West Nile virus (WNV), a member of the Flaviviridae family of RNA viruses, was first introduced to the United States in 1999 with rapid transmission across a variety of hosts throughout the continental states. Genetic research to identify genes involved in resistance and susceptibility to WNV began in mice, where it was observed that natural populations were resistant or fatally susceptible. Further investigation led to the identification of the Flavivirus resistance gene as the oligoadenylate synthetase 1b gene in mice. A nonsense mutation was found within the coding region of this gene that associated absolutely with susceptibility to WNV. A two-stage association study was conducted to identify similar genetic associations to West Nile encephalitis in naturally susceptible and resistant populations of horses in the United States. Genomic sequence of a majority of the equine 2’,5’-oligoadenylate synthetase 1 (OAS1) gene was assembled by shotgun-sequencing CHORI BAC 100:I10 (3.95X). A contig map spanning the entire gene was constructed, including 8 kilobases of promoter sequence upstream of the first exon. Coding regions of equine OAS1 and ribonuclease L (RNASEL) genes, as well as the OAS1 promoter, were screened for mutations from a random sample of horses of multiple breeds. Numerous polymorphisms were identified for case-control analyses. Analysis using Fisher’s Exact test identified allelic and genotypic associations. Odds ratios were also determined to measure strength of the associations. Case-control analysis of haplotype frequencies identified significant differences in haplotype frequencies between populations and association to West Nile encephalitis. A conserved interferon-stimulated response element was mapped to within 518 basepairs upstream of the transcription start site of OAS1. Promoter polymorphisms were not found to affect induction by interferon-tau; however, additional analyses are necessary to further characterize the equine OAS1 promoter and the host factors involved in regulating expression. Statistical analyses of the genotype data from the case and control populations identified significant associations between polymorphisms of the OAS1 and RNASEL genes with severe West Nile encephalitis. The similarity between human and horse OAS immunity genes suggests that the horse may provide a genetic model to further investigate mammalian SNP-associated viral susceptibility.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-23T21:39:24Z No. of bitstreams: 1 Luciane Amorim Santos Uso de ferramentas de bioinformática....pdf: 5329323 bytes, checksum: b477ef604e538000e5f8d84e1188ae91 (MD5) / Made available in DSpace on 2012-07-23T21:39:24Z (GMT). No. of bitstreams: 1 Luciane Amorim Santos Uso de ferramentas de bioinformática....pdf: 5329323 bytes, checksum: b477ef604e538000e5f8d84e1188ae91 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / As ferramentas de bioinformática tem sido amplamente utilizada para o melhor entendimento de diversos microorganismos. Neste trabalho foram realizados três estudos utilizando estas ferramentas para avaliar diferentes questões biológicas. No primeiro estudo realizou-se uma caracterização molecular de 57 sequências do gene pol, provenientes de pacientes infectados pelo HIV-1 de Salvador, Bahia, Brasil. Para identificar os subtipos e formas recombinantes do HIV-1 circulante na cidade de Salvador foi realizado análises filogenéticas, e através do algoritmo do banco de dados Stanford HIV resistance as mutações associadas à resistência aos ARVs foram detectadas. Entre as 57 sequências analisadas foram identificados neste estudo 45 (77,2%) pertencem ao subtipo B, 11 (21,0%) recombinantes BF e uma (1,8%) do subtipo F1. Além disto, uma alta frequência de eventos de recombinação entre os subtipos B e F foram detectados com 5 padrões de recombinação, duas intergênicas e três intragênicas, mostrando uma alta diversidade. As mutações encontradas com uma maior prevalência foram: I54V (PI) em 7,0%; M184V (NRTI) em 14,0% e K103N (NNRTI) em 10,5% das sequências analisadas. Estes resultados contribuem para traçar o perfil da epidemiologia molecular e diversidade do HIV-1 em Salvador. O segundo estudo avaliou a filodinâmica do HIV-1 em pares de mãe e filho infectados, e em diferentes fases da infecção, três pares na fase aguda e um na fase crônica, e que apresentavam sequências de diferentes tempos. Para este fim foi realizado inferências filogenéticas bayesianas, onde a hipótese do relógio molecular e de diferentes crescimentos populacional foram testadas. Não foi possível observar uma diferença entre a dinâmica da população viral da mãe e a encontrada no filho. Porém, quando observamos o crescimento populacional e o tamanho da população efetiva, ao longo do tempo, sequências provenientes de pares em fase crônica da infecção tem um crescimento mais constante, enquanto as sequências dos pares na fase aguda da infecção se observa uma dinâmica das populações virais, provavelmente devido à pressão do sistema imune e a não adaptação destes vírus. No terceiro estudo, 104 sequências do genoma completo do WNV, disponíveis no Genebank, foram estudadas para identificar a região genômica que apresenta máximo poder interpretativo para inferir relações temporais e geográficas entre as cepas do vírus. Alinhamentos de cada gene foram submetidos à avaliação do sinal filogenético através do programa TREEPUZZEL. As regiões NS3 e NS4 apresentaram um sinal filogenético acima de 70%, sendo as regiões mais indicadas para construção filogenética. Além disto, árvores bayesianas foram inferidas utilizando as regiões NS3, NS5 e E, onde os clados das árvores NS3 e NS5 apresentaram um maior suporte e estrutural temporal geográfica, diferente da região E. Estes achados mostram que os genes NS3 e NS5 são os mais indicados para análises filogenéticas. Neste trabalho foi demonstrando o uso de ferramentas de bioinformática para a melhor caracterização da diversidade, epidemiologia molecular, dinâmica populacional e determinação das relações temporal e geográfica dos vírus. / The bioinformatics tools have been widely used for better understanding of several microorganisms. Here three studies were performed using these tools to answer different biological questions. In the first study, it was conducted the molecular characterization of 57 HIV-1 pol gene sequences from infected patients from Salvador, Bahia, Brazil. To identify the HIV-1 subtypes and recombinants forms, phylogenetics analyses were performed and the Stanford HIV resistance Database were used to analyze the antiretroviral susceptibility. Among all analyzed sequences, 45 of them were (77.2%) subtype B, 11 (21.0%) were BF recombinant and one sequence was (1.8%) subtype F1. Furthermore, a high frequency of recombination events between subtypes B and F was detected with five different patterns: two intergenic and three intragenic. The mutations found with higher prevalence were: I54V (PI) in 7.0%; M184V (NRTI) in 14.0% and K103N (NNRTI) in 10.5% of the analyzed sequences. These results contribute for the knowledge of the molecular epidemiology and diversity of HIV-1 in Salvador. The second study have evaluated the HIV-1 phylodynamics in mother and child infected pairs in different stages of infections: three pairs acutely infected and one chronically infected. Phylogenetic inference was performed using the Bayesian framework were the molecular clock and different population growth models hypothesis were tested. We did not find any difference of the population dynamics between mother and child. However, when observing the population growth and the effective population size through time, the chronically infected pair sequences showed a constant growth, while the acutely infected pair sequences showed a more dynamic population growth, probably due to the immune system selective pressure. In the third study, 104 WNV full genome sequences were selected from Genbank, to identify the best genomic region, which could provide the maximal interpretative power to infer temporal and geographic relationships among the virus strains. The phylogenetic signal was evaluated using the TREEPUZZEL program. The results showed that the NS3 and NS5 regions are the best ones to infer phylogeny since their phylogenetic signal was higher than 70%. Furthermore, Bayesian trees were constructed using the NS3, NS5 and E regions, and the NS3 and NS5 tree clades showed a higher support and a temporal geographic structure, different from the E region. These findings show that the NS3 and NS5 genes are the most informative genes for phylogenetic analyses. These studies demonstrated the use of bioinformatics tools for the better characterization of the virus diversity, molecular epidemiology, and population dynamics.

No description available.

This thesis describes the West Nile virus (WNV) epidemic in horses by exploring all aspects: sub-clinical infection, development of clinical disease and case fatality. All of the collected data were then compiled to create predictive risk maps of WNV infection for the province of Saskatchewan. <p>During the 2003 season, 133 clinical cases were documented with laboratory testing. Week of onset of clinical signs, gender, and coat color were significant predictors of whether the horse died or was euthanized due to severity of clinical signs. Studies of the serological response to vaccination and natural infection were examined to interpret the lab results from over 1100 samples taken from approximately 875 horses in 2003. A serologic study involving 212 horses on 20 farms determined the prevalence of sub-clinical infection (55.7% (95%CI, 44.9% to 65.8%)) and identified risk factors for infection. The study found risk of infection was highest in the Grasslands ecoregions compared to the Boreal Transition ecoregion. A case control study looked at risk factors for development of clinical disease. The study followed 23 case farms and control farms with a total of 300 horses sampled. This was the first field study to show that vaccination was efficacious in preventing the development of clinical signs. <p>The inclusion of horse surveillance data in the Saskatchewan Health WNV Integrated Surveillance Initiative was useful; however, it was discontinued due to time constraints, logistics, and declining monetary resources. <p>Since West Nile Virus is a mosquito-borne disease it is highly influenced by environmental changes, spatially and temporally. Discriminant analyses were used to partition Saskatchewan rural municipalities (RM) into categories of risk of infection with WNV based on acquired horse data and different environmental and meteorological data derived from both satellites or climate stations. The result was the creation of yearly predictive risk maps defining low to high risk of infection with WNV for each RM. <p>The 2003 epidemic provided a novel opportunity to study an important zoonotic disease emerging in a new environment. The information gathered will further the knowledge base upon which decisions for prevention of infection and clinical disease are made.

This thesis describes the West Nile virus (WNV) epidemic in horses by exploring all aspects: sub-clinical infection, development of clinical disease and case fatality. All of the collected data were then compiled to create predictive risk maps of WNV infection for the province of Saskatchewan. <p>During the 2003 season, 133 clinical cases were documented with laboratory testing. Week of onset of clinical signs, gender, and coat color were significant predictors of whether the horse died or was euthanized due to severity of clinical signs. Studies of the serological response to vaccination and natural infection were examined to interpret the lab results from over 1100 samples taken from approximately 875 horses in 2003. A serologic study involving 212 horses on 20 farms determined the prevalence of sub-clinical infection (55.7% (95%CI, 44.9% to 65.8%)) and identified risk factors for infection. The study found risk of infection was highest in the Grasslands ecoregions compared to the Boreal Transition ecoregion. A case control study looked at risk factors for development of clinical disease. The study followed 23 case farms and control farms with a total of 300 horses sampled. This was the first field study to show that vaccination was efficacious in preventing the development of clinical signs. <p>The inclusion of horse surveillance data in the Saskatchewan Health WNV Integrated Surveillance Initiative was useful; however, it was discontinued due to time constraints, logistics, and declining monetary resources. <p>Since West Nile Virus is a mosquito-borne disease it is highly influenced by environmental changes, spatially and temporally. Discriminant analyses were used to partition Saskatchewan rural municipalities (RM) into categories of risk of infection with WNV based on acquired horse data and different environmental and meteorological data derived from both satellites or climate stations. The result was the creation of yearly predictive risk maps defining low to high risk of infection with WNV for each RM. <p>The 2003 epidemic provided a novel opportunity to study an important zoonotic disease emerging in a new environment. The information gathered will further the knowledge base upon which decisions for prevention of infection and clinical disease are made.

West Nile virus (WNV) belongs to the Flaviviridae family, a virus family of which many members are known as human pathogens. WNV has a worldwide distribution and strains that cluster in lineage II is endemic to sub-Saharan Africa. The complete nucleotide sequence of four lineage II West Nile virus strains, isolated in South Africa from patients with mild or severe WNV infections, were determined. Using a murine model, these strains had been shown to produce either highly or less neuroinvasive infections and induced similar genes to corresponding highly or less neuroinvasive lineage I strains. Nucleotide and amino acid sequence comparison between highly and less pathogenic lineage II strains demonstrated that the non-structural genes and in particular the gene coding for the NS5 proteins were the most variable. All the lineage II strains sequenced in this study were found to possess the E-protein glycosylation site previously postulated to be associated with virulence. Comparison of the signalase cleavage sites suggested that lineage II strains may be cleaved slightly more efficiently than lineage I strains in the C-prM junction, but less efficiently between prM and E genes. Relative to the highly neuroinvasive strains sequenced in this study major deletions were found in the 3’ noncoding region of 2 lineage II strains shown in previous studies to be either less- or not at all neuroinvasive. This is the first report of full genome sequences of highly neuroinvasive lineage II WNV strains. Currently available commercial WNV ELISA kits were developed with lineage I WNV strains and are expensive to use. For these reasons the development of a potential ELISA diagnostic assay based on the South African lineage II strain, H442, was envisaged. Such assay, if reliable and efficacious would be a useful tool towards WNV surveillance. The prM and E genes were selected to be expressed as recombinant antigens because of their co-expression nature and because the envelope protein is the principal target for neutralization. After cloning of the respective genes and verification of integrity, a mammalian expression system was utilized. Different mammalian cells and transfection media were tested and BHK 21 cells with SuperFect transfection medium were found to be best. Attempted expression of proteins was tested with immunofluorescent antibody testing as well as SDS-PAGE and Western blot analysis. Expression of recombinant WNV antigens were also tested in indirect and sandwich ELISA’s systems. It was however not possible to perform these two ELISA systems at a satisfactory level or clearly indicated if expression of proteins was successful. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

West Nile Virus (WNV) forms part of the Japanese encephalitis serocomplex in the genus Flavivirus, family Flaviviridae. This enveloped positive single-stranded RNA (+ssRNA ) virus is the etiological agent of West Nile fever, and in more severe cases WNV neuroinvasive disease, in both humans and animals. WNV is distributed worldwide and is phylogenetically classified into five distinct lineages. The WNV genome is ~11 Kb in length and encodes a single open reading frame (ORF) that is post-translationally cleaved into three structural proteins and seven non-structural proteins. In this study, two contemporary and two historic South African WNV strains were genetically characterised as lineage 2 strains based on complete genome sequences. Genetic change as a result of passage number and propagation system was quantified on both the consensus genome- and quasispecies level. A lack of variation was observed amongst the consensus genome sequences of WNV strains subject to changes in propagation system from BHK-21 cell culture to mouse brain and vice versa. In contrast, variation amongst the latter was observed on the quasispecies level. Genome-wide single nucleotide polymorphism (SNP) profiles as well as full-length haplotype sequences reconstructed from ultra deep sequence data indicated that high levels of quasispecies diversity persists, particularly in the capsid gene region, during changes in propagation environment. The changes in frequency of variants were consistent throughout isolates propagated in different systems. The increased variation in the capsid gene region may result from selective pressures brought about by differences in host cell type between propagation systems. This study is the first to demonstrate quasispecies dynamics resulting from changes in propagation system of a lineage 2 WNV based on the reconstruction of full-length haplotype sequences from ultra deep sequence data. The approach demonstrates a cost-effective alternative to the estimation of viral population structure in light of viral evolutionary dynamics, which may in turn be assessed by the single plasmid reverse genetic system designed in this study. Although early attempts at rescuing an infectious WNV clone were unsuccessful, the system shows promise in the application of future studies concerning vaccine and diagnostic development, virulence studies and disease control. / Dissertation (MSc)--University of Pretoria, 2013. / gm2013 / Zoology and Entomology / Unrestricted

No description available.

Recently, non-cellular viral agents became the focus of a large number of scientific groups. A prominent and widespread group of these viruses are flaviviruses, which include, for example, Zika virus, Dengue fever virus, tick-borne encephalitis virus and West Nile virus. There is a considerable diversity among these viruses, however, highly conserved proteins can be found throughout this viral genus. The largest and most conserved protein encoded by flaviviruses is the nonstructural NS5 protein. Its N-terminal domain bears the methyltransferase (MTase) activity. Thanks to the methylation of its genome, it allows the virus to initiate translation and at the same time mask it from the host's immune system. By blocking the active site of this enzyme with a small molecule, viral infection could be stopped not only in one flavivirus, but, due to the high conservation of MTases, in all other flaviviruses. This diploma thesis deals with the aforementioned MTase domain of the NS5 protein, specifically of the West Nile virus (WNV). After designing an insert encoding the WNV MTase domain, amplifying it and ligating it into the vector, the MTase domain was prepared by a recombinant expression, followed by purification. Subsequently, complexes of the protein with small molecules (MTase ligands) were formed, in...

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.