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Pesquisa do vírus da raiva em quirópteros no Estado de Roraima pelo método de RT-PCRJames Rodrigues de Souza 31 August 2011 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A raiva é uma enfermidade infectocontagiosa causada por um Lyssavirus, que acomete os mamíferos, inclusive o homem, está presente em todos os continentes com exceção da Antártida. Os cães ainda são considerados os principais responsáveis pela manutenção e transmissão da raiva para o homem. Porém, nos últimos anos os morcegos hematófagos e não hematófagos têm ganhado destaque como potenciais transmissores de raiva para animais e humanos nas Américas. Em 2010, o Brasil registrou três casos de raiva humana, sendo um causado por agressão de morcego. Recentemente, várias epidemias de raiva humana transmitida por morcegos hematófagos foram relatados na região Amazônica. No estado de Roraima até a presente data não há registro de casos de raiva humana. O presente estudo teve o objetivo de detectar a presença e circulação do vírus rábico em quirópteros no estado de Roraima, bem como identificar as espécies de morcegos envolvidas na pesquisa. A técnica Transcriptase Reversa seguida da reação em Cadeia pela Polimerase foi utilizada para a detecção do vírus rábico, utilizando tecido cerebral de morcegos que foram coletados pelas equipes de vigilância epidemiológica e ambiental, da Secretaria de Saúde e Agência de Defesa Sanitária de Roraima. Os morcegos foram identificados utilizando chaves dicotômicas disponíveis para morcegos do Brasil e de outros países sul americanos. No total foram analisadas 94 amostras de morcegos, as quais apresentaram resultados negativos para raiva pela técnica da RT-PCR, no entanto, não é possível afirmar que o vírus rábico não circule em Roraima. Por outro lado, o presente estudo identificou 19 espécies de morcegos distribuídas em seis famílias, sendo uma família (Vespertilionidae) e cinco espécies de quirópteros (Diaemus youngi, Noctilio albiventris, Myotis nigricans, Eptesicus diminutus e Cynomops planirostris) ainda sem relato de ocorrência para Roraima. Morcegos hematófagos foram identificados em cinco municípios. Ressaltando que este trabalho foi um passo inicial e que novos estudos precisam ser desenvolvidos, aprimorando as estratégias de coletas a fim de monitorar a presença do vírus da raiva no Estado. / Rabies is an infectious disease that affects mammals, including human beings. Present on all continents except Antarctica. It is caused by a Lyssavirus. Dogs are considered responsible for the maintenance and transmission of rabies to humans. But in recent years the bats have become a potential source of transmitting rabies to animals and human beings in the Americas. In 2010, Brazil recorded three cases of human rabies. One of them was caused by an attack of bat. Recently, several outbreaks of human rabies transmitted by vampire bats were reported in the Amazon region, so far, in the state of Roraima there is no record of cases of human rabies. This study is aimed to detect the presence and circulation of rabies virus in bats in the state of Roraima, as well as to identify the species involved, it includes, also, the necessity of strengthen the network of epidemiological and environmental surveillance of rabies. The technique followed by reverse transcriptase polymerase chain reaction (RT-PCR) was used for virus research involving brain tissue of bats that were collected by teams of environmental and epidemiological surveillance, belonging to the Department of Health and the health protection agency of Roraima. Species of Bats were identified using dichotomous keys available for bats in Brazil and other Latin American countries. In total of 94 bat samples were analysed. The samples tested were negative for rabies. It can not be said, however, that the rabies virus does not circulate in Roraima. This research identified 19 species of bats distributed in six family. On the other hand, the research points to a richness and abundance of species of bats. This study identified one family (Vespertilionidae) and five species of bats (Diaemus youngi, Noctilio albiventris, Myotis nigricans, Eptesicus diminutus e Cynomops planirostris) not yet reported to the State. Vampire bats were identified in five municipalities. Considering the epidemiological and environmental importance of bats for ecosystems, this study is contributing to the increase of knowledge about both environmental surveillance of rabies and diversity of bats.
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Caracterização molecular de arbovírus isolados da fauna diptera nematocera do Estado de Rondônia (Amazônia ocidental brasileira). / Molecular characterization of arboviruses isolated from mosquitoes fauna (Diptera: nematocera) Rondonia state (western brazilian Amazon).Dyana Alves Henriques 16 December 2008 (has links)
Rondônia apresenta área com rica diversidade de artrópodes, porém pouco se conhece sobre a transmissão de arbovírus por estas espécies. O presente trabalho visou detectar arbovírus, por meio da RT-PCR e da Duplex RT-PCR, nas espécies de dipteros coletados no Estado, bem como caracterizá-los pela reação de sequenciamento. A RT-PCR e a Duplex RT-PCR detectaram as suspensões dos vírus Mayaro e Oropouche até 104 e 101 TCID50/mL, respectivamente, porém o vírus Dengue 2 em pools contendo menos de três mosquitos infectados foi negativa. O controle endógeno foi detectado em 66,8 % das amostras, sendo que, em pools contendo entre um e três mosquitos, a detecção foi aproximadamente metade da detecção nos pools contendo entre 11 e 15. Em 0,66 % dos pools foi encontrado o vírus Oropouche e em outros 0,66 %, o vírus Cacipacoré. O vírus Oropouche foi detectado em Coquillettidia sp. e Deinocerites sp., enquanto o vírus Cacipacoré foi encontrado em Anopheles sp. e Culex sp. As técnicas possibilitaram a detecção dos arbovírus pesquisados nos pools coletados em Rondônia. / The Rondônia state has an area with rich arthropods diversity although the knowledge about the arboviruses transmition for these species is poor. The present work aimed to detect arboviruses through RT-PRC and RT-PCR Duplex in the diptera species collected in the region as well as their characterization through the sequence reaction. The RT-PRC and RT-PCR Duplex detected the Mayaro and Oropouche virus suspensions until 104 e 101 TCID50/mL respectively, although it was negative for the Dengue 2 virus in pools containing less than three infected mosquitoes. The endogenous control was detected in 66,8 % of samples and from pools containing from one to three mosquitoes the detection rate was approximately half from that obtained from pools with 11 to 15 mosquitoes. Oropouche virus was found in 0,66 % of pools and Cacipacore virus also in 0,66 % of pools. Oropouche virus was detected in Coquillettidia sp. and Deinocerites sp. while Cacipacoré virus was found in Anopheles sp. and Culex sp. The techniques allowed the detection of examined arboviruses in the pools collected from Rondonia.
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FACTORS INVOLVED IN THE EVOLUTION OF BROAD BEAN WILT VIRUS 1 AND TOBACCO MOSAIC VIRUSFerriol Safont, Inmaculada 06 June 2012 (has links)
Los virus producen graves pérdidas económicas en la agricultura. Esta problemática es muy dinámica ya que cada año aparecen nuevas virosis y es frecuente los fenómenos de emergencia con una rápida expansión de los virus. El control de las enfermedades víricas resulta poco eficaz en muchos casos porque la población viral es capaz de evolucionar y superar dichas estrategias. Por ello es clave entender la dinámica de las poblaciones y los factores implicados en la evolución de los virus con respecto a distintos aspectos de su biología del ciclo viral: replicación, movimiento dentro de la planta, respuesta a los mecanismos de defensa de la planta, transmisión a otras plantas, etc.
El objetivo de esta tesis ha sido el estudio de los factores implicados en la evolución de dos virus que difieren en su variabilidad genética y gama de huéspedes: i) el Virus 1 del marchitamiento del haba (Broad bean wilt virus 1, BBWV-1), del género Fabavirus; y ii) el Virus del mosaico del tabaco (Tobacco mosaic virus, TMV) del género Tobamovirus.
Primero se han desarrollado una serie de herramientas metodológicas que han permitido la detección rápida de BBWV-1 mediante hibridación molecular de improntas, la detección y cuantificación de BBWV-1 y TMV y su diferenciación de otras virosis del mismo género mediante RT-PCR cuantitativa a tiempo real. Se ha llevado a cabo la construcción de clones de cDNA del genoma completo de BBWV-1 para obtener transcritos infecciosos que puedan ser usados para estudiar la biología molecular, evolución y epidemiología.
Una vez desarrollado esta metodología se ha usado para evaluar la eficacia biológica de BBWV-1 en el huésped y el efecto de algunos factores: concentración del inóculo, estado de desarrollo de la planta, tipo de huésped, aplicación de un activador de la defensa de la planta, y la infección con otro virus. Así mismo se han estudiado los factores relacionados con la eficacia biológica del virus durante su transmisión por pulgones: título viral / Ferriol Safont, I. (2012). FACTORS INVOLVED IN THE EVOLUTION OF BROAD BEAN WILT VIRUS 1 AND TOBACCO MOSAIC VIRUS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16000 / Palancia
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Environmental sampling for detection of norovirus using a real-time RT-PCR Assay: A Tool for Foodborne Outbreak InvestigationsFowler, Jana Margaret 01 July 2012 (has links)
This project was designed to develop a method for the collection of environmental samples during prolonged Norovirus (NoV) outbreak investigations, and to develop real-time RT-PCR assays to analyze environmental samples for GI and GII noroviruses. The collection and processing of environmental samples could provide epidemiological data to facilitate investigations of prolonged NoV outbreaks and could guide public health NoV intervention strategies. Real-time RT-PCR assays for the detection of GI and GII NoVs were developed by adapting the State Hygienic Laboratory clinical GI and GII assays to the AB 7500 Fast platform. Analysis of the GI assay performance yielded a dilution curve slope = 3.28, R2 = 0.999 and a calculated amplification efficiency of 102%. The GII assay yielded a dilution curve slope = 3.39, R2 = 0.999 and a calculated amplification efficiency of 97%. Amplification efficiencies determine the sensitivity and the limit of detection of real-time RT-PCR assays. Optimum efficiencies range from 95%-105%, with a 100% efficiency indicating exponential amplification of targeted nucleic acid.
To develop a method for the collection of environmental samples, multiple swab types were tested to determine their ability to recover NoV from laboratory spiked environmental surfaces. It was determined that foam swabs moistened with viral transport media were most effective in recovering NoV from spiked surfaces. A field test of the environmental sampling method was conducted by sampling environmental surfaces in four restaurants in one Iowa community. NoVs were not detected in the environmental samples. The collection and processing of environmental samples when conducting an investigation of a prolonged NoV outbreak could provide additional information on the epidemiology of NoV transmission and infection.
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Epidemiología y variabilidad patogénica del virus del mosaico del pepino dulce (Pepino mosaic virus). Nuevas enfermedades asociadas a su presencia (torrao o cribado)Alfaro Fernández, Ana Olvido 03 February 2010 (has links)
El Pepino mosaic virus (PepMV) es un Potexvirus que fue descrito por primera vez en pepino dulce (Solanum muricatum Ait.) en Perú (Jones et. Al., 1980). En 1999 se detectó en Holanda, infectando a tomate (Van der Vlugt et al., 2000) mostrando una variada sintomatología. Desde entonces, el PepMV se ha expandido rápidamente por las principales áreas productoras de tomate del Mundo. Este virus se ha convertido en uno de los principales problemas en la producción de tomate en Europa donde produce importantes pérdidas económicas.
La rápida expansión del PepMV en las zonas afectadas ha estado facilitada por su eficaz transmisión mecánica con las operaciones de cultivo y mediante los insectos polinizadores (Lacasa et al., 2003), sin embargo no se ha detectado la existencia de ninguna especie de insecto capaz de actuar como vector del mismo. El PepMV podría permaneces en el campo de una cosecha a la siguiente infectando a la flora arvense que podría actuar como reservorio de la enfermedad. Asimismo se ha comprobado la transmisión por semilla que podría constituir la forma de dispersión del virus a la larga distancia (Córdoba et. A., 2007).
Actualmente se conocen diferentes aislados del virus que afectan al tomate. En primer momento, debido a las diferencias biológicas y moleculares observadas entre el aislado del PepMV que infectaba a tomate y el original de pepino dulce, distintos autores consideraron el aislado de tomate como un aislado diferente, denominándolo aislado tipo tomate (Van der Vlugt y Beredsen, 2002). Estudios posteriores demostraron que los aislados de PepMV de Europa, América del Norte y Canadá comparados con el aislado original de pepino dulce presentaban diferencias evidentes en sintomología, así como estructura poblacional del PepMV en cultivo de tomate en España, analizando y secuenciando tres zonas distintas del genoma del virus. / Alfaro Fernández, AO. (2009). Epidemiología y variabilidad patogénica del virus del mosaico del pepino dulce (Pepino mosaic virus). Nuevas enfermedades asociadas a su presencia (torrao o cribado) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/7027 / Palancia
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Investigation into the variation of infectious bronchitis virus serotypes in KwaZulu-Natal poultry flocksKnoetze, Adrian David January 2013 (has links)
Infectious bronchitis virus (IBV) is a member of family Coronaviridae and is classified into group 3 of the Coronaviruses. The virus is a single-stranded positive-sense RNA virus with a genome of 27kbp. IBV is a highly infectious disease of chickens that results in high morbidity with moderate to severe mortality depending on the strain involved, age of the birds, and immune status of the chickens. Multiple worldwide investigations indicate that differentiation within the S1 glycoprotein gene can lead to serotype variation within the IBV species. In this study 46 isolates collected over two years from broiler and broiler breeder flocks and eight historical isolates were analyzed. Forty one isolates originated from the KwaZulu-Natal region whilst the remaining thirteen were isolated from 4 other poultry-dense provinces. The S1 gene was sequenced and compared to determine variation between South African isolates, as well as global sequences submitted to Genbank. The results indicate the division of isolates analyzed into 2 different clades of IBV within the province. The most prevalent genotype was similar to IBV Mass strain detected in 79% of the full S1 sequences. Variation up to 22.3% was detected within local strains, supporting the hypothesis that multiple IBV serotypes may co-circulate in the same region simultaneously. Additionally, more conservation was observed among Mass serotypes versus QX-like serotypes, implying that vaccine use can influence the variability within the IBV population. Higher variability was found in the first half of the S1 gene in comparison to the last half of the S1 gene. This is in agreement with previous findings that the hypervariable regions of the S1 gene are located within the first 450 base pairs. This study offers the first published consolidation of IBV isolates from South Africa and identifies variation within the IBV population of the SA broiler flock. Previous publications list four or five IBV isolates whilst this study describes variation found in 54 isolates spanning 32 years. In addition this study provides the insight into the prevalence of IBV variation in poultry flocks due to the large number of isolates. The comparative use of geno- and serotyping for South African IBV isolates is also described for the first time in this study. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted
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Unlocking SARS-CoV-2 detection in low- and middle-income countriesAlcántara, Roberto, Peñaranda, Katherin, Mendoza-Rojas, Gabriel, Nakamoto, Jose A., Martins-Luna, Johanna, del Valle-Mendoza, Juana, Adaui, Vanessa, Milón, Pohl 22 November 2021 (has links)
Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 102 ge/μL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%–88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%–100%. The specificity was 96%–100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions. / Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica / Revisión por pares
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Gewebeverteilung und Lokalisation des Transportproteins für reduzierte Folate (RFC1) der RatteHinken, Matthias 10 October 2007 (has links)
Der Folsäureantagonist Methotrexat (MTX) wird zur Behandlung onkologischer und rheumatoider Erkrankungen eingesetzt. Die Aufnahme des Methotrexats in die Zielzelle ist dabei Vorraussetzung für die Bindung an seine intrazellulären Zielstrukturen und erfolgt über verschiedene Transportsysteme. In diesem Zusammenhang ist bei entsprechenden Plasma-konzentrationen von MTX der Reduced Folate Carrier (RFC1) von besonderer Bedeutung. 1994 konnte erstmals die cDNA dieses Transporters aus Maus- und Hamstergewebe isoliert werden. Die cDNA für einen mit dem RFC1 identischen hepatozellulären MTX-Transporter der Ratte wurde 2000 kloniert. Vorhergehende Gen-Expressionsstudien zeigten, dass die RFC1-mRNA ubiquitär gebildet wird. Die Proteinexpression wurde jedoch bisher nur in ausgewählten Geweben der Maus untersucht. Systematische Arbeiten, in denen in vergleichender Weise sowohl die RFC1 Gen- als auch die Proteinexpression in allen Geweben mit einer möglichen Relevanz für die Folat- und Antifolataufnahme, Speicherung und Eliminierung untersucht werden, fehlten bisher. Insbesondere die Expression des RFC1-Proteins der Ratte (rRFC1) mittels immunologischer Verfahren ist bisher nicht beschrieben worden. Ziel dieser Arbeit war es daher, die Gen- und Proteinexpression des rRFC1 in ausgewählten Geweben der Ratte darzustellen. Dieses schließt die Generierung spezifischer Antiseren gegen den rRFC1 als ersten Schritt mit ein. Es wurden geeignete antigene Aminosäuresequenzen des rRFC1 bestimmt und die entsprechenden cDNA Sequenzen wurden amplifiziert und in einen geeigneten Expressionsvektor kloniert. Rekombinante rRFC1 Fusionsproteine konnten mittels E. coli Zellen hergestellt und anschließend aufgereinigt werden. Nachfolgend wurden entweder die rRFC1 Fusionsproteine oder die rRFC1 spezifischen Peptide, welche von dem Affinitätspeptid separiert worden waren, für die Immunisierung von Kaninchen verwendet Drei Antiseren mit ausreichender Reaktivität und Spezifität konnten gewonnen und mittels Affinitätschromatographie aufgereinigt werden. Die erhaltenen Antiseren sind gegen die intrazellulären N- und C-terminalen Regionen (ID1, ID7) bzw. gegen die erste extrazelluläre Schleife (OD1) gerichtet. In Western-Blot Studien konnte mittels dieser Antiseren für den rRFC1, der in transfizierten Nierenepithelzellen (MDCK-rRFC-HA) stabil exprimiert wurde, ein Molekulargewicht von 71 kD für die glykosylierte Form und von 53 kD für die unglykosylierte Form ermittelt werden. Weiter konnte belegt werden, dass das Protein in MDCK-rRFC1-HA Zellen überwiegend in der glycosylierten Form vorliegt. Mittels RT-PCR Analysen wurde die Genexpression des rRFC1 in allen untersuchten Geweben nachgewiesen. Besonders hohe mRNA-Gehalte waren in Thymus, Niere und Milz vorhanden, während in Herz- und Muskelgewebe sowie in Leukozyten nur ein Signal nahe der Nachweisgrenze detektierbar war. Durch immunhistologische Untersuchungen konnten die rRFC1 Proteinexpression und beträchtliche Unterschiede in der Signalintensität bestätigt werden. Zusätzlich konnten neue Informationen über die unterschiedliche subzelluläre Lokalisation gewonnen werden: so konnte eine starke Expression des Transporters in der apikalen Membran von Dünn- und Dickdarmmukosa dargestellt werden, während die ebenfalls starke Färbung in der Niere auf den Bereich der basolateralen Membran der Tubuli beschränkt war. In der Leber war eine Expression mittlerer Intensität im Bereich der Lebertrias erkennbar. Während in der Milz nur in der roten Pulpa das RFC1-Protein detektiert wurde, konnten im Thymus sowohl in der Rinde als auch im Mark positive Zellen nachgewiesen werden. Im Hoden konnte der Transporter in den Sertoli-Zellen dargestellt werden. Eine starke Expression des Transporters wurde im Gehirn im Bereich der apikalen Membran der Ependymzellen des Plexus choroideus nachgewiesen. In der Skelettmuskulatur und im Herzgewebe beschränkte sich die Expression des rRFC1 auf das Perimysium des Muskelgewebes und auf kleinere Gefäße des Muskel- und Herzgewebes. In dieser Arbeit konnte somit gezeigt werden, dass der RFC1 der Ratte ubiquitär exprimiert wird, wobei die Expressionsstärke jedoch stark variiert. Die beobachtete Gewebslokalisation des RFC1 belegt sowohl dessen zentrale Rolle in der Folathomöostase als auch in der MTX vermittelten Organtoxizität und Pharmakokinetik, insbesondere bei der intestinalen Resorption sowie der hepatischen und renalen Exkretion.
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Entwicklung einer Reversen Transkription-Polymerase-Kettenreaktion (RT-PCR) zum Nachweis der Persistenz von Rotaviren beim SchweinSchwarz, Bernd-Andreas 05 September 2003 (has links)
Im Graduiertenkolleg Schlachttierbelastung und Produktsicherheit der Veterinärmedizinischen Fakultät der Universität Leipzig sollten in interdisziplinärer Zusammenarbeit Erkenntnisse zum Verhalten transportbelasteter Schlachtschweine in Bezug auf bakterielle Translokationsprozesse erarbeitet werden. Es wurden Mastschweine aus herkömmlichen Mastbetrieben definierten Belastungssituationen ausgesetzt , geschlachtet und untersucht. Dabei sollten physiologische, pathologisch-anatomische, immunologische, lebensmittel- und fleischhygienische, bakteriologische, virologische sowie ethologische Fragestellungen bearbeitet werden. Ziel war es festzustellen, ob eine Belastung der Tiere (u.a. durch den Transport) Auswirkungen auf die Produktqualität hat und ob durch eine Translokation pathogener Erreger ein Risiko für die Gesundheit des Verbrauchers besteht. Im Teilprojekt des Institutes für Virologie wurde untersucht, ob Rotavirus-Infektionen von Schlachtschweinen unter der Problematik der Belastung ein mögliches Infektionsrisiko für den Menschen darstellen. Um die zu erwartende niedrige Konzentration von Rotaviren in Organen von Schlachtschweinen nachweisen zu können, wurde eine kompetitive RT-PCR zum Nachweis von Rotaviren der Gruppe A verschiedener Spezies entwickelt. Dazu wurde ein sogenannter Kompetitor synthetisch hergestellt, welcher als interne oder externe Reaktionskontrolle eingesetzt wurde. Zum einen diente er der Überprüfung des ordnungsgemäßen Verlaufes einer RT-PCR, zum anderen wurde er zur Herstellung von Standards verwendet. Die RT-PCR wurde anschließend in eine Real time RT-PCR umgewandelt. Sowohl mit der herkömmlichen als auch mit der Real-time RT-PCR konnten 10 spezifische RNA-Moleküle in einer Probe nachgewiesen werden. In einer SPF-Schweineherde, welche einer Belastung infolge eines Tiertransports ausgesetzt war, konnten mit Hilfe der RT-PCR klinisch gesunde intermittierende Rotavirus-Ausscheider entdeckt werden. Bei einigen dieser Tiere gelang der Nachweis der Virusausscheidung über einen Zeitraum von drei Monaten. Nach der Schlachtung wurden in Organen des lymphatischen Systems bei zwei Schweinen sehr geringe Konzentrationen an rotavirus-spezifischer RNA detektiert. Infektiöses Virus konnte daraus allerdings nicht isoliert werden. Auch in einer Mastschweineherde konnte bei einigen Tieren Rotavirus-spezifische RNA im Kot nachgewiesen werden. Ein Infektionsversuch dieser Tiere mit Salmonella typhimurium konnte keine Reaktivierung der Rotavirus-Infektion auslösen. Aufgrund des Zoonose-Potentials von Rotaviren kann nach den Untersuchungen ein Infektionsrisiko für den Verbraucher durch eine endogene Kontamination von Schlachttieren mit Rotaviren nicht sicher ausgeschlossen werden. Die Untersuchungen zeigten auch, dass intermittierende Rotavirus-Ausscheider ein Infektionsrisiko für den Verbraucher darstellen, wenn z.B. bei der Schlachtung der Tiere oder bei der Verarbeitung des Fleisches dieser Tiere hygienische Grundregeln verletzt werden. Besonders gefährdet wären hierbei Neugeborene, Kinder, Senioren und immunsupprimierte Personen. Ein Ort einer Viruspersistenz in Organen konnte auch nach diesen Untersuchungen nicht gefunden werden. Dennoch scheint es, dass Rotaviren in der Natur oder in einer Population von Menschen oder Tieren persistieren. Durch ständige Neuinfektionen bzw. Reinfektionen empfänglicher Organismen haben Rotaviren so ihre Erhaltung gesichert. / Within the graduate programme Schlachttierbelastung und Produktsicherheit of the Veterinary Faculty of the University of Leipzig, the behaviour of slaughter swine exposed to the stress of transport was observed in an interdisciplinary collaboration concerning the translocation processes of bacteria. Fattened pigs from conventional pig fattening units were exposed to particular stress situations, and then slaughtered and examined. The following aspects of this process were investigated: physiology, pathological-anatomy, immunology, food and meat hygiene, bacteriology, virology and ethology. The aim of this study was to verify whether exposing the animals to stress situations (such as transport) influences the quality of the product and whether the translocation of pathogens represents a risk for consumer health. Within the sub-project of the Institute for Virology, analyses were made to verify whether rotavirus infections of slaughter swine exposed to stress situations represents a potential contamination risk for humans. In order to detect the expected low concentration of rotaviruses in the organs of slaughtered pigs, a competitive RT-PCR was developed as a test of rotaviruses for various group A species. To do this, a so-called competitor was synthetically created, which was used as an internal and external reaction control. On one hand, it was used to verify the regular course of an RT-PCR reaction, and on the other hand, it was implemented to develop standards. RT-PCR was then modified by means of a real time RT-PCR. Both with the conventional and with the real time RT-PCR, it was possible to detect 10 specific RNA molecules/ sample.With this new very sensitive and specific amplification process, it was possible to detect rotavirus-specific RNA in the excrement of people and of pigs, cows, horses, rabbits and monkeys. the evidence of the virus excretion was produced over a time period of three months. After slaughtering, low amounts of rotavirus specific RNA were found in the organs of the lymphatic system. There were no indications that any of these organs were infectious. Rotavirus specific RNA was also found in the excrement of some fattened pigs. An attempt to infect these animals with Salmonella typhimurium was unable to cause any reactivation of the rotavirus infection. An infection risk for the consumer through an endogenous rotavirus contamination of fattened pigs cannot be excluded with any degree of certainty on the basis of these analyses due to the zoonotic potential of rotaviruses. The analyses also showed that intermittent rotavirus excretors represent an infection risk for the consumer, if for example basic hygiene rules are broken during the slaughter or meat processing of these animals. At special risk may be new-borns, children, youth, the elderly and people suffering from immunodeficiency. These examinations could not find a specific place in the organs where the virus persists. Nevertheless, it seems that rotaviruses persist in the environment or in a population of people or animals. With constant new infections or re-infections of receptive organisms, rotaviruses seem to have assured their survival.
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Characterization of a Novel Water Stress Related Protein from Geranium (<em>Pelargonium hortorum</em>).Ashley, David W 01 December 2001 (has links) (PDF)
Searches for sequence similarity against a previously isolated and known ABA regulated partial geranium cDNA on GenBank and TIGR databases gave results indicating sequence similarities to both firefly luciferase and 4-coumarate:CoA Ligase (4-CL). To determine whether or not this geranium cDNA, and a root mRNA from Arabidopsis that hybridized with the geranium cDNA, is 4-CL, specific Arabidopsis 4-CL primers were used for expression analysis by RT-PCR of Arabidopsis 4-CL mRNA from Arabidopsis root tissue. The expression pattern of the Arabidopsis 4-CL mRNA was identical with the expression pattern of the mRNA that hybridized with the geranium cDNA. This matching expression pattern gives very strong evidence that our geranium cDNA is 4-CL like and that Arabidopsis 4-CL is downregulated during water stress by ABA. This is significant in that it has not previously been reported. Northern analysis and PCR product sequence analysis further confirmed this expression data.
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