o estabelecimento da vigilância epidemiológica e molecular desses vírus. Com esta finalidade, foram analisadas 2265 amostras de fezes enviadas pelo Laboratório Central do Rio Grande do Sul (LACEN-RS) do estado ao Laboratório de Virologia Comparada e Ambiental (LVCA) provenientes de 741 surtos ocorridos neste período. Para detecção simultânea dos NoV GI e GII foi realizada a reação em cadeia da polimerase precedida de transcrição reversa (RT-PCR), tendo como alvo de amplificação a região da RNA polimerase viral RNA dependente (RdRp), conhecida como região B NoV foi detectado em 36,1% (817/2265) das amostras estudadas, sendo associado a 327 (44,1%) surtos, dos quais 60,5% (109/180) ocorridos no ano de 2006. A analise dos aspectos epidemiológicos revelou uma tendência no aumento da taxa de infecção de acordo com o aumento do grupo etário, assim como uma sazonalidade dos virus nos meses de primavera e inverno, com taxas de detecção de 41,9% e 43%, respectivamente. Em relação aos aspectos clinicos, vômito e dor abdominal foram as manifestações mais observadas nos indivíduos infectados. Para a caracterização molecular dos vírus, de cada um dos oito anos de estudo, foram selecionados aleatoriamente 142 vírus representativos das sete mesorregiões que compõem o estado. A caracterização em GI e GII foi realizada pelo seqüenciamento parcial dos nucleotideos pela amplificação genomica de duas regiões que codificam a proteína do capsideo viral (VP1), denominadas região C e D. Deste total, 110 vírus foram caracterizados como GII (77,4%) e duas como GI (1,4%), sendo o GII.4 (72,3%) o genótipo mais detectado, seguido pelo GII.6 (9,8%). Em menor número também foram caracterizados GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, GII.15, GII.17, GII.21, GI.1 e GI.3. Com o subsequente sequenciamento da região P2, também localizada na VP1, foram identificadas cinco variantes do GII.4 circulando no estado, nomeadas: 2003, 2004, 2006a, 2006b e 2010. A variante 2006b foi detectada em 47,8% (22/46) das amostras, seguida da variante 2010 com 41,3% (19/46) de detecção entre os GII.4. A grande diversidade de genótipos encontrados e alta prevalência do genótipo GII.4 e suas variantes corrobora a necessidade de se manter uma vigilância epidemiológica e molecular desses vírus no país, principalmente devido a associação do surgimento de novas variantes a surtos de de NoV de dimensões globalizadas / Viruses of the genus Norovirus belongs to the family Caliciviridae and are divided into five genogroups (G) and 35 genotypes, GI, GII and GIV able to infect humans and the genotype GII.4 and its variants has the highest epidemiological impact. The norovirus (NoV) are the main agents of outbreaks of acute gastroenteritis (AG) worldwide, infecting individuals of all age groups. In Brazil, the importance of these viruses in sporadic cases and outbreaks of AG has been demonstrated by the prevalence and diversity of circulating viruses in some states, although there is no system of routine surveillance to investigate the prevalence of these viruses across the country. The absence of data on the molecular characterization of NoV in the state of Rio Grande do Sul (RS) stands out by the number of outbreaks of AG reported in recent years. The aim of this dissertation was perform a study of the epidemiology and molecular characterization of NoV from cases of AG due to outbreaks in the RS state from 2004 to 2011, contributing to the establishment of epidemiological and molecular surveillance of these viruses. For this purpose, we analyzed 2265 stool samples sent by the Central Laboratory of RS (LACEN-RS) of the state to Virology Laboratory Comparative and Environmental (LVCA) from 741 outbreaks in this period. For simultaneous detection of GI and GII NoV was performed polymerase chain reaction reverse transcription (RT-PCR), aiming the target amplification the region of viral RNA dependent RNA polymerase (RdRp), known as Region B. NoV was detected in 36,1% (817/2265) of the samples studied, and linked to 327 (44,1%) outbreaks, of which 60,5% (109/180) occurred in 2006 The analysis of epidemiological revealed a tendency in the increased rate of infection in accordance with increasing in the age, as well as seasonality of virus in the months of spring and winter, with detection rates of 41,9% and 43%, respectively. Regarding clinical aspects, vomiting and abdominal pain were the most observed manifestations in infected individuals. For the molecular characterization of the viruses, each of the eight years of the study, were randomly selected 142 representative viruses of the seven mesoregions belonging to state. The characterization in GI and GII was performed by partial sequencing of genomic amplification of nucleotides by two regions that encodes viral capsid protein (VP1), termed region C and D. Of this total, 110 viruses were characterized as GII (77,4%) and two as GI (1,4%), being the GII.4 (72,3%) genotype mostly detected, followed by GII.6 (9,8%). In lower number were also characterized GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, GII.15, GII.17, GII.21, and GI.1 GI.3. With the subsequent sequencing of the P2 region, also located in VP1, we identified five variants of GII.4 circulating in the state, namely: 2003, 2004, 2006a, 2006b and 2010. The variant 2006b was detected in 47,8% (22/46) samples, then the variant 2010 with 41,3% (19/46) between the GII.4 detection. The great diversity of genotypes found and high prevalence of genotype GII.4 and its variants supports the need to maintain a molecular and epidemiological surveillance of these viruses in the country, mainly due to the association of the emergence of new variants of NoV with outbreaks of globalized dimensions

No description available.