Made available in DSpace on 2015-07-08T12:28:16Z (GMT). No. of bitstreams: 2 10.pdf: 3053603 bytes, checksum: 24679f8c07fde204c2748c429bad6849 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A poliomielite é uma infecção causada por um dos três sorotipos de poliovírus (sorotipos 1, 2 e 3) e teve sua incidência diminuída drasticamente (>99%) pelo programa de erradicação global da doença, iniciado em 1988. Atualmente, estes vírus permanecem endêmicos em três países e casos de re-emergência por importação têm sido relatados em países livres da doença. O presente trabalho teve como objetivo estabelecer a vigilância ambiental pelo isolamento e caracterização molecular dos poliovírus e enterovírus não-pólio circulantes, a partir de amostras de esgoto coletadas na Estação de Tratamento de Esgoto (ETE) Alegria/Cedae, localizada no município do Rio de Janeiro, no período de dezembro de 2011 a junho de 2012 e de setembro a dezembro de 2012. Diferentes métodos de concentração viral foram avaliados (separação em duas fases; precipitação por polietilenoglicol (PEG) e adsorção por sílica) e 31 amostras foram coletadas durante o estudo. O isolamento em culturas de células RD e L20B, seguido de RT-PCR foi capaz de detectar enterovírus em 87% das amostras (27/31). Poliovírus foram isolados em 29,6 % (8/27), sendo Sabin 1 - n= 1; Sabin 2 - n= 5 e Sabin 3 - n= 2, enquanto que os demais isolados foram identificados como enterovírus não-pólio. Todos os poliovírus isolados foram submetidos ao sequenciamento nucleotídico completo do gene VP1 e apresentaram mutações sinônimas e não-sinônimas, sendo algumas em aminoácidos específicos relacionados ao fenótipo atenuado. Todavia, não apresentaram mutações suficientes para serem caracterizados como poliovírus derivado da vacina (PVDV), sendo, portanto, classificados como vacinais ou Sabin like. / Poliomyelitis is an infection caused by one of three poliovirus serotypes (serotypes 1, 2 and 3) and its incidence decreased dramatically (> 99%) by the global polio eradication initiative, started in 1988. Currently, wild polioviruses remain endemic in three countries and cases of re-emergency by import of these viruses have been related in countries free of the disease. This study aimed to establish the environmental surveillance by the isolation and molecular characterization of circulating poliovirus and non-polio enteroviruses from wastewater samples collected at the Station of Sewage Treatment Alegria/ Cedae, located in Rio de Janeiro city, in the period from December 2011 to June 2012 and from September to December 2012. Different methods of viral concentration were evaluated (two-phases separate, polyethylene glycol (PEG) precipitation and silica adsorption) and 31 samples were collected during the study. The isolation in cell cultures RD and L20B, followed by RT-PCR was able to detect enteroviruses in 87% samples (27/31). Poliovirus was isolated in 29.6% samples (8/27) being Sabin1 - n= 1, Sabin 2 - n= 5 and Sabin 3 - n= 2, while the remaining isolates were non-polio enteroviruses. All poliovirus isolates were subjected to complete nucleotide sequencing of the VP1 gene and presented non-synonymous and synonymous mutations, being some mutations in specific amino acids related to the attenuated phenotype. However, showed no sufficient mutations for to be characterized like vaccine-derived poliovirus (VDPV), being classified as vaccine or Sabin like poliovirus. By the partial nucleotide sequencing of the VP1 gene analysis, the non-polio enteroviruses have been identified: 1 Echovirus 3; 11 Echovirus 6, 7 Echovirus 7, 2 Echovirus 20; 4 Coxsackievirus B4 and 2 Coxsackievirus B5. Environmental surveillance has been utilized successfully in monitoring the circulation of enterovirus and it is of crucial importance in the final stages of the global eradication initiative of polio by Word Health Organization (WHO). These results show the continuous circulation of Sabin like poliovirus and non-polio enteroviruses in the analyzed area during the study period.

Alkaline glutaraldehyde (GTA) has been used as a high level chemical disinfectant and sterilant for many years and is known to kill a broad spectrum of organisms ranging from vegetative eukaryotes to bacterial endospores. Although the mechanism of sporicidal action has been studied on numerous occasions, GTA's exact mechanism(s) of action are still debated. In addition to the uncertainty of GTA's mechanism(s) of action, GTA has also shown significant variability in the time required to kill endospores and naked viruses. A better knowledge of the lethal mechanism(s) of GTA is needed to understand this discrepancy in kill times for GTA against spores of different species. Similar trends have been observed in GTA's activity against non-enveloped viruses. Based on previous work, one proposed major mechanism of GTA's sporicidal activity is related to the number of available primary amines located on the surface of microbes. In this study, we have compared the efficacy of GTA on spores from 5 Bacillus species. We have also developed a method for staining these spores with amine reactive dyes to create fluorescent profiles correlating to the abundance of free amino groups on each spore type. We also describe a method for staining non-enveloped viruses to identify exposed primary amino groups on capsid proteins that may act as targets for GTA, using amine reactive Gold nanoparticles. We found that GTA 6-Log10 reduction times for various spore types varied at both the batch and species level. Spore coat thickness and fluorescence were useful tools in predicting the susceptibility of spores to GTA. Amine reactive gold particles (AuNPs) also proved useful in identifying virus susceptibility to GTA. Ultimately, more reliable disinfection testing methods are needed, and caution should be used when trying to extrapolate data generated from surrogate organisms to other species.

Die Eradikation des Poliovirus wurde 1988 von der WHO beschlossen. Hierzu wurde die Global Polio Eradikation Initiative ins Leben gerufen. Mit hohem Aufwand und großen Geldsummen wird seitdem das Ziel verfolgt die Poliovirus Wildtypen 1-3 zu eradizieren. Vorläufer dieser Bemühungen war die Eradikation des Pockenvirus, welche innerhalb weniger Jahre gelang. Ursprünglich sollte bereits im Jahr 2000 das Virus erfolgreich eradiziert sein, dieses ehrgeizige Ziel konnte jedoch nicht erreicht werden und wird auch nicht vor 2021 erreicht. Erschwerend kommt hinzu, dass keineswegs nur das WPV Typ 1-3 Fälle von Poliomyelitiden hervorruft, sondern auch Impfviren, welche, insbesondere in Gebieten mit schlechter Durchimpfungsrate, über einen langen Zeitraum zirkulieren und ihre Pathogenität durch Rückmutation wiedererhalten können. Je länger der Zeitraum der Zirkulation umso höher das Risiko der Bildung dieser Revertanten. Ein weiterer, seltener, aber nicht unwichtiger Faktor sind sogenannte Dauerausscheider – Personen mit Immundefekten, welche infolge einer OPV teilweise über Jahrzehnte hinweg das Polioimpfvirus ausscheiden. Auch hier besteht die Gefahr, dass die Viren ihre Pathogenität durch Mutation zurückerhalten. In Deutschland und anderen westeuropäischen Ländern ist die Basisreproduktionszahl des Poliovirus R0 4, d.h. um im Fall einer Infektion eine Epidemie zu verhindern ist eine Durchimpfungsrate von mindestens 75 % notwendig. Nur dann ist davon auszugehen, dass es im Falle einer Erkrankung eine Ausbreitung der Viren lokal begrenzt bleibt oder im besten Fall gestoppt wird. Zuverlässige Datensätze über den Impfstand der Bevölkerung gibt es derzeit lediglich über die Datenerhebung im Rahmen der Schuleingangsuntersuchungen und der KVImpfsurveillance. Diese Daten geben keine Auskunft, wie es mit der Immunität der erwachsenen Bevölkerung steht. In dieser Arbeit soll gezeigt werden, ob in ausreichendem Maße Immunität unter den werdenden Müttern besteht um einerseits von einem ausreichenden Herdenschutz auszugehen und andererseits einen Nestschutz der Kinder zu gewährleisten. Zur Bestimmung der Prävalenz von Antikörpern ist der Neutralisationstest das Verfahren der Wahl. Die Neutralisationstests haben gezeigt, dass nur 1,7 % der Frauen keinen Schutz gegen alle drei Serotypen des Poliovirus aufweisen. 78,9 % der Frauen sind immun gegen alle drei Serotypen des Poliovirus. Die Seroprävalenz von Antikörpern gegen die einzelnen Serotypen des Poliovirus lag zwischen 80,5% und 94,4%. Nichtsdestotrotz haben einzelne Individuen keinen Schutz und sind dem Risiko der Infektion und Erkrankung ausgesetzt. Ebenso sind deren Kinder, zumindest bis zur Impfung und der Bildung eigener Antikörper, ungeschützt. In Zeiten der Globalisierung und Menschenbewegungen besteht keine Garantie auf eine „sichere Zone“. Die Verbreitung der Viren kann, aufgrund der meist asymptomatischen Infektion lange unbemerkt erfolgen. Zudem könne Menschen, welche ihren Schutz durch die inaktivierte Polioimpfung erhalten haben, aufgrund der fehlenden IgA-Bildung in den Schleimhäuten des Magen-Darm-Trakts, infektiös sein und lebende, infektiöse Viren ausscheiden ohne selbst zu erkranken.:1 Inhaltsverzeichnis 2 Abkürzungsverzeichnis 3 Einführung 3.1 Virale Struktur und Replikation 3.2 Poliovirus: Geschichte, Epidemiologie und Relevanz 3.3 Poliomyelitis: Pathogenese und Krankheitsbild 3.4 Diagnostik von Polio-Virus-Infektionen und Impfung 4 Zielstellung 5 Material und Methoden 5.1 Materialien 5.2 Methoden 5.3 Statistik und Datenverarbeitung 6 Ergebnisse 7 Diskussion 8 Zusammenfassung 9 Literaturverzeichnis 10 Tabellen-, Abbildungs- und Formelverzeichnis 11 Selbständigkeitserklärung 12 Danksagung 13 Lebenslauf

Human enteric viruses are present at low levels in sources of potable water. Due to the low minimum infective dose required for such viruses, these low levels are of public health significance and will become more important as water reuse increases. The effectiveness of alum coagulation for virus removal was evaluated using a synthetic water seeded with monodispersed poliovirus LScl. Experiments were done using 10(3),10(4),10(6) Plaque Forming Units (PFU) per liter. A microporous filter concentration technique was used post treatment to increase the virus titer to make possible virus enumeration by plaque assay on BGM cell cultures. Preliminary work was done to determine the efficiency of the concentration technique at 10(3),10(4),10(6)and PFU per liter. An average of 30.9 per cent of the original PFU were recovered. Two coagulation test methods were used at the lower virus titers. In one, virus was added to the system after the coagulant during the flash mix period and in the other, the coagulant was added to water already seeded with virus. Removals were similar for both conditions. Alum coagulation was found to remove 95 percent of the virus present at the low titers. This efficiency of virus removal is similar to that observed at high initial virus titers. Therefore, it appears that alum coagulation is an effective virus removal method. / Master of Science

BACKGROUND: The type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-β. In this study the impact of poliovirus infection on the type I interferon response has been examined. METHODS: The type I IFN response was assessed by measuring IFN-β mRNA levels using qRT-PCR and normalizing to levels of β-actin mRNA. The status of host factors involved in activation of the type I IFN response was examined by immunoblot, immunofluorescence microcopy and qRT-PCR. RESULTS: The results show that poliovirus infection results in induction of very low levels of IFN-β mRNA despite clear activation of NF-κB and ATF-2. In contrast, analysis of IRF-3 revealed no transcriptional induction of an IRF-3-responsive promoter or homodimerization of IRF-3 indicating it is not activated in poliovirus-infected cells. Exposure of poliovirus-infected cells to poly(I:C) results in lower levels of IFN-β mRNA synthesis and IRF-3 activation compared to mock-infected cells. Analysis of MDA-5 and IPS-1 revealed that these components of the RLR pathway were largely intact at times when the type I IFN response was suppressed. CONCLUSIONS: Collectively, these results demonstrate that poliovirus infection actively suppresses the host type I interferon response by blocking activation of IRF-3 and suggests that this is not mediated by cleavage of MDA-5 or IPS-1.

Diversos trabalhos têm demonstrado a presença de peptídeos bioativos em hemolinfa de insetos e seu potencial uso como agentes terapêuticos. Este trabalho buscou identificar e isolar proteínas da hemolinfa de Lonomia obliqua com ação antiviral. A adição de hemolinfa antes da infecção reduziu o título de poliovírus de 1,5x107 TCID50/mL no cultivo controle para 4x105 TCID50/mL e em aproximadamente 100 vezes o título do sarampo. Após cromatografia de gel filtração, foram obtidos 3 pools de proteína. O pool responsável pela ação antiviral foi identificado (Pool 2), uma vez que reduziu em 87 vezes o título de poliovírus, e de 1,6x106 TCID50/mL para 2,7x105 TCID50/mL o título do sarampo. O Pool 2 foi fracionado por cromatografia de troca iônica. A fração responsável pela ação antiviral da hemolinfa de Lonomia obliqua frente ao poliovírus e sarampo foi identificada (RQ 3-4), esta proteína, de aproximadamente 20 kDa, tem ação sobre vírus envelopado (sarampo) e vírus desprovido de envelope viral (poliovírus). / Several works have demonstrated the presence of bioactive peptides in insect hemolymph and their potential use as therapeutic agents. This work sought to identify and purify proteins from Lonomia obliqua hemolymph with antiviral activity. The analyses demonstrated that the best time for hemolymph addition in the cell culture is before the infection, the poliovirus titer had reduced from 1.5x107 TCID50/mL to 4x105 TCID50/mL and for measles, the virus titer was about 100 times lower than the control. After gel filtration chromatography, the hemolymph was divided into 3 pools of protein. The pool responsible for the antiviral activity was identified (Pool 2), once it reduced to 87 times the poliovirus titer, and the measles titer from 1.6x106 TCID50/mL to 2.7x105 TCID50/mL. The Pool 2 was fractionated by ion-exchange chromatography. The fraction from Lonomia obliqua hemolymph responsible for the antiviral activity was identified - RQ 3-4, this protein of approximately 20 kDa, has antiviral effect on enveloped virus (measles) and on non enveloped virus (poliovirus).

The CDC estimated that foodborne infections resulted in approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths per year in the United States (Mead, 1999). There are over 200 known diseases caused by viruses, bacteria, parasites, toxins, metals, or prions that can be transmitted through food. Of these illnesses caused by foodborne disease, the CDC estimates that 38.6 million cases are from identifiable pathogens and 30.9 million of these cases are caused by viruses. Hence, approximately 80% of foodborne illnesses of known etiology result from viral transmission (Mead, 1999). Viral gastrointestinal illness may be caused by virus families such as: enterovirus, rotavirus, calicivirus, astrovirus, or norovirus. These viruses are highly contagious and are spread through the fecal-oral route; transmission vehicles include contaminated food or beverages, infected food handlers, fomites or close contact with an infected individual (FDA Bad Bug Book, 2003). Until recently, there have been few studies concentrating on viruses found in or on foods. There are several technical difficulties that hinder progress in detecting viral agents from foods. One of these problems is the presence of matrix inhibitors. Substances responsible for matrix inhibition include humic acid, polysaccharides, myoglobins, metal ions, glycogen, and lipids (Monpoeho, 2001). These substances in foods produce smearing of the RT-PCR amplicon bands on agarose gels. Several methods to reduce inhibitory compounds utilize multiple toxic reagents in the procedure. In this study, varying centrifugal forces were tested at different steps of the virus extraction/concentration procedure to reduce matrix inhibitory effects for molecular detection of norovirus and poliovirus seeded onto food surfaces. This method incorporates the rapid detection capabilities of RT-PCR with the ability to reduce or eliminate matrix inhibitors present in food, by altering the centrifugal force. Results for both viruses showed that band intensity decreased as the viral concentration decreased and no one method was superior for all food matrices. This investigation showed that matrix specific modifications to the basic protocol are required to efficiently extract viruses from the surface of foods. Each food should be assessed to determine modifications to the standard method that would be optimal for viral concentration and extraction.

With global freshwater supplies under pressure, viable water reuse methods are being examined to assist in improving water supplies. Municipal effluent is an ideal source for water reclamation as it is consistent in quality and quantity. The health aspects of water reuse have been identified as an issue of concern, in particular the potential presence of enteric viruses. Managed Aquifer Recharge (MAR) is a method that can aid water reclamation by recharging water such as treated effluent into a suitable aquifer. Research into the removal of pathogenic contaminants by natural processes within aquifers, namely the action of autochthonous bacteria, has led to the consideration that MAR could be used to assist in the removal of microbial pathogens. Pathogens have been demonstrated to be removed during residence in groundwater, but the presence of active autochthonous groundwater bacteria are required for significant removal rates to occur. The aim of this research was to investigate the interaction between autochthonous groundwater bacteria (AGB) and the enteroviruses Poliovirus type 1, Coxsackievirus B3 and Adenovirus B41. It was established that these viruses decrease in number in the presence of AGB but the mechanisms causing this decrease are poorly understood. Experiments were designed to examine how the individual AGB caused decay of the viruses. In this study AGB were isolated and tested for their ability in increase the decay of the viruses. It was determined that 27 % (17/63) of the isolated AGB influenced viral decay. The AGB isolates varied in their influence with only 3 out of 17 isolates being able to cause of the decay of both poliovirus and coxsackievirus. Similar variations in decay were observed for adenovirus. Decay times for all three viruses varied amongst the AGB and between the viruses. Experiments were undertaken to characterise the mechanism causing the antiviral activity of four groundwater isolates (1G, 3A, 4B and 9G) under varying conditions and treatments to give insight into the compounds or mechanisms responsible for viral decay. This would indicate whether compounds produced by the AGB responsible for viral decay were closely associated to bacterial cells (perhaps membrane bound), independent of metabolic activity, heat labile or were enzymatic in nature. The influence of enzyme inhibiters and heat treatment indicated that viral degradation is caused by compounds that are enzymatic in nature. As viral numbers were monitored by nucleic acid copy numbers rather than via infectivity assays, the viral protein coats must be the first step in degradation followed by the removal of the viral nucleic acid. This two step process would require both protease and nuclease enzymes to result in loss of viral numbers as measured by RT-PCR/PCR. Further characterisation and identification of these four bacterial isolates was also carried out. Three out of the four isolates were sequenced and analysed using partial 16S rRNA gene sequences to determine their phylogenetic relationships compared to related organisms. Isolate 3A was placed in the order Burkholderiales. Isolate 4B was placed in the family Xanthomonadaceae. Isolate 9G was placed in the family Rhizobiaceae. Isolate 1G was only partially sequenced and preliminary identification placed it in the phylum Bacteriodetes. Understanding of the processes carried out by AGB within an aquifer during MAR using reclaimed waters will aid in increasing the viability of this water reuse process. If important natural processes could be utilised to remediate any potential pathogens, the health concerns with reclaimed waters could be addressed and solved simply through prescribed retention times within the aquifer. Key species of AGB may even be utilised as markers to assess the suitability of an aquifer for MAR.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 198-225). Online version available via ProQuest Digital Dissertations.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 126-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;