Search

OBJECTIVES: Measles and rubella are nationally notifiable, vaccine-preventable diseases targeted for elimination by the Pan American Health Organization (PAHO). To support national and international elimination efforts, surveillance optimization is important to ensure rapid case detection, document endemic transmission interruption, identify susceptible populations and inform immunization strategies. While current national surveillance captures confirmed-case data, its performance cannot be assessed using PAHO-recommended surveillance indicators as suspect-case investigation data are required for their estimation. In Canada, the investigation of clinically-suspect measles-like illness (MLI) is highly dependent on laboratory evidence, providing an opportunity to use laboratory data to estimate MLI investigation rates. The Measles and Rubella Surveillance (MARS) pilot project was developed to address existing surveillance challenges with the central hypothesis that (I) ‘it is feasible to develop and implement a real‐time, web‐based measles and rubella surveillance system in the Canadian setting’, and the following sub‐hypotheses: (II) ‘implementation of real‐time surveillance in MARS pilot provinces will result in increased timeliness of national measles and rubella surveillance when compared with established confirmed-case surveillance’, and (III) ‘it is possible to use augmented laboratory data to estimate the performance of national measles and rubella surveillance using adapted PAHO indicators’. METHODS: A MARS application was designed to support centralized real-time measles/ rubella investigation reporting and alerting with integration of non-nominal laboratory and epidemiological data, then developed and piloted using the web-based Canadian Network for Public Health Intelligence platform in British Columbia, Alberta and Newfoundland from June/2011-May/2012. Pre- and post-pilot laboratory surveys were conducted to retrospectively assess national surveillance per

For three decades, CD8+ T cells have been implicated in the control of HIV, ever since early studies revealed that a temporal correlation exists between the emergence of CD8+ T cells and the decline of viral loads in HIV infection. Subsequently, a large body of research focusing on the impact of CD8+ T cell responses on HIV has been produced. The central aim of this thesis was to investigate the relationship between CD8+ T cells and control of HIV, with a focus on the differences in CD8+ T cell responses to consensus HIV epitopes and their naturally occurring variants, as well as CD8+ T cell-mediated infection inhibition in disease progression groups. Previous work has indicated that mutations in HIV epitopes of just one or two amino acids can have a drastic impact on the resulting CD8+ T cell response. Considering the extreme genetic diversity of the virus, understanding how CD8+ T cell responses differ to these common natural variants is essential when trying to elucidate what the best targets for an HIV vaccine would be. It was hypothesized that CD8+ T cell responses to consensus HIV epitopes, as a consequence of them being more common in nature, would be more frequent, polyfunctional, and proliferative than responses to their less common variants, as well as being associated with better disease outcomes. After assessing these functional parameters in response to four consensus HIV epitopes and their natural variants, this hypothesis was rejected, and it was determined that the consensus status of an epitope could not reliably dictate the resulting CD8+ T cell response. Rather, it seems more likely that the particular epitope being presented, combined with the HLA allele presenting it and the particular TCRs binding to it, have a much larger impact on the CD8+ T cell response. In the course of this study, the Gag TL9 T variant epitope was identified as stimulating a CD8+ T cell response that is considered to be beneficial in HIV infection. Responses to this epito

Listeria monocytogenes is a Gram-positive bacterium that is found ubiquitously throughout nature and is the etiologic agent of listeriosis. The majority of human listeriosis is foodborne, resulting from the consumption of unpasteurized and ready-to-eat foods that are contaminated during food processing. During the 2008 nationwide outbreak, the Gilmour laboratory performed the first real-time application of high-throughput whole-genome sequencing (WGS) of outbreak strains. Within this genomic data, the 77 kb plasmid, pLM5578, was newly identified in a clinical isolate, and additional Listeria plasmids (the 80 kb pLM5026 and the 60 kb pLM0813) were subsequently identified after WGS was completed on an expanded panel of outbreak isolates. Little was known regarding how plasmids contribute to persistence and virulence of L. monocytogenes, and to investigate these potential relationships, a panel of 147 L. monocytogenes food, environmental, and clinical isolates from Canadian public health events from the last three decades was selected for further study of the plasmids they might contain. Strain carriage of plasmids was determined using conventional PCR targeting known plasmid gene targets. Bioinformatic analyses were then used to predict the functions of individual genes encoded by each sequenced plasmid. These analyses were then used to direct experiments investigating the functions and associated phenotypes conferred by plasmid carriage. Phenotypic analyses included antimicrobial susceptibility testing, heavy metal resistance, and biofilm formation assays. Finally, WGS analyses was performed on isolates with plasmid screening patterns that indicated carriage of potential novel plasmids. Screening revealed that 75 of 147 isolates were positive for the presence of a plasmid, for which WGS analysis identified 24 unique newly identified L. monocytogenes plasmids. Phenotypically, 15 of these plasmids were found to contribute to a decreased susceptibility to the heavy meta

Influenza is a common and potentially life threatening infection. The constant evolution of the virus poses challenges on the cross-reactive response of the immune system, and emergence of new strains renders the antibody-mediated protection insufficient. Cellmediated immunity (CMI) may attenuate the severity of illness and provide better heterosubtypic coverage. A myriad of underlying comorbidities affect the outcomes of influenza infection; however, such known risk factors fail to explain a significant proportion of severe influenza infections. To investigate cross reactive antibody and cell-mediated responses and predictors of disease severity we employed several projects and distinct cohorts- after natural infection; live- attenuated vaccine and inactivated vaccine. The main contributions of this project were the development of assays to measure antibody responses to multiple influenza strains, using a microbead based assay and application of phenotypic and functional assays to the study of influenza specific responses. Using these methods in healthy volunteers it was shown that repeated vaccination using a recurring strain failed to elicit increased antibody or cytotoxic T cell (CTL) responses. The administration of live attenuated influenza vaccine (LAIV) resulted in generation of measurable cross-reactive antibody responses. The study showed that even in a vaccine naïve adult population, LAIV resulted in limited generation of CD4 or CD8 responses. Furthermore, the microbead assay was applied to the study of prevalence rates of 2009 H1N1 pandemic during the first wave, demonstrating acceptable specificity with increased sensitivity along with the added benefits of high throughput and ability to simultaneously study responses to multiple strains of influenza. The study of severe influenza infection during II the 2009 pandemic was able to characterize the profile of several pro-inflammatory cytokines and chemokines that trended towards higher concentrations in t

To enhance outbreak investigations of Shiga toxin-producing Escherichia coli O157:H7, PulseNet Canada has recently applied Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) as a supplemental subtyping tool in combination with the gold standard subtyping method Pulsed-field Gel Electrophoresis (PFGE) for enhanced resolution of isolates exhibiting indistinguishable/highly similar PFGE patterns. The objective was to assess the discriminatory power and level of specificity MLVA offers for outbreak detection and response. Results demonstrate that MLVA provides a statistically significant increase in discriminatory power for outbreak investigations (0.998) compared to PFGE alone (0.993). MLVA was able to provide additional resolution over PFGE analysis and generally agreed with PFGE when isolates were identical and epidemiologically linked. MLVA shows great promise as a molecular epidemiological tool to complement PFGE, as it improves case categorization during outbreak investigations, and the greatest benefits of MLVA may be realized during routine surveillance, when epidemiological information is not available.

In Canada, and more specifically in Canadian-born Aboriginals and foreign born populations, high incidence of tuberculosis (TB) causes significant morbidity and mortality. The presence or absence of specific killer immunoglobulin-like receptor (KIR) genes, individually or in conjunction, may be associated with tuberculosis (active, latent, or uninfected disease status) as well as ethnicity of an individual. It is hypothesized that the differences in KIR profiles, gene frequencies, and/or haplotypes in Canadian-born Aboriginal, Canadian-born non-Aboriginal, and foreign born individuals elicits a differential activation or inhibition profile, resulting in differential cytokine expression and eventually contributes to the outcome of TB infection. In this study we examined the enrichment or depletion of KIR genes in different ethnic populations in Manitoba with special focus on active, latent, and uninfected TB status. In addition, we sought to explore the statistical correlation between TB status and inhibitory/stimulatory KIR haplotypes.

HIV in Karnataka, south India disproportionately burdens female sex workers (FSWs) and men who have sex with men (MSM). The best long-term strategy for managing the global HIV epidemic might involve a preventive vaccine; however, vaccine availability cannot guarantee its acceptability. An exploratory, cross-sectional study was conducted among frontline health service providers (HSPs) working with MSM/FSWs in relation to HIV-related health services in Karnataka. Face-to-face structured interviews were performed to better understand potential barriers/facilitators to acceptability/uptake of a future HIV vaccine among MSM/FSW communities. Descriptive analyses explored HSPs’ perceptions of vaccine acceptability/uptake and likelihood to recommend an HIV vaccine. Although HSPs mentioned numerous potential barriers to future HIV vaccine acceptability/uptake, most believed that MSM/FSWs would be willing to receive the vaccine to protect their health and avoid HIV. HSPs reported being very likely to recommend the vaccine, however young age of potential vaccine recipients negatively affected likelihood to recommend.

Zaire ebolavirus is a long filamentous single-stranded RNA virus belonging to the family Filoviridae. Due to the virus’ high mortality rate, lack of an approved vaccine, and potential use as a bioterrorism weapon, research on this topic has been of high demand. To address this issue, several vector platforms have been investigated as vaccine candidates. DNA and adenovirus vaccine platforms are known to elicit robust cellmediated immune responses, while adeno-associated virus and vesicular stomatitis virus vaccines are recognized for strong humoral responses. The leading hypothesis of the present project was to determine whether these four vaccination platforms, in a heterologous prime-boost regimen, increase survival and the breadth of the immune response. To test this hypothesis, the main objectives were to evaluate the cell-mediated and humoral immune responses, as well as correlate the induced immunity to protection against MA-EBOV. The heterologous pairings were strategically designed to induce both arms of the immune response. An optimized Zaire ebolavirus glycoprotein was inserted into each of the vaccine platforms and evaluated against mouse-adapted Zaire ebolavirus. Serum obtained from vaccinated mice was analyzed by a neutralizing antibody assay and IgG ELISA to determine the humoral response. The cell-mediated immune response was monitored via ELISPOT. Collectively, the data indicates that regardless of whether homologous or heterologous, a more robust immune response was observed in prime-boost strategies compared to an individual vaccination alone. In addition, the cell-mediated and humoral data show that heterologous combinations induce higher IgG specific titers in comparison to homologous regimens. As expected and consequent with immune responses, survival studies demonstrate that prime-boost III vaccinations, heterologous or homologous, are superior to vaccination regimens involving only one strategy. This data supports further evaluation of the prim

The 1918 “Spanish Flu” pandemic was cause by an Influenza A virus that infected 500 million people and nearly 50 million people died. Influenza viruses utilize a RNA-Dependant RNA Polymerase (RdRp) complex, composed of the PA, PB1 and PB2 proteins along with the viral nucleoprotein (NP) to mediate viral transcription and replication. The 1918 PB1 gene has been linked to increased virulence in mice and ferrets. We have investigated the role of PB1 in the transcription and replication of the 1918 virus and its relation to pathogenicity by comparing its RdRp to the RdRp of a low virulence conventional H1N1 human virus isolate, A/Canada/RV733/2007 (RV733). Contrary to previous studies, our dual-luciferase reporter assay revealed the 1918 RdRp had lower transcriptional activity than the RV733 RdRp in vitro. The 1918 NP seems to be the key determinant for the difference in transcriptional activity of the 1918 and RV733 RdRp complexes. The 1918 PB1 in the RV733 RdRp maintained high reporter expression while the RV733 PB1 in the 1918 RdRp abolished reporter expression. 1918/RV733 chimeric PB1 proteins were also generated and evaluated with the reporter assay. Recombinant RV733/1918 viruses were generated by reverse genetics and we determined that PB1 is a key determinant of the high growth phenotype of the 1918 virus, but only a minor contributor to pathogenicity. A novel role for the 1918 NP in the high growth phenotype and pathogenicity of the 1918 virus is also described.

Influenza A virus is well known for its severe clinical consequences. Structurally, this virus is made up of a lipid bilayer embedded with HA, NA and M2 proteins and a core containing eight viral ribonucleoprotein (RNP) complexes. In a typical RNP complex, the nucleoprotein binds with RNA in a non specific manner. The nucleoprotein plays a vital role in transcription, replication, and packaging of RNA during infection. This study aims that NP of A/PR/8/34(H1N1) virus and A/NY/55/2004(H3N2) virus interact with different host proteins depending on cell lines and virus strains. Monoclonal antibodies targeting the nucleoprotein of these viruses have been used for immunoprecipitation and the interacting proteins were identified by mass spectrometry. Tow proteins from the cytoplasm (elongation factor 1 sigma, and Mov10 protein) and 3 proteins from the nucleus (heat shock protein70, hnRNP K protein, and anti alpha actinin 4) were found in all the viral infected cells, and were chosen for validation study. This study will help to understand the virus-host interactions in a better way and may open the gateway for the synthesis of new antiviral drugs which can block these interactions, hence controlling the infection.

Influenza virus is a globally significant infectious agent with the potential to cause catastrophic pandemic outbreaks. Present treatment of influenza infections is restricted to only four anti-viral drugs, but there are increasing global reports of anti-viral resistance in several seasonal strains and also the 2009 pandemic swine-origin influenza virus H1N1. Possible future pandemic outbreaks, emerging new strains and drug resistance underscore the need to understand this complex virus and its pathogenicity with the goal that novel targets can be uncovered for future therapeutic development. Extensive lung tissue damage during influenza virus infection is proposed to contribute to the development of aberrant host immune responses. Strong evidence now demonstrates the significance of the cellular death pathway in promoting efficient influenza virus replication and disease progression. Viruses rely heavily on the machinery of their host for productive replication, which is also an Achilles’ heel that could be targeted for treatment. In pursuit of unraveling the complex nature of influenza virus replication, I carried out a global shRNA screen to identify specific host factors and signaling pathways that are involved in influenza-induced cell death and replication. In this study I identified 138 genes required for influenza viruses to induce infected host cell death. These genes were found to be involved in Protein Kinase A, NF-kB and PI3K signaling cascades. These signaling pathways are well known regulators of cell death and survival, which suggests influenza viruses may carefully regulate these pathways to reach a balance that suit their requirements for efficient proliferation, eventually at the cost of the host cell. I chose five candidate genes—BAD, MxB, TNFSF12-13,TNFSF13, and USP47—that were associated with apoptosis and the major signaling pathways determined in my network analysis to further verify the genome-wide screen as well as elucidate the role of thes

Herpes simplex virus type 1 (HSV-1) is a double stranded DNA virus that causes epithelial skin infections and persists through the life of the host by infecting neurons, where it can switch to a latent state to evade an immune response. In rare cases during primary infection or after reactivation, instead of undergoing lytic infection at the epithelial surface, it instead travels to the brain and causes herpes simplex virus encephalitis (HSVE) which can have a ≥70% mortality rate if untreated. As the virus takes over its host cell, it gains control of the host cell machinery and manipulates host gene expression in order to evade the immune system and to pool its resources into the replication of the virus. One aspect of the dysregulated gene expression involves microRNAs (miRNAs). MiRNAs are short, non-coding RNAs that bind to the 3' untranslated region (3'UTR) of messenger RNAs (mRNAs), leading to translational repression of the target. Dysregulated miRNAs are often down-regulated during infection as the virus takes over, but many miRNAs have also been found to be up-regulated as well1–5. The aim of this study is to observe the full cellular miRNA changes in the context of an acute viral encephalitic infection using HSV-1, and to further characterize selected up-regulated miRNAs to determine their function in the context of the disease state. Of particular note were miR-141 and miR-200c which showed anti-apoptotic effects on neuronal cell culture and did not impact cell viability during an over-expression of the miRNAs. MiR-141, miR-183 and miR-200a expression was enriched within specific areas of the brain during infection. In addition, the potential for miR-150 to bind to a bioinformatically predicted target site within the shared 3'UTR of the HSV-1 UL18, UL19 and UL20 genes was explored. Examining the changes in expression of this class of regulatory RNAs and investigating their potential functions may yield new insight into the relationship between host and vir

Infection with challenge virus standard-11 (CVS) strain, a laboratory fixed rabies virus strain, induces neuronal process degeneration in both in vivo and in vitro models. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal staining indicating a critical role of oxidative stress. Mitochondrial dysfunction is one of the most important causes of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection increased electron transport chain capacity, Complex I and IV activities, but did not affect Complex II-III, citrate synthase, and malate dehydrogenase activities. CVS maintained normal oxidative phosphorylation capacity and proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced Complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by high NADH/NAD+ ratio. CVS infection was associated with a higher rate of hydrogen peroxide production. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration, oxidative stress and neuronal process degeneration.

Methicillin resistant Staphylococcus aureus presents clinical challenges. The ability of S. aureus to cause human disease is correlated with the nematocidal activity of that isolate. This thesis investigates the relationship between SCCmec type, colony spreading, and the presence of the fudoh/psm-mec region of MRSA isolates to their ability to kill Caenorhabditis elegans. PCR was used to test for the fudoh/psm-mec region in 420 CMRSA2 isolates and 155 CMRSA1 isolates. Spread plate assays were performed on 149 CMRSA2 isolates to determine correlation between SCCmec type and colony spreading. Fudoh/psm-mec was correlated with the infection/colonization status of a patient. SCCmec type IV was correlated with increased spreading ability. SCCmec type, PFGE type, spa type, and colony spreading ability of 40 MRSA isolates were determined; these isolates were characterized using a nematode killing assay. It was found that the colony spreading ability of an isolate correlated with the nematocidal ability of that isolate.

The genus Enterococcus has an exceptional ability to acquire and transmit antibiotic resistance genes and is considered to be a major vector in their dissemination. Enterococci are part of the normal gut microbiota of humans and animals and are frequently encountered in food products including dry fermented sausage. Since fermented sausages are not heat-treated before consumption they might be a vehicle for transmitting resistance and virulence traits of enterococci by conjugation with commensal bacteria present in the human gut and pathogenic bacteria that might be present, such as Listeria species. A PCR-based assay was developed to detect enterococci in dry fermented sausage meat at the generic level by targeting a 16S rRNA sequence and a total of 29 Enterococccus strains (15 E. faecalis, 13 E. faecium, and one E. gallinarum) were identified. The susceptibility of these enterococci to antibiotics was tested and it was found that 27/29 were resistant to more than one antibiotic and possessed antibiotic resistance determinants. All strains were positive for at least one virulence gene. Strong biofilm formation occurred at lower than optimum temperature in all three species of enterococci and probably contributed to their survival in the harsh conditions experienced during dry sausage fermentation and drying. SmaI pulsed-field gel electrophoresis (PFGE) patterns exhibited genomic heterogeneity within and between the two larger groups of isolates. In spite of this heterogeneity, the phenotypic similarities observed suggested that food could still be a vehicle for distribution of antibiotic resistant bacteria among humans. In vitro conjugation experiments demonstrated transfer of the tetracycline resistant determinant, tet(M), from E. faecium S27 isolated from fermented sausage to clinical isolates of both E. faecium and E. faecalis. The streptomycin resistance of E. faecium S27 was also transferred to a clinical strain, E. faecalis 82916, which was confirmed by the p

Rift Valley fever virus is a zoonotic pathogen that is transmitted between mosquitoes and mammals such as sheep, cattle and humans. It is an enveloped negative-sense single-stranded RNA virus, which is a member of the family Bunyaviridae and the genus Phlebovirus. During replication, RVFV produces a 78 kDa glycoprotein NSm1 of unknown function, believed to be nonstructural. Here I show that NSm1 is incorporated into RVFV virions assembled in C6/36 mosquito cells, but not in virions assembled in Vero E6 mammalian cells. The presence of NSm1 in insect-amplified virions was demonstrated through repeated immunoblots of purified virions and further supported by mass spectrophotometric confirmation of the identity of immunoblot-positive protein bands. This research appears to be the first evidence that distinct viral protein profiles are correlated to the host cell in which replication occurred.

Viruses are obligate parasites that use the host cellular machinery to produce progeny virions. The host responds to this invading pathogen by induction of the immune system; however, the virus employs a variety of strategies to overcome these attacks. The complexity of the virus-host interaction is of great interest to researchers with aims to both characterize the relationship and target steps of the viral life cycle to hinder infection. Many targeted tactics employ single protein analysis; however, approaches that examine the whole set of virus/host interactions are available. Transcriptional alterations within host cells have been determined for many virus- host interactions by micro-array techniques; however little is known about the effects on cellular proteins. This study uses a quantitative mass spectrometric-based method, SILAC, to study differences in a host cell's proteome with infection by a virus. Mammalian reoviruses and herpes simplex viruses are prototypical viruses commonly studied to determine virus life cycle and interactions with hosts. Using three strains of reoviruses and one HSV1 strain, cells were infected to identify differentially regulated proteins at different times. Thousands of proteins were identified for each virus type, some up or down regulated after infection. Biological functions and network analyses were performed using online networking tools. These pathway analyses indicated numerous processes including cell death and inflammatory response are affected by T1L reovirus infection. Comparing reovirus strains revealed a greater overall proteomic change in host function when infected with the more pathogenic T3DC strain. For the HSV infection, host proteins altered during the different immediate early, earlyand late phases of infection helped characterize the host-virus interaction parallel to the virus life cycle. Overall, my study has characterized proteomic changes in different virus infection systems, identifying numerous novel

The transfer of a desired gene to several types of target tissues has been accomplished successfully in the past using existing Adeno-associated viruses (AAVs). Also, it has recently been shown that AAV can stimulate robust antibody responses due to long-term transgene expression or abolishment of transgene product by cell-mediated immune responses, suggesting the potential use of AAVs as vaccines. Most humans already have pre-existing immunity to common AAV serotypes making novel AAVs of low seroprevalence attractive as gene transfer or vaccine vehicles. This thesis describes my primary research objectives that included the isolation of novel AAV serotypes based on AAV DNA sequences from porcine tissues, novel AAV vector production, and biological characterization of porcine AAVs in vitro and in vivo. This was followed by evaluating immune responses in mice vaccinated with porcine AAV vectors expressing the hemagglutinin (HA) from the avian influenza A/Hanoi/30408/2005 (H5N1) strain. These findings show that low seroprevalence porcine AAV vectors were able to efficiently transduce a wide range of cells and tissues. The porcine vectors also performed well as vaccine candidates and were efficient at stimulating host immune responses. Although porcine vectors were successful as vaccines, further studies involving long term gene expression by porcine AAVs is still necessary to confirm their role as gene therapy vehicles.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

Influenza H5N1 poses a significant threat to global health in both agricultural world and general populations due to the highly virulent nature of this virus. The potential of mutations or recombination’s of this virus with other Influenza strains could lead to the generation of pandemic strain of the virus capable of bridging the infectivity divide between avian species to human. Currently, for all Influenza infections, vaccination remains the primary strategy for prevention and control. Many vaccine strategies have been developed in an attempt to combat the threat from avian flu. Codon optimization has been used to improve vaccine efficacy for many vaccines, which rely on in vivo expression of a protein antigen. In this study, two types of vaccine platforms were used to evaluate the differential effect of codon optimization of the hemagglutinin (HA) gene of the highly pathogenic avian influenza H5N1 virus, A/Indonesia/5/05 (Ind05) on the level of protection and immune response provided against lethal challenge. Taking advantage of the degenerate nature of codon usage, a codon optimized gene was synthesized to enhance the use of codons represented by the most abundant tRNAs. The codon optimized HA gene produces a protein, which remains identical to the wild type protein regardless of codon changes but is theoretically expressed at higher level based on codon usage. The synthesized genes were cloned into a DNA plasmid based expression vector as well as a replication competent VSV viral vector. In vitro expression studies, using both a HA-expression plasmid and a recombinant VSV HA-virus, compared expression between optimized- and wild type-HA constructs. Vaccination with both optimized HA and wild type HA DNA vaccine platforms and recombinant VSV HA viruses, followed by lethal challenge with Ind05, was then used to determine the relative efficacy of each vaccine and subsequent immune response in a mouse model.

Listeria monocytogenes was the causative agent of the nationwide 2008 outbreak associated with contaminated ready-to-eat meat products. Within the whole genome DNA sequences of the outbreak isolates we previously identified a novel 50kb genomic island, designated as Listeria Genomic Island 1 (LGI1). LGI1 is predicted to contribute to Listeria pathogenesis and/or environmental persistence because it encodes genes related to known virulence factors and mobilization functions, including a putative type IV secretion system and a putative small multidrug resistance efflux pump. The distribution of LGI1 in Canadian L. monocytogenes isolates was determined by PCR screening for LGI1 within 126 isolates from 1987 to 2010 that represented different serotypes and pulsed-field gel electrophoresis (PFGE) patterns. To assess the evolutionary history and genetic diversity of this island, total LGI1 sequences from 15 whole-genome sequences were compared, and from the full study panel of isolates,PCR screening for the chromosomal insertion site and multiple LGI coding sequences were performed. LGI1 was detected almost exclusively in serotype 1/2a isolates, and within those, the isolates predominantly had the same PFGE patterns. These LGI1-encoding isolates also exclusively belonged to the multi-locus sequence typing (MLST)clonal complex 8. LGI1 was highly genetically conserved and it was inserted at the same location within the genome in 65 of the 67 isolates that harboured the island. To study the function and expression of LGI1, antimicrobial susceptibility assays,bioinformatic analyses and real-time reverse-transcription PCR were used. Isolates encoding LGI1 had an increased tolerance to quaternary ammonium compounds commonly used in sanitizing agents (benzalkonium chloride (BCl) and benzethonium chloride (BeCl)) compared to isolates lacking LGI1 (but still highly related by MLST and PFGE). LGI1 is also tightly regulated, with expression of 13 of 16 tested coding sequences only b

The purpose of this thesis was to molecularly characterize macrolide-resistant S. pneumoniae (SPN) isolates in Canada between 1998 and 2008. The characterization involved looking at the multi-drug resistant phenotype (MDR), the mechanisms of macrolide resistance, the genetic relatedness, the serotype distribution and PCV7 vaccine coverage as well as the determination of presence of pili-virulence factors. The hypothesis of the study was that macrolide-resistant SPN will growingly be MDR, genetically related, piliated and consisting of serotypes not found in PCV7 vaccine. Over 1500 macrolide-resistant SPN isolates collected between 1998 and 2008 were studied. Macrolide-resistant isolates came from patients from all regions of Canada, and from all age groups. They came from slightly more males (60%) and slightly more in-patients (62%). Macrolide resistant SPN remained low at 8% during the first 4 years of the study, and started to increase reaching 22% by the end of the study in 2008 (p=0.001). Overall the most common mechanism of resistance was efflux mediated by mef(A) (51%), followed by target site modification mediated by erm(B) (36%). The efflux mediated macrolide resistance in S. pneumoniae was predominantly due to the presence of subtype E (95%), which was resistant to more antibiotic classes, and was genetically and serotypically more diverse than the A subtype. Isolates carrying both erm(B) and mef(A) macrolide resistance genes increased overtime from 1% (1998) to 19% (2008) (p=0.002). Serotype distribution showed a decrease in PCV7 vaccine coverage from 67% to 31% (p=0.0072). Isolates with non-PCV7 serotypes increased overtime from 33% to 57% (p=0.0152). Isolates with serotype 19A increased by 15% (p=0.005). They were found to be multi-drug resistant, carried both erm(B) and mef(A) subtype E macrolide resistance genes, and were genetically related. The presence of virulence factor pili-type 1 (PI-1) and pili-type 2 (PI-2) was found associated with these isol

Currently, the HIV pandemic remains a major global health challenge. In order to effectively control and cure HIV-1 infection, it is necessary to perform greater research on host-HIV interactions and develop novel preventive and therapeutic approaches. The human cytidine deaminase APOBEC3G (A3G) is the first identified host restriction factor, which can serve as an initial line of defense against HIV-1 by inducing lethal mutations on proviral DNA and disrupting viral reverse transcription and integration. In order to better understand the action of A3G on HIV-1 replication, my study was focused on characterizing the interplay between A3G and HIV-1 reverse transcriptase (RT). The results indicated that A3G directly bound to RT, which contributed to A3G-mediated inhibition of viral reverse transcription. Overexpression of the RT-binding polypeptide A3G65-132 was able to disrupt wild-type A3G and RT interaction, consequently attenuating the anti-HIV effect of A3G on HIV replication. While the potent antiviral activities of A3G make it an attractive candidate for gene therapy, the actions of A3G can be counteracted by HIV-1 Vif during wild-type HIV infection. In order to overcome Vif-mediated blockage and maximize the antiviral activity of A3G, this protein was fused with a virus-targeting polypeptide (R88) derived from HIV-1 Vpr, and various mutations were then introduced into R88-A3G fusion protein. Results showed that Vif binding mutants R88-A3GD128K and R88-A3GP129A exhibited very potent antiviral activity, and blocked HIV-1 replication in a CD4+ T lymphocyte cell line as well as human primary cells. In an attempt to further determine their potential against drug resistant viruses and viruses produced from latently infected cells, R88-A3GD128K was chosen and delivered by an inducible lentiviral vector system. Expression of R88-A3GD128K in actively and latently HIV-1 infected cells was shown to be able to inhibit the replication of both drug sensitive and resistant s

Group A rotaviruses are a major cause of acute gastroenteritis in children. Almost all children are infected by the age of 5 years old. Rotavirus disease causes around 600,000 deaths per year. VP4 (P) and VP7 (G) genotypes were analyzed for prevalence and potential antigenicity, as they are known to elicit a neutralizing antibody response during infection. This study predicted the effectiveness of two recently licensed rotavirus vaccines based on Canadian surveillance. 271 out of 348 diarrhea samples from 8 paediatric hospital were successfully genotyped by PCR. Canadian rotavirus genotypes were found to be mostly G1P[8] followed by G3P[8], G2P[4], G9P[8], G4P[8], and G9P[4], between 2007 and 2010. Reassortment and motif analysis was done with a subset of rotavirus-positive samples. There were no unusual reassortment events found in Canadian strains. Variations amongst strains were commonly genotype-specific, but otherwise rare. In conclusion, rotavirus vaccine escape is presently unlikely amongst Canadian strains.

Emergent multidrug resistant Escherichia coli increase clinical challenges. This thesis describes the resistance patterns, molecular epidemiology and mechanisms, for 315 E. coli from patients in the Vancouver Coastal Health Region for 2006/2007. Automated susceptibility testing was confirmed via E-test® for AmpC and/or ESBL production. PFGE, RFLP and PCR were used to assess genotypic relationships, and plasmid character. AmpC production was facilitated mainly by promoter mutations (54.5%). The principal ESBL detected was CTX-M-15 (49.5%). An unidentified ESBL-producer, with a pI near 8.3, was detected. A plasmid displayed variant resistance phenotypes dependent on selective growth media. A positive correlation between ST131 with CTX-M-15 and CIPR indicated the dissemination of companion phenotypes. Ciprofloxacin resistance resulted mainly (98.0%) from a double gyrA mutation. Overall fluoroquinolone resistance was not assessable due to exclusive selection parameters in this evaluation. Fluoroquinolone resistance factors require further examination to understand what causes resistant phenotypes exclusive of chromosomal mutations. ii

First in 1997, and later re-emerging in 2003, highly pathogenic avian influenza A virus, subtype H5N1, has spread from wild bird reservoirs to domestic bird flocks. As a result, cross-transmission has been confirmed in people living or working in close contact with infected birds. H5N1 virus infection is associated with a high mortality rate (>60%) in humans and the rapid evolution of the virus suggests that it could potentially develop into a new, and possibly severe, pandemic influenza virus. To-date, conventional inactivated and live-attenuated vaccine strategies offers the best protection against influenza virus infection; however, poor immunogenicity and weaker efficacy have been observed against H5N1 viruses. It was hypothesized that experimental adenovirus-based vaccines based on human adenovirus serotype 5 (AdHu5) or porcine adenovirus serotype 3 (PAV3) can offer protection against a broad range of avian influenza, subtype H5N1, viruses. Ad vaccine vectors are highly immunogenic and have demonstrated protective efficacy against several disease models. However, natural immunity against AdHu5 can interfere with vector efficacy. The nonhuman PAV3 was not neutralized by pooled human serum from 10,000-60,000 individuals and offers a promising alternative to AdHu5-based vectors. Systematic antigen screening using DNA vaccines identified the hemagglutinin (HA) glycoprotein as the most immunogenic H5N1 antigen. HA was then inserted directly into PAV3 or AdHu5. Comparable immune responses were observed between both vectors but, interestingly, the PAV3-based vaccine generated stronger T-cell responses and better rapid protection 8 days following immunization. Additionally, better long-term protection 1 year following vaccination was observed with the PAV3-HA vaccine. The co-administration of multiple H5N1 antigens was also screened to improve protection against divergent H5N1 challenge. Combinations of DNA vaccines expressing (HA+NA) and (HA+NP) offered the best promi

Entrotoxigenic Escherichia coli (ETEC) K88 is a causative agent of post-weaning diarrhea (PWD) in early-weaned pigs. This study investigated the efficacy of two alternative diets, charcoal (0.1, 0.5, 1, and 2%) and a low crude protein (CP) diet (17%) supplemented with probiotic E. coli strains (UM2 and UM7), against PWD infection in ETEC K88 challenged piglets. The present study found that charcoal had no effect on the challenged piglets’ performance, ileal and colonic microbiota or their fermentation end products. There was, however, a correlation between charcoal dosage and fecal consistency score. Charcoal reduced the ileal mucosal attached ETEC K88. Feeding a low-CP diet resulted in a lower ileal ammonia concentration. The low-CP diet reduced the E. coli populations in the ileal digesta as well as lowered mRNA expression of the IL-1ß. We concluded that the use of both 1-2% charcoal diet and a low-CP diet supplemented with probiotic E. coli strains were effective in reducing the incidence and severity of PWD infection.

Objective: Serpins are serine protease inhibitors that are involved in a wide variety of biological functions in nature. They are known to regulate inflammation processes as well as provide host defense against microorganisms. Recent evidence has associated many types of mucosal serpins with a protective phenotype against HIV infection in women. Our hypothesis is that serpins with known antiviral activity against HIV-1 are correlated with protection in a group of HIV exposed seronegative individuals (HIV-resistant) from the Pumwani sex worker cohort. Study design: Cervico-vaginal lavage (CVL) fluid was collected from 66 HIV-positive, 82 HIV-negative and 84 HIV-resistant sex workers from the cohort. Clinical and epidemiological information was recorded at the time of sample collection. CVL protein levels were determined by BCA assay and serpin (A1 and A3) concentrations by a commercially available ELISA kit. Mucosal serpin concentrations were compared against clinical and epidemiological factors as well as sexual practices. Results: Serpin A1 was significantly higher in the HIV-resistant group compared to the HIV-negative controls (Anova: p=0.0470*). Total concentration of serpin A3 did not reach statistical significance between groups. Serpins did not correlate with age, sexual practices, contraceptive use or number of pregnancies. Serpins were differentially abundant during different stages of the menstrual cycle whereas serpin A1 was elevated during the proliferation phase but not in secretory phase (p=0.0275*). Conclusion: Serpin A1 was correlated with HIV-protection in this group of HESN women. This work will contribute to a more complete understanding of mechanisms of resistance and susceptibility to HIV infection.

Prion diseases are invariably fatal proteinaceous neurodegenerative disorders of the central nervous system. The infectious agent is the host encoded prion protein which has undergone a post-translational refolding from a predominantly alpha-helical to highly beta-sheet containing structure. The mechanism of prion-induced neurotoxicity remains elusive in large part due to the absence of a sufficiently neurotoxic cell culture assay. A modern technique for identifying previously unrecognized mediators of a biological pathway is to screen a commercially available library of gene silencing molecules targeting all known open reading frames. Synthetic gene silencing molecules, such as short hairpin RNA (shRNA), employ the endogenous gene silencing pathway to inhibit protein synthesis. To date, no publication has described the implementation of a large-scale library to screen for genetic mediators of prion neurotoxicity. This project was aimed at developing a cell culture model of acute prion neurotoxicity and screening a library of shRNA molecules in order to identify previously unrecognized gene targets essential to prion-induced neurotoxicity. Using a fragment of the prion protein (PrP106-126 peptide) to mimic prion neurotoxicity, human neuroblastoma cells transduced with a retroviral shRNA library were screened for resistance. Involvement of a subset of library identified gene targets in prion disease was assessed in vivo by quantitative real-time PCR (qPCR) analysis. Validation of the protection conferred by reducing expression of a gene target of interest was accomplished using individual lentiviral vectors expressing shRNA. Of the approximately 54,000 shRNA sequences screened, 80 different shRNA sequences recovered from neurotoxic prion peptide-resistant cells were considered to be of interest. Of these, 49 corresponding gene targets were assessed in vivo by qPCR with the majority demonstrating significant differential expression in brains of prion infected mice. Va

APOBEC3 proteins, in particular APOBEC3G/F, are important innate host factors that contribute to protection from HIV-1 infection by inducing high levels of guanine to adenine nucleotide substitutions (termed hypermutation) during HIV-1 viral replication. These nucleotide substitutions occur at different rates and locations across the HIV-1 genome and are thought to be particularly more frequent in the pol region. The virus has evolved ways to counteract these host factors by inducing degradation of APOBEC3G/F proteins through protein interactions with HIV-1 Vif. The aim of this thesis is to characterize and investigate the role of APOBEC3G/F-mediated hypermutation in the HIV-1 genome. We identified a subset of women from the Pumwani Commercial Sex Worker (CSW) cohort with significantly higher rates of hypermutated proviruses in pol. This degree of hypermutation correlated to less severe HIV disease progression as measured by CD4+ T cell count. This was in agreement with previous studies that evidence of APOBEC-mediated hypermutation correlate with reduced disease progression, confirming APOBEC3G/F proteins role in HIV-1 disease. Furthermore, we investigated the in vitro and ex vivo interaction between HIV-1 Vif and APOBEC3G from subjects infected with hypermutated and non-hypermutated proviruses. In vitro studies indicated that HIV-1 Vif protein expression in subjects with hypermutated proviruses were quite divergent and levels of APOBEC3G also differed between subjects. Ex vivo studies in subjects with hypermutated proviruses indicated that endogenous APOBEC3G expression was greater than in subjects with hypermutated proviruses. Both studies suggest that host and viral factors such as APOBEC3G and HIV-1 Vif are playing an influential role in HIV-1 pathogenesis. Further investigations into these interactions may lead to novel strategies into the development of therapeutic drugs for the fight against HIV-1.

Although the demand for high scale production of mammalian orthoreoviruses (T3D in particular) is mounting to advance the research studies and clinical trials associated with their oncolytic capacity, very few reported studies have tried to understand the factors that affect the its production and subsequently optimize them to maximize the virus yield in cell culture systems. For this study, we manipulated several growth parameters (multiplicity of infection, cell density, cell feeding, media pH, and flask size) sequentially to select optimal conditions. Manipulation of cell density, whether cells were fed or not, and flask size all led to moderate changes in progeny virus titer. Altered media pH led to dramatic (more than 100 fold) virus replication changes. We conclude that cell physiological status has a key impact on infection progression and virus production. Optimal parameters consisted of infection at MOI of 0.1, cell density at infection of 95%, media re-feeding, media pH 7.0-8.5, and the smaller the flask size the better.

The importance of arenavirus glycoprotein processing has only been understood within the past decade, with the majority of work focused on the Old World arenaviruses. Evidence has shown that SKI-1/S1P (subtilisin kexin isozyme-1/site 1 protease) is the cellular protease responsible for glycoprotein cleavage in Old and New World arenaviruses. Furthermore, glycoprotein cleavage is shown to be necessary for the production of infectious virus particles in Lassa and Junín viruses. In this thesis, evidence is provided that the recently emerged Chapare virus (New World) is also processed by SKI-1/S1P. Additionally, novel serpin-based SKI-1 inhibitors were shown to effectively prevent SKI-1 mediated cleavage. Using a wide panel of recombinant vesicular stomatitis viruses expressing New World arenavirus glycoproteins, these inhibitors were capable of significantly reducing viral titres. This provides strong evidence that SKI-1 inhibitors can be used as an effective treatment against the majority of New World Clade B arenaviruses and LASV in vivo.

Ricin toxin is a very deadly plant protein that is synthesized by the plant Ricinus communis. The molecular structure of ricin toxin places it in a group of similar proteins classified as a Type II RIP due to its heterodimeric construction; it is composed of a toxic A-chain possessing enzymatic action, and a receptor binding B-chain. Monoclonal antibodies were obtained with binding activities against either the A-chain or B-chain, and a surrogate non-toxic ricin analogue, TST10114, was determined to be suitable for characterization of the anti-ricin monoclonal antibodies. One potent anti-ricin A-chain neutralizing monoclonal antibody was chosen for chimerization, RAC18, which exhibited strong binding affinity and neutralizing properties. The constant regions of a human immunoglobulin G2 (IgG2) were used as the backbone for the recombinant chimeric antibody. The resulting chimeric RAC18-huG2 was transiently expressed in human-derived HEK 293F cells, purified, and assessed for binding characteristics and functional attributes.

Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.