Abstract Background: Gastroenteritis is a common infection and the leading cause of morbidity worldwide and is mostly caused by viruses. Outbreaks appear in both developed and developing countries and result in large economic costs. Rapid detection is important for appropriate treatment, control and to prevent the spread of infection.  Objective: Evaluation and performance comparison between the BioFire®FilmArray® Torch System gastrointestinal panel and the Molecular BD MAXTMenteric viral panel to indicate a multiplex method for viral gastroenteritis diagnostic in a routine laboratory setting.  Material and methods: In this study, 58 different samples were used which consisted of selected stool specimens from patients who were tested and treated for gastroenteritis infection at Uppsala Academic Hospital and Norrlands University Hospital in Umeå during 2018-2021, samples from Quality control for molecular diagnostics viral gastroenteritis EQA pilot study during 2018-2019 and cultivated strains of different adenovirus species from 2018. All samples were analyzed with both systems for comparison of detected pathogens.  Results: Sensitivity and specificity values were 95% and 100% respectively for the BioFire®FilmArray®Torch System and 100% and 93.3% for the BD MAXTMSystem.   Conclusions: Bothsystems are rapid and adequate diagnostic tools. The BioFire®FilmArray®Torch System with greater coverage has the ability of detecting more pathogens and is more promising particularly in the occasional infection circumstance. The BD MAXTMSystem demonstrated almost the same results and seems to be a better option in times of an outbreak when the numbers of patients are significantly higher.

No description available.

No description available.

Mucosal passive immunization is the transfer of active antibodies from one organism to the mucosal surfaces of another organism for preventing or treating infectious diseases. Mucosal passive immunization has a great potential for the prevention and treatment of enteric infections like Rotavirus, which causes more than 114 million episodes of diarrhoea annually with a death toll of more than 450.000 per year. However, the high cost of recombinant antibodies with the current manufacturing systems based on mammalian cells hampers the production of the high antibody quantities required for passive immunization strategies. Alternative expression platforms such as plants could provide higher scalability and reduced costs. Moreover, the use of edible plant organs, which are Generally¿Regarded¿As¿ Safe (GRAS), could reduce manufacturing costs even further by easing the requirements for antibody purification. We analyze here the feasibility of utilizing fruits as inexpensive biofactories of human antibodies that can be orally delivered as crude extracts or partially purified formulations in mucosal passive immunization strategies. In the first section of this thesis, the construction of tomato plants producing a model human Immunoglobulin A (IgA) against rotavirus in their fruits is described. As a result, an elite homozygous line was obtained whose fruits produced on average 41 ¿g of IgA per gram of fresh weigh, equivalent to 0.69 mg IgA per gram of dry tomato powder. Minimally processed products derived from IgA¿expressing tomatoes were shown to strongly inhibit virus infection in an in vitro neutralization assay. Moreover, in order to make IgA¿expressing tomatoes easily distinguishable from wild¿type tomatoes, they were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. The resulting transgenically¿labelled purple tomatoes contained not only high levels of recombinant neutralizing human IgA but also increased amounts of anthocyanins. In the second section of the thesis the composition of IgA¿expressing tomatoes was analyzed in search of possible unintended effects that could compromise the GRAS status of the final product. To this end, transgenic IgA¿tomatoes were compared with wild type tomatoes and also commercial tomato varieties using proteomic and metabolomic approaches. 2D¿DIGE gels coupled with LC¿MSMS for protein identification showed that all the uptrend differential proteins detected corresponded only to immunoglobulin chains or antibody fragments. On the other hand, non¿targeted metabolite data obtained by UPLC¿MS / Juárez Ortega, P. (2014). Production of recombinant Immunoglobulin A in plants for passive immunotherapy [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37015

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

No description available.

This thesis is concerned with the level of parents´ awareness and attitudes to above-standard vaccination of children from 0-3 years in Příbram. Among the optional vaccination of children from 0-3 years belong vaccination against pneumococcal disease, rotavirus infections, meningococcal invasive disease (caused by meningococcal of group C, A+C, or A, C, W 135 and Y, now also of group B), tick-borne encephalitis, varicella smallpox, influenza and hepatitis A (or a combination of type A + B). The first part deals with the issue in a theoretical perspective. It foreshadows the basic characteristics of the diseases against which the premium vaccination of children from 0-3 years is offered. Further it is focused on vaccines against these diseases, which can occur in the Czech Republic. The survey is summarized in the research. There were parents of children attending one of the 12 kindergartens in Příbram in the sample of the research. tely 40% of the parents (or mothers) of these children in each kindergarten.The results were summarized in schedules of absolute and relative frequencies, or graphs. It was set three basic goals that were met with five hypotheses. The first of these was to monitor the attitudes of parents to above-standard vaccination of children from 0-3 years in Příbram. To this target relate hypothesis H1: Parents are interested in above-standard vaccination of children from 0-3 years in Příbram, H2: Parents with higher educational level have significantly higher interest in above-standard vaccination of children from 0-3 years and H3: Parents are statistically significantly more interested in vaccination against tick-borne encephalitis than other extra vaccination of children from 0-3 years, due to an endemic area of tick-borne encephalitis in Příbram. These hypotheses were not confirmed statistically. The second aim investigated the main reason for the possible lack of interest in extra vaccination of children from 0-3 years in Příbram. The target was filled with hypothesis H4: The main reason for the possible lack of parents´ interest in extra vaccination of children from 0-3 years is high price of vaccines, which was statistically refuted. The third goal was to explore parents' knowledge about diseases against which the extra vaccination of children from 0-3 years is offered. To this goal was set the hypothesis H5: Parents have sufficient information about the diseases against which the extra vaccination of children from 0-3 years is offered. This hypothesis was confirmed. The hypotheses were verified by ?chi-square? test at a significance level of 5%, which is an instrument of verification or falsification of hypotheses. This work may be used in practice as a preview to the parents´ awareness and interest in extra vaccination of children from 0-3 years. As well as cumulative information materials for professionals and the public about the above-standard vaccination of children from 0-3 years.

Le Canada est l’un des plus grands pays exportateurs de porc du monde et le Québec à lui seul compte pour 6% de ce commerce mondial. Pour conserver sa compétitivité et l’excellence de ses produits, une connaissance approfondie des agents infectieux circulant au sein des troupeaux est primordiale. Plusieurs pathogènes sont peu étudiés et pourtant retrouvés chez les porcs à travers la planète. Cette étude avait pour but l’évaluation de la prévalence des astrovirus porcins, calicivirus, kobuvirus porcin, rotavirus, torque teno sus virus ainsi que le virus de l’hépatite E lors du suivi de porcelets dans un réseau de production porcine, de la maternité jusqu’en fin d’engraissement. Nous voulions brosser un portrait de l’excrétion de ces virus à travers les différentes étapes de production des animaux. L’échantillonnage de porcelets sains et en diarrhée en pré-sevrage a permis de déterminer lesquelles de ces infections virales constituaient des facteurs de risque pour la diarrhée à ce stade de production. Le virome intestinal, la partie virale du microbiome, a également été caractérisé permettant de connaître la diversité des virus entériques porcins aux différentes étapes de production, ainsi qu’entre les porcelets sains et en diarrhée. De plus, la dissémination du virus de l’hépatite E dans l’environnement ainsi que les sources probables de contamination ont été décrites à l’aide d’échantillons provenant des environnements intérieurs et extérieurs de fermes d’engraissement, de la cour d’un abattoir et de transporteurs d’animaux. Les résultats obtenus ont permis de décrire les dynamiques temporelles d’excrétion de ces virus entériques porcins en fonction des stades de production des porcs, démontrant une différence dans l’excrétion de ces virus en fonction de l’âge. Les calicivirus, ainsi que les astrovirus porcins groupes 3 et 5 étaient des facteurs de risque de diarrhée en maternité. Pour la première fois au Canada, la détection et la caractérisation des souches du kobuvirus porcin ont été réalisées, permettant de mieux comprendre leur diversité et leur persistance à travers les stades de production. La diversité du virome entérique porcin a été analysée avec la plateforme de séquençage MiSeq et cette diversité était différente entre les porcelets sains et en diarrhée, ainsi qu’entre les stades de production. Cependant, les traitements enzymatiques utilisés pour le prétraitement des échantillons fécaux ne permettaient pas le séquençage de certains virus à ARN simple-brin. Des souches similaires du virus de l’hépatite E étaient présentes dans l’environnement des fermes, ainsi qu’aux endroits communs à forte circulation des intervenants du réseau. Les activités dans la cour de l’abattoir pourraient donc être impliquées dans la dissémination de ce virus. Cette étude a permis de mieux connaitre la prévalence et la distribution des virus entériques infectant les porcs. De plus, certains des virus entériques étudiés ont été reconnus comme facteurs de risque de la diarrhée en co-infections et devront être étudiés en détail pour comprendre leurs mécanismes en relation avec la diarrhée néonatale. Des interventions plus spécifiques lors d’éclosion de diarrhées porcines, dont l’étiologie est inconnue, pourront donc être réalisées, ainsi que l’élaboration de mesures de biosécurité plus adaptées en fonction du stade de production des porcs. / Canada is a major pork exporter around the world and the province of Quebec alone accounts for 6% of this trade. To maintain the province’s competitiveness and the excellence of its products, a comprehensive understanding of the infectious agents circulating in herds is essential. Several pathogens have been intensively studied, while others have yet to be investigated even though they have been reported in pigs all around the world. This study evaluated the prevalence of porcine astroviruses, calicivirus, porcine kobuvirus, rotavirus, torque teno sus virus and hepatitis E virus, monitored in a pig production network, from the nursing farms to the end of the fattening farms to portray the excretion patterns of these viruses through the different life stages of pigs. The sampling of healthy piglets alongside piglets with diarrhea in the nursing farms allowed to determine which viruses, or co-infections of viruses were factors of diarrhea at this life stage. The intestinal virome, the viral part of the microbiome, was characterized and viral diversity of porcine enteric viruses at different life stages, as well as between healthy and diarrheic piglets were evaluated. Moreover, the dissemination of the hepatitis E virus in the farm environment, as well as the possible sources of contamination were described, from the indoor and outdoor environment of fattening farms, the slaughterhouse yard and animal transporters. The results obtained in this study described the temporal excretion dynamics of these porcine enteric viruses according to the life stages of the pigs, demonstrating the difference in the excretion of the studied viruses according to the life stage. The calicivirus, as well as the porcine astrovirus groups 3 and 5 were found to be risk factors for diarrhea in the nursing farms. For the first time in Canada, the detection and characterization of porcine kobuvirus strains were evaluated and provided a better understanding of their diversity and the persistence of these strains in the network. The porcine enteric virome diversity was analyzed on a MiSeq sequencing platform and this diversity was different between healthy and diarrheic piglets, as well as between the different life stages. However, the different enzymatic treatments used as pretreatments for fecal samples altered the ability to detect certain single-stranded RNA viruses. Similar strains of the hepatitis E virus were present in the indoor and outdoor environment of the fattening farms, as well as in common places of high circulation from the various stakeholders in the pig production network. The activities in the slaughterhouse yard could therefore be involved in the spread of this virus. This study shed light on enteric viruses infecting pigs. In addition, some of the infections from enteric viruses studied were risk factors for diarrhea in co-infections and will need to be studied in more details to understand their mechanisms, in relation to neonatal diarrhea. More specific interventions during outbreaks of porcine diarrhea of unknown etiology could be carried out, as well as the development of more adapted biosecurity measures according to the life stage of the pigs.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-10-23T16:32:24Z No. of bitstreams: 1 carmen_b_vieira_ioc_bcm_0002_2010.pdf: 1656859 bytes, checksum: 62b8e8b68ef938a3f60ee88a24b5056c (MD5) / Made available in DSpace on 2012-10-23T16:32:24Z (GMT). No. of bitstreams: 1 carmen_b_vieira_ioc_bcm_0002_2010.pdf: 1656859 bytes, checksum: 62b8e8b68ef938a3f60ee88a24b5056c (MD5) Previous issue date: 2010 / Dra. Ana Maria Coimbra Gaspar / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O aumento das atividades recreacionais na água tem contribuído para a transmissão de doenças de veiculação hídrica. Os parâmetros de balneabilidade avaliados para exposição humana em águas naturais incluem indicadores bacteriológicos. Entretanto, a presença de vírus nestas águas não é considerada. Neste contexto, os vírus excretados nas fezes de pessoas infectadas são importantes contaminantes de águas superficiais em função do contínuo despejo de esgoto doméstico. Este estudo tem como objetivo avaliar a contaminação pelos principais agentes etiológicos da gastroenterite viral aguda, rotavírus grupo A (RV-A) e norovírus (NV), em águas superficiais da Lagoa Rodrigo de Freitas pela detecção, quantificação e caracterização molecular destes agentes, correlacionando-os com parâmetros microbiológicos e físico-químicos de qualidade da água. A Lagoa é um ponto turístico e de lazer de grande expressão na cidade do Rio de Janeiro, tem sua água classificada como água de recreação de contato primário e, atualmente, está passando por um programa de despoluição para reverter o seu estado de degradação ambiental. Entre Agosto de 2007 e Julho de 2008, 2L de água superficial foram coletados mensalmente em 12 pontos, incluindo 10 pontos na Lagoa Rodrigo de Freitas, um no Rio dos Macacos, que desemboca na Lagoa, e um na praia do Leblon, onde a água da Lagoa é escoada, totalizando 144 amostras. As amostras foram concentradas 1000X pelo método de adsorção-eluição utilizando uma membrana carregada negativamente e reconcentradas a uma volume final de 2mL em Centriprep®YM-50. RV-A e NV foram detectados e quantificados pelas técnicas de reação em cadeia pela polimerase convencional e quantitativa (cPCR / qPCR) precedidas por transcrição reversa (RT). Pela análise conjunta destas metodologias, RV-A e NV-GII foram detectados em 24,3% (35/144) e 18,8% (27/144) das amostras estudadas, respectivamente. A quantificação de RV-A e NV- GII variou de 3,34 a 4680 cg/100mL e 1,57 a 26,5 cg/100mL, respectivamente. As amostras positivas de RV-A foram caracterizadas pelo sequenciamento parcial do segmento 6 (VP6) como subgrupo I e genótipo I2 segundo a nova classificação proposta para estes vírus. E.coli foi quantificada por Kit Colilert-18Kit Quanti-Tray®/ 2000 em cada ponto de coleta como um indicador bacteriológico de contaminação fecal e 87,5% (126/144) das amostras de água foram caracterizadas como próprias conforme estabelecido pela legislação vigente. RV-A e/ou NV-GII foram detectados em 38,1% (48/126) dessas amostras, evidenciando a presença de vírus em águas que estão dentro dos padrões aceitáveis para E.coli, não sendo observada a associação entre este parâmetro e a detecção destes vírus. Adenovirus humano (HuAdV) foram pesquisados como possíveis marcadores virais de contaminação fecal humana, sendo detectados em 16,7% (24/144) das amostras, apresentando distribuição não homogênea em relação aos resultados de E.coli, RV-A e NV-GII. Parâmetros físico-químicos como salinidade, temperatura, pH, cloro e turbidez foram determinados, sendo demonstrada uma distribuição não homogênea entre RV-A e turbidez e NV-GII e pH. Os dados obtidos neste estudo enfatizam a necessidade do estabelecimento de parâmetros virais para a avaliação da qualidade da água e a necessidade de se disponibilizar protocolos de detecção viral que auxiliem para a adoção de medidas de controle de contaminação ambiental pelas autoridades Municipal e Estadual. / The increase of recreational activities in water has contributed to the transmission of waterborne diseases. The bathing parameters evaluated for human exposure in natural waters include bacteriological criteria. However the presence of virus is not considered. In this context, viruses excreted in feces of infected people are important contaminants of surface water due to the continuous discharge of domestic sewage.This study aims to evaluate the contamination by the main etiologic agents of viral acute gastroenteritis, group A rotavirus (RV-A) and norovirus (NV), in surface waters of the Rodrigo de Freitas Lagoon by detection, quantification and molecular characterization of these agents, correlating with the microbiological and physico- chemical standards for water quality. From August 2007 to July 2008, 2L of surface water were monthly collected at 12 sites, including 10 sites in the Rodrigo de Freitas Lagoon, one in the Macacos River, which flows into Lagoon, and one at Leblon Beach, where water Lagoon is drained, totalizing 144 samples. The samples were concentrated 1000X by an adsorption-elution method using a negatively charged membrane and reconcentrated to a final volume of 2mL in Centriprep ®YM-50 concentrator. RV-A and NV were detected and quantified by conventional and quantitative polymerase chain reaction (cPCR/qPCR) preceded by reverse transcription (RT). For the joint analysis of these methodologies RV-A and NV-GII were detected in 24,3% (35/144) and 18,8% (27/144) of the studied samples, respectively. Quantification of RV-A and NV-GII ranged from 3,34 to 4680 cg/100mL and 1,57 to 26,5 cg/100mL, respectively. The RV-A positive samples were characterized by partial sequencing of segment 6 (VP6) as subgroup I and I2 genotype according to the new classification proposed. E.coli was quantified using Colilert®-18Kit Quanti-Tray®/2000 in each collection site as bacterial standard of fecal contamination and 87.5% (126/144) of water samples analyzed were characterized as suitable for bath as established by legislation. RV-A and/or NV-GII were detected in 38,1% (48/126) of these samples, showing the presence of viruses in waters that are within the standards for acceptable E.coli and there was no association between this parameter and viral detection. Human adenoviruses (HuAdV) were investigated as possible viral markers of human fecal contamination and were detected in 16,7% (21/144) of the samples showing non-homogeneous distribution in relation to the results of E. coli, RV-A and NV-GII. Physico-chemical parameters, like salinity, temperature, pH, chlorine and turbidity, were determined in loco. It was demonstrated a non-homogeneous distribution of positive and negative samples between RV-A and turbidity and NV-GII and pH. Data obtained in this study emphasize the need for the establishment of viral parameters for the assessment of water quality and the need to provide viral detection protocols that lead to the adoption of measures to control environmental contamination by municipal and state authorities