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Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Résumé: Introduction : Le rotavirus est la principale cause de gastro-entérite aiguë (GEA) chez les tout-petits à travers le monde. En 2011, le vaccin antirotavirus monovalent (RV1) a été introduit dans le programme de vaccination universel du Québec afin de réduire la morbidité reliée à la gastro-entérite à rotavirus (GERV). Ce mémoire avait pour objectif de décrire les taux d’hospitalisation pour GEA et GERV avant et après l’implantation du programme chez les jeunes enfants estriens (étude d’impact) ainsi que la couverture vaccinale et d’évaluer l’efficacité vaccinale (EV) du RV1 (étude d’efficacité). Méthode : Le jumelage d’une banque de données hospitalières avec le registre régional de vaccination a permis d’extraire une cohorte d’enfants nés au Centre hospitalier universitaire de Sherbrooke (CHUS), vivant en Estrie et âgés de moins de cinq ans durant la période d’étude de juin 2004 à mai 2014 (n = 37 757). Cette cohorte a été suivie de façon rétrospective afin d’examiner les taux annuels d’hospitalisation pour GEA et GERV des années pré- (2004/2005-2010/2011) et post-implantation (2011/2012-2013/2014), globalement et selon diverses caractéristiques socioéconomiques. De plus, l’EV du RV1 a été calculée à l’aide de trois cohortes d’enfants : (1) les enfants vaccinés nés en 2011-2013 (n = 5 033), (2) les enfants non vaccinés nés en 2011-2013 (n = 1 239) et (3) les enfants non vaccinés nés en 2008-2010 (n = 6 436). Résultats : Le taux d’hospitalisation pour GEA a évolué de 81/10 000 enfants de moins de cinq ans en période pré-implantation à 46/10 000 en période post-implantation (réduction relative = 43 %, p < 0,001). Suite à l’implantation du programme, la couverture vaccinale a rapidement augmenté pour atteindre 81 %. Malgré une couverture vaccinale similaire parmi les différents groupes, les plus faibles réductions relatives ont été observées chez les groupes défavorisés. L’EV ajustée pour une série complète était de 62 % (intervalle de confiance [IC] 95 % : 37-77 %) et de 94 % (IC 95 % : 52-99 %) contre les hospitalisations pour GEA et GERV, respectivement. Les enfants vivant dans des quartiers ayant une proportion élevée de familles à faible revenu avaient une EV plus faible contre les hospitalisations pour GEA (30 % contre 78 %, p = 0,027). Conclusion : Trois ans après son introduction dans le programme universel, le RV1 a réduit de façon significative les gastro-entérites sévères chez les jeunes enfants estriens. Ce vaccin est très efficace pour prévenir les hospitalisations pour GERV, particulièrement chez les groupes plus aisés. D’autres études en contexte similaire sont nécessaires pour déterminer les facteurs reliés à une plus faible EV chez les groupes vulnérables. / Abstract: Introduction: Rotavirus is the main cause of acute gastroenteritis (AGE) among young children worldwide. In 2011, the monovalent rotavirus vaccine (RV1) was introduced into the Quebec universal immunization program to reduce morbidity related to rotavirus gastroenteritis (RVGE). This thesis aimed to examine AGE and RVGE hospitalization rates before and after implementation of the program in young children from the Eastern Townships (impact study) and the vaccine coverage, and to assess vaccine effectiveness (VE) of the RV1 (effectiveness study). Methods: The pairing of a tertiary hospital database with the regional immunization registry allowed to extract a cohort of children born at the Centre hospitalier universitaire de Sherbrooke (CHUS), living in Eastern Townships and aged less than five years during the study period from June 2004 to May 2014 (n= 37,757). This cohort was retrospectively followed-up to examine AGE and RVGE annual hospitalization rates of pre- (2004/2005-2010/2011) and post-program years (2011/2012-2013/2014), globally and according to several socioeconomic characteristics. Moreover, RV1 VE was calculated using three children cohorts: (1) vaccinated children born in 2011-2013 (n=5,033), (2) unvaccinated children born in 2011-2013 (n=1,239), and (3) unvaccinated children born in 2008-2010 (n=6,436). Results: AGE hospitalization rates evolved from 81/10,000 children aged less than five years in pre-program period to 46/10,000 in post-program period (relative reduction=43%, p<0.001). Following implementation of the program, vaccine coverage rapidly increased to reach 81%. Despite similar vaccine coverage among different groups, lowest relative reductions were observed in disadvantaged groups. Adjusted VE of a complete series was 62% (95% confidence interval [CI]: 37%-77%) and 94% (95% CI: 52%-99%) against AGE and RVGE hospitalizations, respectively. Children living in neighbourhoods with higher rates of low-income families had lower VE against AGE hospitalizations (30% vs. 78%, p=0.027). Conclusion: Three years following its introduction into the universal vaccination program, RV1 significantly reduced severe gastroenteritis in young children in the Eastern Townships. This vaccine was highly effective to prevent RVGE hospitalizations, particularly among the most well-off. Further studies in similar setting are needed to determine factors related to lower VE among vulnerable groups.

La gastroenteritis ocasionada por rotavirus se encuentra entre las principales causas de morbilidad y mortalidad infantil. En el Perú rotavirus representa el 30% de las muertes por diarrea y 4% del total de defunciones, afectando principalmente a niños menores de 5 años. Desde el año 2009 el Perú implementó la vacuna contra rotavirus en su programa de vacunación universal, sin embargo, debido a la aparición de genotipos emergentes, es importante monitorear la diversidad del virus para evaluar los posibles efectos sobre la eficacia de la vacuna. Pese a esto, existen escasos reportes de la epidemiología de rotavirus en nuestro país. El objetivo del presente estudio es determinar la presencia y los genotipos G y P de Rotavirus en muestras provenientes de niños con gastroenteritis aguda atendidos en un hospital de Lima entre Octubre del 2013 y Octubre del 2014. Como metodología se utilizó el RT-PCR en tiempo real (qRT- PCR) para la detección del rotavirus y el RT PCR convencional para la genotipificación de las muestras positivas, asimismo se secuenciaron algunas muestras para determinar los genotipos finales. De las 448 muestras analizadas en el estudio, 45 (10%) tuvieron resultado positivo para rotavirus. Entre los genotipos identificados, G12 (40.9%), G2 (25%) y P[8] (77.3%) fueron los más frecuentes y la combinación G/P más dominante fue G12P[8] (54.5%). Los resultados evidenciaron una alta prevalencia de G12P[8]entre los casos positivos, genotipo no reportado previamente en el Perú. Se recomienda realizar nuevos estudios para evaluar las variaciones en la diversidad de rotavirus circulantes en el Perú y los posibles efectos ante la presión selectiva de la vacuna.Rotavirus gastroenteritis is one of the leading causes of morbidity and mortality in children. In Peru rotavirus represents 30% of deaths due to diarrhea and is responsible of 4% of the total deaths, affecting mostly children under 5 years. Peru implemented the rotavirus vaccine in its universal vaccination program since 2009. Due to the appearance of emerging genotypes, it is important to evaluate the diversity of the virus in order to determine possible effects on vaccine efficacy. However there are few reports on the molecular epidemiology of rotavirus in our country. The aim of this study was to determine the presence and Rotavirus G and P genotypes in samples from children with acute gastroenteritis attended in a hospital in Lima between October 2013 and October 2014. The method usedfor the detection of rotavirus was real time RT-PCR (qPCR) and conventional RT-PCR to determine the genotype of positive samples. Additionally, sequencing was used to confirm the final genotype of some samples. Of the 448 samples analyzed, 45 (10%) were positive for rotavirus.G12 (40.9%), G2 (25%) and P[8] (77.3%) were the most frequent genotypes and the most prevalent G/P combination was G12P[8] (54.5%). The results showed a high prevalence of G12P[8] among the positive cases. G12P[8] has not been previously reported in Peru. We recommend more in deep studies to identify the diversity of circulating genotypes in Peru.

Gastrenterites virais em cães são doenças transmissíveis infecciosas com importância para a saúde animal, como as causadas por Parvovírus canino e Coronavírus canino (CCoV) e saúde pública, como no caso dos rotavírus. Rotavírus em cães são encontrados com baixa freqüência, tanto em cães com diarréia quanto sadios, mas sua importância como reservatório para a rotavirose humana já é conhecido. O CCoV, pertencente ao grupo 1 do gênero Coronavirus, ocorre sob a forma dos genótipos I e II, amplamente distribuídos mundialmente e implicados em diarréia moderada, mas podendo levar a elevada letalidade no caso de patótipos altamente patogênicos. No Brasil, a ocorrência de rotavírus do sorogrupo A em cães é um fato conhecido, mas, no caso do CCoV, existe, até o momento, apenas uma investigação relatando sua ocorrência, sem dados de diversidade molecular. A presente investigação teve por objetivos avaliar o papel de cães jovens com enterite sintomática, bem como sadios, como reservatórios de rotavírus, estudar a freqüência de ocorrência de Coronavírus canino (CCoV) em amostras fecais destes animais e estudar a diversidade molecular das amostras de CCoV encontradas. Para tato foram colhidas 100 amostras fecais de cães não vacinados, entre 1 e 180 dias de idade entre 2007 e 2008, sendo 50 com diarréia e 50 sem diarréia no momento da colheita, nos Municípios de São Paulo, São Bernardo do Campo, Santo André, São Caetano do Sul, Taboão da Serra, Itapecerica da Serra e uma aldeia indígena em Parelheiros. Às amostras foi aplicada a técnica de eletroforese em gel de poliacrilamida (PAGE) para a detecção de rotavírus e uma RT-PCR dirigida ao gene da proteína de membrana M do CCoV (nucleotídeos 337 a 746) para a detecção deste vírus, sendo os fragmentos detectados submetidos a seqüenciamento de DNA. As seqüencias obtidas, traduzidas em aminoácidos, foram utilizadas para a construção de uma árvore genealógica enraizada de distância com o algoritmo Neighbor-Joning e modelo de Poisson com 100 repetições de bootstrap. Nenhuma das amostras resultou positiva para rotavirus, enquanto que 47 foram positivas para CCoV, com freqüência significativamente superior nos animais com diarréia. Vinte e dois dos 47 fragmentos de DNA obtidos resultaram em seqüências viáveis de DNA, sendo 12 classificadas como CCoV Tipo I e 10 como Tipo II, tendo sido encontrada uma sublinhagem exclusivamente brasileira para o Tipo II. Em relação à amostra vacinal de CCoV submetida ao seqüenciamento de DNA, a maior identidade ocorreu com o grupo Tipo II sublinhagem 01, com um valor de 100%, seguido de 97,2% para o Tipo II sublinhagem 02 (a linhagem brasileira) e 93,2% para o Tipo I. Sugere-se que a diversidade de CCoV encontrada seja derivada da elevada freqüência de ocorrência deste vírus, o que pode aumentar a probabilidade de divergências e de possíveis falhas vacinais por diferenças entre a amostra vacinal (Tipo II) e as amostras de campo (Tipos I e II), e, dessa forma, a vacina não diminuiria a transmissão e novas linhagens de CCoV emergiriam. Conclui-se que cães jovens com enterite sintomática, bem como sadios, não tiveram papel como reservatório para rotavírus, considerando-se a região geográfica e o período de colheita de amostras. O CCoV ocorreu com uma freqüência de 47% na população canina estudada, com freqüência estatisticamente significativamente superior naqueles com diarréia do que naqueles sem diarréia. Finalmente, amostras brasileiras de CCoV, com base em seqüenciamento parcial do gene codificador da proteína de membrana M, ocorrem tanto como tipo I quanto II, sendo que, para o tipo II, há uma lihangem tipicamente brasileira. / Viral canine gastroenteritis is infectious transmissible diseases with importance for animal health, as those caused by Canine parvovirus and Canine coronavirus (CCoV) and public health, as in the case of rotavirus. Canine rotavirus occurs at low frequencies, both in diarrheic and health dogs, but the importance of dog as reservoirs for human rotaviruses is known. CCoV belongs to group 1 of the genus Coronavirus and occurs as genotypes I and II, worldwide distributed and implicated in mild diarrhea, but high pathogenic types might lead to high lethality. In Brazil, the occurrence of serogroup A rotavirus in dogs is already known but, in the case of CCoV, theres a single report on the occurrence of this virus, with no data on its molecular diversity. The aims of the present investigation were to evaluate the roles of diarrheic and health young dogs as reservoirs of rotavirus, to study the occurrence of CCoV in these animals and to assess the molecular diversity of the strains found. One hundred fecal samples were collected from unvaccinated dogs between 2007 and 2008 (50 with diarrhea and 50 health dogs) in the Municipalities of São Paulo, São Bernardo do Campo, Santo André, São Caetano do Sul, Taboão da Serra, Itapecerica da Serra and in an indian community in Parelheiros. The samples were submitted to polyacrilamide gel electrophoresis (PAGE) for rotavirus detection and to an RT-PCR targeted to the membrane M protein gene (nucleotides 337 to 746) of CCoV for the detection of this virus; amplicons were then submitted to DNA sequencing and the putative amino acids sequences were used to build a rooted distance genealogic tree with the Neighbor-Joinng algorithm and he Poisson correction with 1,000 bootstrap replicates. No sample was positive to rotavirus, while 47 out of the 100 samples were positive for CCoV, with a statistically significative higher frequency for the dogs with diarrhea. Twenty-two out of the 47 ampicons resulted in viable sequences, being 12 classified as CCoV Type II and 10 as Type I; besides, and exclusively Brazilian sub lineage was found for Type II. Regarding the vaccine strain, the highest identity was found to Type II sub linage 02 (10%), followed by 97.2% for Type II sub linage II (the Brazilian sub linage) and 93.2% for Type I. Its suggested that the high diversity for CCoV detected is a consequence of the high frequency of occurrence of this virus, what might increase the probability of the emergence of divergence and possible vaccine failures due to differences amongst the vaccine strain (Type II) and field strains (Types I and II) and thus vaccination would not decrease the transmission and new lineages would emerge. It can be concluded that both health and diarrheic young dogs have played no role as reservoirs for rotavirus taking into account the geographic area and the time of samples collection. CCoV ocurred at a frequecy of 47%, with a higher frequency in the diarreic animals. Finally, Brazilian strains of CCoV, based on partial M gene sequences, occur as both type I and II, while, for Type II, a typical Brazilian lineage was described.

O coronavírus bovino (BCoV) e rotavírus (RV) são importantes agentes da diarréia neonatal bovina, causando grandes perdas econômicas. O BCoV é também responsável pela disenteria de inverno em vacas adultas e por desordens respiratórias e o RV possui um aspecto zoonótico de importância na saúde pública. Este estudo teve o objetivo de desenvolver uma multiplex hemi-nested RT-PCR para detecção simultânea de BCoV e RV do grupo A com a utilização de um controle interno. Para tanto, foram desenhados três primers dirigidos ao gene N de BCoV (306pb) e três primers dirigidos ao gene VP1 do RV do grupo A (228pb); para o controle interno, foram utilizados dois primers dirigidos ao mRNA do gene mitocondrial bovino ND5 (191pb). Foram utilizados como controles positivos a amostra Kakegawa de BCoV (título HA de 256), a amostra 8209 de rotavírus do grupo A (título viral de 101.66TCID50%/200µL) e suspensão de células MDBK para o controle interno. A extração de RNA foi realizada com o reagente comercial TRIzol, de acordo com as recomendações do fabricante. Foram realizados gradientes de temperatura para a determinação da temperatura ótima de hibridação para cada par de primers, resultando em 50ºC para PCR e 55ºC para a hemi-nested. Doze protocolos com diferentes concentrações de Taq DNA polymerase, MgCl2, primers e DNA-alvo foram testados. Para determinação do limiar de detecção, o protocolo final foi utilizado em diluições dos dois vírus em suspensão de amostras fecais negativas para BCoV e RV adicionadas de 10% de suspensão de células MDBK, obtendo o limiar de detecção de 10-8 para os dois vírus e aplicado em amostras fecais de bezerros (53) e vacas adultas (22). Todas as amostras foram previamente testadas para BCoV por uma nested RT-PCR dirigida ao gene RdPd, sendo 15 positivas, e para rotavírus pela PAGE, sendo 3 positivas. Para a multiplex, 15 amostras foram positivas para BCoV e 6 para rotavírus. O seqüenciamento de DNA das amostras positivas para multiplex demonstrou a especificidade do teste. Uma concordância ótima foi encontrada para BCoV (0,833) e substancial para rotavírus (0,648) calculada pela estatística kappa, comparando-se com os testes de referência. Estes resultados demonstram que a padronização da multiplex foi eficiente para a detecção de BCoV e RV, com elevadas sensibilidade e especificidade. Além disso, o diagnóstico simultâneo dos dois agentes permite um diagnóstico rápido e a um menor custo devido à utilização de reduzidas quantidades de reagentes e mão de obra, fornecendo uma importante ferramenta para o diagnóstico e estudo da etiologia da diarréia em bovinos. / Bovine coronavirus (BCoV) and rotavirus (RV) are important agents of neonatal diarrhea in cattle, causing large economic losses. BCoV is also responsible for winter dysentery in adult cattle and respiratory disorders and RV has a zoonotic aspect of importance in public health. This study aimed to develop a multiplex hemi-nested RT-PCR for simultaneous detection of BCoV and RV of group A with an internal control. For this purpose, three primers were designed targeting the N gene of BCoV (306pb) and three primers targeting to the VP1 gene of group A RV (228pb); for internal control, two primers targeting to the mRNA of ND5 bovine mitochondrial gene (191pb) were used. Positive controls were BCoV Kakegawa strain (HA titer 256), group A bovine rotavirus strain 8209 (viral titer of 101.66TCID50%/200µL), and MDBK cells suspension for internal control. The RNA extraction was performed with the commercial reagent TRIzol. Temperature gradients were carried out for each primers pair for the determination of the optimal hybridization temperatures, resulting in 50°C for PCR and 55°C for the hemi-nested. Twelve protocols were tested with different concentrations of Taq DNA polymerase, MgCl2, primers and target DNA. The final protocol was used in 10-fold dilutions of the two viruses in suspension of fecal sample negative for BCoV and RV added to 10% of MDBK cells suspension, obtaining the detection limit of 10-8 for the two viruses, and applied to 75 fecal samples from calves (53) and cows (22). All samples were previously tested for BCoV by a nested RT-PCR to RdPd gene, being 15 positive; and for rotavirus by PAGE, being 3 positive. For multiplex, 15 samples were positive for BCoV and 6 for RV. The DNA sequencing of multiplex-positive samples substantiates the specificity of the test. A great agreement was found for BCoV (0.833) and substantial for RV (0.648) by calculating the kappa statistic in comparison to the reference tests. These results demonstrate that the multiplex standard was effective in the detection of BCoV and RV, with high sensitivity and specificity. In addition, simultaneous diagnosis of both agents allows a faster and at lower cost diagnosis due to use of smaller quantities of reagents and labor, providing an important tool for the diagnosis and study on the etiology of diarrhea in cattle.

As diarréias neonatais constituem-se em um dos mais importantes fatores econômico e sanitário nas granjas suínas, quer pela mortalidade, quer pelas perdas agregadas ao atraso no desenvolvimento dos leitões, à profilaxia e ao manejo. Os rotavírus ocupam lugar de destaque pela rápida disseminação dentro do plantel, bem como pelo potencial zoonótico, dada a probabilidade de rearranjo ou recombinação genética entre amostras humanas e animais O objetivo deste trabalho foi detectar a presença de rotavírus a partir de 277 amostras fecais de leitões com quadro clínico de diarréia, provenientes dos Estados do Rio Grande do Sul (RS), Santa Catarina (SC), Paraná (PR), Mato Grosso do Sul (MS), Mato Grosso (MT), São Paulo (SP), Rio de Janeiro (RJ) e Minas Gerais (MG) entre os anos de 2009 e 2011 e analisar o perfil eletroforético de migração dos segmentos genômicos bem como diferenças de eletroferótipos nas amostras positivas, pela técnica de eletroforese em gel de poliacrilamida (PAGE). Das 277 amostras fecais diarréicas de leitões analisadas, 25 foram positivas (25/277= 9%). De conformidade com os Estados de origem, foram verificadas as frequências de 20% (1/5) no RS, 11,1% (1/9) em SC, 12,5% (1/8) no PR, 15,3% (6/39) em MS, 14,2% (3/21) em MT, 6,7% (5/74) em SP, 0% (0/7) no RJ e 7% (8/114) em MG. Pela análise da migração eletroforética dos segmentos genômicos, todas as 25 amostras positivas apresentaram perfil eletroforético compatível com o RV-A, tal como a amostra padrão NCDV com migração característica em quatro classes ou agregados [4-2-3- 2]. Foram observadas pequenas diferenças na velocidade de migração de um ou mais segmentos dentro da mesma classe. Estes resultados evidenciam a importância da PAGE como metodologia de diagnóstico e de investigações epidemiológicas nas rotaviroses suínas. / The neonatal diarrhea constitute one of the most important economic and health factors in pig farms either by death or by the aggregate losses to the delay in the development of piglets, prophylaxis and management. Rotaviruses have a prominent role by the rapid spread within the herd, as well as the zoonotic potential, given the likelihood of genetic reassortment or recombination between human and animal samples. The objective of this study was to detect the presence of rotavirus from fecal samples from 277 piglets with clinical cases of diarrhea, from the states of Rio Grande do Sul (RS), Santa Catarina (SC), Paraná (PR), Mato Grosso do Sul (MS), Mato Grosso (MT), São Paulo (SP), Rio de Janeiro (RJ) e Minas Gerais (MG) between the years 2009 and 2011 and to analyze the electrophoretic migration of genomic segments and electropherotype differences in positive samples, by polyacrylamide gel electrophoresis (PAGE) technique. From 277 piglets diarrheal stool samples analyzed, 25 were positive (25/277 = 9%). In accordance with the States of origin, were observed frequencies of 20% (1/5) in the RS, 11.1% (1/9) in SC, 12.5% (1/8) in PR, 15.3 % (6/39) in MS, 14.2% (3/21) in MT, 6.7% (5/74) in SP, 0% (0/7) in Rio de Janeiro and 7% (8/114) in MG. For the analysis of the electrophoretic migration of genome segments, all 25 positive samples showed electrophoretic profile compatible with the RV-A as a standard sample NCDV, showing characteristic fourth class or aggregates [4-2-3-2]. We observed small differences in the migration speed of one or more segments within the same class. These results highlight the importance of PAGE as a method of diagnosis and epidemiological investigations in the porcine rotavirus.