Inhibition of the interferon-mediated antiviral response is a major determinant of virulence in influenza A virus. The NS1 protein of influenza A virus has been identified as the IFN antagonist. However, the specific mechanisms for IFN antagonism are not known. NS1 binds both the PKR as well as single and double stranded RNA to inhibit activation of PKR. Adaptation of human influenza virus to the mouse lung may likewise involve mutations that affect IFN antagonism. The prototype human influenza A virus A/HK/1/68 (H3N2 subtype) was utilized for this project. Six mouse-adapted variants possessing four different point mutations on NS gene were obtained from independently derived mouse-adapted variants. The mutations were located in the RNA binding domain and several sites in an 8 amino acid region from as 98 to 106. Mutant HKMA20c was of interest because it possessed a mutation in common with highly pathogenic avian influenza virus H5N1 (Leucine on AA 103 on NS1 protein). Base on these phenomena, a hypothesis was brought out that this region may encode a site of interaction with a host or viral factor and that mutation(s) on it may enhance the ability of NS1 protein to function as an IFN antagonist. The approach was to first characterize the IFN resistance and IFN induction properties of HK mouse-adapted mutants as well as pathology in mice lung, followed by the generation of defined recombinant viruses possessing desired mutation(s), then analysis of these recombinant viruses for IFN induction and IFN resistance. Rescuing of the HK wild type NS gene and those of HK mouse-adapted NS mutant genes into the backbone of parental HK genome discovered that NS20, NS20c and NS411 produced attenuating phenotypes on their own. Finally, all these mutant NS genes were inserted into backbone of WSN (lab adapted strain, A/WSN/33, H1N1) genome to construct recombinant viruses. Using recombinant viruses that differ due to individual NS1 gene showed that all of the NS1 mutations increased resistance to IFN in mouse cells. The extent of IFN resistance due to individual mutation was influenced by cell types: epithelium versus fibroblast, as well as host type: mouse versus human. Interestingly most of the NS1 mutations attenuated growth of virus which suggests that resistance to IFN involves changes in host interaction that are not optimal for growth in the absence of IFN. Infection assay showed mouse-adapted mutant NS genes conferred resistance to mouse IFN and vulnerability to human IFN. And mutant NS1 protein NSMA20c had the most potent ability to resist human and mouse IFN. IFN assay showed that mutant NS1 protein NSMA20c had the highest ability to induce mouse IFN. Furthermore, all synthetic recombinant viruses induced low amounts of IFN in human cells. Immunopathology of infected lungs showed that mouse adapted progeny virus HKMA20C had acquired a crucial ability to spread to and infected alveoli which may be influenced by IFN resistance. Mouse-adapted variants possess mutations that increase IFN resistance that in some instances leads to higher IFN induction. One of the key discussions was that most of these mutations cluster in a small region that has been previously mapped to involve a region of host protein (eIF4GI) interaction.

To date, the effect of HIV-1 infection on CD8+ T-cells remains poorly studied. Previous studies have shown that the effector functions of CD8+ T-cells diminish during AIDS. We postulated that CD8+ T-cell functions decrease due to the tropism of HIV-1 for CD8+ T-cells. Therefore we examined whether CD8+ T-cells provide suitable targets for HIV-1 infection and the mechanism(s) by which the virus enters the cells. We hypothesized that HIV-1 entry into these cells may be facilitated through access to extracellular receptors. Ex vivo experiments were performed using blood samples from a cohort of HIV-1 infected patients attending the Ottawa Hospital Immunodeficiency Clinic. Frequency of HIV-1 infection was monitored by flow cytometric detection of HIV-1 p24 antigen and viral production from separated CD8+ and CD4+ T-cell lineages was assayed by the quantitation of viral RNA transcripts. Primary CD8+ T-cells and CD8+ T-cell clones used in the in vitro studies were isolated from the peripheral blood of healthy volunteers. HIV-1 infection was monitored by both ELISA and flow cytometric analyses. Similarly, receptor analysis was performed by flow cytometry and confirmed by RT-PCR analysis. There was a significantly higher frequency of CD8+HIV+ cells than CD4+HIV+ cells found in the ex vivo studies of patient samples, however, viral production from the CD8+HIV+ subset was 2-3 logs lower than that found in CD4+HIV+ T-cells. In addition, CD8+ T-cells served as suitable targets for productive HIV-1 infection in vitro and preferential HIV-1 replication occurred in the memory T-cell subset. Interestingly, the HTLV-I transformed CD8+ T-cell clones exhibited HIV-1 production 20-fold greater than CD4+ T-cells. Our research also demonstrated that during the course of infection, there was a decrease in mean expression of the CD8 and CXCR4 cell-surface molecules in the HIV-1 infected CD8+ T-cell clones. Accordingly, the use of antibodies to the CD8 or CXCR4 molecules eradicated viral adsorption and replication in the CD8+ T-cell clones. Our research was the first to demonstrate the significance and susceptibility of CD8+ T-cells to HIV-1 infection through both ex vivo and in vitro analyses. We conclude, with multiple lines of evidence detecting and measuring HIV-1 infection of CD8+ T-lymphocytes, that this cellular target and reservoir may be central to HIV-1 pathogenesis. By identifying a novel target of HIV-1 and potential cellular receptors used by the virus, future therapeutic strategies may be designed to help prevent and treat HIV-1 infection.

Ciliates provide a powerful system within microbial eukaryotes in which molecular genealogies can be compared to detailed morphological taxonomies. Two groups with such detailed taxonomies are the Colpodea and the Halteriidae. There are about 200 described Colpodea species that are found primarily in terrestrial habitats. In Chapters 1 and 2, taxon sampling is increased to include exemplars from all major subclades using nuclear small subunit rDNA (nSSU-rDNA) sequencing. Much of the morphological taxonomy is supported, but extensive non-monophyly is found throughout. The conflict between some nodes of the nSSU-rDNA genealogy and morphology-based taxonomy suggests the need for additional molecular marker. In Chapter 3, character sampling is increased using mitochondrial small subunit rDNA (mtSSU-rDNA) sequencing. The nSSU-rDNA and mtSSU-rDNA topologies for the Colpodea are largely congruent for well-supported nodes, suggesting that nSSU-rDNA work in other ciliate clades will be supported by mtSSU-rDNA as well. Chapter 4 compares the underlying genetic variation within two closely related species in the Halteriidae with increased taxon and molecular sampling using nSSU-rDNA and internally-transcribed spacer (ITS) region sequencing. The morphospecies Halteria grandinella shows extensive genetic variation that is consistent with either a large effective population size or the existence of multiple cryptic species. This pattern contrasts with the minimal of genetic variation in the morphospecies Meseres corlissi. Chapter 5 discusses the congruence and incongruence among morphological and molecular data in ciliates. Most of the incongruence occurs where there is little statistical support for the molecules, or where molecular data is consistent with alternative morphological hypotheses. Chapter 6 reviews the data for sex, or lack thereof, in the Colpodea, a potentially ancient asexual group where sex was regained in a derived species. In Chapter 7, four ciliate clades are redefined using the PhyloCode.

The genome of Hz-2V, which is sexually transmitted and causes sterility of Helicoverpa zea moths, was sequenced. It is composed of 231,621 bases with G+C content of 41.9%, and contains 113 putative open reading frames (ORFs). Homologies were found to 38 of the 113 ORFs predicted for genes with known functions. Hz-2V shares 16 of the 30 baculovirus conserved core genes, including RNA polymerase genes, and per os infectivity factor genes. Partially conserved baculovirus core cluster structure was found in Hz-2V suggests that the Hz-2V is distantly related to baculovirus. Interstingly, Hz-2V showed 93.5% of sequence homology with Hz-1V and with other nudiviruses, hytrosaviruses, and polydnaviruses. Based on the genome sequencing results, an analysis of structural proteins and prediction of putative microRNAs of Hz-2V was performed. Four of structural genes, p11.7, p15.1, p28.4, and p31.7 were identified by matrix assisted laser desorption/ionization time-of-flight mass spectroscopy. Two transmembrane domains were predicted in p11.7, and a DNA binding motif was found in p31.7 by amino acid sequence analysis, therefore p11.7 and p31.7 were predicted to be envelope and capsid protein, respectively. A putative late gene promoter motif sequence, AGTAT was found in the 3' upstream sequences of these genes. Twenty putative pre-microRNA sequences were predicted in Hz-2V by searching stem-loop structures with less than -25kcal/mol of free energy, and one of the microRNA candidates was predicted to target the Hz2V007 gene transcript, which codes for a putative juvenile hormone esterase.

Inflammation in the female genital tract (FGT) is associated with increased HIV-1 viral replication, HIV-1 transmission and HIV-1 acquisition. The optimal commensal Lactobacillus bacterial species is associated with reduced inflammation in the FGT and dampened immune responses to non-optimal bacteria in vitro. Using a transcriptomics approach, this research aimed to investigate gene expression patterns in the FGT of HIV-infected women compared to peripheral blood. Furthermore, transcriptomics was used to investigate interactions between different vaginal Lactobacillus species and the host to elucidate its immunomodulatory mechanisms. Cervical cytobrushes and blood samples were collected from chronically HIV-infected South African women. Cervical and peripheral blood mononuclear cells (CMCs and PBMCs) were isolated and mRNA was extracted for microarray analysis using the Illumina HumanHT-12 v3 Expression BeadChip system. Eight Lactobacillus isolates, two of each L. jensenii, L. mucosae, L. crispatus and L. vaginalis species were included in this study. The effects of these lactobacilli on cytokine production by vaginal epithelial (VK2) cells stimulated with Gardnerella vaginalis (ATCC 14018) were tested in vitro, RNA was extracted and used for Affymetrix Genechip whole transcript microarray analysis. This study found that significantly over-expressed genes in CMCs compared to PBMCs were mapped to proinflammatory signaling pathways (including Nuclear factor kappa B (NFκB), Tumor necrosis factor (TNF), Toll-like receptor (TLR) and Nucleotide-binding and oligomerization domain (NOD)-like receptor). Concurrently, a signature of reduced potential for adaptive immunity was observed in CMCs compared to PBMCs, as evidenced by underrepresentation of the T cell receptor signaling and natural killer cell mediated cytotoxicity pathways. G. vaginalis induced a potent proinflammatory cytokine response by VK2 cells in vitro. Over-expressed genes in G. vaginalis-stimulated VK2 cells compared to unstimulated VK2 cells were mapped to inflammatory signalling pathways. In contrast, 3/8 Lactobacillus isolates, including two L. mucosae and one L. vaginalis species, reduced inflammatory cytokine production by VK2 cells in response to G. vaginalis and were thus termed “cytokinesuppressive”. Several genes, 7/8 of which are involved in inflammation, were downregulated in VK2 cells co-cultured with lactobacilli and G. vaginalis in combination compared to coculture with G. vaginalis only. Futhermore, when gene expression changes were investigated in cells cultured with cytokine-suppresive lactobacilli versus non-cytokine-suppressive lactobacilli, it was found that SAMD9L, DDX58, IFIT1 gene expression was downregulated exclusively in VK2 cells co-cultured with cytokine-suppressive lactobacilli and G. vaginalis compared to co-culture with G. vaginalis only. The findings of this study have identified distinct gene expression patterns in the FGT compared to peripheral blood. Furthermore, key genes that may play a critical role in the immunomodulatory effects of vaginal lactobacilli were identified, motivating for further confirmatory research.

During the past 30 years, there have been several Flaviviridae threats. Among them, Hepatatis C virus (HCV) emerged in the Western hemisphere as the previously unidentified etiological agent of non-A non-B hepatitis in transfusion patients. Less than twenty-five years after the 1989 discovery of HCV, several high-efficacy direct acting antivirals (DAAs) boasting a >95% cure rate were approved for treatment of HCV infected patients. While the advent of these DAAs has revolutionized the prognosis for chronically infected HCV patients, a high level of HCV-induced disease burden remains due to low rate of diagnosis (% here) and the high cost of therapy. Without treatment, 80% of chronically infected hepatitis C individuals exhibit hepatic intracellular lipid accumulation, termed steatosis. This liver damage can eventually contribute to cirrhosis (20% of individuals) and hepatocellular carcinoma (5% of individuals), necessitating a liver transplant for patient survival. Hepatitis C virus (HCV) infection perturbs host lipid metabolism through both cellular and viral-induced mechanisms, with the viral core protein playing an important role in steatosis development. In the first three years of my Ph.D., I sought to identify mechanisms contributing to HCV-induced steatosis. This work was published and Spotlighted in Journal of Virology. In this study, we identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as a HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. We then further investigated the biological significance and molecular mechanism of this downregulation. Importantly, we validated our in vitro finding with an in vivo model system, and saw that HCV infection in a live mouse model downregulated CIDEB in the liver tissue. We also found that CIDEB gene knockout in a hepatoma cell line resulted in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very low density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis was further supported by the requirement of HCV core and its LD localization for CIDEB downregulation, which utilized a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB. In late 2015, the global scientific community became aware of the emergence and threat of another Flaviviridae virus called Zika virus (ZIKV) in Brazil. The previously obscure ZIKV, which had laid relatively dormant for the previous 70 years, began spreading rapidly through the Western hemisphere, thus prompting the World Health Organization (WHO) to declare a public health emergency in February 2016. In response to the global health emergency posed by the ZIKV outbreak and its link to microcephaly and other neurological conditions, we established a collaboration with Dr. Wei Zheng at the National Center for Advancing Translational Sciences (NCATS) at this time, with whom we together performed a drug repurposing screen of ~6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds. We reported these results in Nature Medicine in August 2016. From this initial research, we identified 37 lead compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. We found that emricasan, a pan-caspase inhibitor, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. We then continued our work to identify additional compounds by refining our high-throughput assay. We developed a high-throughput ZIKV-NS1 based FRET detection assay to rescreen all 6,000 compounds, and then validated 256 hits by a semi-automated viral titer assay we developed in our lab in collaboration with NCATS. From this combinatorial approach, we identified an additional 117 compounds for use in further antiviral development. Among these, we found a conserved role of proteasome inhibitors in inhibiting ZIKV infection, and identified additional lead compounds including emetine, an anti-protozoal small molecule compound. In collaboration with NCATS and Dr. Anil Mathew Tharappel who completed mouse studies, we found that emetine is effective in a live animal model at reducing Zika viral load and likely inhibits viral replication via a direct block on the ZIKV NS5 RNA dependent RNA polymerase. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / April 16, 2018. / Antiviral, Drug discovery, Flavivirus, Hepatitis C Virus, Steatosis, Zika virus / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Yi Ren, University Representative; Thomas C. Keller, Committee Member; Fanxiu Zhu, Committee Member; David Meckes, Committee Member.

Almost all DNA viruses must deliver their genomes to the nucleus in order to initiate infection. The route by which they accomplish that goal varies greatly and in many cases is not well characterized. SV40 is a non-enveloped virus and therefore does not have the option of fusing with cellular membranes in order to gain entry into a cell or its nucleus. It must solve the problem of having to cross at multiple cellular membranes before it can initiate infection. SV40 enters host cells via caveolae, and is then transported along the microtubules to the endoplasmic reticulum (ER). Within the lumen of the ER, the viral capsid undergoes structural modification followed by the translocation of the virus across the ER membrane and into the cytoplasm. In the final steps in the pathway to infection, the SV40 disassembly intermediate delivers the viral genome to the nuclear membrane where the DNA enters the nucleus and initiates an infection. The research presented here investigates the state of the SV40 particles as well as attempts to clarify the route that the virus takes as it delivers its genome into the nucleus. We propose that the SV40 route to infection is a dynamic process in which the virus undergoes multiple disassembly steps while traversing through the host cell. The first disassembly event occurs within the ER which exposes the internal capsid proteins VP2, VP3 to detection by indirect immunofluorescence. Directly following the viral exit from the ER, the second capsid disassembly step takes place in the cytoplasm. The additional capsid modification within the cytoplasm allows for the detection of the viral genome by multiple detection methods. However, the DNA of the viral disassembly intermediates found within the cytoplasm retains a close association with VP2/3 and VP1 until the particles reach the nucleus. Upon reaching the nuclear membrane, the viral genome dissociates from the disassembly intermediate and the DNA enters the nucleus without VP2/3. In conclusion, this research adds to our current understanding of viral infection pathway, the nature of the viral particle during an infection, and finally investigates viral entry into the nucleus.

Kaposi sarcoma herpes virus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement-binding protein, KCP) that binds complement proteins and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Kaposi's sarcoma is an angiogenic skin lesion that has been recognized as one of the most abundant tumours found in many parts of Southern Africa and which can occasionally become highly invasive, aggressive and capable of causing death, particularly amongst AIDS patients. It is of major significance to understand how complement control proteins (CCPs) such as KCP perform their biological functions at the molecular and structural levels, because of their potentials as therapeutic agents, their implications in the pathology and importance in the etiology of many disease conditions. This study was therefore undertaken to characterise the structure-function relationship of KCP. Based on primary sequence analysis and comparison to other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by PCR, three regions of the predicted ORF 4 from human herpes virus-8 (llliV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris and to produce separately, the 4 N-terminal Sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane binding domain (KCP-M, medium) and the full-length protein (KCPF, full). Expression of the viral proteins was confirmed by SDS-PAGE and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. All the KCP proteins migrated electrophoretically as higher bands compared to their expected sizes. The lower mobilities of the proteins may be due to g1ycosy1ation since there are potential N-and O-glycosylation sites in the protein's primary sequence. Also, diffused bands were obtained in all the electrophoretic gels and Western blots carried out, which is characteristic of glycoproteins. Furthermore, the antibody recognized several larger and smaller bands that may represent aggregates and/or degradation products respectively. Both partially purified KCP-S and KCP-S directly from expression media were able to inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice more efficient in inhibiting the classical pathway-mediated lysis of erythrocytes than the vaccinia virus complement control protein (VCP), which also contains 4 Sushi domains. The KCP-F and KCP-M proteins did not show any significant complement inhibitory activities. Preliminary immunohistochemical studies using the same antibody were carried out to determine the expression and distribution of KCP proteins in Kaposi's sarcoma.

Acinetobacter baumannii is a major pathogen causing nosocomial infections, particularly in critically ill patients. This organism has acquired the propensity to rapidly develop resistance to most antibiotics. At several hospitals within Cape Town, tobramycin and colistin remain frequently the only therapeutic options. The Vitek2 automated susceptibility testing (AST) is used in the clinical laboratory to determine selected susceptibility profiles. The suspicion of a possible AST-related technical error when testing for susceptibility to tobramycin in A. baumannii precipitated this study.

This study constitutes the first report on type-specific human papillomavirus (HPV) concordance and transmission in heterosexually active couples that are human immunodeficiency (HIV)-seronegative, HIV-seropositive or HIV-discordant and in which 71% of female participants have normal cervical cytology.