abstract: Flavivirus infections are emerging as significant threats to human health around the globe. Among them West Nile(WNV) and Dengue Virus (DV) are the most prevalent in causing human disease with WNV outbreaks occurring in all areas around the world and DV epidemics in more than 100 countries. WNV is a neurotropic virus capable of causing meningitis and encephalitis in humans. Currently, there are no therapeutic treatments or vaccines available. The expanding epidemic of WNV demands studies that develop efficacious therapeutics and vaccines and produce them rapidly and inexpensively. In response, our lab developed a plant-derived monoclonal antibody (mAb) (pHu-E16) against DIII (WNV antigen) that is able to neutralize and prevent mice from lethal infection. However, this drug has a short window of efficacy due to pHu-E16's inability to cross the Blood Brain Barrier (BBB) and enter the brain. Here, we constructed a bifunctional diabody, which couples the neutralizing activity of E16 and BBB penetrating activity of 8D3 mAb. We also produced a plant-derived E16 scFv-CH1-3 variant with equivalent specific binding as the full pHu-E16 mAb, but only requiring one gene construct for production. Furthermore, a WNV vaccine based on plant-derived DIII was developed showing proper folding and potentially protective immune response in mice. DV causes severe hemorrhaging diseases especially in people exposed to secondary DV infection from a heterotypic strain. It is hypothesized that sub-neutralizing cross-reactive antibodies from the first exposure aid the second infection in a process called antibody-dependent enhancement (ADE). ADE depends on the ability of mAb to bind Fc receptors (FcγRs), and has become a major roadblock for developing mAb-based therapeutics against DV. We aim to produce an anti-Dengue mAb (E60) in different glycoengineered plant lines that exhibit reduced/differential binding to FcγRs, therefore, reducing or eliminating ADE. We have successfully cloned the molecular constructs of E60, and expressed it in two plant lines with different glycosylation patterns. We demonstrated that both plant-derived E60 mAb glycoforms retained specific recognition and neutralization activity against DV. Overall, our study demonstrates great strives to develop efficacious therapeutics and potent vaccine candidates against Flaviviruses in plant expression systems. / Dissertation/Thesis / M.S. Applied Biological Sciences 2014

Limited research has been performed in Ontario to ascertain risk factors for West Nile Virus (WNV) and to develop a unified risk prediction strategy. The aim of the current body of work was to use spatio-temporal modelling in conjunction with surveillance and environmental data to determine which pre-WNV season factors could forecast a high risk season and to explore how well mosquito surveillance data could predict human cases in space and time during the WNV season. Generalized linear mixed modelling found that mean minimum monthly temperature variables and annual WNV-positive mosquito pools were most significantly predictive of number of human WNV cases (p<0.001). Spatio-temporal cluster analysis found that positive mosquito pool clusters could predict human case clusters up to one month in advance. These results demonstrate the usefulness of mosquito surveillance data as well as publicly available climate data for assessing risk and informing public health practice.

<div> <div> <div> <p>Flaviviruses is a genus within the family Flaviviridae. The genus consists of more than 70 viruses, including important threatening human pathogens such as dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). These viruses are causative agents for a range of mild to lethal diseases and there are currently no US- licensed therapeutic treatments for infection. The virus genome is a positive-sense, single-stranded RNA, encoding ten viral proteins. Of the ten flavivirus proteins, Non- Structural protein 1 (NS1) remains the most elusive in terms of its functions. To date NS1 has been linked to disease pathology and progression and plays roles in virus replication and assembly. However, little is understood how NS1 orchestrates these functions and how NS1 from different viruses function distinctively from one another. Moreover, flavivirus NS1 has a peculiar ability to associate with lipid membranes. During the life cycle of NS1, the protein travels through the classical secretory path- way, similar to infectious virus particles, and is secreted into the extracellular space as mostly hexameric oligomers containing a lipid core. How the protein binds to lipids and whether such lipid binding is important for NS1 functions and overall flavivirus pathology remain unknown. Using structure-based mutagenesis, we found a group of mutants on WNV NS1, which particularly altered the viral specific infectivity but maintained wild-type level of virus replication. Purified mutated virus particles revealed that the specific infectivity alteration was not because of the particle but interaction of the virus particles and NS1 mutated proteins. Here we demonstrated that specific residues on NS1 were responsible for distinctly roles in NS1 functions and the virus specific infectivity was regulated by NS1 protein. In other structure-base study, we focused on the membrane association ability of NS1. All structure-predicted regions on NS1 were examined for its contribution for the membrane/lipid binding function. This interaction was required for NS1 biology activities including intracel- lular trafficking, oligomerization, and endocytosis. The lipidomes from deletion of each membrane association region revealed differences in lipid classes binding to each region and the composition flexiblity of the lipid cargo of NS1 hexamer. </p> </div> </div> </div>

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

Natural products offer many possibilities for the treatment of disease. More than 70% of the Earth’s surface is ocean, and recent exploration and access has allowed for new additions to this catalog of natural treasures. The Central Red Sea off the coast of Saudi Arabia serves as a newly accessible location, which provides the opportunity to bioprospect marine sponges with the purpose of identifying novel antiviral scaffolds. Antivirals are underrepresented in present day clinical trials, as well as in the academic screens of marine natural product libraries. Here a high-throughput pipeline was initiated by prefacing the antiviral screen with an Image-based High-Content Screening (HCS) technique in order to identify candidates with antiviral potential. Prospective candidates were tested in a biochemical or cell-based assay for the ability to inhibit the NS3 protease of the West Nile Virus (WNV NS protease) as well as replication and reverse transcription of the Human Immunodeficiency Virus 1 (HIV-1). The analytical chemistry techniques of High-Performance Liquid Chromatograpy (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR) where used in order to identify the compounds responsible for the characteristic antiviral activity of the selected sponge fractions. We have identified a 3-alkyl pyridinium from Amphimedon chloros as the causative agent of the observed WNV NS3 protease inhibition in vitro. Additionally, we identified debromohymenialdisine, hymenialdisine, and oroidin from Stylissa carteri as prospective scaffolds capable of HIV-1 inhibition.

Culex pipiens, in the family Culicidae, has emerged as one of the biggest vectors for West Nile virus. It has two bioforms, pipiens and molestus, which differ from each other regarding habitat, diapause, and prey. Pipiens prefers to bite birds, and molestus prefers to bite humans. There is to some extent hybridization between the two, which creates a bridge-vector between birds and humans. One way to study the relationships and spreading of the mosquitos is using the intracellular bacteria Wolbachia pipientis which is present in at least 99% of al Culex mosquitoes. The bacteria have two fast evolving genes, pk1 and ank2 which are suitable for this. Not only are the bacteria suitable for genetics, but it is also manipulating the reproductive system of the mosquitoes through Cytoplasmic Incompatibility, which changes structures of populations and allows for the bacteria to spread fast and efficient. We wanted to investigate levels of Wolbachia in different populations, as well as if the two bioforms prefer a prey, together with mapping the relationships between populations using the two genes. We found that Wolbachia was present in all tested mosquitoes, with higher levels of it in the abdomen than in the thorax. We also found that the theory of a preferred prey was false within the tested populations, since both bioforms preferred birds. Lastly, we could identify five different strains of Wolbachia pipientis concentrated to different locations. This study has shown that Wolbachia is present in all tested mosquitoes and is a useful tool to determine relationships within and between populations. This is important to be able to gain understanding of the spread of West Nile virus and other vector borne diseases spread by Culex pipiens mosquitoes.

Culex quinquefasciatus has been identified as one of the most prominent vectors of West Nile virus (WNV) in Southern California. WNV is a zoonotic disease that is endemic in North America and is known to primarily cause flu-like symptoms in humans, and in rare cases, life-threatening conditions. The goal of this study was to identify which animal species are most frequently fed upon by these mosquitoes in this region. To examine the relationship between blood-feeding patterns and West Nile virus activity in San Bernardino County, the feeding patterns of Cx. quinquefasciatus are determined in a variety of habitat types, which was the primary focus of this study. Furthermore, potential shifts in seasonal blood-feeding patterns of this population of Cx. quinquefasciatus towards increased mammalian feeding was examined. The WNV activity in the county during 2011 was also analyzed. Over 740 Cx. quinquefasciatus samples were collected by West Valley Mosquito and Vector Control District in San Bernardino County during 2011 from 34 different sites. DNA from the bloodmeals was extracted and purified, and a 658-base pair region of DNA located in the mitochondrial gene cytochrome c-oxidase I (COI) was amplified. This was followed by DNA sequencing of the PCR product, and identification of the individual sequences using the Bar Code of Life Data Systems. A total of 683 bloodmeals were successfully identified. These bloodmeals belong to 29 vertebrate species across four different habitats. It was found that species richness was not significantly different between habitats, even though the sample sizes for each habitat varied. Across habitats, the highest percentage of avian bloodmeals were taken from House Sparrows and House Finches. Bloodmeals were identified from five mammalian species which included Humans. A seasonal shift towards increased mammalian bloodmeal prevalence was observed in urban habitats. It was found that WNV activity during 2011 in San Bernardino County was relatively low when compared to the following six years.

No description available.

No description available.

Insecticide resistance in disease-transmitting arthropods has become a serious hindrance for successful vector control. Mosquitoes, in particular, are notorious vectors of potentially deadly diseases like malaria, dengue fever, and West Nile virus. Anopheles gambiae and Culex quinquefasciatus are just two examples of mosquito vectors that possess genetic mutations (denoted kdr and ace-1 ) and/or enhanced detoxifying enzymes (oxidases, esterases, and glutathione-s-transferases) that confer insecticide resistance. Culex tarsalis, a primary vector for West Nile virus among other arboviruses in Northern California, is a target for insecticide application and is under constant insecticide pressure, making it likely to adapt resistance mechanisms like kdr or ace-1 or increased detoxifying enzymes. Culex tarsalis adult females were collected from Yolo and Sutter counties. A bottle bioassay was completed to determine prevalence of resistance to Sumithrin (a pyrethroid; N=217) and Naled (an organophosphate; N=154). A susceptible lab-reared colony was used for comparison. Microplate assays were completed to investigate elevated levels of detoxification enzymes present as well as AChE. PCR was used to amplify the VGSC and ace-1 genes. Amplicons were sequenced and aligned to determine if mutations were present. No evidence of the ace-1 mutation was found in any mosquitoes, but the kdr mutation was seen in all semi-resistant and resistant individuals exposed to Sumithrin. Microplate data revealed significant differences between certain detoxifying enzymes within mosquitoes collected from Sutter and Yolo Counties exposed to both Sumithrin and Naled. The data obtained from this study suggests that resistance to Sumithrin in both populations is carried out by both metabolic and target site insensitivity, while resistance to Naled is caused by metabolic resistance only.