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Lyophilization maximises vaccines’ stability, shelf life; maintains chemical or biological functions; enables easier transportation and storage compared to a cold chain. Success of vesicular stomatitis virus (VSV) based vaccines has been highlighted by its increased adoption by pharmaceutical companies and most importantly, the recently approved vaccine for high consequence Ebola virus (EBOV). Lujo virus (LUJV) and Lassa virus (LASV), both arenaviruses, are still without licensed vaccines, however, researchers are exploring VSV-vectored vaccines. Many of these highly pathogenic viruses are endemic in tropical regions like Africa where slight breach in cold chain may affect stability of vaccines. It was established that slight changes in cold chain conditions do not affect efficacy of VSV-based vaccines, however, it is imperative to optimise thermostability of VSV-based vaccines using lyophilization. The primary hypothesis of this study is that lyophilized VSV-LASV and VSV-EBOV vaccines exposed to sub-optimal temperature will retain infectivity in cell culture and protect against LASV challenge in inbred Hartley guinea pigs. Different combinations of commercially available stabilisers were used to assess the thermostability of VSV-based vaccines at 4, 21 and 37 °C of storage temperatures. Lyophilized VSV-LASV and VSV-EBOV were recoverable in Vero cells, the recovery titer showed an inverse relationship with exposed temperature and duration of storage of 1, 7, 35 and 90 days. Furthermore, lyophilized VSV-LASV affords 100% protection and sterilising immunity against LASV infection in inbred Hartley guinea pigs.

Blastomycosis is a pulmonary disease caused by Blastomyces dermatitidis, a dimorphic fungus endemic to Manitoba and northwestern Ontario. Immunosuppression is a major risk factor affecting disease susceptibility, yet host immunity is not well understood. Genetic immunodeficiencies can also influence disease, with variants in IL6, GATA2, and VDBP shown to influence susceptibility. However, additional genetic factors in disease susceptibility and severity remain undetected. Our study seeks to establish what is known about susceptibility to blastomycosis and explore genetic risk factors in a case-control cohort. We conducted a scoping review to establish current knowledge on blastomycosis immunity and a literature review to identify candidate genes influencing susceptibility to fungal and mycobacterial infections. Exomes from 18 blastomycosis cases and 9 controls were sequenced, variants were identified, and filtered according to best practices. We performed candidate gene prioritization and variant aggregation to identify genetic associations and explored the full exome dataset. The scoping review included 58 articles on susceptibility to blastomycosis. TNF-a, GM-CSF, CD4+ deficiency, and the IL-12-IFN-y pathway had the most evidence as susceptibility factors. The literature review identified 86 candidate genes relevant to fungal and mycobacterial infections. One hundred and three genetic variants in 42 candidate genes were identified in the exome dataset. No variants associated with susceptibility were identified in a single-variant analysis although two non-synonymous variants in TYK2 were enriched among cases suggesting a possible role in susceptibility. Gene-based association analysis found TLR1 and GATA2 enriched in controls (p = 0.024 and 0.051, respectively) suggesting a possible protective effect, although GATA2 has previously been associated with blastomycosis susceptibility. Gene cluster analysis identified genetic variants in genes of chromatin remodeling

Recent mumps outbreaks have been observed in vaccinated young adults due to MuV genotype G, whereas the current vaccine is a mixture of two genotype A strains. These outbreaks could be attributed to waning vaccine immunity or antigenic differences between the vaccine and circulating MuV, HN, and F glycoproteins. These glycoproteins are essential targets for the immune system, and antigenic variations may reduce the recognition of mumps antibodies, rendering the population susceptible to MuV. We established stable cell lines expressing the MuV glycoproteins to study cross-reactivity between genotype A and genotype G. Cross-reactivity of antibodies between genotypes was assessed by IFA analysis using patient sera from vaccinated individuals without genotype G infection (vaccinated-only), unvaccinated individuals with a history of genotype G infection (infected-only), and vaccinated individuals with genotype G infection (vaccinated-infected). A limiting dilution assay was used to determine antibody titer, defined as the reciprocal of the last serum dilution at which fluorescence could be detected, and the geometric mean titer (GMT) was calculated. Titer ratios were calculated by dividing the genotype A titer by the genotype G titer, and the arithmetic mean was then calculated. Titer ratios showed that the vaccinated-only individuals had a higher titer for genotype A than for genotype G. In contrast, the infected-only individuals had a lower titer for genotype A compared to genotype G. Notably, no difference in titer ratio was observed for individuals vaccinated and infected with mumps. Additionally, a comparison of GMT in different groups showed that vaccinated-only individuals had lower titers for genotype G than infected-only and vaccinated-infected individuals. These results show a differential antibody response to vaccine vs. wild-type strains of MuV, which may explain the increased susceptibility of mumps in vaccinated young adults. However, there is no correlatio

Prions are infectious proteins that cause rare but invariably fatal neurodegenerative diseases in humans and other mammals. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of human prion disease, with an annual prevalence of 1-2 cases per million individuals. Though extremely rare, sCJD is devastating. This disease is rapid, with most patients dying within 5 months from the onset of symptoms and no available treatments. sCJD is heterogeneous, with diverse clinical signs, brain lesion profiles, and disease durations. It is believed this variability is attributed to different prion strains, which are influenced by host genetics and the shape of the misfolded protein. Strain diversity is poorly understood and remains to be explored experimentally. For these reasons, we have developed methodologies to exploit strain-specific properties and applied them in a retrospective analysis of 31 sCJD cases in Canada. We characterized the biochemical properties of the pathogenic prion proteins from these cases using thermal denaturation assays, capillary-electrophoresis immunoassays, and protein seeding kinetic assays. Atypical cases were inoculated into a novel animal model to analyze strain-specific properties such as incubation periods, clinical signs, and brain lesions. Within our cohort of 31 sCJD patients, we identified two atypical sCJD cases and two cases of a rare strain of sCJD called variably-protease sensitive prionopathy. We propose the two atypical sCJD cases represent novel strains of sCJD in Canada. Additionally, we have developed a sCJD strain baseline through which atypical cases could readily be identified in the future. Understanding prion strains has implications for the detection of zoonotic transmission as well as for the development of therapeutics.

Acinetobacter spp. are a group of Gram-negative bacteria found in a variety of different environments such as hospitals, food, soil and water sources. In fact, investigation of Acinetobacter spp. has been declared a One Health crisis. highlighting that not just clinical, but also animal and environmental samples are important to our understanding and resolution of these infections. Many strains of the most notorious member, Acinetobacter baumannii are multi-drug resistant (MDR), and virulent, causing infections in humans including soft tissue and wound infections, ventilator-associated pneumonia and even bacteremia. Various antibiotic resistance genes (ARGs) are common in Acinetobacter spp. In Chapter 3, the diversity of Acinetobacter spp. is highlighted with the discovery of novel sequence types and therefore novel clonal complexes from non-clinical sources. We found that the most common ARGs are efflux pumps and the presence of ARGs did not necessarily determine antibiotic susceptibility. There was also no correlation between virulence and geographic origin or isolation source. In Chapter 4, we explore clinical MICs of colistin resistance observed in clinical isolates of Acinetobacter courvalinii and Acinetobacter pittii, and in non-clinical isolates of Acinetobacter seifertii. Again, with Chapter 3 as a basis, Chapter 5 describes the discovery of an ADC-5 cephalosporinase encoding gene from a tank milk isolate of A. baumannii, the first to be observed in a non-clinical isolate. Efflux pumps were the most commonly detected ARG and this was studied in more detail in Chapter 6. Determinants of substrate selectivity using molecular genetic techniques. Finally, because of the classification of A. baumannii as a One Health challenge. understanding the impact of a wide-spread environmental non-antibiotic compound, SA, and its effects on antibiotic susceptibility and virulence are explored in Chapter 7. Studying the impact of SA on ARGs and virulence genes (VGs) using a

With the widespread use of antiretroviral therapy, HIV drug resistance (HIVDR) rates are increasing globally. Many reported cases of HIV are found in areas without readily accessible testing centres capable of processing blood samples. Blood samples received from these areas can become degraded due to storage conditions during transportation to another facility or country capable of testing. Degraded samples present challenges for HIVDR testing, due to compromised viral RNA integrity interfering with RNA amplification and sequencing. Similarly, archival samples and dried blood spots (DBS) can also pose challenges. DBS samples are a highly accessible and cost-effective sample collection method used for HIVDR testing in resource-limited settings within Canada and across the world; however, they can be difficult to process through traditional methods due to susceptibility to degradation and low input volumes. To address this, we have developed a multiplex, amplicon-based, direct-to-sequencing HIVDR testing protocol compatible with degraded samples. This approach uses a hemi-nested approach with primer pools spanning the entire pol region of the HIV-1 genome. Protocol primers also incorporated Illumina P5/P7 adaptors, single index barcodes, and unique molecular identifiers (UMIs or Primer ID). This approach allow direct sequencing of amplicons, decreasing overall cost, mitigating PCR biases, and increasing clinical throughput. Here, we show the utility of this approach, successfully targeting the entire pol gene region of the HIV-1 subtype B genome in degraded and DBS samples. Preliminary work suggests the protocol may be adaptable for other major subtypes circulating in Canada as data shows the primer pools can amplify other major HIV-1 subtypes, albeit this may require further optimization. This protocol provides a potential option for NGS-based HIV drug-resistance testing from degraded samples and phylogenetic studies, while also building towards the ultimate goal of

HIV disproportionately affects adolescent girls and young women in Sub-saharan Africa and having a diverse microbiome has been associated with an increased risk of HIV acquisition. Among African women studies have shown that most women harbour diverse microbiome groups, and this is coupled by high recurrence rates even after successful treatment of BV. We hypothesize that sexual intercourse and sex work has a negative impact on the immunological and microbial milleu of the female genital tract and is associated with increased sexually transmitted infection risk in adolescent and young girls from Mombasa. Adolescent girls and young women were recruited from hotspot in Mombasa, Kenya and they self-triaged into 3 distinct groups using a triage questionnaire: Non-sex worker group, transactional group and female sex worker group. Cervicovaginal lavage and urine were collected from the study participants. The CVL sample was processed into a supernatant which was used to measure cytokines and soft cup pellet which was used to for microbiome sequencing and gene expression assays. The urine was used for qPCR of sexually transmitted infections. In the first sub study we analysed microbiome data for 168 samples and found that molecular-BV was common among the participants, and it was highly correlated with cytokines associated with increased risk of HIV acquisition (chapter 4). We further assessed cytokine receptor expression of the samples above and found that most receptors were downregulated especially those that contained diverse microbiome (chapter 5). In this chapter we further assessed whether there was a correlation between barrier marker E-cadherin and, receptors and epidemiological variables and found that participants in the female sex worker and transactional groups have lower levels of E-cadherin (p=0.015). A negative correlation was also observed between E-cadherin and some of the receptors; IL10RA (p=0.027, R= -0.259*), IL1RAP (p=0.046, R= -0.234*), GMCSFRA (p=0

At the onset of the COVID-19 pandemic, researchers around the globe joined forces to study the evolutionary biology of SARS-CoV-2 and identify approaches to minimize its spread in the population. Whole genome sequencing (WGS) quickly became the gold standard for monitoring SARS-CoV-2 viral evolution and how it may impact disease severity, transmission, and vaccine efficacy. As a result, researchers demonstrated concerted efforts to develop, optimize, and validate methods for WGS of the novel virus. This research herein optimized wet-lab sequencing protocols developed by the international research group using reverse transcription PCR-tiling and nanopore sequencing technologies to sequence the whole genome of SARS-CoV-2. As a part of the Canadian COVID Genomics Network (CanCOGeN), the work presented in this thesis outlines materials, methods, and results that contributed to the optimization and validation of a Canadian-specific SARS-CoV-2 WGS approach. To achieve this goal, the investigation included identifying the most economical reverse transcriptase, DNA polymerase, PCR primer schemes, library preparation conditions, and comparison of Nanopore and Illumina sequencing platforms. The optimized WGS protocol was shared with the CanCOGeN partners across Canada to increase Canada’s SARS-CoV-2 sequencing capacity towards a sustainable national genomic surveillance program. Throughout this project, the WGS protocol was validated to ensure that emerging variants of concern were detectable. In summary, the current research contributed to operationalizing the first national genomic surveillance program for viral pathogens in Canada. The developed protocol remains in use across Canadian partners and contributes data to support evidence-based decisions for implementing public health measures. Thanks to our work through the CanCOGeN project and its network of expertise, Canada is well-positioned and prepared to perform genomic surveillance of emerging and re-emerging pathogens

SARS-CoV-2 and Nipah virus are two zoonotic viruses responsible for global and local outbreaks of high consequence, respectively, putting a strain on capacity for rapid testing and diagnostic testing. Developed by Joung et al. 2020, STOPCovid.v2 combines chemical RNA extraction with loop-mediated isothermal amplification and Cas12b-mediated detection, capable of detecting SARS-CoV-2 nucleic acid. However, the assay sensitivity is not as good as the gold standard RT-qPCR. Moreover, an assay of this sort does not exist to detect Nipah virus. In this investigation, various RT-LAMP and Cas12b optimization strategies, based on extensive literature reviews, were employed to examine their effects on the assay to potentially improve kinetic and detection performance. It was found that substituting Tween-20 with Brij-35 in the reaction buffer, increasing the MgSO4 concentration, and adding dithiothreitol to the reaction only slightly improved assay characteristics. A rapid enrichment-extraction protocol using minimal commercially available reagents and resources was developed herein. Beyond this, a previously published Nipah virus RT-LAMP primer set combined with an in-house-designed sgRNA was developed and found to detect the Nipah virus N gene, improving the sensitivity 2000-fold compared to the published colorimetric RT-LAMP assay. These findings show that combining previously developed isothermal amplification with CRISPR-based detection can improve pathogen detection, paving the way for developing CRISPR-based point-of-care diagnostics.

Introduction: In 2021, 1.5 million new Human Immunodeficiency Virus (HIV) infections occurred. Inflammation increases HIV risk; decreased inflammation is associated with protection in HIV-exposed seronegative (HESN) individuals who remain HIV seronegative despite repeated exposures. Understanding how behaviours/interventions alter inflammation is crucial and is the basis of this thesis. Aim 1: The Sniffing Around the Issue Study (SUS, 2015-2023) assessed the effect of solvent use on inflammation. Aim 2: The Inducing Immune Quiescence Study (IIQ, 2013-2022) assessed the ability of anti-inflammatory drugs, acetylsalicylic acid (ASA) and hydroxychloroquine (HCQ), to induce the HESN-like phenotype. Methods: Blood/plasma were collected in both aims. Aim 1: SUS enrolled 27 individuals – 14 solvent users and 13 community-matched controls. Immune cell phenotypes/functions, markers of microbial translocation, and cytokines/chemokines/hormones were measured. Aim 2: IIQ enrolled women (ASA:38, HCQ:39) with samples pre-/post-drug. Immune cell phenotypes/functions, antibody levels, oxylipin levels, and in vitro phosphorylation and cell migration were assessed. Both studies utilized integrated knowledge translation and community engagement which required pre-study/mid-study/post-study engagement. Results: Aim 1: Solvent users had increased natural killer (NK) cell activation, exhaustion, cytokine production, and degranulation with reduced KIR expression following stimulation. T cells exhibited altered cytokine response (increased on Th1 and Th1/Th17 and reduced on Th2 and CCR4+CCR6+ T cells) post stimulation. Solvent use reduced free thyroid hormone T3/T4 levels. For those in the IIQ study HCQ altered T cell trafficking, suppressed recall memory T cell responses, and decreased total IgG levels. ASA reduced T cell trafficking markers and total IgG levels. ASA enhanced recall memory T cell responses and NK activation without affecting NK function. ASA reduced levels of cyclooxygena

The introduction of the Measles, Mumps, and Rubella (MMR) vaccine to Canada in 1983 successfully eradicated measles and rubella, though the mumps virus (MuV) continues to persist. Recently an increase in mumps outbreaks due to genotype G mumps infections was observed in vaccinated young adult populations. Characteristics of the mumps outbreaks in Canada were explored using data from outbreaks in Manitoba, Newfoundland, and Nova Scotia. A new method of whole genome sequencing (WGS) using the pull-down of mumps-specific sequences was developed that allows for direct sequencing from clinical samples with a success rate over 70%. This method obtained 299 whole genome sequences from provincial outbreaks that were analyzed using Bayesian Evolutionary Analysis by Sampling Trees. Several conclusions can be reached from this analysis: i) WGS is a superior method to resolve and explore mumps outbreaks when compared to traditional SH gene sequencing; ii) The outbreaks are due to the endemic circulation of mumps in Canada; iii) endemic circulation of mumps likely occurs freely within both Canada and the USA. Using epidemiological data available for Manitoba, it was determined that the risk of being infected with a mumps strain that contained a non-synonymous mutation compared to a mumps genotype G consensus was eight times higher in those who only had one dose of an MMR vaccine. This suggests some single nucleotide variants might be linked to vaccine escape factors. A cross-reactivity study was conducted between the vaccine strain (genotype A) and the circulating strain (genotype G) to investigate potential reasons for waning vaccine immunity. The study used convalescent sera from mumps cases in vaccinated or unvaccinated individuals and vaccinated individuals with no mumps history. Multiple approaches were employed, including western blot, ELISA, and an attempt at Plaque Reduction Neutralization Test. The overall results were inconsistent, possibly due to a lack of cross-react

Tuberculosis (TB) is primarily a respiratory disease caused by the bacterium Mycobacterium tuberculosis (MTB) and accounted for the deaths of 1.6 million people in 2021. TB is typically a treatable disease but drug resistance has become a major public health threat since the 1990s. Current drug susceptibility testing of MTB relies on culture which is slow, labour intensive, and requires specialized infrastructure that may not be available in some regions. Rapid detection of MTB from patient sputum using whole genome sequencing could provide high discriminatory information, while reducing diagnostic turn-around-times thereby preventing costly and ineffective treatments. The aim of this study was to develop a culture-free genomic method to identify MTB and predict drug resistance from sputum using whole genome sequencing. A validation study was done in two stages: (1) MTB-negative sputum spiked with Mycobacterium bovis BCG, (2) MTB positive sputum. Sputum is the primary clinical specimen for MTB testing but presents challenges due to the presence of both high host and microbial DNA compared to MTB. Sputum was decontaminated using standard methods to liquefy sample and reduce host and bacterial presence. Samples were enzymatically treated to degrade host DNA. Prior to sequencing, extracts were subjected to two amplification methods: an in-house developed multiplex-PCR targeting known MTB resistance markers and a random approach using GC-rich primers. Sequences obtained from Illumina MiSeq and Oxford Nanopore Technologies (ONT) were subjected to quality analysis in Galaxy (version v20.01) before being submitted to bioinformatics pipelines: Kraken2, Mykrobe Predictor and BioHansel which were used to assess bacterial and human presence, predict antimicrobial resistance (AMR) determinants and species identification, respectively. Results indicated that amplification methods improved both DNA concentrations and genome coverage by increasing mycobacterial DNA abundance. The

HIV viral load (VL), measured as RNA copies/mL of plasma, is a predictor of disease progression and transmission potential. Therefore, the restriction of VL is key to achieving the “Undetectable=Untransmittable” and UNAIDS “95-95-95” targets to reduce the amount of people living with HIV and AIDS. VL is variable between individuals, with host genetics, viral fitness, and the environment contributing to this variability. The HLA region has been consistently associated with HIV protection or susceptibility, based on the ability of an HLA allele to restrict HIV. However, HIV escape from HLA is well described, with some viral mutations completely abrogating the protective effect of HLA alleles. A previous genome wide association study (GWAS) of HIV VL in individuals of African ancestry identified a locus on chromosome 1, near the protein coding gene CHD1L, that has a novel association with control of HIV replication. However, not all of the individuals carrying protective alleles in this locus maintained low VL. In addition, this regions’ impact on viral evolution has not been investigated. To address this, we conducted an analysis in 174 people living with HIV (PLWH), from South Africa and Kenya, with both human and viral sequence data available. Logistic regression was used to test the association between HIV amino acid variants in reverse transcriptase (RT), protease, integrase (IN), gag, and nef with host alleles near CHD1L and in HLA. We observed associations between the CHD1L variants rs77029719, rs7519713, rs59784663 and rs73004025 with codon 248 of HIV RT. We also observed associations between the CHD1L variant rs7519713 and codons 18 and 147 of HIV gag, and codons 60 and 216 of IN. These associations are consistent with viral escape from CHD1L pressure. In addition, we observed associations between HLA B*81 and HLA C*18 with RT codon 4 and HLAB*58 with RT codon 196. Lastly, we conducted a HIV VL GWAS in 552 PLWH from South Africa, a population that has not been

Disease-associated changes to the gastrointestinal (GI) microbiome have been detected in a variety of chronic human illnesses. Currently, GI microorganisms are thought to play a major role in systemic health and homeostasis through interactions with the immune system of their host. In recent years, the available technology for capturing and characterizing the GI microbiome has expanded greatly, resulting in a rapid expansion of the field. In particular, culture-independent technologies allowing for direct analysis of microbial genes, transcripts, proteins, and other metabolites (collectively referred to as meta-omics) from stool samples show potential for personalized disease detection and monitoring through non-invasive screening. However, due to the high degree of variability inherent in microbiome profiles, establishing a consensus for microbial signatures or biomarkers of disease across studies is difficult. Differences in study methodology can further complicate comparisons, since the laboratory protocols for sequence-based data capture and analysis are varied, and there are several commercial kits and reagents available for the storage and isolation of microbial DNA and RNA from stool for downstream microbiome profiling. Research has shown that the choice of nucleic acid extraction kit and storage method can affect resulting nucleic acid quality. In the current methodological study, a single stool sample from a healthy donor is divided and processed using multiple commercially available methods for nucleic acid stabilization and isolation in order to evaluate the effect of processing on observed sequence-based microbiome profiles. The research herein demonstrates that the experimental methodology used to stabilize and isolate nucleic acids from human stool samples can significantly impact the ability to capture GI microbiome diversity from metagenomic and metatranscriptomic data. Notably, GI microbiome characteristics commonly used as health markers in disease

Introduction: C. difficile spores are dispersed throughout the environment and can asymptomatically colonize and/or infect animals. Previous studies have shown that C. difficile spores can be isolated from commercially available beef, veal, pork, vegetables, and seafood. However, a definitive link has yet to have been made between food contamination and hospitalized cases. This study aims to isolate C. difficile from retail meat samples and compare them to human isolates. Methods: Frozen retail pork, beef, and veal samples were obtained from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) program and FoodNet Canada. These samples were analyzed for C. difficile contamination by direct plating on selective media and by inoculation in enrichment broth. Suspected C. difficile colonies were confirmed by polymerase chain reaction (PCR). Toxigenic C. difficile isolates were molecularly characterized by ribotyping and pulsed-field gel electrophoresis (PFGE). Antibiotic susceptibility was determined by ETEST® strips. Whole genome sequencing (WGS) was performed on all C. difficile isolates from retail meats and from select human cases. Results: Overall, toxigenic C. difficile was isolated from 10 of 644 retail meat samples (1.6%). All 10 isolates were A/B toxin positive. Additionally, 2 isolates were found to harbor binary toxin. All retail meat isolates were susceptible to vancomycin, metronidazole, tigecycline, rifampin, and clindamycin, excluding 1 NAP1 isolate that was resistant to moxifloxacin. Molecular typing revealed strain types commonly found in human clinical isolates (e.g. NAP1 (RT027), NAP4 (RTNS195), and NAP11 (RT106)). The closest related human clinical isolate to the C. difficile isolates from retail meats differed by 8-34 SNVs when analyzed at the highest resolution (84.54%-95.02% core genome). Conclusion: A low percentage of retail meats (1.6%) were contaminated by C. difficile. All ribotypes and NAP types of C. difficile i

Helicobacter pylori is a Gram-negative, flagellate, microaerophilic member of the Epsilonproteobacteria, with a spiral or curved bacilli morphology (Marshall and Warren 1984). The bacterium resides in the human stomach, and infections can span decades, often going undetected due to minor pathogenesis (Kusters et al. 2006). Infections frequency is higher in the Global South, and is associated with low socioeconomic status, education, remoteness, and inaccessibility to healthcare (Hooi et al. 2017; Bruce and Maaroos 2008). This infection is a public health concern because H. pylori infection increases the risk of adenocarcinoma of the gastric mucosa and gastric B-cell mucosal-associated lymphoid tissue (MALT) lymphoma, as well as peptic ulcer disease (Haley and Gaddy 2015). Indigenous communities in Canada experience increased rates of H. pylori infection, which is associated with poverty, crowded living conditions, and inaccessible health care. Infection is a concern for Indigenous communities given the bacterium's implication in the development of associated cancers, which is a severe health burden that too disproportionately affects Indigenous peoples. This project builds upon ongoing community-driven research by the Canadian North H. pylori (CANHelp) Working Group investigating the impact of H. pylori infection. In this research project we present the sequence H. pylori genomes of 57 CANHelp isolates sampled from the Western Canadian Arctic communities of Aklavik, Inuvik, Old Crow, Ross River, Teslin, and Fort McPherson and a comparator cohort of 73 southern Manitoban isolates. I developed and implemented a contamination detection pipeline to ensure only H. pylori sequence reads were used in genome assembly. The genomes were analyzed for their presence of: known markers associated with antimicrobial resistance to clarithromycin, metronidazole, levofloxacin, and amoxicillin; virulence factors like cagA, the cagPI, vacA, outer membrane proteins such as babA, and the

Coronavirus outbreaks have been increasing in both frequency and magnitude for the last 20 years. Prior to 2002, there were two identified human coronaviruses. Now in 2022 there are seven, potentially eight. From severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, to Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 and SARS-CoV-2 in 2019, coronavirus outbreaks have continued to escalate, causing more infections and more deaths with each successive outbreak. SARS-CoV-2 was declared a pandemic on March 11, 2020, by the World Health Organization. To date, there have been around 460 million cases and over 6 million deaths, worldwide. The need for therapeutic interventions for SARS-CoV-2 is continuing, but even greater is the need for broad spectrum coronavirus therapeutics that may be used for this and other coronavirus outbreaks. Due to the critical role of ACE2 in the replication of SARS-CoV, SARS-CoV-2 and human coronavirus (HCoV)-NL63, ACE2 has become a prime target for therapeutic intervention. Groups have tested chimerics of ACE2 fused to an Fc peptide: ACE2-Fc as a decoy molecule that would bind the receptor binding domain (RBD) of SARS-CoV-2 with the same or greater affinity than ACE2 on the surface of cells. Here is described the production and evaluation of seven ACE2-Fc chimerics, and the down-selection to one lead candidate as a potent inhibitor of SARS-CoV-2 cell entry. All ACE2-Fc chimerics were sequence verified, their expression was confirmed in HEK293Ts using western blots and their relative neutralization capacity was tested in in vitro neutralization assays. Evaluation of these constructs led to the identification of a lead construct, which was a potent inhibitor of a SARS-CoV-2 D614G pseudotype luciferase reporter virus. Additional testing of the lead ACE2-Fc construct in wild-type SARS-CoV-2 also demonstrated potent neutralization. Further evaluation of this protein in vivo will allow for determination of dose range, and

Copper is an antimicrobial metal used in sink drains that is becoming ineffective due to copper tolerance of species such as Pseudomonas aeruginosa enhanced by the presence of the genomic island GI-7. Studies of copper tolerance associated with GI-7 are hindered by the lack of accurate planktonic and biofilm testing methods to determine copper susceptibility, and a lack of knowledge regarding the prevalence of this sequence amongst Canadian Pseudomonas isolates. In chapter 3, we developed a new deep well biofilm device that provided increased surface area for biofilm biomass formation. This method can be used to screen antimicrobial susceptibility in a 96-well high throughput format. The increased surface area allowed for greater biofilm biomass per mm2 as compared to the standard device and revealed plastic preferences in biofilm biomass formation by P. aeruginosa and Escherichia coli. Both devices produced different minimum biofilm eradication (MBEC) values for benzalkonium chloride and bleach, yet both were suitable for biofilm cultivation. In chapter 4, we examined 2467 genome sequenced Pseudomonas isolates collected from a 2017-2019 Ontario hospital intensive care unit study. Isolates from patients and hospital rooms with regular and copper sink drains were collected and used to explore if GI-7 presence was associated with copper sink drains. This analysis revealed that GI-7 is widely spread across clinical and environmental isolates of certain multi-locus sequence types, but statistical analyses did not show significant associations between GI-7 and copper sink drains due limited sampling from copper sink drains. Some correlation trends amongst GI-7 isolates were noted and discussed. Finally, phenotypic characterization of P. aeruginosa GI-7 copper tolerance through planktonic and biofilm culturing methods was examined with various P. aeruginosa strains. Copper salt addition to media caused lethal acidification that inhibited both planktonic and biofilm cultur

Background: Over 296 million people worldwide are living with chronic hepatitis B (CHB) infection who require monitoring of viral activity and disease progression. Serum HBV RNA is a promising, although poorly characterized in its encapsidated form, new biomarker in CHB management. No standardized method for serum HBV RNA quantification has been established. Aims: The project aims to characterize the HBV serum transcriptome in order to better understand its composition and to develop and validate, both analytically and clinically, a 3′ RACE RT-qPCR assay for quantification of relevant transcripts within serum HBV RNA. Methods: Nanopore long read sequencing and Northern blotting were employed to characterize the HBV RNA in the serum of 13 patients in different phases of CHB. Sequencing data analysis was done using three isoform detection workflows. A 3′ RACE RT-qPCR method was developed using published primers for the most relevant RNA species determined by serum HBV RNA characterization. The analytical limit of detection and quantification, linearity, inter- and intra-assay repeatability, and clinical specificity and sensitivity were evaluated using synthetic pre-genomic RNA (pgRNA) and specimens from various patient populations. Intra- and inter-laboratory ring trials involving three laboratories were completed. Results: Long read sequencing found higher proportions of spliced variants in patients with HBV genotype B and C, and in HBeAg positive patients with ALT ≤ ULN (upper limit of normal). All chosen isoform detection workflows showed high agreement in most samples, and pgRNA was the most abundant isoform in most patients. Northern blotting was ineffective at detecting serum HBV RNA. The 3′ RACE RT-qPCR assay performed well during analytical and clinical validation and inter- and intra-laboratory analysis demonstrated moderate to high agreement among participants. Conclusions: Long read sequencing is a promising tool for the characterization of the serum HBV tr

Higher microbial load and antibiotic delay are associated with increased morbidity and mortality in septic shock. Antibiotic combination is the basis of empiric treatment for sepsis and septic shock, current recommendations suggest the use of a combination of antibiotics to broaden the initial spectrum of coverage to treat some multi-resistant bacteria. Antibiotic combination therapy leads to more rapid pathogen clearance, which may translate into improved patient outcome. The systematic review in this thesis based on the available randomized clinical trials, does not support the use of combination therapy as the initial treatment for sepsis and septic shock. Although, this could have been due to study design differences, variable degree of severity and even sequence of antibiotic administration. While antimicrobial synergy has been established for beta-lactam combinations with aminoglycosides or fluoroquinolones, antimicrobial sequence has only been studied in vitro. These studies suggest that giving the beta-lactam first or at the same time as an aminoglycoside improves bacterial clearance. Herein we sought to confirm this in the first in vivo study of antimicrobial sequence in septic shock. This thesis confirmed better bacterial clearance using antibiotic combination therapy, but failed to show significant differences in bacterial clearance depending on the sequence of administration of antibiotics in our E. coli and S. pneumoniae models of septic shock. This study sheds light on sequence of administration of antibiotics in sepsis, helping to inform further research.

Introduction: Pneumonia is a major respiratory infection that significantly strains Manitoba’s healthcare system, resulting a substantial number of hospitalizations and fatalities. Understanding the transmission dynamics and risk factors associated with pneumonia in this population is crucial for targeted interventions. Spatial variability in pneumonia hospitalizations has been observed in Manitoba, where various risk factors contribute to pneumonia infection. Therefore, it is essential to investigate the determinants of pneumonia, including spatial aspects and disease transmission rates. Methods: We applied a spatial Poisson regression model that incorporates the Intrinsic Conditional Autoregressive model to explore region level potential risk factors. To understand the influence of comorbidity, we utilized a mixed-effects Poisson regression model. Our analysis focused on hospital data spanning the years 2015 to 2019, and encompassing 96 Manitoba health regions. Moreover, to investigate disease transition rates, we employed both the Susceptible-Exposed-Infected-Recovery (SEIR) model (excluding reinfection) and the Susceptible-Exposed-Infected-Recovery-Susceptible (SEIRS) model (including reinfection). These compartmental models considered data from both hospital and physician-reported pneumonia cases during August 2017 to July 2018. Results: The raw incidence rate of pneumonia infection exhibited significant variation across different regions, ranging from 2 to 55 cases per 1000 population. After adjusting for potential risk factors, the incidence rate ratios ranged from 0.38 to 6.63, indicating substantial variations in incidence rates among regions. Factors such as age, immigration status, and comorbidities, notably Chronic Obstructive Pulmonary Disease (COPD) and Cardiovascular Disease (CVD), were identified as significant contributors to the risk of pneumonia infection. Conversely, vaccination was found to exert a protective effect, especially among individuals

The geographical range of Ebola virus (EBOV) outbreaks overlaps with the habitat of diverse ecosystems that are home to many potential reservoir species. Although researchers have discovered EBOV viral RNA and serological evidence in different rodents and bats, live virus has not been isolated from any species. Identifying the host reservoir is only the first of many challenges; predicting and preventing spillover events requires research on population distribution, ecology, host metabolism and immunology. The EBOV field is clearly missing critical knowledge about where the virus hides, either from being overlooked during sampling, being rarely encountered and/or specific conditions are required that regulate viral expression. To this end, we investigated EBOV infection in cryptic tissues in vitro and in vivo and found that adipose tissue (AT), plays a significant role in both acute and prolonged infections. AT is a dynamic endocrine organ that helps regulate homeostasis, and coordinating energy metabolism, as well as neuroendocrine and immune functions; however, its role in infectious disease has been less thoroughly studied. To assess the role of AT during EBOV infection, we used immortalized and primary brown adipose cultures in vitro as well as developed in vivo reservoir models using Golden Syrian hamsters (Mesocricetus auratus) and Deer mice (Peromyscus maniculatus). Wild type EBOV infected rodents showed no clinical signs of disease nor detectable virus in commonly sampled tissues and swabs. However, EBOV RNA and viral antigen was detected in AT up to 56 days post infection. Furthermore, we tested numerous metabolic agents and found that administration of epinephrine significantly increased viral replication in adipose depots and spillover into the blood. These results show the importance of AT in long-term infection and how the metabolism of this tissue may play a role in viral transmission between reservoir species and/or zoonotic spillover events. Addition

Municipal wastewater effluents discharged into the Red and Assiniboine Rivers of Winnipeg may not be fully treated as traditional methods that monitor the microbial quality of wastewater focus solely on the detection of fecal indicator bacteria. There is also a lack of current wastewater system effluent regulations at the federal and provincial level. Since viral DNA and RNA sequences in current genomic databases are classified as unknown, the objective of this thesis is to characterize viral DNA and RNA community structures using metagenomics and quantitative-PCR, for the purpose of establishing the virome distribution in aquatic environment’s receiving wastewater discharge. Environmental water samples were collected at 11 locations along the Red and Assiniboine rivers during the Spring, Summer and Fall 2021. Samples were filtered and underwent skimmed milk flocculation for viral concentration. Total nucleic acids were extracted, separated into half and enriched enzymatically for viral DNA and RNA to carry out culture independent approaches. Kraken 2 taxonomic sequence classification system and MG-RAST metagenomic analysis server were used to identify the taxonomic classification and functional potential of assembled reads as DNA and RNA viruses. The taxonomic classification of DNA viruses identified from the RefSeq database (available from MG-RAST) and Kraken 2 Viral Genome database were predominately DNA bacteriophages (Myoviridae, Podoviridae and Siphoviridae) belonging to the order Caudovirales which accounted for approximately 90 % of each aquatic sample location along the Red and Assiniboine rivers. Furthermore, phage related functionalities such as phage tail fiber proteins, phage replication, and phage packaging machinery accounted for 40 % of each aquatic samples collected which possibly correspond to the DNA phages that were previously identified. RNA phages (Cystoviridae and Leviviridae) were identified to a lesser extent accounting for approximately 3 %

Prion diseases are rare neurodegenerative disorders that are deadly, incurable, and caused by misfolded prion proteins (PrPSc) in the brain leading to neuropathological changes and rapid cognitive decline. The paucity of current approaches to prion disease research has hampered progress and so here we address two technical challenges that have limited research tools. Firstly, human prion isolates are incompatible with most cellular prion infection models, limiting their clinical relevance. Secondly, genome-wide transcriptional studies of bulk brain tissue cannot resolve the contribution of different brain cell types to prion pathogenesis. This makes it difficult to understand the relationship between prion accumulation, pathogenesis, and neurotoxicity. In the first part of this thesis we set out to develop novel in vitro cellular models of human prion infection using two approaches: (a) the prion organotypic slice culture assay (POSCA) and (b) an immortalized human neural progenitor cell line (ReN). In these two experimental paradigms, prion replication was sensitively tracked over several weeks through measurements of amyloid seeding activity with real time quaking induced conversion (RT-QuIC). Deer mouse cerebellar slice cultures and PrPC-overexpressing ReN cultures were found to resist infection with human prion isolates. While we were unable to establish an in vitro model of human prion infection, our findings reflect the inherent difficulty of establishing clinically relevant cellular models. In the second part of this thesis, we compared prion-elicited transcriptional responses between mouse brain cell types to shed further insight into prion neuropathology. Using bulk RNA sequencing, gene expression was examined longitudinally in microdissected brain tissues from an in vivo mouse model of RML Scrapie infection. We then applied single-cell RNA sequencing to brain cells isolated at the clinical endpoint of RML Scrapie infection. A single-cell atlas of nearly 15

Multiple sclerosis (MS) is a chronic, progressive, and debilitating disease of the central nervous system. This disease is thought to have multiple predisposing factors, including genetic risk factors, vitamin D deficiency, and infectious agents. Although a fungal presence in MS has long been suspected, only recently did studies associate the fungal gut microbiome—the mycobiome—with MS in adult individuals. However, investigations of the mycobiome’s association with disease have been hampered by a lack of standardized methodologies. Both the methods for sequencing and bioinformatic characterization of the mycobiome require systematic assessment in order to standardize such methodologies. Furthermore, although alterations in the mycobiome have been associated with MS in adults, no studies to date have examined if the same case can be made for pediatric-onset MS (POMS) individuals. We set out to evaluate the performance of different internal transcribed spacer (ITS) amplicon sequencing and bioinformatics approaches by varying the PhiX concentration and assessing the abilities of different analytical pipelines to characterize a predefined fungal community. We also used the most optimal methods resulting from our evaluation to examine the gut mycobiome in persons with and without POMS. Our methodology evaluation finds that a 50% PhiX spike-in increased the sequence quality, with 15% more bases at a Phred score of 30 or greater (≥ 99.9% accuracy) but decreased the overall number of reads by more than half when compared to a 25% PhiX spike-in. Of the three fungal metabarcoding bioinformatics pipelines we examined, LotuS classified the highest number of expected species. Although we did not find a significant difference in the fungal community between POMS and unaffected control individuals, we found that the genus Agaricus was highly abundant in POMS individuals; we also found an association of the species Candida albicans with POMS.

The Burkholderia cepacia complex (Bcc), a group of Gram-negative multi-drug resistant pathogens are notorious for causing persistent lung infections in people with cystic fibrosis and new antibiotics are needed to treat these infections. One way to find new antimicrobials is to repurpose old drugs for a new use. One such example of this auranofin, which has antimicrobial activity against Gram-positive bacteria, with low to no activity against Gram-negatives. Thus, to create novel antimicrobials for Gram-negatives, specifically Burkholderia cenocepacia, we synthesized chemical analogs of auranofin. I hypothesize auranofin analogs that will be bactericidal against B. cenocepacia K56-2 and other CF pathogens and that the mechanism of action of these antimicrobials can be determined through randomly barcode transposon sequencing (BarSeq). Two auranofin analogs with bactericidal activity against B. cenocepacia determined their mechanism of action. The two auranofin analogs are bactericidal, do not select for resistance, kill persisters, and are well-tolerated by C. elegans and Galleria in toxicity tests. They also inhibit the enzyme thioredoxin reductase. Interestingly, they do not inhibit the structurally and functionally similar protein glutathione reductase. The compounds also cause an increase in reactive oxygen species upon exposure. BarSeq identified the mechanism of action of these compounds include the glutathione system, potentially by protecting from the increase in reactive oxygen species, and purine metabolism, by altering the metabolic state of the bacterium. Thus, we have determined the mechanism of action of these novel therapeutics. We have also shown that BarSeq is able to determine the mechanism of action of novel antimicrobials.

Salmonella enterica serotype Heidelberg is the third most frequently isolated serotype in Canada and is of particular interest due to its resistance to cephalosporin-class antimicrobials. Through national surveillance of Canadian S. Heidelberg strains isolated from poultry sources and human infections, we previously demonstrated that human and animal-derived isolates were genetically similar (ST15) and that structurally similar IncI1 plasmids harbouring the blaCMY-2 gene were predominantly responsible for cephalosporin-resistance. Here, we focus on the impact of two variants (pCMYN13-02944A and pCMY12-2460C) of a well-characterized and widely disseminated IncI1plasmid on the core physiology of an S. Heidelberg strain isolated from Canadian national surveillance. The plasmids were transferred via conjugation to a susceptible and previously whole-genome sequenced (WGS) S. Heidelberg isolate (N13-01291) to form isogenic strain pairs. Illumina WGS was performed on the parent and transconjugants to monitor genetic alteration introduced during conjugation. Plasmid maintenance and viability were assessed using the PMAxx™ (Biotum, Inc., Hayward, CA, USA) photoreactive viability dye followed by quantitative polymerase chain reaction (PCR). Growth curves and Biolog Omnilog (Biolog, Hayward, CA, USA) phenotypic microarrays were performed to analyze the effects of the plasmids of interest on fitness and growth kinetics. The presence of either plasmid resulted in observable changes in growth kinetics compared to N13-01291. Biofilm assays were performed using 0.1% (w/v) crystal violet staining which determined that N13-01291, N13-01291/pCMYN13-02944A, and N13-01291/pCMY12-2460C are poor biofilm producers. Phylogenomic analysis of the isogenic strain pairs indicated the presence of a shared single nucleotide variant (SNV) and a unique SNV in the presence of either plasmid which affected various changes in growth kinetics. Proteomics were analysed using tandem mass tags and LC- MS

CD8+ T cells are key in mediating immune responses to viral infections. Viral infections can be acute, in which the infection is resolved, or chronic, when antigen persists. Several studies have illustrated that CD8+ T cells adopt distinct functional states in each infection type. In acute infections, naïve CD8+T cells differentiate into cytotoxic CD8+ T cells and memory CD8+ T cells. In chronic infections, activated CD8+ T cells adopt an altered state known as exhaustion. Exhausted CD8+ T cells lose effector functions and show high expression of inhibitory receptors such as PD-1. Several studies have characterized CD8+ T cell exhaustion at late time-points after a persistent infection has already been established. Recent studies demonstrate that CD8+ T cell exhaustion may be specified during earlier phases of a developing chronic infection. However, these studies do not address the effect of biological sex on early CD8+ T cell responses to chronic infection. Sex differences in susceptibility and outcomes of viral infectious diseases are well-appreciated. Still, the role of sex in the development of CD8+ T cell exhaustion and in the molecular programs underlying CD8+ T cell responses to acute and chronic viral infections remains undefined. This study, therefore, set out to investigate early and sex-based differences in the transcriptional profiles of CD8+ T cells that underlie their responses to acute versus chronic viral infection. The mouse model of acute and chronic lymphocytic choriomeningitis virus infection was used to comparatively analyze transcriptional signatures of single antigen-specific CD8+ T cells during acute versus chronic infection at multiple time-points post-infection. Next sequencing coupled with bioinformatics-based approaches were used to analyze our single-cell RNA sequencing data. Our results reveal an early transcriptional divergence of CD8+ T cells responding to acute versus chronic viral infection. We show that there are sex-specific diff

Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete and leaves a pool of chronically infected cells. Understanding of the persistence mechanisms is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells at the site of infection has been proposed to prevent parasite clearance in vivo. In the presented thesis, I used a novel TCR transgenic mouse model, where CD4+ T cells recognize an immunodominant peptide derived from Leishmania- glycosomal phosphoenolpyruvate carboxykinase (PEPCK), as a sensitive tool to characterize the dynamics of anti-L. major CD4+ T cell responses and to characterize mechanisms which restrain their effector function. Intravital microscopy studies characterizing L. major-specific CD4+ T cell migration dynamics within skin lesions directly in live mice show a significant recruitment of adoptively transferred effector T cells to the lesion site in vivo, displaying cellular behaviors consistent with antigen recognition at early and late stages of infection. However, cellular dynamics are augmented at the healed stage, indicating a fundamentally altered environment. I show that Leishmania-specific Tregs display higher suppressive activity compared to polyclonal control Tregs, and that this suppression is mediated through IL-10 and not through disrupting cell-cell contacts or antigen presentation. Challenge of healed mice with L. major antigen results in expansion of endogenous Leishmania-specific Tregs that lead to loss of lesion control in an IL-10 dependent manner. Lack of PEPCK antigen during challenge does not supress effector Th1 response and parasite control. My data proposes a stochastic model of parasite survival, where inflammatory factors that control parasite numbers are counterbalanced by Leishmania-specific immunosuppressive factors that facilitate parasite persistence.

Antiretroviral therapy (ART) has helped Human Immunodeficiency Virus (HIV) become a manageable chronic infection by repressing viremia to undetectable levels in People living with HIV (PLWH). However, this treatment is not curative as treatment interruption causes viremia to rebound to pre-treatment levels due to viral latency, a state of reversible non-productive infection of individual cells established early during acute infection. These latently infected cells serve as HIV reservoirs, residing in various anatomical sites and presenting a significant obstacle to achieving a complete HIV cure. Notably, central and effector memory CD4+ T cells are regarded as the major component of the long-lived HIV reservoirs. Memory CD4+ T cells play an essential role in the adaptive immune response. Guided by chemokine cues, memory CD4+ T cells continually recirculate between lymphoid and non-lymphoid tissues, enabling them to carry out immunosurveillance of the body for pathogens. These long-lived cells are sustained by tonic signalling through the T cell receptor (TCR) and cytokines within the secondary lymphoid organs (SLOs), where they can undergo clonal expansion. Therefore, the SLOs are important tissue reservoirs that allow for persistent viral replication in PLWH under suppressive ART. However, an unaddressed question is whether latently infected memory CD4+ T cells retain their ability to recirculate between lymphoid and non-lymphoid organs, allowing them to seed various tissues and access the same survival signals as uninfected memory CD4+ T cells to persist. Therefore, to address this question, I developed a fluorescent protein-based HIV reporter system that allows for the assessment of the phenotypic and migratory capacity of latently infected CD4+ T cells. The full-length, R5-tropic dual-fluorescent HIV reporter, HIV TosZy, encodes two fluorescent markers: Nef-ZsGreen fusion protein under the control of the HIV-LTR and dTomato protein driven by constitutively expre

Antimicrobial resistance (AMR) is a growing global health problem, exacerbated by the widespread use of antimicrobials in healthcare, agriculture, and industrial settings. Cationic antimicrobials (CAs) such as the therapeutic antibiotic colistin (COL) and antiseptics cetrimide (CET) and chlorhexidine (CHX) exert their mechanism of action by disrupting bacterial membranes, leading to cell content leakage and death. Tolerance to CAs is rapidly increasing and of the many known AMR mechanisms, the role of outer membrane vesicle (OMV) formation is least understood. In this thesis, we examined OMV isolation methods in a tolA deletion mutant to understand how OMV properties differ when using two common methods (Chapter 3). Using the most robust OMV isolation method, OMV production from a set of CA- tolerant strains were compared to a wild-type (WT) E. coli K-12 BW25113 strain (Chapter 4). OMVs from WT and ∆tolA strains were isolated from culture supernatants by ultradiafiltration and ultracentrifugation, then analyzed with a light scattering based single particle tracking analysis (NTA) to compare OMV isolation differences. We characterized the morphology of OMVs isolated from each strain by cryo- transmission electron microscopy, compared proteomes using liquid chromatography-mass spectrometry, and evaluated susceptibility changes by antimicrobial susceptibility testing (AST). In Chapter 3, we demonstrated that deletion of the IM protein tolA in E. coli, an integral part of the membrane integrity Tol- Pal system, resulted in increased vesicle formation and morphological changes to vesicles, including a new kind of vesicle that we coined “G-OMVs”. In Chapter 4, we found that the CA-adapted strains all had increased OMV formation as compared to WT, where each strain had distinctive morphological alterations; CET-OMVs were encapsulated and aggregated, CHX-OMVs were multilamellar and COL-OMVs were large and amorphous as compared to WT-OMVs. Proteomic analysis highlighted an i

Abrin toxin, located in the seeds of Abrus precatorius, is a potent Type II Ribosome-Inactivating Protein (RIP) A-B subunit toxin that has acquired a heightened biothreat profile over the past decade. Significant research efforts must be made in order to develop abrin bioforensics diagnostic response capabilities to the level which is currently in place for ricin, another Type II RIP toxin with similar molecular structure and mechanism of toxicity. To serve this endeavor, three interrelated investigations have been carried out. The first study addresses the need to expand the supply of anti-abrin monoclonal antibody (mAb) reagents. Monoclonal antibodies were developed from hybridomas immunized with synthetic peptides derived from in silico prediction of abrin B-cell epitopes. Two epitopes produced nine abrin-reactive mAbs. Secondly, an attenuated, antigenically-faithful recombinant proabrin holotoxoid was expressed and purified using the baculovirus system for purposes as both an immunogen in future hybridoma experiments, as well as a non-toxic ‘safe antigen’ diagnostic reagent standard. The reduction in toxicity of proabrin relative to wild-type abrin was 108-fold in cell-free translation assay, and 291- to 302-fold in cytotoxicity assays. Finally, the third study combined shotgun proteomics with multivariate analysis differentiation methods in order to retrospectively identify six toxin extraction protocols with further sub-variation of reagent source. Differentiation was based solely on forensic proteomic signatures of carryover seed proteins. A 5-way hierarchical sPLS-DA model correctly classified samples into 8 extraction categories with 100 percent accuracy. Continued assessment of FASP LC-MS/MS and sPLS-DA modelling for attribution of plant toxin extraction methods may lead to establishment of a novel bioforensics diagnostic platform. Together, these three studies serve to advance Canadian bioforensics capabilities and to provide a foundation for future work.

Newly re-emerging viruses are major sources of concern around the world, especially when no treatment options are available during an outbreak. ZIKV is a neurotropic virus that causes congenital abnormalities in babies when they are infected in utero. Some studies have reported that these congenital abnormalities result from ZIKV attacking neural progenitor cells within the brain that differentiate into neurons, oligodendrocytes, and astrocytes. Each of these glial cells plays an important role in fetal brain development. Newborns infected with ZIKV suffer from microcephaly and delayed neurodevelopment, but the underlying causes at the proteomic level are largely unknown. In addition to congenital defects caused by ZIKV, infected patients have been documented to suffer gastrointestinal problems such as diarrhea, nausea, vomiting, and abdominal discomfort, among other things. My PhD thesis for the first time identified host proteins, which we predicted to be linked in the development of; 1) neurosensory alterations that have been reported to occur in babies born to ZIKV infected pregnant mothers and 2) gastrointestinal complications reported in ZIKV infected patients. Invitro proteomic analysis of ZIKV-induced changes was performed in Vero and Caco-2 cells, which are known to be permissive to ZIKV infection. Tandem mass tag mass spectrometry- based proteomic and the SOMAScan were used in monitoring these cells infected with ZIKV. Thousands of host proteins were identified to be dysregulated across selected multiple time point post ZIKV infection. Many protein candidates were linked to neurodevelopmental processes, including the development of the auditory and visual/retinal system as well as gastrointestinal complications as predicted after bioinformatics analysis by IPA. The role of these dysregulated neurodevelopmental associated, as well as gastrointestinal related host proteins for ZIKV propagation, needs to be validated to gain more understanding of ZIKV biology

The overuse of antimicrobials in medicine and agriculture has led to the emergence of antimicrobial resistant bacteria, which are difficult to treat and incur significant healthcare costs. The purpose of this thesis was to characterize important antimicrobial resistance genes (fosA and mmr) from two organisms identified as pathogens of concern by the World Health Organization; Escherichia coli and Mycobacterium tuberculosis. The goal of these two studies was to distinguish the antimicrobial resistant phenotypes of these genes to clinically relevant antimicrobial agents and learn more about their drug selectivity, their structure-function, and their phylogenetic relationships to previously characterized representative homologs. The first objective was to characterize the fosfomycin resistance (fosA) genes from three Canadian clinical E. coli isolates with high-level fosfomycin resistance (MIC >512 µg/mL). Whole genome sequencing was performed on these isolates which uncovered fosA3, fosA8, and a novel gene fosA7.5. The fosA3, fosA8, and three fosA7.5 variants (fosA7.5WT, fosA7.5Q86E, fosA7.5W92G) were individually cloned and over-expressed in E. coli K-12 BW25113. Antimicrobial susceptibility testing using Clinical and Laboratory Standards Institute (CLSI) methodology was performed to confirm the role of these fosA genes in conferring fosfomycin resistance. Each gene was found to confer high level fosfomycin resistance, with the exception of fosA7.5W92G which did not confer resistance to fosfomycin. Phylogenetic comparison, protein sequence alignment, and homology modelling of the FosA7.5 variants identified amino acid residues that distinguish this sub-family and play an important role in the active site of these enzymes. The second objective was to characterize the small multidrug resistance (SMR) family efflux gene mmr (Rv3065) from M. tuberculosis H37Rv using M. smegmatis as a model organism for expression. The mmr gene was electroporated into M. smegmatis mc2451