A diarréia neonatal é o principal problema sanitário que acomete os bezerros nas primeiras semanas de vida, causando grandes prejuízos devido à morbidade, mortalidade, custos com tratamento e atraso no desenvolvimento. Os rotavírus são os mais importantes agentes virais causadores de gastroenterites em crianças e diarreia em diferentes espécies animais. Além do seu impacto econômico na produção animal, os bovinos podem atuar como reservatórios da diversidade genética e antigênica para humanos. Assim, o diagnóstico deste agente é fundamental para o desenvolvimento de medidas profiláticas mais especificas visando o seu controle. O objetivo deste trabalho foi desenvolver um método de PCR em Tempo Real para a detecção de rotavírus bovinos em sistema Taqman® tendo como alvo, a proteína não-estrutural 5 (NSP5). Para isso, 113 amostras de fezes bovinas foram coletadas de reabanhos do estado de São Paulo, e previamente testadas por PCR convencional. Para a padronização da PCR em Tempo Real, a estirpe padrão foi clonada, gerando um plasmídeo com 3x1010 cópias/reação. Como controle exógeno foi utilizado β-actina. O limite de detecção foi determinado por diluições seriadas na base 10, detectando até 6x101 cópias/µl. A curva padrão da PCR em Temo Real para a detecção do segmento codificador da proteína NSP5 teve como resultados, uma eficiência de 100,47%; com slope igual a -3,18 e R2 de 1,0. De um total de 113 amostras testadas pela PCR convencional 63 delas (55,7%) foram consideradas positivas para rotavírus. Dessas mesmas amostras testadas, 5 não amplificaram para β-actina e não foram incluídas nas análises posteriores. Para a PCR em Tempo Real o limite de detecção foi considerado o valor de 6x101 cópias/reação, sendo definido o ponto de corte o ciclo (Ct) de número 36 para o teste com a amostra viral a partir do DNA ligado em vetor plasmidial. Considerando-se o ponto de corte de 60 (6x101) cópias/reação, das 108 amostras testadas, 63 (58,3%) foram consideradas positivas ao teste. O valor de concordância obtido através do teste Kappa, a um intervalo de confiança de 95%, a partir dos resultados gerados entre os testes de PCR convencional e PCR em foi de 0.279 (baixa concordância). Os resultados obtidos nesse estudo demonstrou que o teste foi eficiente podendo ser utilizado para um diagnóstico rápido e eficiente do rotavirus do grupo A, aumentando assim o repertório dos testes já estabelecidos / Neonatal diarrhea is the main health problem affecting the calves in the first weeks of life, causing major losses due to morbidity, mortality, treatment costs and delayed development. Rotaviruses are the most important causative agents of viral gastroenteritis in children and in different animal species. In addition to its economic impact on livestock, cattle can act as reservoirs for genetic and antigenic diversity for human samples. Therefore, the diagnosis of this agent is critical to the development of more specific preventive measures for their control. The objective of this work is to develop a method of PCR for rotavirus detection in cattle using TaqMan® system targeting the 5 nonstructural protein (NSP5). For this, 113 samples of cattle feces were collected from farms of São Paulo, and previously tested by convencional PCR. To PCR standardization, the standard sample was cloned, generating plasmid 3x1010 copies/reaction. The β-actin was usede as exogenous control. The limit of detection was determined by serial dilutions, to detect 6 x101 copies/µl. The standard curve of PCR to encoding segment detection NSP5 protein had as a result, an efficiency of 108.5%; with slope equal to -3.18 and R2 equal 1.. From a total of 113 samples tested by conventional PCR 63 (55.7%) were positive for rotavirus. From these samples 5 not amplifyed for β-actin gene and were not included in subsequent analyzes. For detection limit of Real-Time PCR was considered the amount of 6x101 copies / reaction, the cut-off being defined cycle (Ct) number 36 to the test with a viral sample from the DNA ligated into plasmid vector. Considering the cut-off 60 (6x101) copies/reaction of the 108 samples tested, 63 (58.3%) were positive to the test. The correlation value obtained by the Kappa test, at a 95% confidence interval, based on results generated between the conventional and PCR testing PCR was 0.279 (low agreement). The results of this study demonstrated that the probe can be efficiently used for a fast and efficient diagnosis of rotavirus of group A, thereby enhancing the repertoire of the established tests

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No. of bitstreams: 2 Dissertação - Tâmera Nunes Vieira Almeida - 2011.pdf: 3539556 bytes, checksum: 0d3229ddfa2244e57c236faf04b328e8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2011-04-16 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The group A rotaviruses (RVA) are recognized as the main viral agents of acute childhood gastroenteritis, and due to the high morbidity-mortality rates vaccination has being considered the best alternative for prevention and control of RVA. In Brazil, in March, 2006 the Ministry of Health included the human rotavirus oral vaccine – VORH, which was developed from a monovalent attenuated strain G1P[8], in the National Immunization Program. In this context, the present study aimed at the investigation of the occurrence of RVA infections in the city of Goiânia after the implementation of the VORH, as well as the comparative analysis of the RVA circulating strains during the pre- and post-vaccination periods. For the RVA identification, 65 fecal samples obtained from children with acute gastroenteritis, in the period from 2008 to 2009, were tested by an immunoenzymatic assay and by polyacrilamide gel electrophoresis, with a total detection rate of 16.9% (11/65). After molecular characterization, the G2 genotype was identified in 10 samples, and four of those were considered as G2P[4] genotype. For the comparative analysis, the G2P[4] samples, as well as other 15 samples, obtained in the pre- and post-vaccination periods, were submitted to genomic sequencing of the coding regions for the proteins VP6, VP7 and NSP4. The molecular characterization of the VP7 gene showed that the G1 samples belonged to lineages I and II, sublineages d and b, respectively, and that all the G2 samples belonged to lineage II, with the differentiation of three sublineages, a, c and d, which were correlated with the collection periods. Regarding the VP6 genogroups and the NSP4 genotypes, a predominance of genogroup I and genotype A in postvaccination period was observed, whereas a predominance of genogroup II and genotype B was identified in the period before de vaccine implementation. The association between the G and P genotypes with VP6 genogroups and NSP4 genotypes revealed the predominance of the G1-P[8]-II-B combination in the pre-vaccination period, and the association G2-P[4]-I-A in the post-vaccination period, which suggests the substitution of these combinations after the implementation of the VORH. / Rotavírus do grupo A (RVA) são reconhecidos como os principais agentes virais da gastroenterite aguda infantil e, devido aos relevantes índices de morbi-mortalidade, a vacinação tem sido considerada a melhor alternativa para a prevenção e o controle de RVA. No Brasil, em março de 2006 o Ministério da Saúde adicionou ao Programa Nacional de Imunização a Vacina Oral de Rotavírus Humano (VORH), a qual foi desenvolvida a partir de uma amostra monovalente atenuada G1P[8]. Neste contexto, o presente estudo objetivou a investigação da ocorrência das infecções por RVA na cidade de Goiânia após a implantação da VORH, bem como proceder a uma análise comparativa das amostras de RVA circulantes nos períodos pré e pós-vacinal. Para identificação de RVA, 65 espécimes fecais coletados no período de 2008 a 2009 de crianças com gastroenterite aguda, foram submetidos ao Ensaio Imunoenzimático e Eletroforese em Gel de Poliacrilamida, sendo observado um índice total de detecção de 16,9% (11/65). Após caracterização molecular, o genótipo G2 foi identificado em 10 amostras, sendo que quatro destas foram definidas como G2P[4]. Para análise comparativa, as amostras G2P[4], bem como outras 15 amostras coletadas nos períodos pré e pós-vacinal, foram submetidas ao sequenciamento genômico para as regiões codificantes das proteínas VP6, VP7 e NSP4. A caracterização molecular do gene de VP7 mostrou que as amostras G1 pertenciam às linhagens I e II, sublinhagens d e b, respectivamente, e que todas as amostras G2 pertencem à linhagem II, com a diferenciação de três sublinhagens, a, c e d, as quais foram correlacionadas com os períodos de coleta. Considerando os genogrupos de VP6 e genótipos de NSP4 identificados, observou-se predominância para genogrupo I e genótipo A no período pós-vacinal, enquanto, genogrupo II e genótipo B foram identificados com maior frequência antes da implantação da vacina. A associação entre os genótipos G e P com genogrupos de VP6 e genótipos de NSP4 revelou predominância da combinação G1-P[8]II-B no período pré-vacinal e da associação G2-P[4]-I-A no período pós-vacinal o que sugere uma substituição destas combinações após a implantação da VORH.

Made available in DSpace on 2015-04-22T22:14:08Z (GMT). No. of bitstreams: 1 DISSERTACAO-CYNTHIA PINTO.PDF: 1386521 bytes, checksum: cd8c6e6b087f4a1705287014f9b5ac34 (MD5) Previous issue date: 2009-07-20 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Human rotavirus constitutes one of the most important causes of diarrheic disease in children less than five years in the whole world. In 2006, the National Program of Immunization of Health Minister introduced a monovalent vaccine against rotavirus objecting the reduction of prevalence of rotaviruses. With the object of verifying the occurrence of genotypes of human rotavirus in children of 0 the 12 months vaccinated and non vaccinated residents in the city of Manaus in Amazon, had been analyzed during a year (may/2007 may/2008), a total of 294 feces diarrheic samples that had been colleted of children attended hospital of children in the city of Manaus. This analyzes was carried through by the detention of the viral genome through the techniques of PAGE (Polyacrilamide Gel Eletrophoresis) and RT-PCR (Reverse transcription Polymerase Chain Reaction). Through the PAGE method can be observed a positivity of 9.5% of the human rotavirus samples, and of these samples 36% were of vaccinated children against rotavirus. All of the positive samples of human rotavirus had been typed by the method of RT-PCR with use of specific primers for genotypes G (G1, G2, G3, G4 and G9) and genotypes P (P[8], P[4], P[6], P[9] e P[10]). By the typing, it was detected genotype G in 42.9% of the positive samples for rotavirus with detection only of genotype G2 and genotype P was detected in 94% of positive samples with detection only of P[4]. The combinations of genotypes G and P appeared in 50% of the positive samples for G and P, and all of these combinations were G2P[4]. It was observed that 17% of the positive samples for genotype G and 0,6% of the positive sample for genotype P had not presented characteristics for none of searched genotypes G and P. The number of positive samples for human rotavirus had been similar in such a way in children of the masculine sex how much in children of the feminine sex, being that these samples had predominated in children with age between 7 and 12 months. The positive samples of human rotavirus had been found in every months of had lasted the study with exception of august of 2007 and march of 2008. The vaccination reduce the human rotavirus incidence in children that had been take the two doses of vaccine, perhaps the emergency of the genotype G2P[4] observed in this study could not be associated to the vaccine. / Os rotavírus humanos constituem uma das mais importantes causas de doença diarréica em crianças menores de cinco anos no mundo. Em 2006, o Programa Nacional de Imunização do Ministério da Saúde introduziu uma vacina monovalente contra o rotavírus visando à redução da prevalência das rotaviroses. Com o objetivo de verificar a ocorrência de rotaviroses em crianças de 0 a 12 meses vacinadas e não vacinadas atendidas nos pronto-socorros infantis do município de Manaus no Amazonas, foram analisadas durante 13 meses (maio/2007 a maio/2008), um total de 294 amostras de fezes diarréicas coletadas de crianças atendidas em prontos socorros infantis do município de Manaus. Esta análise foi realizada pela detecção do genoma viral através das técnicas de EGPA (Eletroforese em Gel de Poliacrilamida) e RT-PCR (Reação da Polimerase em Cadeia com Transcriptase Reversa). Por meio do método da EGPA pode-se observar uma positividade de 9,5% das amostras para rotavírus humano, sendo que destas amostras, 36% eram de crianças vacinadas contra rotavírus. Todas as amostras positivas na EGPA para rotavírus humano foram tipadas pelo método de RT-PCR com utilização de primers específicos para os genótipos G (G1, G2, G3, G4 e G9) e genótipos P (P[8], P[4], P[6], P[9] e P[10]). Pela tipagem foi detectado o genótipo G em 42,9% das amostras positivas para rotavírus com detecção apenas do genótipo G2 e o genótipo P foi detectado em 94% das amostras positivas para rotavírus com aparecimento apenas do genótipo P[4]. As combinações dos genótipos G e P apareceram em 50% das amostras positivas para G e P, sendo todas da combinação G2P[4]. Foi observado que 17% das amostras positivas para o genótipo G e 0,6% das amostras positivas para o genótipo P não apresentaram características de nenhum dos genótipos G e P pesquisados. O número de amostras positivas para rotavírus humano foi semelhante tanto em crianças do sexo masculino quanto em crianças do sexo feminino, sendo que estas amostras predominaram em crianças de 7 a 12 meses. As amostras positivas para rotavírus humano, assim como para os genótipos G e P foram encontradas em todos os meses do estudo, com exceção de agosto de 2007 e março de 2008. A vacinação reduz a incidência de rotavírus humano em crianças que tomaram as duas doses da vacina, porém a emergência do genótipo G2P[4] observada neste estudo pode não estar associada à vacina.

During the 20th century, significant strides were made in curtailing the burden of childhood diarrhea, including advances in vaccine research, the advent of antibiotics, improved water and sanitation, and expanded access to health information across the globe. Despite this progress, today diarrhea ranks second only to pneumonia as a leading cause of mortality in children under five years, with a disproportionate burden of 90% of diarrheal deaths in South Asia and Sub-Saharan Africa. Additionally, substantial morbidity due to diarrhea persists in young children, with more than 45 million disability-adjusted life years (DALYs) lost due to diarrhea in 2015. Long-term consequences of childhood diarrhea include undernutrition, impaired gut function, altered gut microbiota, and compromised cognitive development. The 21st century presents an opportunity to eliminate the health disparity affecting millions of children suffering disproportionately from preventable diarrheal diseases. Recent advances in molecular laboratory technology have enabled detailed assessment of diarrheal burden and etiology, illuminating the highest burden pathogens for focused interventions. Among the top diarrheal pathogens, rotavirus (RV) is the leading cause of diarrhea-attributable death in the first year of life. While we have vaccines against RV, these vaccines consistently underperform in low and middle-income countries (LMICs) with efficacy of 18% to 61% compared to > 85% efficacy in high income countries. Reasons for rotavirus vaccine underperformance remain unclear, and no vaccines are available for other high burden diarrheal pathogens. This requires consideration of complementary and alternative interventions for diarrhea prevention. To assess factors related to rotavirus vaccine performance, we enrolled a 700-infant birth cohort in an urban slum of Dhaka, Bangladesh, in the Performance of Rotavirus and Oral Polio Vaccines in Developing Countries (PROVIDE) study: a randomized controlled trial of a 2-dose monovalent oral rotavirus vaccine (RV1). With a primary outcome of any rotavirus diarrhea (RVD) post-vaccination to one year, we conducted biweekly home-based diarrhea surveillance with rotavirus antigen detection in diarrheal stools by ELISA. We found RV1 efficacy of 51% (95% CI 33.8–63.7) in per protocol analysis. Importantly, among 12 explanatory variables tested for association with RVD, serum zinc concentration (SZC) in infants at week 18 associated with risk of RVD up to one year (OR 0.77, 95% CI 0.66–0.91), independent of vaccination status. This finding led to broader investigation of the relationship between zinc status and diarrhea in the PROVIDE cohort. Among 577 PROVIDE infants, 16.5% were zinc deficient at week 18 (SZC < 65μg/dL). By logistic regression, zinc deficient infants had increased odds of diarrhea in the first year of life compared to zinc replete infants (OR 2.76, 95% CI 1.08–7.04), and they were nearly 4 times more likely to have diarrhea of viral etiology (OR 3.94, 95% CI 1.55–10.03). Furthermore, in Kaplan Meier analysis we found a strong correlation between zinc deficiency and time to first episode of viral diarrhea (median survival 27 vs 33 weeks in zinc deficient vs non-deficient infants, p Our results indicate further consideration of zinc as a critical and modifiable co-factor in ameliorating the burden of childhood viral diarrhea. Carefully designed trials of zinc supplementation interventions could determine whether zinc may fill the gap in protection against childhood viral diarrhea, and inquiries into the zinc-diarrhea molecular pathway could elucidate mechanisms for focused development of future interventions.

Group A rotaviruses are a major cause of acute gastroenteritis in children. Almost all children are infected by the age of 5 years old. Rotavirus disease causes around 600,000 deaths per year. VP4 (P) and VP7 (G) genotypes were analyzed for prevalence and potential antigenicity, as they are known to elicit a neutralizing antibody response during infection. This study predicted the effectiveness of two recently licensed rotavirus vaccines based on Canadian surveillance. 271 out of 348 diarrhea samples from 8 paediatric hospital were successfully genotyped by PCR. Canadian rotavirus genotypes were found to be mostly G1P[8] followed by G3P[8], G2P[4], G9P[8], G4P[8], and G9P[4], between 2007 and 2010. Reassortment and motif analysis was done with a subset of rotavirus-positive samples. There were no unusual reassortment events found in Canadian strains. Variations amongst strains were commonly genotype-specific, but otherwise rare. In conclusion, rotavirus vaccine escape is presently unlikely amongst Canadian strains.

Group A rotaviruses are a major cause of acute gastroenteritis in children. Almost all children are infected by the age of 5 years old. Rotavirus disease causes around 600,000 deaths per year. VP4 (P) and VP7 (G) genotypes were analyzed for prevalence and potential antigenicity, as they are known to elicit a neutralizing antibody response during infection. This study predicted the effectiveness of two recently licensed rotavirus vaccines based on Canadian surveillance. 271 out of 348 diarrhea samples from 8 paediatric hospital were successfully genotyped by PCR. Canadian rotavirus genotypes were found to be mostly G1P[8] followed by G3P[8], G2P[4], G9P[8], G4P[8], and G9P[4], between 2007 and 2010. Reassortment and motif analysis was done with a subset of rotavirus-positive samples. There were no unusual reassortment events found in Canadian strains. Variations amongst strains were commonly genotype-specific, but otherwise rare. In conclusion, rotavirus vaccine escape is presently unlikely amongst Canadian strains.

Ypač svarbią reikšmę Lietuvos žemės ūkiui turi viena iš gyvulininkystės šakų – galvijininkystė. Jos plėtrai didelę žalą daro galvijų užkrečiamosios, ypač virusinės, ligos, todėl būtina greitai, tiksliai ir efektyviai diagnozuoti galvijų virusines ligas, užkirsti kelią jų plitimui Lietuvoje. Kvėpavimo ir virškinimo trakto ligomis galvijų prieauglis serga visose pramoninės gyvulininkystės šalyse, dėl šių ligų galvijai gaišta žymiai dažniau, nei nuo reprodukcijos, medžiagų apykaitos ligų ar mastitų. Didesnį veršelių susirgimų skaičių ir gaišimą nuo enteritų bei respiratorinių ligų galima būtų paaiškinti dideliu virusinių ligų – GVD, IGR, PG-3, RSV, adenovirusų ,RV, CV ir bakterinių infekcijų išplitimu. Mūsų darbo tikslas buvo atlikti galvijų užkrečiamųjų virusinių ligų paplitimo analizę, ištirti CV, RV, RSV ir adenovirusų paplitimą galvijų bandose. Dėl rota ir korona virusų buvo ištirti 56 išmatų mėginiai, dėl antikūnų prieš adenovirusą – 20, RS virusą 28 kraujo serumo mėginiai. Šiems tyrimams buvo naudojami komerciniai standartizuoti imunofermentinės analizės (IFA) rinkiniai. Veršelių 30 kraujo serumo mėginių buvo ištirti pusiau kiekybiniu natrio sulfito precipitacijos metodu, dėl imunoglobulinų kiekio nustatymo. Tyrimai atlikti LSMU VA Užkrečiamųjų ligų katedroje ir Nacionalinio maisto ir veterinarijos rizikos vertinimo instituto Virusologijos skyriuje 2013 metais. Įvertinus padėtį 3 ūkiuose, kur buvo atlikti ligų tyrimai, nustatyta, kad rotavirusine infekcija vidutiniškai... [toliau žr. visą tekstą] / Cattle take a very important place in Lithuanian agriculture. Since the development of livestock can be seriously affected by contagious diseases, especially viral ones, it is highly important quickly, accurately and efficiently diagnose viral disease of cattle, and to prevent its spreading in Lithuania. In all livestock farming industry countries, cattle offspring are suffering from respiratory and digestive tract diseases. They are dying from these diseases more often than from reproductive, metabolic diseases and mastitis. Increasing calves’ cases and mortality rate from enteritis and respiratory diseases can be explained by the high viral diseases such as GVD, IGR, PG-3, RSV, adenovirus, RV, CV and bacterial infections spread. The goal of our study was to analyze the spread of livestock infectious viral diseases and examine the CV, RV, RVS and adenovirus prevalence in the cattle herds. 56 fecal samples were tested for the rotavirus and coronavirus, 20 – for antibodies against adenovirus, and 28 blood serum samples for RS virus. To test these samples, we used standardized commercial enzyme-linked immunosorbent assay (ELISA) kits. 30 blood serum samples of calves were analyzed by quantitative sodium sulfite precipitation method for the determination of immunoglobulin. The tests took place at LSMU VA Department of Infectious Diseases and National Food and Veterinary Risk Assessment Institute, Department of Virology in 2013. Cattle from 3 farms were assessed for anticipated... [to full text]

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.