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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
91

Long-term Consequences of West Nile Virus in Virginia.

Ocampo, Diana Cruz 01 January 2005 (has links)
Objective: The purpose of this investigation was to describe the long-term effects and functional outcomes of patients in Virginia who were reported to the Virginia Department of Health with West Nile virus (WNV) non-neuroinvasive and neuroinvasive disease. The study identified the duration of symptoms after initial illness, the number of persons who fully recovered versus the number who continue to be symptomatic and how patients' quality of life differed after illness.Methods: The study population was drawn from 60 human cases that met the surveillance case definition for non-neuroinvasive and neuroinvasive WNV illness in Virginia between 2002-2004. Information was collected during personal interviews using a standard questionnaire. The questionnaire included questions on demographics, clinical signs and symptoms, existing medical conditions and the respondents' personal assessment of health. Statistical analysis were used to compare pre and post illness symptoms, respondents vs. non-respondents, and non-neuroinvasive respondents vs. neuroinvasive respondents. Results: Thirty-four patients were enrolled in the study. Five (14.7%) respondents had non-neuroinvasive disease and 29 (85.2%) had neuroinvasive disease. Thirty respondents (88.2%) reported being hospitalized. Respondents with non-neuroinvasive disease spent a median of 3.5 (range, 0-7) days in the hospital and were unable to resume normal activities for a median of 17 (range, 7-365) days. Respondents with neuroinvasive disease spent a median of 7.5 (range, 0-82) days in the hospital and were unable to resume normal activities for a median of 127.50 days (range, 0-1023). Two (40%) of the respondents that suffer from non-neuroinvasive illness were unable to resume normal activities for at least 90 days. Fifteen (51.7%) respondents with neuroinvasive disease were unable to resume normal activities for at least 90 days. At the time of the interview, 20% of respondents with non-neuroinvasive disease reported fatigue, tremors, arthralgia, paralysis and memory problems. Respondents with neuroinvasive disease reported fatigue (58.5%), weakness (51.7%), myalgias (37.9%), confusion (41.4%), and memory loss (55.2%). Conclusion: WNV illness, including non-neuroinvasive illness, may be more serious and prolonged than generally thought. Neuroinvasive disease resulted in long-term morbidity and non-neuroinvasive disease resulted in work absenteeism and extended recovery periods. The mortality rates and potential long-term effects associated with non-neuroinvasive and neuroinvasive illness emphasizes the importance of continuing to develop effective methods of targeting preventive education to high-risk populations while continuing to pursue longer-term solutions such as vaccines to prevent emerging infection. Further research is needed to document the long-term effects of WNV, especially in areas with a high number of WNV human cases with more non-neuroinvasive patients. WNV is an emerging infectious disease with a wide clinical spectrum and variable long-term effects; thus a public health concern.
92

Maladies infectieuses émergentes au sein des zones humides méditerranéennes dans le contexte des changements globaux / Climate changes and emerging infectious diseases in the Mediterranean wetlands

Vittecoq, Marion 23 November 2012 (has links)
L'émergence de maladies telles que le SRAS et le SIDA au cours des dernières décennies a fait prendre conscience des liens étroits existant entre santé animale, santé humaine et santé des écosystèmes. En effet, les pathogènes émergents ont pour la plupart une origine zoonotique (i.e. ils circulaient à l'origine au sein des populations animales). Les risques sanitaires associés à ces émergences sont en constante évolution sous l'influence des changements globaux qui modifient les écosystèmes et les contacts entre les hôtes. La prévention et le contrôle des maladies infectieuses émergentes nécessitent la compréhension de leur dynamique dans l'ensemble des compartiments dans lesquels elles circulent. Le travail présenté ici avait pour objectif d'améliorer cette compréhension au sein des zones humides méditerranéennes en ce concentrant sur deux pathogènes émergents : les virus Influenza A (VIA) et le virus West Nile. Il a été structuré selon trois axes de recherche : i) Utiliser la surveillance épidémiologique de l'avifaune sauvage pour comprendre la circulation du virus West Nile dans le bassin méditerranéen ii) Comprendre la dynamique des VIA au sein des différents compartiments où ils circulent et à leur interface iii) Comprendre le rôle des conditions environnementales dans la dynamique des VIA notamment au sein des populations humaines. Nos résultats mettent en évidence l'intérêt de mener des études multidisciplinaires sur le long terme pour comprendre l'épidémiologie des maladies émergentes. Ils soulignent également le rôle des activités anthropiques et des conditions environnementales dans la dynamique de ces maladies. Nos études apportent des éléments de réflexion pour allier gestion des risques d'émergence et gestion des écosystèmes et des populations. Elles encouragent à développer ce type d'approche afin de relever le défi de la prévention et du contrôle des pathogènes émergents. / During the last decades, the emergence of numerous infectious diseases such as SARS and AIDS has raised awareness of the close links that exist between animal health, human health and ecosystem health. Many of the emerging pathogens have a zoonotic origin (i.e. they originally circulated among animal populations). The health risks associated with the emergence of these diseases are progressing under the influence of global changes that affect ecosystems and contacts between hosts. The prevention and control of emerging infectious diseases require an in-depth understanding of their dynamics in all the compartments in which they occur. The aim of the present work is to improve our understanding of these phenomena within the context of Mediterranean wetlands by focusing on two emerging pathogens: Influenza A viruses (IAV) and West Nile virus. The thesis is structured around three research axes i) Using epidemiological surveillance of wild birds to investigate the circulation of West Nile virus in the Mediterranean Basin ii) Exploring IAV dynamics in the different compartments in which they circulate and at their interface iii) Determining the role of environmental conditions in IAV dynamics, especially within human populations. Our results highlight the value of long-term interdisciplinary studies for the understanding of the epidemiology of emerging diseases. They also emphasize the role of human activities and environmental conditions in the dynamics of these diseases. Our studies open up perspectives for combining emerging disease risk management and the management of ecosystems and populations. They also argue in favour of further developing this type of approach in order to meet the challenge of emerging pathogen prevention and control.
93

Functional Analyses of West Nile Virus (WNV) Bicistronic Replicons Containing Different Sequence Elements and of Simian Hemorrhagic Fever Virus (SHFV) Polyprotein Processing

Radu, Gertrud Ulrike 29 November 2007 (has links)
The flavivirus West Nile virus (WNV) encodes a single polyprotein that is processed into three structural and seven nonstructural proteins. Various WNV bicistronic replicons that direct cap-dependent translation of an N-terminal viral capsid or capsid/Renilla luciferase fusion protein as well as IRES-dependent translation of the nonstructural proteins were constructed. An original replicon consisting of the WNV 5' NCR, the 5' 198 nts of the capsid coding sequence, which included the 5' cyclization sequence (Cyc), and an EMCV IRES followed by the WNV nonstructural genes and 3' NCR was generated. Real time qRT-PCR analysis of intracellular levels of this replicon RNA showed a 4 fold increase by 96 hr after transfection of BHK cells. Increasing the distance between the 5' Cyc and IRES by insertion of a 5' IRES flanking sequence alone or together with a Renilla luciferase reporter did not increase RNA replication. Addition of only a reporter decreased RNA replication. The insertion of an extended capsid coding sequence also did not enhance RNA replication, but did enhance both cap- and IRES-dependent translation of replicon RNA, as indicated by immunofluorescence and Western blot analysis. These results suggest the presence of a translation enhancer in the 3' portion of the capsid coding region. Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, order Nidovirales. SHFV is unique among Nidoviruses in having three instead of two papain-like cysteine protease (PCP) motifs designated alpha, beta, and gamma, within the N-terminal region of its ORF1a. Mutations of putative PCP cleavage sites showed that the most efficient cleavage was by PCP beta at its downstream cleavage site. A large deletion located between the two catalytic residues of PCP alpha was hypothesized to render this protease inactive. However, processing was observed at the cleavage site following PCP alpha. Mutational analyses confirmed that PCP alpha is an inactive protease, and that the cleavage sites downstream of PCP alpha are cleaved by PCP gamma. When the catalytic residues of PCP gamma were mutated, PCP beta was also able to back cleave at these sites. This "back" cleavage is a previously unreported activity for an arterivirus PCP.
94

The ecology of infectious pathogens in a long distance migratory bird, the blue-winged teal (Anas discors): from individuals to populations

2013 May 1900 (has links)
The aim of this study is to improve our understanding of the ecology, spatiotemporal patterns, and risk of infectious pathogens of migratory waterfowl, using the blue-winged teal (Anas discors, BWTE), as a model. From 2007-2010, 1,869 BWTE were sampled in the prairie provinces (Alberta, Saskatchewan and Manitoba, Canada) to examine infection status and/or evidence of previous exposure to avian influenza virus (AIV), West Nile virus (WNV), and avian paramyxovirus-1 (APMV-1), in relation to host demographic variables (age, sex, body condition, exposure to other pathogens), other ecological variables such as local waterfowl breeding population density and local pond density, and year. The probability of AIV infection depended on an interaction between age and AIV antibody status. Hatch year birds with antibodies to AIV were more likely to be infected, suggesting an antibody response to an active infection. After hatch year birds with antibodies to AIV were less likely to be infected, suggesting immunity resulting from previous exposure. AIV infection was positively associated with local BWTE density, supporting the hypothesis of density dependent transmission. Exposure to WNV and APMV-1 were also associated with age and year. Furthermore, the probability of WNV exposure was positively associated with local pond density rather than host population density, likely because ponds provide suitable breeding habitat for mosquitoes, the primary vectors for transmission. We also investigated large-scale spatiotemporal trends in apparent prevalence of AIV across Canada and the United States throughout the year, using data from national avian influenza surveillance programs in Canada and the US in 2007-2010. Our analyses revealed that age, sex, year of sampling, flyway, latitude, and season (categorized by stages of the BWTE annual life cycle) were all important variables in predicting probability of AIV infection. There was an interaction between age and season. During late summer staging (August) and fall migration (September-October), hatch year birds were more likely to be infected than after hatch year birds, however there was no difference between age categories for the remainder of the year (winter, spring migration, and breeding season). Probability of infection increased non-linearly with latitude, and was highest in summer, corresponding to the beginning of fall migration when densities of birds and the proportion of susceptible hatch year birds in the population are highest. Birds in the Pacific, Central and Mississippi flyways were significantly more likely to be infected compared to those in the Atlantic flyway. Observed trends in seasonal, annual, and geographic patterns of AIV infection in BWTE across Canada and the US were primarily driven by the dynamics of AIV infection in hatch year birds. Our results demonstrate demographic as well as seasonal, latitudinal and flyway trends across Canada and the US. This research provided further evidence for the role of wild dabbling ducks, particularly BWTE, in the maintenance and ecology of AIV. This improved understanding of the role of BWTE as natural hosts, and the geographic, demographic and temporal variables that affect infection and transmission parameters, moves us closer to deciphering the overall ecology of the virus and its transmission and transportation pathways at the individual, population and continental levels. This knowledge, in turn, will permit development of better tools to predict and perhaps to prevent possible outbreaks in domestic animals as well as in humans.
95

Regulation of Interferon Stimulated Genes in West Nile Virus Infected Mouse Embryofibroblasts

Pulit-Penaloza, Joanna A 05 May 2012 (has links)
The induction of type I interferon (IFN) and subsequent activation of interferon stimulated genes (ISGs) represent a first line of defense against viral infection. Typically type I IFN signaling leads to the phosphorylation of the STAT1 and STAT2 transcription factors (TFs) which then form a trimetric complex with IRF-9 and translocate to the nucleus to induce ISG expression. However, the results of this study showed that IFN-mediated upregulation of the ISG Oas1b, the product of which confers resistance to flavivirus induced disease, can be induced in a STAT1-independent manner. Since numerous ISGs have antiviral functions, many viruses have evolved strategies to disrupt the type I IFN-signaling pathway. In cases when STAT1 activation is blocked by a viral infection, STAT1-independent upregulation of ISGs provides an additional strategy for the cell to mount an effective antiviral response. Infection of mouse embryofibroblasts (MEFs) with West Nile virus (WNV) induced the production of IFN beta and STAT1 and STAT2 phosphorylation but blocked nuclear translocation and binding of these TFs to the promoters of the ISGs, Oas1a, Oas1a, Irf7 and Irf1. However, each of these antiviral ISGs was efficiently upregulated in infected cells and IRF-9 was shown to be crucial for the upregulation of Oas1a, Oas1b and Irf-7. IRF-3 or IRF-7 was needed to maintain the upregulation of these genes at later times of infection. In contrast, the upregulation of Irf1 by WNV infection did not depend on the tested IRFs but was reduced by inhibition of the p38 or NF-kappa B pathways. Although Irf1 mRNA was efficiently upregulated in WNV-infected cells IRF-1 protein synthesis was blocked. The precise mechanism of the IRF-1 translational suppression is not yet known, but the suppression was shown not to be due to increased proteasomal degradation of IRF-1 nor to alternative splicing of Irf1 mRNA. Preliminary results suggest miRNAs may play an indirect role in regulating IRF-1 translation. The results of this study expand knowledge about the strategies evolved by viruses to evade host cell antiviral responses and also provide valuable insights about alternative mechanisms utilized by the host cell to counteract viral infections.
96

Assessment of U.S. Agriculture Sector and Human Vulnerability to a Rift Valley Fever Outbreak

Hughes, Randi Catherine 2011 May 1900 (has links)
Foreign animal disease outbreaks can cause substantial economic losses. Policy makers need information on both the vulnerability of the food supply to disease epidemics and the impacts of alternative protection actions. This research focused on the assessment of the U.S. agricultural sector and human vulnerability to a Rift Valley Fever (RVF) outbreak and the value of a select set of alternative disease control strategies. RVF is a vector-borne, zoonotic disease that affects both livestock and humans; thus both animal and human consequences of an outbreak were examined. This research was conducted in two parts. Livestock impact assessment used an integrated epidemic/economic model to examine the extent of RVF spread in the animal population and its consequences plus the outcome of implementing two different control strategies: emergency vaccination and larvicide vector control. The number of infected, aborted, and dead animals is best controlled by coupling vaccination along with larvicide, but results in the second highest median national welfare loss. Therefore, careful decisions must be made as to what actions should be taken. Total national producer welfare is reduced with each scenario, and is more severe than the total national welfare loss (producer, consumer, and processor together). Consumer welfare is increased with each scenario due to a drop in prices of some commodities, and in some instances, an increase in supply as well. The majority of the national welfare loss can be attributed to the producers' and processors' loss in welfare. The highest damages are seen in the regions of the outbreak such as the South Central (SC). Other regions such as the Corn Belt, Lake States, and South East regions also see high damages due to price changes. The outbreak did not have substantial price effect on dairy products, but did have noticeable price changes for live cattle such as heifer calves, stocked yearling, and dairy calves. Prices for substitutes such as pork, chicken, and turkey experienced a price reduction, which can also be a factor resulting in consumer welfare gains. Human impact assessment utilized an inferential procedure for estimating the human consequences which comprise of a cost of illness calculation to assess the dollar cost of human illnesses and deaths, as well as a Disability Adjusted Life Year calculation to give an estimate of the burden of disease on public health as a whole. With potential costs above $2 billion for human illness, and with this number not accounting for loss or damages to other sectors of the economy, it can be highly probable that investing in a human vaccination campaign can be cost-effective and possibly cost-reducing. This cost along with the economic loss of the agriculture sector suggests substantial potential losses to the U.S. if this hypothetical situation were to become reality. Combining total loss estimates from the cost of illness and ASM models, potential damage of a RVF outbreak could range from 121 million to 2.3 billion US 2010$. The results of this study show the economic damages of an outbreak in the livestock population being much greater relative to the outbreak in the human population (roughly 16 times greater). It should be pointed out that both cost estimates are most likely under estimated. The animal outbreak is not incorporating all susceptible livestock (e.g. hogs and goats), and the human illness is not incorporating other damages to society (e.g. damages due to loss of tourism). By providing estimates on the potential economic outcomes, policy makers can better choose where, when, and how to invest their resources.
97

A comparison study of gravid and under house CO2 mosquito traps in Harris County, Texas

White, Stephanie Lyn 10 October 2008 (has links)
Harris County Mosquito Control Division (HCMCD) is responsible for surveillance of mosquito species that are vectors of St. Louis Encephalitis (SLE) virus and West Nile Virus (WNV) within Harris County, Texas, including the Houston metroplex. The metroplex area has some unique attributes and a vast variety of environmental habitats that are attractive to vectors of arboviruses and for the transmission of arboviruses to the human population. Data describing the efficacy of Gravid (GV) and Underhouse (UH) CO2 traps were analyzed to determine if there is a significant difference between these two trap types with respect to the number of mosquitoes and the variety of mosquito species caught. This study was conducted during the off-peak HCMCD trapping season, to gain information in preparation for a yearround trapping program utilizing Underhouse CO2 traps for WNV and SLE virus surveillance. Adjusting for the week of collection, results suggest that Gravid traps caught significantly (P = 0.009) more mosquitoes (mean = 23.134 per trap) in the study area than Underhouse traps (mean = 3.616 per trap), and that Underhouse Traps caught a larger variety of mosquito species (n = 13) than Gravid Traps (n = 11), out of 15 total different species caught. Gravid and Underhouse traps caught 9 out of 15 of the same mosquito species during the study period. Culex quinquefasciatus mosquito catches in Gravid traps and temperature were strongly correlated (Spearman's Correlation Coefficient = 0.707, P = 0.005). Geographic Information System spatial analysis indicated clustering of Culex quinquefasciatus mosquito catches in both Gravid traps, week 9 and 21 (Moran's I = 0.69, P = 0.040 and 0.74, P = 0.021, respectfully ) and Underhouse traps, week 13 and 19 (Moran's I = 0.92, P = 0.002, and 0.89, P = 0.011, respectfully). It is recommended that Harris County Mosquito Control Division continue to utilize gravid traps as a primary method of surveillance. Gravid traps (16,194) caught 85% more mosquitoes than Underhouse traps (2,531) over the fourteen week study period. Their overall success far outweighs the additional materials or labor required for their use in a successful surveillance program.
98

Cytotoxic T lymphocyte Responses Against Japanese Encephalitis Virus In Mice: Specificity And Immunotherapeutic Value

Krishna, Kaja Murali 10 1900 (has links)
Cytotoxic T Lymphocytes (CTL) are known to play an important role in clearing infectious virus from infected hosts in a variety of viral infections. Depending on the type of virus and mode of virus entry both class I and class II restricted CTL can contribute to protection from virus-induced disease. Although CD8 positive CTL are associated with virus elimination and control in many viral infections, elimination of neurotropic viruses from the Central Nervous system (CNS) is more complex due to the lowered expression of MHC antigens on neuronal cells. This failure to constitutively express high levels of MHC antigens by neurons could serve as an advantage to avoid damage to this differentiated and non-renewable tissue. However, abnormal induction of MHC antigens in the CNS mediated by CD4 positive lymphocytes or by astrocytes have also been shown to cause destructive inflammation in the CNS. The present study deals with CTL responses against one such neurotropic virus called Japanese Encephalitis Virus (JEV). JEV is a positive-stranded RNA virus that belongs to the flavivirus group, a group that is among the most important agents causing human encephalitis worldwide. Although passive transfer of monoclonal antibodies against this virus has been shown to confer protection of mice from lethal challenge with virus, neither the presence of CTL against this virus nor its role in conferring protection has been reported so far. Understanding the CTL responses against these viruses acquired importance in light of recent reports that neurovirulence of JEV and yellow fever viruses can be enhanced by the administration of virus specific antibodies. Hence this study was undertaken to examine the possibility of raising CTL specific to JEV. The specificity of the CTL raised, their therapeutic value and the ability of different lymphocyte subsets to mediate protection in vivo are dealt with in this study. Generation of CTL against JEV The generation of CTL against JEV in BALB/c mice, requires MHC defined cell lines that not only support virus infection but are also histocompatible. Several cell lines were initially examined for their ability to support JEV infection as a prc-rcquisitc before their utilization in in vivo and in vitro stimulation protocols aimed at generating JEV-specific CTL. Virus infection was monitored by immunofluorescence using JEV envelope-specific monoclonal antibodies as well as by titration of virus produced from infected cells by plaque assays. These different cell lines that were characterised for their ability to support JEV infection were then utilised to generate and monitor antiviral CTL. Several in vivo immunisation protocols were examined initially find out which of these infected cells prime BALB/c mice efficiently for generation of virus-specific CTL upon secondary stimulation in vitro with infected syngeneic cells. Immunisation of mice with infected cells per se was preferred over free virus since this was thought to facilitate priming against some viral non-structural proteins preferentially found on infected cells in addition to other viral structural proteins. It was observed that not only infected syngeneic and allogeneic cells but also infected xenogeneic cells prime BALB/c mice for the generation of JEV- specific CTL upon secondary restimulation in vitro. An optimal protocol was standardised for the generation of CTL against JEV. This included primary in vivo immunisation of mice followed by secondary in vitro restimulation of splenocytes with infected syngeneic cells. Either immunisation alone or in vitro stimulation of naive splenocytes alone was unsuccessful. The effector cells generated specifically lysed JEV-infccted P388D1 targets but not uninfected P388D1 or YAC-1 targets suggesting that the lysis on infected targets is not mediated by Natural Killer activity. Specificity and MHC restriction of anti JEV Effectors Cell depletion studies using complement mediated lysis were performed to examine the phenotype of the cells mediating virus specific lysis of infected targets. Depletion of Lyt 2.2+ or Thy 1+ but not L3T4+ sub-populations of effector cells inhibited lysis of infected targets showing that the effectors mediating virus-specific lysis were Lyt-2+ T cells. Examination of target specificities and MHC restriction of the antiviral CTL generated showed that although infected xenogeneic cells were used for immunisation, the effector cells recognised only infected syngeneic (P388D1, Sp2/0) and semisyngeneic (Neuro 2a, YAC-1) cells. Virus-specific recognition was found to be class I Kd and class I Dd restricted. These effector cells were also found to recognise cells infected with a closely related flavivirus, West Nile Virus (WNV) suggesting that they were crossreactive to some degree. Based on the consensus motif that has been established for H-2Kd associated peptides, several nonamers were predicted as possible CTL epitopes by scanning the deduced amino acid sequences of three strains of JEV and WNV. Among several predicted nonamers, three peptides were examined for their ability to reconstitute lysis of uninfected targets by polyclonal anti JEV CTL populations. Results demonstrate that peptides derived from NS1 and NS3 but not NS5 protein of JEV were able to partially reconstitute lysis of uninfected targets by effectors when pulsed with the appropriate peptide. Protective ability of the CTL raised against JEV To examine whether anti-JEV effectors raised in vitro could confer protection from intracerebral challenge with JEV, these effectors were adoptively transferred into adult BALB/c mice intracerebrally along with 10 x LDJ0 dose of JEV. More than 55% of these animals were protected from death and survived beyond 100 days after JEV challenge demonstrating that adoptively transferred anti-JEV effectors could indeed confer protection from lethal challenge with JEV. However, adoptive transfer of effectors by either intravenous or intraperitoneal routes did not protect adult mice from the lethal effects of intracerebral challenge with JEV. In contrast to adult mice, newborn mice were not protected from death by the adoptively transferred effector cells. This was also supported by experiments where a correlation was observed with the increasing age of mice and the success of protection conferred by the adoptively transferred effector cells. To establish the identity of cell subsets responsible for protection, Lyt 2, L3T4 or Thy 1 positive cells were specifically depleted from the polyclonal CTL by multiple cycles of complement mediated lysis and the remaining cells were adoptively transferred intracerebrally along with 10 x LD of JEV. These results demonstrate that both Lyt 2 and L3T4 positive T cells present in the effector population were necessary to confer protection of adult mice. Examination of virus-specific neutralising antibodies in the sera of protected and unprotected mice revealed that presence of L3T4 positive cells in the adoptively transferred population increases virus-specific neutralising antibodies. However presence of neutralising antibodies alone was not sufficient to confer protection. The protection required both Lyt-2 and L3T4 positive cells together. These studies could in the long term throw some light on similar observations about age dependant susceptibility to JEV in humans.
99

Eine Studie zum Vorkommen des West-Nil-Virus in der Wildvogelpopulation Deutschlands

Prell, Juliane 14 November 2013 (has links) (PDF)
In den letzten Jahren erreichten viele neue (emerging) Viren Europa, die zum Teil (z.T.) zoonotisch auf den Menschen übertragbar sind. So musste man sich mit Geflügel- und Schweinegrippe, Blauzungenkrankheit, Infektiöser Anämie der Einhufer oder auch SARS (severe acute respiratory syndrome) auseinandersetzen. Bedingt durch verschiedene Faktoren, wie Klimawandel oder zunehmende Globalisierung und damit einhergehendem Verkehr zwischen den Kontinenten verbesserten sich auch die Bedingungen für die Virusverbreitung, so dass viele für Deutschland untypische Krankheitserreger auch hier auftraten. Das West-Nil-Virus (WNV) ist in Europa bereits endemisch verbreitet und könnte somit eine besondere Gefahr für Deutschland darstellen. Es ist ein bekannter Zoonose-Erreger, und sein Eintrag und die rasche Verbreitung des Virus in Amerika 1999 zeigten wie gefährlich neue Viren in naiven Populationen sein können. Über die Verbreitung des Virus in Deutschland gibt es nur wenige Studien z.B. des Robert-Koch-Instituts (LINKE et al. 2007a) und des Friedrich-Loeffler-Instituts (SEIDOWSKI et al. 2010), wobei in keiner Studie tote Vögel als Untersuchungsmaterial genutzt wurden. Da das WNV in Amerika mit einem auffälligen Vogelsterben einherging, ist es naheliegend, den Virusnachweis zuerst bei toten Vögeln zu erbringen.
100

Expression diagnostisch verwendbarer Antigene zum Nachweis West-Nil-Virus-spezifischer Antikörper

Delker, Anna Maria 26 March 2014 (has links) (PDF)
Grundlage der vorliegenden Arbeit ist die Überlegung, dass eine Möglichkeit, die Spezifität der bisher angewendeten Verfahren zur West-Nil-Virus-Diagnostik zu verbessern, in der Anwendung rekombinanter WNV-spezifischer Antigene besteht. Die unter anderem auf bioinformatischen Methoden basierende Identifikation von potenziellen B-Zell-Epitopen und Auswahl entsprechender Sequenzabschnitte richtete sich dabei gezielt auf immunogene Bereiche, die innerhalb der Gruppe der Flaviviren einen ausreichenden Sequenzunterschied zu allen weiteren sequenzverwandten Erregern, zusammengefasst im Japanische Enzephalitis-Serokomplex, boten. Drei ausgewählte Bereiche innerhalb der Strukturproteinsequenz, bezeichnet als prM, Cnat und Cme, sollten mit Hilfe des Expressionssystems Pichia pastoris bzw. Escherichia coli rekombinant exprimiert werden. Nach Erarbeitung optimaler Expressionsbedingungen folgte die affinitätschromatografische Reinigung der im weiteren Verlauf zur Immunisierung von Balb/c-Mäusen eingesetzten Polypeptide. Die gewonnenen Seren der nach verschiedenen Immunisierungsprotokollen geimpften Mäuse wurden im Anschluss immunologisch untersucht. Es zeigte sich, dass die rekombinanten Derivate des Capsid-Proteins eine deutliche Serokonversion hervorriefen. Analysen der mit Cnat und MBP-Cme immunisierten Mausseren wiesen vorhandene peptidspezifische sowie virusspezifische Antikörper nach. Der Einsatz dieser gewonnenen Peptidantigene im indirekten ELISA-Testsystem zur Detektion WNV-spezifischer Antikörper unter Verwendung humaner WNV-IgG-positiver Serumproben zeigte positive Resultate. Im Gegensatz hierzu führte die Immunisierung mit prM lediglich zu einer unspezifischen murinen Antikörperbildung. Die Unterscheidung zwischen WNV-positiven und WNV negativen Humanseren war unter Verwendung des rekombinanten Antigens prM nicht möglich. Im Ergebnis zeigten zwei der drei in dieser Arbeit rekombinant erstellten Strukturproteinabschnitte ihr immunologisches Potenzial in der Generierung muriner WNV spezifischer Antikörper. Zudem konnte mit der Expression der WNV-spezifischen C Protein Antigene ein Beitrag zur Etablierung eines indirekten ELISA-Testsystems zur Detektion WNV-bedingter Humaninfektionen geleistet werden.

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