MSc (Microbiology) / Department of Microbiology / BACKGROUND: Human norovirus (NoV) is the etiological agent associated with acute gastroenteritis (AGE) in both children and adults worldwide. Children of <5 years of age, the elderly and individuals suffering from chronic diseases are potentially at high risk of NoV-associated illness. High morbidity and mortality rate associated with NoV have been reported worldwide. In children under the age of 5 years about 1.8 million death cases have been reported in developing countries alone. Despite the fact that the virus is affecting people of all age groups, there is lack of data to elucidate the importance and the role of NoV in children of the age above 5 years and adults. OBJECTIVE: To characterize human norovirus in patients with diarrhoea in rural communities of Vhembe district, Limpopo province. MATERIALS AND METHODS : From August 2017 to October 2018, outpatient between 5 and 68 years of age from rural communities of Vhembe district, Limpopo province were recruited for this study. A total of n=80 stool samples were collected from patients with diarrhoea and were kept at 4˚C throughout the transportation to the laboratory and refrigerated at - 20˚C prior to RNA extraction. Stool samples were tested for norovirus using the RIDA©GENE NOROVIRUS I & II real-time RT-PCR. The RNA extracts tested positive for norovirus were subjected to RT-PCR amplification. The RT-PCR products of the amplified fragments were sequenced, and phylogenetic trees were constructed by the neighbor-joining method using MEGA 7 software. RESULTS: NoV was detected in 13(16%) out of 80 stool samples collected, of which 6 (46%) strains belonged to norovirus GII and 7 (54%) strains to norovirus GI. A total of 5 genotypes were detected (GII.Pg, GII.1, GII.2, GII.4 Sydney 2012). The phylogenetic analysis revealed circulation of NoV genotypes with considerable diversity. CONCLUSION: This study illustrates NoV prevalence and substantial genetic diversity in patients above 5 years of age living in rural communities of Vhembe district, Limpopo province. Continued systematic surveillance to evaluate norovirus association with diarrhoea is needed to have a full picture on the epidemiology and disease burden in people of all the age groups. / NRF

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-04-25T15:22:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Alana_Tavares.pdf: 523511 bytes, checksum: 0e4a085043934dadf9915c93d3f924f5 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-25T17:21:22Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Alana_Tavares.pdf: 523511 bytes, checksum: 0e4a085043934dadf9915c93d3f924f5 (MD5) / Made available in DSpace on 2017-04-25T17:21:22Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Alana_Tavares.pdf: 523511 bytes, checksum: 0e4a085043934dadf9915c93d3f924f5 (MD5) Previous issue date: 2015-02-23 / Sem bolsa / Este trabalho foi realizado com o objetivo de determinar as possíveis fontes de contaminação de Yersinia enterocolitica em diferentes pontos do processo de ordenha de vacas leiteiras em oito propriedades da região de Pelotas, RS, distribuídos em quatro períodos diferentes ao longo de um ano. Y. enterocolitica é uma bactéria psicrotrófica, responsável por causar gastroenterite em humanos, principalmente em bebês e crianças, a partir do consumo de água ou alimentos contaminados, sendo o leite cru um destes. Foram analisadas amostras de leite cru de conjunto logo após a ordenha, água de estábulo leiteiro, mão de ordenhador, balde de recolhimento do leite e insuflador de teteiras. As amostras de leite cru e água foram coletadas em frascos estéreis e as amostras de mão de ordenhador, balde e teteiras foram coletadas com o auxilio de zaragatoas estéreis. As amostras de leite cru foram submetidas a um pré-enriquecimento em água peptonada tamponada a 4°C durante 30 dias, sendo posteriormente incubadas em caldo PSTA, adicionado de ampicilina, a 28°C durante 48h. As amostras de água foram filtradas em membrana de éster de celulose e incubadas em caldo TSB a 30°C durante 24h. As amostras de leite após incubação em PSTA, as membranas utilizadas na filtragem da água incubadas em TSB, bem como o material de mãos, balde e teteiras coletadas nas zaragatoas, foram semeadas em ágar MacConkey e incubados a 30°C durante 24h, para a obtenção de colônias. Colônias características foram analisadas através de duplex PCR para confirmação da espécie. Os perfis moleculares dos isolados de Y. enterocolitica foram comparados utilizando a técnica de rep-PCR. Y. enterocolitica foi isolada de 9,37% (3/32) das amostras de leite, 6,25% (2/32) das amostras de água e 12,5% (4/32) das amostras de mão. Não houve similaridade no perfil de bandas dos isolados encontrados, entretanto foi identificada a presença de cepas diferentes na mesma amostra, demonstrando uma variedade grande de cepas distribuídas no ambiente. A presença de Y. enterocolitica em leite cru no Brasil é preocupante, já que uma quantidade considerável do produto ainda é comercializado de forma clandestina, expondo o consumidor ao risco de infecção pela bactéria, ao consumi-lo sem tratamento térmico adequado. / This work was performed in order to determine the possible Yersinia enterocolitica contamination sources at different points of the dairy cows milking process in eight properties of Pelotas, RS, over four different periods distributed in a year. Y. enterocolitica is a psychrotrophic bacterium responsible for causing gastroenteritis in humans, especially in infants and children, from the consumption of contaminated water or food, and raw milk. Raw milk samples were analyzed immediately after milking, water of milking parlor, milker’s hands, milk collection bucket and inflator liners. The samples of raw milk and water were collected in sterile bottles and milker hand samples, bucket and liners were collected with sterile swabs. The raw milk samples were subjected to a pre-enrichment peptone water buffered at 4°C for 30 days and subsequently incubated in PSTA broth ampicillin added at 28°C for 48h. Water samples were filtered through cellulose ester membrane and incubated in TSB medium at 30°C for 24h. The milk samples after incubation in PSTA, the TSB where the membranes used in water filtration were incubated and the material of the hands material, bucket and liners collected in the swabs were plated on MacConkey agar and incubated at 30°C for 24h, to obtain colonies. Characteristics colonies were analyzed by duplex PCR to confirm the species. The molecular profiles of Y. enterocolitica isolates were compared using rep-PCR. Y. enterocolitica was isolated from 9,37% (3/32) of milk samples, 6,25% (2/32) of water samples and 12,5% (4/32) of hand samples. There weren’t similarities in the band profile of the isolates found, however showed the presence of different strains of the same sample, demonstrating a variety of strains distributed in the environment. The presence of Y. enterocolitica in raw milk in Brazil is dangerous, considering that the product is sold clandestinely, exposing consumers to the risk of infection by the bacterium, to consume it without proper heat treatment.

Les rotavirus et les norovirus sont les principaux agents étiologiques des gastro-entérites en Tunisie. Pendant l’hiver 2011-2012, nous avons collecté les selles et les salives de 114 enfants âgés de moins de 6 ans, souffrant de gastro-entérites et admis à l’Hôpital Fattouma Bourguiba de Monastir. L’analyse des salives a montré que la cohorte se répartissait entre 79% de sécréteur et 21% de non-sécréteur (absence d’antigène dans les salives). Parmi les sécréteurs, les individus du groupe O étaient les plus représentés (42%) suivis des groupes A (30%), B (21%) et AB (7%) alors que 96% des patients étaient positifs pour l’antigène Lewis. Pour 98 patients, l’analyse génétique du sang a montré que le gène FUT2 se répartissait entre 77.6% de sécréteur (Se+/Se+ et Se+/se-) et 22.4% de non-sécréteurs (se-/se-, N=22).L’analyse des fèces a montré que les rotavirus et les norovirus étaient responsables respectivement de 22% et 31% des cas, les infections mixtes représentant 6% des cas. Parmi les norovirus, le génotype GII.3 était prédominant (69% de tous les NoV) tandis que le génotype G9P[8] était le plus fréquemment détecté de tous les rotavirus (37,5%). Les rotavirus ont été détectés chez les individus sécréteurs (N=28) mais aussi chez 4 patients non-sécréteurs (3 souches G9P[8] et une souche G3P[8]). Nous n’avons pas observé de distribution particulière des rotavirus en fonction des antigènes A, B et H parmi les enfants sécréteurs. En revanche, nous avons constaté que l'infection à rotavirus ne s’était produite que chez les individus positifs pour l’antigène Lewis (P=0.017). La présence de génotype P[8] chez des non-sécréteurs est inédite, elle a été confirmée par le séquençage du segment correspondant à VP8* de ces rotavirus.La majorité des infections à norovirus a été détectée chez les patients sécréteurs et cela sans distribution particulière en fonction des antigènes A, B, H et Lewis. Cinq GII.3, un GII.1 et un norovirus de génotype GII.7 ont été détectés chez les non-sécréteurs, Lewis-positifs. La production de particules de synthèse (VLP) de norovirus GII.3 en baculovirus à partir des selles d’un des patients non-sécréteurs nous a permis de tester les échantillons salivaires de toute la cohorte. L’absence d’attachement de ces VLP sur les salives des non-sécréteurs montre que la présence ou l’absence des antigènes de groupe ne reflète pas nécessairement le pouvoir infectieux des norovirus chez les jeunes enfants. Les résultats obtenus sur les rotavirus et les norovirus suggèrent qu’ils existent des voies alternatives aux antigènes de groupe sanguin pour l’attachement des rotavirus et des norovirus dans l’intestin. / Rotavirus and norovirus are the main aetiological agents of gastroenteritis in Tunisia. Stool specimens and saliva were collected from children younger than 6 years of age, admitted to the Fattouma Bourguiba Hospital (Monastir, Tunisia) for gastroenteritis during the winter 2011-12. Saliva analysis showed that 79% and 21% patients had secretor and non-secretor phenotypes, respectively. Group O blood type was predominant (42%) followed by groups A (30%), B (21%) and AB (7%), whilst 96% of the patients were positive for Lewis antigen. For 98 patients, blood samples were available and were used for FUT2 genotyping. 77.6% of the cohort were secretor (Se+/Se+ and Se+/se-) and 22.4% were non-secretor (se-/se-).Rotavirus and norovirus were found alone in 22% and 31% of the stool specimens, respectively. Mixed rotavirus-norovirus infections accounted for 6% of the cases. GII.3 noroviruses were predominant among the noroviruses whilst the G9P[8] genotype was predominant for the rotaviruses.Rotaviruses were detected in secretor (N=28) as well as in non-secretor individuals (three G9P[8] strains and one G3P[8]). No significant association was found between ABO antigens or the secretor status and RV infection. Inversely, we observed that RV infection always occurred in Lewis-positive patients (P=0.017). The presence of the P[8] genotype was confirmed by sequencing part of the VP8* coding region.There was no significant association between norovirus infection and ABO antigens and the FUT2 genotype. Five GII.3, one GII.1 and one GII.7 noroviruses were found in Lewis-positive non-secretor patients. Virus-like particles from a GII.3 norovirus infecting a non-secretor patient from the cohort were expressed in baculovirus and used for binding assay with the 114 saliva samples of the study group. VLP binding with non-secretor saliva was negative and suggested that saliva binding assay might not reflect norovirus infectivity. Overall, our data suggested that rotavirus and norovirus infection might involve non-HBGA binding pathways as well as the canonical HBGA ligands.

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.

O mamastrovirus 5 (MAstV5) é classificado no gênero Mamastrovirus da família Astroviridae, sendo associado com surtos agudos de gastroenterite transitória em filhotes de cães ao redor do mundo. O objetivo desta dissertação foi detectar e analisar a variabilidade genética dos MAstV5 circulantes em cães no Brasil. Para isto, amostras de suabe retal foram coletadas de 269 cães de diferentes regiões do Brasil no período de 2008-2014, dos quais 26,39% foram positivos para MAstV5 através de RT-PCR convencional e de RT-Hemi-nested PCR, amplificando porção conservada do gene do capsídeo e do gene da polimerase, respectivamente. Quatro destas cepas tiveram seu genoma parcialmente sequenciado, caracterizado e analisado filogeneticamente. A caracterização dessas amostras revelou uma notável heterogeneidade genética entre as cepas de MAstV5. A baixa identidade entre as sequências do gene do capsídeo (<85%) indicaria uma possível nova classificação entre a espécie MAstV5 em dois genótipos. Conclue-se que o MAstV5 ocorre em cães no Brasil e as cepas circulantes possuem uma grande diversidade genética. / The Mamastrovirus 5 (MAstV5) is classified in the genus Mamastrovirus of the Astroviridae family, being associated with acute episodes of transient gastroenteritis in puppies around the world. The aim of this work was to detect and analyze the genetic variability of circulating MAstV5 in dogs in Brazil. For this, rectal swab samples were collected from 269 dogs from different regions of Brazil in the 2008-2014 period, of which 26.39% were positive for MAstV5 by conventional RT-PCR and RT-Hemi-nested PCR, amplifying conserved portion of the capsid gene and polymerase gene, respectively. Four of these strains had its genome sequenced partially characterized and analyzed phylogenetically. The characterization of these samples revealed a remarkable genetic heterogeneity among strains of MAstV5. The low identity between the sequences of capsid gene (<85%) indicate a possible new classification between the two genotypes MAstV5 species. We conclude that the MAstV5 occurs in dogs in Brazil and circulating strains have a high genetic diversity.

O mamastrovirus 5 (MAstV5) é classificado no gênero Mamastrovirus da família Astroviridae, sendo associado com surtos agudos de gastroenterite transitória em filhotes de cães ao redor do mundo. O objetivo desta dissertação foi detectar e analisar a variabilidade genética dos MAstV5 circulantes em cães no Brasil. Para isto, amostras de suabe retal foram coletadas de 269 cães de diferentes regiões do Brasil no período de 2008-2014, dos quais 26,39% foram positivos para MAstV5 através de RT-PCR convencional e de RT-Hemi-nested PCR, amplificando porção conservada do gene do capsídeo e do gene da polimerase, respectivamente. Quatro destas cepas tiveram seu genoma parcialmente sequenciado, caracterizado e analisado filogeneticamente. A caracterização dessas amostras revelou uma notável heterogeneidade genética entre as cepas de MAstV5. A baixa identidade entre as sequências do gene do capsídeo (<85%) indicaria uma possível nova classificação entre a espécie MAstV5 em dois genótipos. Conclue-se que o MAstV5 ocorre em cães no Brasil e as cepas circulantes possuem uma grande diversidade genética. / The Mamastrovirus 5 (MAstV5) is classified in the genus Mamastrovirus of the Astroviridae family, being associated with acute episodes of transient gastroenteritis in puppies around the world. The aim of this work was to detect and analyze the genetic variability of circulating MAstV5 in dogs in Brazil. For this, rectal swab samples were collected from 269 dogs from different regions of Brazil in the 2008-2014 period, of which 26.39% were positive for MAstV5 by conventional RT-PCR and RT-Hemi-nested PCR, amplifying conserved portion of the capsid gene and polymerase gene, respectively. Four of these strains had its genome sequenced partially characterized and analyzed phylogenetically. The characterization of these samples revealed a remarkable genetic heterogeneity among strains of MAstV5. The low identity between the sequences of capsid gene (<85%) indicate a possible new classification between the two genotypes MAstV5 species. We conclude that the MAstV5 occurs in dogs in Brazil and circulating strains have a high genetic diversity.

O mamastrovirus 5 (MAstV5) é classificado no gênero Mamastrovirus da família Astroviridae, sendo associado com surtos agudos de gastroenterite transitória em filhotes de cães ao redor do mundo. O objetivo desta dissertação foi detectar e analisar a variabilidade genética dos MAstV5 circulantes em cães no Brasil. Para isto, amostras de suabe retal foram coletadas de 269 cães de diferentes regiões do Brasil no período de 2008-2014, dos quais 26,39% foram positivos para MAstV5 através de RT-PCR convencional e de RT-Hemi-nested PCR, amplificando porção conservada do gene do capsídeo e do gene da polimerase, respectivamente. Quatro destas cepas tiveram seu genoma parcialmente sequenciado, caracterizado e analisado filogeneticamente. A caracterização dessas amostras revelou uma notável heterogeneidade genética entre as cepas de MAstV5. A baixa identidade entre as sequências do gene do capsídeo (<85%) indicaria uma possível nova classificação entre a espécie MAstV5 em dois genótipos. Conclue-se que o MAstV5 ocorre em cães no Brasil e as cepas circulantes possuem uma grande diversidade genética. / The Mamastrovirus 5 (MAstV5) is classified in the genus Mamastrovirus of the Astroviridae family, being associated with acute episodes of transient gastroenteritis in puppies around the world. The aim of this work was to detect and analyze the genetic variability of circulating MAstV5 in dogs in Brazil. For this, rectal swab samples were collected from 269 dogs from different regions of Brazil in the 2008-2014 period, of which 26.39% were positive for MAstV5 by conventional RT-PCR and RT-Hemi-nested PCR, amplifying conserved portion of the capsid gene and polymerase gene, respectively. Four of these strains had its genome sequenced partially characterized and analyzed phylogenetically. The characterization of these samples revealed a remarkable genetic heterogeneity among strains of MAstV5. The low identity between the sequences of capsid gene (<85%) indicate a possible new classification between the two genotypes MAstV5 species. We conclude that the MAstV5 occurs in dogs in Brazil and circulating strains have a high genetic diversity.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-13T10:36:11Z No. of bitstreams: 2 Dissertação - Thairiny Neres Silva - 2017.pdf: 2022674 bytes, checksum: a9ff46169fe46df4f2909e2719e18791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-13T10:36:47Z (GMT) No. of bitstreams: 2 Dissertação - Thairiny Neres Silva - 2017.pdf: 2022674 bytes, checksum: a9ff46169fe46df4f2909e2719e18791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-13T10:36:47Z (GMT). No. of bitstreams: 2 Dissertação - Thairiny Neres Silva - 2017.pdf: 2022674 bytes, checksum: a9ff46169fe46df4f2909e2719e18791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-03 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Sapoviruses are important agents causing acute gastroenteritis worldwide, and are most often detected in children under five years of age. The present study aimed to evaluate the positivity and viral load index of sapovirus in faecal samples and nasopharyngeal swab of children, in association with the symptoms presented by these children. The study included 102 children hospitalized at the Hospital Materno Infantil, presenting symptoms of gastroenteritis, attended between May 2014 and May 2015, with a sample of faeces and a nasopharyngeal swab sample from each of them. Samples of faeces and nasopharyngeal swabs from all children were submitted to extraction of the genetic material from a commercial kit and screened for sapovirus by RT-qPCR. The viral load was determined by constructing a standard curve from recombinant plasmid. A faecal sample and a nasopharyngeal swab sample were obtained and 47% of the children were positive for sapovirus in at least one of the samples collected, being 10.7% positive only in faecal samples and 28.4% positive only in the samples of nasopharyngeal swab. Regarding viral load, a median of 7.77 x 108 CG / mL was found for stool samples. Regarding the viral load of the nasopharyngeal swab samples, a median 1.54 x 108 CG / mL was observed and a statistically significant result was observed (13/14 p = 0.01), for the samples with viral load above the median obtained in the rainy season. The median viral load of samples from positive children in both clinical specimens was 2.33 x 108 CG / mL in the faeces and 2.67 x 108 CG / mL in the nasopharyngeal swab. Two of these children presented a considerably higher viral load when compared to the others. It is hoped that the data obtained may contribute to a better understanding of the molecular epidemiology of sapoviruses, as well as to the development of better preventive measures, in order to limit the risk of transmission of sapoviruses, especially in a hospital environment. This is the first study to carry out the research and detection of sapovirus in a respiratory tract sample, which could suggest a possible alternative route of viral transmission. / Sapovírus são importantes agentes causadores de gastroenterite aguda no mundo todo, sendo mais frequentemente detectados em crianças menores de cinco anos. O presente estudo teve como objetivo, avaliar o índice de positividade e carga viral de sapovírus em amostras de fezes e swab nasofaringeano de crianças, em associação com os sintomas apresentados por essas crianças. Participaram do estudo 102 crianças hospitalizadas no Hospital Materno Infantil, apresentando sintomas de gastroenteríte, atendidas entre o período de maio de 2014 a maio de 2015, sendo obtidas uma amostra de fezes e uma amostra de swab nasofaringeano de cada uma delas. As amostras de fezes e de swab nasofaringeano de todas as crianças, foram submetidas a extração do material genético a partir de kit comerciale triadas para sapovírus por RT-qPCR. A carga viral foi determinada através da construção de uma curva padrão, a partir de plasmídeo recombinante. Foram obtidas uma amostra de fezes e uma de amostra de swab nasofaringeano sendo 47% das crianças foram positivas para sapovírus em pelo menos uma das amostras coletadas, sendo 10,7% positivas apenas nas amostras de fezes e 28,4% positivas apenas nas amostras de swab nasofaringeano. Em relação a carga viral constatou-se uma mediana 7,77 x 10 8 CG/mL para as amostras de fezes. Em relação a carga viral das amostras de swab nasofaringeano, foi observada uma mediana 1,54 x 10 8 CG/mL e um resultado estatisticamente significativo foi observado (13/14 p= 0,01), para as amostras com carga viral acima da mediana obtidas no período chuvoso. A mediana da carga viral das amostras proveniente das crianças com positividade em ambos espécimes clínicos foi de 2,33 x 10 8 CG/mL nas fezes e de 2,67 x 10 8 CG/mL nos swab nasofaringeano. Duas dessas crianças apresentaram carga viral consideravelmente mais elevada, quando comparada com as demais. Espera-se que os dados obtidos possam contribuir para um melhor entendimento da epidemiologia molecular dos sapovírus, bem como para que melhores medidas preventivas sejam desenvolvidas, a fim de limitar os riscos de transmissão dos sapovírus, especialmente em ambiente hospitalar. Este é o primeiro estudo a realizar a pesquisa e detecção de sapovírus em amostra do trato respiratório, o que poderia sugerir uma possível rota alternativa de transmissão viral.

Este trabalho teve por objetivo identificar e estudar a expressão de genes envolvidos na resposta imune a endoparasitas gastrintestinais em bovinos. Contagem periódica de ovos por grama de fezes (OPG) e análises de coproculturas foram realizadas para identificar animais resistentes e susceptíveis a endoparasitas gastrintestinais. A contagem de OPG permitiu identificar estes animais, sendo que o grupo susceptível apresentou uma contagem 20 vezes superior ao grupo resistente (P<0,001). Análises de coproculturas permitiram concluir que a infestação em bovinos foi mista, com a predominância dos gêneros Cooperia e Haemonchus e menor incidência de Oesophagostomum. Para a identificação dos genes, foi utilizada a metodologia EST (Expressed Sequence Tag) e foram construídas duas bibliotecas de clones de cDNA obtidos a partir da mucosa do abomaso, intestino delgado e nódulos linfáticos abomasais de bovinos resistentes (ER1) e susceptíveis (ES1) a nematódeos gastrintestinais. Foram geradas 2496 ESTs de cada biblioteca. Destas, 1664 e 1898 foram válidas para as bibliotecas ER1 e ES1, respectivamente. Dentre as 2323 sequências únicas obtidas foram identificados vários produtos gênicos relacionados à resposta imune, tais como moléculas de classe I e II do MHC, imunoglobulinas, interleucinas, lisozima e pepsinogênio. Para a quantificação da expressão de RNA mensageiro, foi utilizada a metodologia de transcrição reversa e reação em cadeia da polimerase em tempo real, onde foram analisados 10 genes relacionados à polarização da resposta imune (as interleucinas IL- 2, IL-4, IL-8, IL-12p35 e IL-13, as citocinas TNF-&#945;, IFN-&#947;, MCP-1&#945; e MCP-2 e a glicoproteína mucina 1-MUC1). As análises de expressão gênica realizadas na mucosa do abomaso revelaram aumento nos níveis de RNAm para IL-4 (P<0,018), IL-13 (P<0,002) e diminuição de TNF-? (P<0,0001) nos animais resistentes; nos nódulos linfáticos abomasais houve aumento de IL-4 (P<0,019) nos animais resistentes e de IFN-&#947; (P<0,007) nos animais susceptíveis; no intestino delgado houve aumento de IL-4 (P<0,01) e IL-13 (P<0,045) nos animais resistentes, ao contrário de IL-2 (P<0,047), IL-12p35 (P<0,029), IFN-&#947; (P<0,004) e MCP-1 (P<0,03), cujos níveis de RNAm foram maiores nos animais susceptíveis. No abomaso dos animais resistentes houve menor expressão de pepsinogênio (P<0,025) e maior de lisozima (P<0,042). Em conclusão, a estratégia utilizada permitiu a identificação de vários genes envolvidos na resposta imune e a descoberta inédita de que Bos indicus resistentes a parasitas gastrintestinais apresentam uma resposta TH2 polarizada, em contraste aos animais susceptíveis, que apresentam uma resposta TH1. / The aim of this work was identify genes related to immune response to gastrointestinal nematodes in cattle. The periodic counts of eggs per gram (EPG) of feces and coproculture analysis were done to identify the resistant and susceptible animals. The EPG counts allowed us to identify these animals. It was twenty-fold higher in susceptible group (P<0.001). The coproculture analysis allowed us to conclude that the infestation is predominantly characterized byCooperia spp. and Haemonchus spp. and a low incidency of Oesophagostomum. To identify the genes, it was used the EST (Expressed Sequence Tags) methodology and constructed two cDNA libraries from abomasum, abomasum lymph nodules and small intestine from resistant (ER1) and susceptible (ES1) cattle. It was generated 2496 ESTs from each library. From these, 1664 and 1898 ESTs were valids to ER1 and ES1 libraries, respectively. Among the 2323 unique sequences were identifyed several genes related to immune response, such as MHC class I and II molecules, immunoglobulins, interleukins, lysozyme and pepsinogen. To study the gene expression, it was used the reverse transcription and real-time polymerase chain reaction methodology to quantify the messager RNA expression of 10 genes related to polarization of immune response (the interleukins IL-2, IL-4, IL-8, IL-12p35 e IL-13, the cytokines TNF-&#946;, IFN- &#947;, MCP-1&#946; and MCP-2 and the glycoprotein mucin 1-MUC1). The gene expression analysis in the abomasum reveled that IL-4 (P<0,018) and IL-13 (P<0,002) were up-regulated and TNF-&#946; (P<0,0001) was down-regulated in resistant group; in the abomasum lymph nodules IL-4 (P<0,019) and IFN-&#947; (P<0,007) were both up-regulated in resistant and susceptible group, respectively; in the small intestine IL-4 (P<0,01) and IL-13 (P<0,045) were up-regulated in resistant group and IL-2 (P<0,047), IL-12p35 (P<0,029), IFN-&#947; (P<0,004) and MCP-1 (P<0,03) were down-regulated in susceptible one. In the abomasum from resistant group, pepsinogen was down-regulated (P<0,025) and lysozyme was up-regulated (P<0,042). In conclusion, the strategy used allowed us to identify several genes involved in immune response and the inedit discovery that Bos indicus resistant to gastrointestinal nematodes present a TH2-type response, in contrast to susceptible animals that present a TH1-type response.

Parvoviruses is a viral disease characterized by an acute hemorrhagic gastroenteritis, caused by a canine parvovirus (CPV) that is stable in the environment, able to bear pH variations and high temperatures. It is resistant to many common disinfectants and can survive for many months in contaminated areas. There are two common clinical forms of the disease: the myocardial and the gastroenteric. This work had as objective to analyse microscopically the cardiopathy cases, diagnosticated macroscopically during the necropsy of dogs with parvovirus detected in faeces. In the 100 samples send to the Histopathology Laboratory, from the University of Uberaba, they get in the left ventricular myocardium the following alterations: myocarditis 38%, hemorrhage 43%, hyaline degeneration 21% and hyperemia 79%. Having been carried out the Qui-Quadrado test with a significance level of 0,05, we can conclude that there is association (p = 0,02) between the infected animals with the parvoviruses virus and the histopathologyc alterations observed in the left ventricular myocardium. / Parvovirose é uma enfermidade viral caracterizada por gastroenterite hemorrágica aguda, cujo agente etiológico é parvovírus canino (PVC), vírus estável no ambiente, capaz de suportar variações de pH e temperaturas altas, resistente a vários desinfetantes comuns, podendo sobreviver por muitos meses em áreas contaminadas. Há duas formas clínicas comuns da doença: a miocárdica e a gastroentérica. No Brasil a doença eclodiu subitamente na população canina no ano de 1978. Objetiva-se com este trabalho analisar microscopicamente o miocárdio de cães com teste de detecção de antígenos parvovírus nas fezes. Das 100 amostras do miocárdio ventricular esquerdo, enviadas ao Laboratório de Histopatologia da Universidade de Uberaba, foram observadas as seguintes alterações: miocardite 38%, hemorragia 43%, degeneração hialina 21% hiperemia 79%. Ao realizar o teste Qui-Quadrado com nível de significância de 0,05, concluiu-se que existe associação (p = 0,02) entre animais infectados com o vírus da parvovirose e as alterações histopatológicas observadas no miocárdio ventricular esquerdo. / Mestre em Ciências Veterinárias