Made available in DSpace on 2015-11-11T12:09:50Z (GMT). No. of bitstreams: 2 mariela_gomez_ioc_dout_2014.pdf: 8571554 bytes, checksum: 662bf4d26fe89925ff0e648a52a7d4e6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-06-10 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os Rotavírus da espécie A (RVA) são os principais agentes etiológicos causadores de gastroenterite aguda (GA) em crianças \22645 anos. Após a introdução, em março de 2006, da vacina monovalente G1P[8] (Rotarix® - RV1) no Programa Nacional de Imunizações (PNI) pelo Ministério da Saúde (MS) do Brasil, observou-se uma mudança na epidemiologia dos genótipos de RVA circulantes na população. Apesar desta variação dos genótipos circulantes, observou-se no Brasil uma redução de 22-28% de mortes e de 21-25% de hospitalizações em crianças \02C22 anos de idade, particularmente nas regiões Norte e Nordeste. Provavelmente a variação dos genótipos de RVA, tanto no tempo quando nas regiões geográficas, esteja relacionada a diversos fatores. O fato da vacina RV1 apresentar uma constelação genética (11 genes) Wa-like pode estar influenciando na eficácia da mesma frente aos RVA que apresentem constelações diferentes, como vírus DS-1-like e AU-1-like. Neste estudo foi analisada a diversidade genética de RVA de diversos genótipos detectados no Brasil antes e após a introdução da vacina RV1 pelo PNI, tanto em crianças vacinadas quanto não vacinadas, com o intuito de: i) identificar e caracterizar variantes de RVA emergentes e reemergentes; ii) contribuir para um melhor entendimento da dinâmica evolutiva deste vírus; iii) identificar mutações pontuais e genes que foram introduzidos mediante reestruturação genética, que eventualmente posam estar vinculados ao fato de RVA causarem GA inclusive em crianças vacinadas Deve-se ressaltar que este estudo engloba amostras de diferentes regiões geográficas do Brasil, visto que dados epidemiológicos sugerem diversidades genotípicas regionais. Todas as cepas G2P[4] para as quais foram analisados os 11 segmentos gênicos apresentaram uma constelação gênica DS-1-like: I2-R2-C2-M2-A2-N2-T2-E2-H2, apesar de que diversas variantes virais circularam no período estudado. Não foram observadas diferenças nos sítios antigênicos das proteínas VP8* e VP7 entre crianças vacinadas e não vacinadas. As cepas G1P[6] analisadas apresentaram uma constelação gênica Wa-like: I1-R1-C1-M1-A1-N1-T1-E1-H1. As cepas G12P[9] apresentaram uma constelação gênica AU-1-like: I3-R3-C3-M3-A3-N3-T3-E3-H6, enquanto as cepas G12P[8] revelaram uma constelação gênica Wa-like: I1-R1-C1-M1-A1-N1-T1-E1-H1. Além disso, a análise do viéis no uso de códons de RVA genótipo G2P[4] revelou que existe uma correlação entre o viéis no uso de códons e a composição de bases, o que sugere que a pressão mutacional é um fator importante na determinação do viéis no uso de códons em RVA humanos / Specie A rotaviruses (RVA) are the main etiological agents of acute gastroenteritis (AG) in children ≤ 5 years old. After the introduction of monovalent vaccine G1P[8] (Rotarix® - RV1) in the National Immunization Program (NIP) in March 2006, a change in the epidemiology of RVA circulating genotypes in the population was observed in Brazil. Despite this variation, a reduction of 22-28% of deaths and 21-25% of hospital admissions in children ˂ 2 years of age, particularly in the North and Northeast regions, occurred after RV1 introduction. RVA genotypes variation along time and in the different geographical regions might be related to several factors. Since the RV1 vaccine has a Wa-like genetic constellation the effectiveness of this vaccine against RVA showing different genetic constellations, such as DS-1-like and AU-1-like viruses, it might be influenced. In this study we analyzed the genetic diversity of different RVA genotypes detected in Brazil, before and after the introduction of the RV1 vaccine by the NIP, in vaccinated and unvaccinated children in order to: i) identify and characterize RVA emerging and reemerging variants; ii) contribute to a better understanding of the evolutionary dynamics of this virus; iii) identify point mutations and genes that have been introduced by reassortment events and that could eventually be linked to the fact that some RVA are capable to cause AG in vaccinated children. It should be emphasized that this study includes different geographical regions of Brazil, as epidemiological data suggests regional genotypic diversity. All G2P[4] strains analyzed revealed a DS-1-like genome constellation: I2-R2-C2-M2-A2-N2-T2-E2-H2. However, several viral variants circulated during the study period. No differences were observed inside antigenic sites of VP7 and VP8* proteins between vaccinated and unvaccinated children. The G1P[6] strains analyzed showed a Wa-like genome constellation: I1-R1-C1-M1-A1-N1-T1-E1-H1. The G12P[9] strains showed a AU-1-like genome constellation: I3-R3-C3-A3-M3-N3-T3-E3-H6, while G12P[8] strains showed a Wa-like genome constellation: I1-R1-C1-M1-A1-N1-T1-E1-H1. Furthermore, analysis of codon usage bias of RVA G2P[4] genotype revealed that a correlation between the codon usage bias and base composition exists, suggesting that the mutational pressure is the main factor in determining the codon usage bias in human RVA.

Diversas doenças virais emergentes e re-emergentes têm sido descritas em morcegos. As alterações ambientais provocadas por seres humanos associada a adaptação dos morcegos às áreas urbanas aumentam as chances da transmissão dessas doenças para humanos e animais domésticos. O estudo das coronaviroses associadas a esse hospedeiro tem evidenciado que os morcegos atuam como reservatório dessa doença, enquanto o estudo das rotaviroses ainda foi pouco explorado. Este estudo tem como objetivo verificar a ocorrência de coronavírus e rotavírus em diversas espécies de morcegos do Estado de São Paulo, Brasil, e realizar inferências filogenéticas a partir dos genes RdRp e S dos coronavírus, assim como de genes de proteínas estruturais e não estruturais dos rotavírus. Para tanto, foi utilizada a RT-PCR seguida de sequenciamento de DNA. Para análise filogenética e de diversidade molecular foi utilizado critério de otimização de distância e cálculo das identidades de nucleotídeos e aminoácidos entre as sequências obtidas e sequências recuperadas do GenBank. A ocorrência de coronavírus foi de 2,95% (9/305) e a de rotavírus de 9,18% (28/305). De acordo com a análise filogenética do gene da RdRp oito amostras foram classificadas como alphacoronavirus. A análise do gene S dos CoV mostrou que as amostras deste estudo formaram uma linhagem única, segregadas das demais amostras de alphacoronavírus. Em relação aos rotavírus, foi possível a identificação de um genótipo G3-P[3]-IX-RX-CX-MX-AX-NX-T3-E3-H6, similar a encontrada em morcegos, equinos e humanos. Além disso, outra amostra foi classificada como G20, similar ao genótipo encontrado em humano, sendo que os genótipos encontrados para os genes VP4, NSP3 e NSP5 desse vírus podem ser classificados como novos genótipos. Os resultados obtidos mostram que esses animais podem carrear agentes infecciosos de importância na saúde pública, sendo que mais estudos são necessários para o esclarecimento do papel dos morcegos como reservatório e fonte de infecção destas zoonoses virais. / Several viral emerging and re-emerging diseases have been described in bats. Environmental changes caused by humans associated with the adaptation of bats to urban areas increase the chance of transmission of these infectious diseases to humans and domestic animals. Coronaviruses studies associated with bats have shown that these hosts act as reservoirs for these viruses, while rotaviruses has been poorly studied in these hosts. This study aimed to determine the occurrence of Rotavirus and Coronavirus in several species of bats from São Paulo State, Brazil, and to perform phylogenetic inferences from Coronavirus RdRp and S genes, as well as from Rotavirus structural and non-structural proteins genes. To this end, RT-PCR followed by DNA sequencing was used. Optimization criterion of distance and identities calculation of the nucleotides and amino acids among the obtained sequences and sequences retrieved from the GenBank were used for phylogenetic and molecular diversity analysis. The occurrence of Coronavirus was 2.95% (9/305) and of Rotavirus was 9.18% (28/305). According to phylogenetic analysis of the RdRp gene, eight strains were classified as Alphacoronavirus. The analysis of the CoV S gene showed that the starins of this study formed a single lineage, segregated from other alphacoronaviruses lineages. Regarding Rotavirus, it was possible to identify the genotype G3-P [3] -IX-RX-CX-MX-AX-NX-T3-E3-H6, similar to that reported in bats, horses and humans. In addition, another strain was classified as G20, similar to the genotype described in humans, while the genotypes found for VP4, NSP3 and NSP5 genes may be classified as new genotypes. These results show that bats may carry infectious agents of public health interest, but further studies are necessary to clarify the role of these animals as reservoirs and infectious sources of these viral zoonoses.

No description available.

Rotaviruses, members of the family Reoviridae, are the major etiologic agents of severe, acute dehydrating diarrhea in the young of many mammalian species, including humans, calves and foals. Recent estimates indicate an annual death toll of approximately 600,000 infants due to rotavirus, besides inflicting staggering economic burden worldwide. Most of these deaths occur in the developing countries and India is estimated to account for about a quarter of these deaths. Extensive molecular epidemiology studies carried out by our laboratory have revealed many interesting aspects about rotavirus diversity in this country. Molecular epidemiology of rotaviruses causing severe diarrhea in foals in two organized farms in northern India was carried out. These foal rotaviruses exhibited 5 different electropherotypes (E), E1-E5. Strains belonging to E1, E2 and E5 exhibited G10, P6[1]; G3 and G1 type specificities. Though the E1 strains possessed genes encoding G10 and P6[1] type outer capsid proteins, unlike the G10, P8[11] type strain I321, they exhibited high reactivity with the G6-specific MAb suggesting that the uncommon combination altered the specificity of the conformation-dependent antigenic epitopes on the surface proteins. Strains belonging to electropherotypes E3 and E4 were untypeable. Sequence analysis of the VP7 gene from E4 strains (Erv92 and Erv99), revealed that they represent a new VP7 genotype, G16. Nonstructural protein 4 (NSP4) of rotavirus is a multidomainal, multifunctional protein and is the first viral enterotoxin identified. We have recently reported that the diarrhea-inducing and double-layered particle (DLP)–binding properties of NSP4 are dependent on a structurally and functionally overlapping conformational domain that is conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail (Jagannath et al., J. Virol, pp 412-425, 2006). Further, a stretch of 40 amino acids (aa) from the C-terminus is predicted to be unstructured and highly susceptible to trypsin cleavage. We examined the role of this unstructured C-terminus of Hg18 NSP4 and SA11 NSP4 on the biological properties of NSP4 using a series of deletion and substitution mutants of the conserved proline and tyrosine residues in this region. Gel filtration, CD spectroscopy and Thioflavin T binding studies showed that these mutants have altered secondary structural contents and either failed to multimerize efficiently or multimerized with altered conformation. The C-terminal ten residues appear to play a regulatory role on multimerization. Proline 168, tyrosine 166 and methionine 175 appear to be critical determinants of DLP binding activity whereas, proline 165 and tyrosine 85 and 131 appears to determine the affinity of binding to DLP in the context of NSP4 ∆N72. Deletion and substitution mutants exhibited severely reduced diarrhea inducing ability and DLP binding property. Of great biological significance is the drastic decrease in the diarrhea inducing ability of the N- and C- terminal deletion mutant ∆N94 ∆C29 that exhibited about 11,000-fold increase in DD50 than the wild type (WT) ∆N72. These studies revealed that the predicted unstructured C-terminus is an important determinant of biological properties of NSP4. Extensive efforts to crystallize the complete cytoplasmic tail (CT) of NSP4 were unsuccessful and to date, the structure of only a synthetic peptide corresponding to aa 95-135 has been reported. Our recent studies indicate that the interspecies variable regions from aa 135-141 as well as the extreme C-terminus are critical determinants of virus virulence and diarrhea-inducing ability of the protein. Here, we examined the crystallization properties of several deletion mutants and report the structure of a mutant recombinant NSP4 from symptomatic (SA11) and asymptomatic (I321) strains that lacked the N-terminal 94 and C-terminal 29 aa (NSP4: 95-146) at 1.67 Å and 2.7Å, respectively. In spite of the high-resolution data, electron density for the stretch of 9 residues from the C-terminus could not be seen suggesting its highly flexible nature. The crystal packing showed a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization under the experimental conditions. The present structure revealed significant differences from that of the synthetic peptide in the conformation of amino acids at the end of the helix as well as crystal packing owing to the additional space required to accommodate the unstructured virulence-determining region. Conformational differences in this critical region effected by the presence or absence of proline or glycine at specific positions in the unstructured C-terminus, could form the basis for the wide range of variation seen in the diarrhea-inducing ability of NSP4 from different strains in newborn mouse pups. Although symptomatic and asymptomatic strains do not generally differ in the presence or absence of the conserved prolines or glycines, they contain a few additional changes that could alter the unique conformation required for optimal biological activity. In conclusion, we demonstrate that the predicted unstructured C-terminal region is indeed highly flexible and is an important determinant of biological functions of the NSP4, mutations in which probably correlates with the virulence properties of the virus.

Background: Rotavirus (RV) is the leading agent of acute gastroenteritis in children under five years old, especially in children of 6~24 months. RV can lead to serious complications or even death. RV can cause a heavy burden of disease particularly in developing countries such as China. To prevent or reduce rotavirus diarrhea (RVD) and RV infection related diseases, damages or complications in children is an important task of public health. RV can also cause extraintestinal damage and complications. The finding of elevated CK-MB has been reported in a couple of Chinese literatures, but the epidemiology and the potential implication of this phenomenon remain poorly understood. Objective: 1. To study the prevalence of elevated serum CK-MB level among children hospitalized with rotavirus gastroenteritis in Guangzhou. 2. To examine factors associated with an elevated CK-MB level 3. To examine the clinical implication of elevated serum CK-MB level with different clinical symptomatology, severity, and disease outcome of rotavirus gastroenteritis in children. Methods: Aretrospective analysis of hospital records had carried out in Guangzhou Women and Children’s Medical Centre (GZWCMC), China. Hospital records during the period from 1 January 2011 to 31 December 2011 of inpatients from GZWCMC were screened and all inpatients with a diagnosis of rotavirus gastroenteritis in GZWCMC were recruited. Result and conclusion: Our study included418 cases of rotavirus infection hospitalized in GZWCMC in Guangdong in 2011, we found elevation of CK-MB a common phenomenon among patients of RV gastroenteritis, and identified a number of risk factors for elevated CK-MB level. These included patients of aged between 6-24 months, cases occurring in autumn or winter, patients coming from low income families, had never been breast-fed, or having dehydration, vomiting, malnutrition, fever, or having an abnormal blood gas and electrolyte. The elevated CK-MB level was positively associated with a higher occurrence of complications, a longer duration of hospitalization, and higher hospital cost per day. / published_or_final_version / Public Health / Master / Master of Public Health

Thesis (M.P.H.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 42-45).

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 2015-11-04T13:39:09Z (GMT). No. of bitstreams: 2 marcelle_silva_ioc_dout_2014.pdf: 10537387 bytes, checksum: 2d26a95e51c9f156abba0ee948b14523 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-10-29 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os rotavírus da espécie A (RVA) são os principais agentes etiológicos causadores de gastroenterite aguda (GA) em crianças \2264 5 anos e, anualmente, são responsáveis por aproximadamente 196.000 casos de óbito infantil em todo o mundo. Diferentes mecanismos genéticos (mutações pontuais, rearranjos genéticos, reestruturação de segmentos genômicos (reassortment) e recombinação genética) estão envolvidos na geração de variabilidade genética destes vírus. As combinações genotípicas mais encontradas mundialmente são: G1P[8], G2P[4], G3P[8], G4P[8] e G9P[8], entretanto, os países em desenvolvimento apresentam uma maior variabilidade genotípica entre as cepas circulantes. No intuito de se compreender a diversidade dos RVA, em 2011 foi proposto um novo sistema de classificação para os RVA baseado no sequenciamento nucleotídico dos onze segmentos gênicos destes vírus. No presente estudo foram realizadas análises filogenéticas dos onze genes de 28 cepas brasileiras de RVA de genótipo G5P[8] coletadas entre 1986 e 2005, 90 cepas de RVA de genótipo G1P[8] em diferentes regiões brasileiras entre 1986 e 2013, além do perfil de flutuação do genótipo P[8] no Brasil durante as últimas 3 décadas Os resultados demonstraram que as cepas brasileiras de genótipo G5P[8] e G1P[8] possuem uma constelação gênica do genótipo Wa-like (I1-R1-C1-M1-A1-N1-T1-E1-H1), com exceção de duas cepas G1P[8], que apresentaram o genótipo T3 (genótipo AU-1-like), comumente detectado em felinos. Foi proposta a classificação do genótipo G5 em 3 linhagens diferentes (I, II e III), de modo que a linhagem I circulou no Brasil entre 1986 e 2005 e a linhagem que está circulando atualmente entre países dos continentes Africano e Asiático pertencem à linhagem III. Os resultados permitiram demonstrar que a prevalência do genótipo G1P[8] variou anualmente no Brasil e que o genótipo G1 circulou em associação com as linhagens P[8]-1, P[8]-2 e P[8]-3 no país. A vacina RV1 apresenta especificidade P[8]-1, enquanto as cepas que estão circulando atualmente são P[8]-3. A análise do perfil de P[8] no Brasil demonstrou que no país circularam as linhagens P[8]-1, P[8]-2 e P[8]-3 e a variabilidade verificada para a linhagem P[8]-3 permitiu a sua classificação em 6 sublinhagens (P[8]-3.1 \2013 P[8]-3.6). Portanto, a melhoria dos programas de vigilância de RVA através de estudos que incluam a análise dos 11 segmentos gênicos das cepas circulantes no país contribuirá para entender melhor alguns pontos ainda não esclarecidos a respeito da biologia dos RVA, como de que maneira a introdução de um esquema de vacinação em massa pode influenciar na circulação de RVA em humanos e animais e a real frequência de detecção de eventos de reassortment entre cepas de espécies diferentes ou entre cepas selvagem e vacinal na população / Specie A rotaviruses (RVA) are the main etiological agent of acute gastroenteritis (AG) in children ≤ 5 years old and, annually, are responsible for about death of 196.000 children worldwide. Different genetic mechanisms (pontual mutation, genetic rearrangement, reassortment and genetic combination) are involved in the generation of variability of this viruses. The most detected combinations worldwide are: G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8], however developing countries have strains with a greater genotypic variability. In order to unsderstand the RVA diversity, in 2011, was proposed a new classification system for RVA based on the nucleotide sequencing of the 11 gene segments. In the present study were conducted Phylogenetic analyses of the 11 gene segments from 28 brazilian RVA strains genotype G5P[8] collected between 1986 and 2005, 90 RVA strains genotype G1P[8] collected between 1986 and 2013 in different regions of the country, besides the analysis of the fluctuation profile from genotype P[8] during the last 3 decades in Brazil. The results showed that brazilian RVA strains genotypes G5P[8] and G1P[8] have a genectic constellation characteristic of the genotype Wa-like (I1-R1-C1-M1-A1-N1-T1-E1-H1), except for 2 G1P[8] strains, which showed genotype T3 (AU-1-Like), generally detected in felines. Was proposed the classification of genotype G5 in 3 different lineages (I, II and III), lineage I circulated in Brazil between 1986 and 2005, while the lineage actually circulating in African and Asiatic continents is lineage III. The results demonstrated that the G1P[8] prevalence in Brazil changed annually and genotype G1 circulated in Brazil in association to P[8]-1, P[8]-2 and P[8]-3 lineages. RV1 vaccine shows P[8]-1 specificity, whilst the actually circulating strains have P[8]-3 specificity. The analysis of the P[8] genotype profile demonstrated that P[8]-1, P[8]-2 and P[8]-3 lineages circulated in Brazil and the P[8]-3 variability enabled the classification into 6 sublineages (P[8]-3.1 – P[8]-3.6). Therefore, the enhancement of surveillance RVA programs through studies that include analysis of the 11 gene segments of strains circulating in the country contribute to better understand some points that remain unclear about the RVA biology, such as how the introduction of a mass vaccination scheme can influence the circulation of RVA in humans and animals and the actual frequency of reassortment events between strains of different species or between wild and vaccine strains in the population.

Made available in DSpace on 2016-04-15T13:03:38Z (GMT). No. of bitstreams: 2 gentil_vasconcelos_ioc_dout_2015.pdf: 3238898 bytes, checksum: 4b0a8f2422705d1ca8dfa7ae9a2ca1da (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A produção de anticorpos em aves imunizadas seguida da extração desses anticorpos da gema dos ovos (IgY), tem atraído o interesse da comunidade científica, como pode ser demonstrado pelo aumento significativo da literatura sobre a IgY. Esta abordagem, que é apropriada à produção em larga escala, oferece inúmeras vantagens, tais como, baixo custo e alta eficiência da técnica, em vista do extraordinário rendimento de IgY em somente uma ave (20-40 g IgY por ano), e é mais adequada ao conceito bioético quando trata-se da manutenção e do manejo das aves. Destaca-se que a tecnologia da IgY oferece novas possibilidades de aplicação em imunoterapia e métodos de diagnóstico, tanto para aplicação humana quanto veterinária, incluindo estratégias de tratamento de doenças intestinais graves em crianças, particularmente em países pobres. Neste presente estudo, objetivou-se avaliar a eficiência terapêutica da IgY utilizando macacos cynomolgus (Macaca fascicularis) jovens desafiados com o rotavírus do grupo A (RVA) humano, a maior causa de morbidade e mortalidade de crianças em todo o mundo, especialmente em países em desenvolvimento. Para esta proposta, anticorpos IgY específicos contra o RVA foram produzidos em aves, purificados por polietileno glicol, caracterizados por eletroforese em gel de poliacrilamida, western blotting e um teste de neutralização em cultura de células (MA-104). Este experimento preliminar rendeu uma suspensão altamente concentrada de IgY específica anti-rotavírus (IgY anti-RVA) (média de 37 mg/mL) O macaco cynomolgus foi estabelecido como modelo de infecção experimental após uma única administração de suspensão de rotavírus humano (3,1x106 FFU/mL) por sonda gástrica. Os animais foram acompanhados durante onze dias, sendo observadas as manifestações clínicas, cargas virais sérica e fecal, hematologia e dosagem de eletrólitos séricos. O principal sinal clínico (observado em dois dos sete animais inoculados) foi diarreia associada com diminuição dos níveis séricos de potássio durante três dias, seguido de recuperação. O RNA viral foi detectado nas fezes e no soro dos animais infectados, além de partículas infecciosas encontradas nas fezes, sugerindo replicação viral. Na imunoterapia experimental, os macacos foram inoculados com RVA humano (3,1x107 FFU/mL), desafiados com a suspensão de IgY anti-RVA obtida previamente, e foram monitorados durante cinco dias pelos parâmetros observados no experimento anterior. A eficiência terapêutica da imunoterapia com IgY foi confirmada pela ausência de episódios de diarreia, que é reconhecida como \201Cpadrão ouro\201D para eficácia clínica, apesar do RNA viral ter sido detectado nas fezes de 11 de 12 animais inoculados com o RVA. A duração da detecção do RNA foi reduzida em dois dos três grupos de animais tratados com IgY, quando comparado ao grupo controle positivo. Em um animal que foi tratado com IgY pelas vias oral e intravenosa, não foi detectado genoma viral nas fezes. Como conclusão, a aplicação de anticorpos IgY anti-RVA específicos produzidos em aves, apresenta eficácia no tratamento de gastroenterite aguda causada pelo rotavírus do grupo A humano Nossos resultados também confirmam que macacos cynomolgus podem ser considerados hospedeiros suscetíveis à infecção com RVA humano, e apontam para a necessidade de controle sanitário da rotavirose humana em colônias de criação de macacos cynomolgus. Esses resultados preliminares sugerem um papel promissor da imunoterapia passiva utilizando IgY anti-RVA em infecção experimental com o rotavírus do grupo A humano. No entanto, um enfoque direto na patogênese da infecção no trato entérico fornecerá informações adicionais para confirmar a eficácia do tratamento com a IgY / The production of antibodies in chickens and the extraction of specific antibody suspensions from egg yolk (IgY) are increasingly attracting the interest of the scientific community, as demonstrated by the significant growth of the IgY literature. This approach, which is suitable to a large - scal e production, offers several advantages such as the low cost and high efficiency of the technique, in view of the extraordinary yield of IgY by a one hen (20 g – 40 g IgY per year), and its suitability to a more bioethical manner for hen keeping. Of note, the IgY - technology offers new possibilities for application in human and veterinary diagnostics and therapeutics, including strategies for the treatment of severe intestinal diseases in children, particularly in poor countries. In this study, we aimed to e valuate the therapeutic efficacy of the IgY by using young cynomolgus monkeys ( Macaca fascicularis ) challenged with human rotavirus group A (RVA), a major cause of morbidity and mortality in children worldwide, especially in developing countries. For this purpose, specific IgY antibodies against RVA were produced in hens, purified by polyethylene glycol, characterized by polyacrylamide gel electrophoresis, western blotting and a neutralization assay in a cell culture system (MA - 104). This preliminary experi ment has yielded a high concentrated suspension of anti - rotavirus specific IgY (anti - RVA IgY) (average 37 mg/ml). The cynomolgus experimental infection model was established after a single administration of a human rotavirus suspension (3.1x10 6 FFU/ml) by oral gavage. The confined animals were followed during a period of eleven days, observed for clinical signs, measurement of serum and faecal viral load, and evaluation of hematology and serum electrolytes. The main clinical sign (observed in two of the sev en inoculated monkeys) was diarrhea associated with a decrease in serum potassium during three days, followed by recovery. Viral RNA was detected in both serum and faeces of the infected animals, thus suggesting viral replication. In cynomolgus experimenta l immunotherapy, the monkeys were inoculated with human RVA (3.1x10 7 FFU/ml), challenged with the anti - RVA IgY suspension previously obtained, and monitored during five days by using the same clinical and biochemical parameters, as previously established. The therapeutic efficacy of the immunotherapy with IgY was confirmed by the absence of episodes of diarrhea, which is recognized as the "gold standard" for clinical efficacy, although viral RNA had been detected in faeces of all but one of the inoculated m onkeys. The duration of RNA detection was shortened in two of the three groups of animals treated with IgY, when compared to the positive control. One animal, which was orally and intravenously treated with the anti - RVA IgY, had no RNA detected in faeces. In conclusion, the application of specific anti - RVA IgY antibodies, produced in hens, presents efficacy in the treatment of acute gastroenteritis caused by human rotavirus group A. Our results also confirm that cynomolgus monkeys can be considered suscepti ble hosts to infection with human RVA, and pointed to the necessity of sanitary control of human rotavirus disease in the breeding colonies of cynomolgus monkeys. Our preliminary results suggest the promising role of passive immunotherapy using anti - RVA Ig Y in experimental infection with human rotavirus group A. However, a direct approach to the pathogenesis of enteric tract infection will provide additional data to confirm the effectiveness of the IgY treatment.

Enteric microbiota influences enteric viral infections, and host response to these pathogens and vaccines. Using gnotobiotic (Gn) pigs transplanted with human gut microbiota (HGM), we studied the effects of HGM on the immune response to oral rotavirus vaccination and rotaviral disease. We also used HGM transplanted Gn pigs to determine the effects of HGM on human norovirus infection. Despite commercially available vaccines, human rotavirus is a leading acute gastroenteritis in children, especially those in developing countries. Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in all age groups worldwide, and no vaccines are commercially available. Further understanding of how enteric microbiota influences these viral diseases may identify therapeutic targets. In our rotavirus study, pigs were colonized with HGM from an infant with low fecal concentrations of enteropathy biomarkers and responded well to their first dose of oral rotavirus vaccine (healthy human gut microbiota "HHGM"); or pigs were colonized with HGM from an infant with high fecal concentrations of enteropathy biomarkers and a poor response to the first dose of oral rotavirus vaccine (unhealthy human gut microbiota "UHGM"). HHGM colonized pigs had stronger cell-mediated and mucosal immune response to oral rotavirus vaccine compared to UHGM pigs based on the number of rotavirus-specific IFN-γ producing T cells in the ileum, spleen, and blood, and trends towards higher rotavirus specific antibody titers in intestinal contents, respectively. Significant correlations between multiple Operational Taxonomic Units (OTUs) of bacteria and frequencies of IFN-γ producing T cells at the time of human rotavirus challenge existed, suggesting that certain members of the microbiota influenced the immune response to the vaccine. After the vaccinated pigs were challenged with human rotavirus, HHGM pigs had less severe and shorter duration of viral shedding and diarrhea compared to UHGM pigs, suggesting that HHGM facilitated development of stronger protective immunity. These results demonstrated that composition of the enteric microbiota influenced host immune response to oral vaccination. In the norovirus study, Gn pigs were colonized with HHGM to determine the effects of microbiota on HuNoV infection. Colonized pigs shed more virus for a longer duration than non-colonized pigs, and also had higher viral titers in the duodenum and distal ileum. Diarrhea was more severe 4-10 days post-infection and lasted longer in colonized compared to non-colonized pigs. Twenty-seven genes related to the immune system were highly upregulated in HuNoV infected, colonized pigs compared to non-colonized controls. These result showed that HHGM influenced infectivity of HuNoV in the Gn pig model and altered host gene expression related to the immune system. These studies showed that HHGM can improve the host immune response and efficacy of rotavirus vaccine, but it can also enhance infection and clinical disease in HuNoV infected Gn pigs. Depending on the virus, gut microbiota may be beneficial or detrimental to the host. Those developing future treatments aimed at altering microbiota to prevent or ameliorate one viral pathogen need to consider the potential for enhancing a different pathogen. These studies demonstrated the usefulness of HGM transplanted Gn pigs for evaluation of microbiota influence on infection and immunity of enteric viral pathogens. / Ph. D. / Gut microbiota influences intestinal viral infections, and host response to these pathogens and vaccines. Using gnotobiotic (Gn) pigs transplanted with human gut microbiota (HGM), we studied the effects of HGM on the immune response to oral rotavirus vaccination and rotaviral disease. We also used HGM transplanted Gn pigs to determine the effect of HGM on human norovirus infection. Despite commercially available vaccines, human rotavirus is a leading acute gastroenteritis in children, especially those in developing countries. Human norovirus (HuNoV) is a leading cause of vomiting and diarrhea in all age groups worldwide, and no vaccines are commercially available. Further understanding of how gut microbiota influences these viral diseases may identify therapeutic targets. In our rotavirus study, pigs were colonized with HGM from an infant without evidence of intestinal disease based on fecal analysis, and who responded well to the first dose of oral rotavirus vaccine (healthy human gut microbiota “HHGM”); or pigs were colonized with HGM from an infant with evidence of potential intestinal dysfunction and a poor response to the first dose of oral rotavirus vaccine (unhealthy human gut microbiota “UHGM”). HHGM colonized pigs had a stronger immune response to the oral rotavirus vaccine compared to UHGM pigs. Significant correlations between multiple Operational Taxonomic Units (OTUs) of bacteria and frequencies of rotavirus-specific immune cells at the time of human rotavirus challenge existed, suggesting that certain members of the microbiota influenced the immune response to the vaccine. After the vaccinated pigs were challenged with human rotavirus, HHGM pigs had less severe and shorter duration of viral shedding and diarrhea compared to UHGM pigs, suggesting that HHGM enhanced vaccine efficacy. These results demonstrated that composition of the gut microbiota influenced host immune response to oral vaccination. In the norovirus study, GN pigs were colonized with HHGM to determine the effects of microbiota on HuNoV infection. Colonized pigs shed more virus for a longer duration than non-colonized pigs, and also had higher viral titers in sections of small intestine. Diarrhea was more severe 4-10 days after infection and lasted longer in colonized compared to non-colonized pigs. Twenty-seven genes related to the immune system were highly upregulated in HuNoV infected, colonized pigs compared to controls. These result showed that HHGM influenced infectivity of HuNoV in the Gn pig model and altered host gene expression related to the immune system. These studies showed how HHGM improved the host immune response and efficacy of rotavirus vaccine, but conversely enhanced infection and clinical disease in HuNoV infected pigs. Depending on the virus, gut microbiota may be beneficial or detrimental to the host. Those developing future treatments aimed at altering microbiota to prevent or ameliorate one viral pathogen need to consider the potential for enhancing a different pathogen. These studies showed the usefulness of HGM transplanted Gn pigs for evaluation of microbiota influence on infection and immunity of intestinal viruses.