Dengue is an emerging infectious disease and is spreading world-wide at exponential levels. Two billion people in over 100 countries are at risk for infection from one of the four serotypes of the dengue virus. Those infected with dengue may develop diseases such as dengue fever and dengue hemorrhagic fever (DHF) and of the 500,000 cases that progress to DHF each year, more than 22,000 will result in fatality. Discovering new antivirals to treat DHF is essential to reducing this disease burden. Here, we have developed a cytopathic effect-based high-throughput screen (HTS) to discover possible inhibitors of Dengue viral infection of hepatocytes in vitro. Dengue virus infection of hepatocytes induces massive cell death, cytopathic effect (CPE), which we converted into a screening assay whereby inhibitors of Dengue infection prevent cells from dying. In this assay, the viral induced CPE is quantitated by monitoring cellular ATP levels, which positively correlates with cellular viability. ATP in the cell culture will drive the oxidation of luciferin resulting in the emission of light that is quantitated using a luminometer. The assay is simple and highly reproducible yielding a screening window coefficient, Z-factor, of 0.78±0.12 between plates. The Z-factor is a statistical parameter commonly accepted as an assay quality assessment and is reported as a value 0 to 1 and anything over 0.5 is considered excellent quality. This assay is advantageous to current methodology as it simultaneously screens possible inhibitory compounds while controlling for any unwanted toxicity triggered by these drugs. Our initial HTS of a 288 small compound library yielded a total of eleven hits that prevented the CPE of dengue infection. Further evaluation with an immunofluorescence assay showed that two of these compounds, Streptovitacin A and Nagilactone C, are highly potent inhibitors of dengue infection. At effective inhibitory doses, they did not appear to be cytotoxic, and therefore both of these compounds are possible antivirals and could be used to elucidate various cellular mechanisms utilized during the dengue life cycle. The discovery of these two inhibitors demonstrates the efficacy of our newly developed assay and the public health significance of this project.

Lymphatic endothelial cells (LECs) line lymphatic vessels and are present at mucosal portals of entry for many pathogens, including simian immunodeficiency virus (SIV) and human immunodeficiency virus type-1 (HIV-1). Recent studies have shown that LECs express pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), capable of recognizing pathogen-associated molecular patterns (PAMPs). PAMPs are structurally similar molecules expressed by groups of pathogens. LECs have also been shown to express chemokines, a group of small molecules secreted by cells that induce chemotaxis in responsive cells, such as CCL21, which is used by CCR7+ mature antigen presenting dendritic cells (DCs) to migrate to draining lymph nodes (LNs). These previous findings indicate that LECs might play an integral role in innate immune responses to a wide variety of microbes. In this study, I set out to characterize the expression of antiviral restriction factors as well as possible viral entry receptors for SIV/HIV-1 within three populations of human LECs. Real-time RT-PCR and immunofluorescent staining techniques were used to determine the relative expression of the restriction factors BST-2/Tetherin, APOBEC3G, and TRIM5-á. All of these factors have been shown to inhibit the replicative cycle of HIV-1 and have othologs present in nonhuman primates (NHPs). Expression of the viral entry receptors CD4, CXCR4, CCR5, DEC-205/CD205, D6/CCBP2, and CD209 as well as the LEC-specific markers podoplanin and LYVE-1 was also investigated. In addition, LEC populations were exposed to SIV, HIV-1, and markers internalization to determine to what extent LECs interact with virus in vitro. Data from populations exposed to HIV-1 as well as other substrates for internalization of extracellular materials illustrate the ability of LECs to actively monitor the extracellular milieu. LECs exposed to SIV showed multi-spliced viral transcripts possibly due to de novo transcription. Taken together, this study provides evidence that LECs are equipped with tools not only to bind and internalize pathogens, but may also serve as a low-level replicative cellular substrate for virus. Further studies to characterize LECs are of great public health relevance, particularly at mucosa sites of microbial exposure, due to their potential roles during transmission/infection.

Highly active anti-retroviral therapy (HAART) has been successful in delaying the progression to AIDS in HIV infected individuals. Exposure to HAART can result in metabolic side effects such as dyslipidemia and lipodystrophy in a subset of treated patients. We used a custom designed Illumina GoldenGate Genotyping assay to investigate the genetic susceptibility to dyslipidemia attributed to HIV infection and HAART treatment. 1,945 men were selected from the Multicenter AIDS Cohort Study (MACS) for genotyping and phenotypic analysis of serum lipid levels. This population was stratified by biogeographical ancestry and HIV/HAART status. Among men of European ancestry, those who were infected with HIV and receiving HAART had significantly lower serum low-density lipoprotein cholesterol (LDL-C, P = 1.90 x10-4) and high-density lipoprotein cholesterol levels (HDL-C, P < 1.00 x10-7), with significantly higher serum triglyceride (TRIG, P < 1.00 x10-7) levels compared to HIV/HAART (-/-) controls. Among men of mixed African and European ancestry, those who were HIV/HAART (+/+) had significantly lower LDL-C (P = 1.80 x10-4) levels compared to HIV/HAART (-/-) controls. Four SNPs; rs1532624 (P = 1.66 x10-5), rs1532625 (P = 2.36 x10-5), rs711752 (P = 4.48 x10-5), and rs708272 (P = 4.59 x10-5), located in the CETP gene region on chromosome 16 had statistically significant associations with serum HDL-C levels in HIV/HAART (+/+) European men. One SNP, rs261334 (P = 6.53 x10-6), located in the LIPC gene region on chromosome 15 was associated with serum LDL-C levels and another SNP, rs4783961 (P = 9.83 x10-6) located in the CETP gene region, was associated with HDL-C levels in HIV/HAART (+/+) men of mixed African and European ancestry. These results show that dyslipidemia attributed to HAART varies depending on biogeographical ancestry and implicates two genes associated with serum lipid levels in these patients. Understanding the mechanism of HAART-associated dyslipidemia is important to global public health because nearly half of the estimated 30 million individuals infected with HIV are receiving or eligible to receive these drugs and are at risk of these HAART related side effects. Our results can also aid in identifying those individuals at greatest risk of developing HAART-associated dyslipidemia, which could improve monitoring and management of care given to these patients.

ADAR1 is an RNA editing enzyme which acts on completely or partially double-stranded RNA. Since HIV-1 RNA has such secondary structures, we have examined whether ADAR1 exhibits antiviral activity against HIV-1. Our results indicated that ADAR1 inhibited viral replication and infectious HIV-1 production in various cell lines including 293T, HeLa and Jurkat T cells, and was active against a number of X4- and R5-tropic HIV-1 of different clades. Analysis of the level of intracellular HIV-1 RNA showed no change in levels of intracellular gag, pol, and env RNA in the presence of ADAR1 despite a significant inhibition of intracellular and virion associated HIV-1 protein production. Furthermore mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the first exon of rev at positions 5998, 6011, 6017, and 6036 and in the Rev Response Element (RRE) binding region (positions 8413 and 8438) of rev and env RNA. In elucidating the mechanism of ADAR1 inhibition of HIV-1, we observed that A-G mutations in rev have a significant negative effect on the expression of Rev. However, all mutations could be complemented by wild type Rev.Furthermore, these A-G mutations in the RRE binding region of rev inhibited the binding of Rev to the RRE region in env and inhibited transport of primary transcripts like gag, pol and env from the nucleus to the cytoplasm. Introduction of these specific mutations in rev of an infectious molecular clone of HIV-1 by site directed mutagenesis abolished the replication capacity of HIV-1 by inhibiting viral protein synthesis without any effect on viral RNA synthesis, a phenotype exhibited by HIV-1-infected cells exposed to ADAR1. ADAR1 induced mutations in env further attenuated viral infectivity. ADAR1, thus, constitutes a novel class of cellular antiviral proteins with multiple targets in the viral genome thereby providing a new avenue of exploration for therapeutic drugs benefitting public health.

Data from our group and others have demonstrated that tumor-derived factors directly skew T-cell differentiation from an effective tumor fighting Th1 state to a less effective Th2 state, allowing for tumor growth. Why the Th1 response is more effective is largely still unknown. The recently discovered microRNAs (miRNAs) are a large family of small regulatory RNAs that control diverse aspects of cell functions such as cell proliferation, apoptosis, development, differentiation and immune regulation. We thereby sought to examine miRNAs differentially expressed in Th1 and Th2 cells in an effort to better understand the enhanced ability of Th1 cells in tumor immunity. MicroRNA microarray analyses revealed that the miR-17-92 cluster of microRNAs (miR-17-92) is consistently over-expressed in murine Th1 cells compared to Th2 cells. Quantitative RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Furthermore, disruption of IL-4 signaling through either IL-4 neutralizing antibody or knockout of STAT6 reversed the miR-17-92 cluster suppression in Th2 cells. MiR-17-92 expression correlated with differential proliferation capacity as Th1 cells proliferated at higher levels than Th2 cells, dependent on IL-4 and STAT6. Th1 cells consistently expressed lower levels of anti-proliferative transcription factors E2F1 and E2F2, which are the known targets of miR-17-92. Collectively, our data suggests that the Th2 skewing tumor microenvironment can induce the down-regulation of miR-17-92 expression in CD4+T cells, thereby diminishing the effective proliferation of tumor-specific T cells and tumor destruction. This has significant public health relevance as we propose that therapy targeting miR-17-92 cluster may provide enhanced T-cell function and prevent tumor growth.

K65R, a lysine to arginine change at codon 65 of HIV-1 reverse transcriptase (RT), has been selected in vitro by many NRTIs (nucleoside analog RT inhibitors), but until recently, was infrequently detected in patients. Located in the fingers subdomain of RT, K65R causes NRTI resistance through a discrimination mechanism by increasing selectivity for natural deoxynucleotide triphosphate substrates (dNTP) incorporation over triphosphorylated NRTI incorporation. The thymidine analog mutations (TAMs) include the following amino acid changes in RT: M41L, D67N, K70R, L210W, T215F/Y and K219Q. Different combinations of TAMs facilitate AZT resistance by a primer unblocking mechanism, also known as an excision mechanism, in which the chain-terminating NRTI is removed from the nascent DNA strand to allow polymerization to resume. From in vitro and clinical observations, we proposed that K65R and TAMs represent two different pathways of NRTI resistance that exhibit bi-directional phenotypic antagonism and counterselection in vivo. We have generated several lines of evidence in support of this hypothesis: (1) HIV encoding K65R has reduced susceptibility to all NRTIs tested except those with a 3 azido in the pseudosugar component (AZT and AZA); (2) in a large clinical database, K65R is increasing in prevalence in patient isolates, whereas TAMs are decreasing in prevalence; (3) a strong negative relation exists between the frequency of K65R and specific TAMs among HIV-1 isolates in a large clinical database; (4) K65R reverses viral resistance to AZT caused by TAMs and TAMs reverse resistance to abacavir and tenofovir caused by K65R; (5) K65R antagonizes the primer unblocking activity of TAMs and TAMs antagonize discrimination by K65R; and (6) in plasma samples in which both K65R and TAMs were detected by population sequencing, K65R was not found on the same genome with T215F/Y and 2 or more other TAMs, except when the Q151M multi-drug resistance complex was also present. HIV-1 drug resistance is a significant public health problem. This work contributes to the understanding of NRTI resistance and will help to optimize current and future therapy for HIV-1 infection.

Lyme borreliosis (LB), the most common tick-borne disease in Europe and North America, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex. The spirochetes can invade several different organs, thereby causing many different symptoms and signs. Diagnosis of LB relies on patient history, physical examination, and detection of anti-Borrelia antibodies. However, anti-Borrelia antibodies are not always detectable, and they commonly persist even after LB is successfully treated or spontaneously healed. The aim of my work was to study diagnostic aspects on clinical cases of LB and control subjects in an area endemic to LB, with a focus on newly developed anti-Borrelia antibody tests. A total of 617 patients with symptoms and/or signs consistent with LB, as well as 255 control subjects, were studied. The diagnostic panel included the following new LB tests: Immunetics Quick ELISA C6 Borrelia assay kit (C6), invariable region 6 peptide antibody assays (IR6), Liaison Borrelia CLIA (Li) and the chemokine CXCL13. Results were compared with the older Virotech Borrelia burgdorferi ELISA (VT) and with a Western blot method, the Virotech Borrelia Ecoline IgG/IgM Line Immunoblot (WB EL), when appropriate. In general, no significant differences were noted between the C6, VT and Li tests regarding serosensitivity in various LB manifestations. However, the seropositivity rate was lower for the C6 test compared with the VT and Li tests 2–3 and 6 months after diagnosis of erythema migrans (EM), indicating normalization of antibody levels. In addition, EM patients reporting a previous LB episode had a C6 seropositivity rate similar to that of patients without a previous LB episode, and seroprevalence in healthy blood donors was lower in the C6 test than the VT and Li tests. Taken together, these results support the recommendation of the serum C6 test as a Borrelia serological test due to its ability to reflect ongoing or recent infection. Although the majority of EM patients at presentation showed concordant serological responses to IR6 peptides representing the three main Borrelia species and the C6 peptide, there were also clinical EM cases that were C6-negative and could be detected mainly by a seroresponse to a B. burgdorferi sensu stricto-derived IR6 peptide. Thus, an antibody test combining antigens could be of value in the serodiagnosis of LB in Europe. The serosensitivity of the C6 test in cases of Lyme neuroborreliosis (LNB) was shown to be associated with symptom duration. A serosensitivity rate of 93% was found in LNB patients ³ 12 years of age with a symptom duration of more than 30 days. Therefore, a negative C6 test in serum in such a patient argues against an LNB diagnosis. The presence of chemokine CXCL13 in cerebrospinal fluid was confirmed to be a reliable marker of LNB. CXCL13 differentiated LNB from other conditions and also indicated a high probability of LNB in children with short symptom duration where anti-Borrelia antibodies were still lacking in the cerebrospinal fluid. A two-tiered approach (C6 test in combination with WB EL) showed no significant improvement in specificity over the C6 test alone. However, WB EL may be useful in diagnosing suspected cases of acrodermatitis chronicum atrophicans and Lyme arthritis, usually displaying multiple IgG bands. In conclusion, although the serodiagnosis of LB remains to be settled, this thesis provides some practical tools regarding the use and interpretation of Borrelia serology including proposed diagnostic routines.

Perinatal infections are associated with significant morbidity and mortality for both pregnant women and their infants, including while in utero. Prenatal screening for potential infectious diseases can effectively prevent MTCT infections. It allows both timely and suitable medical interventions when required. In Manitoba, prenatal screening for Rubella, Hepatitis B Virus (HBV), Human Immunodeficiency Virus (HIV), Treponema pallidum, Chlamydia Trachomatis (CT) and Neisseria gonorrhoeae (GC) is recommended for all pregnant women and in each pregnancy. The research described in this thesis assesses the current adherence to the Manitoba prenatal screening guidelines. Data consisted of prenatal screening tests conducted at Cadham Provincial Lab (CPL) for the time period of 2006 to 2011. Approximately one fifth of pregnant women did not receive any form of recommended prenatal testings’. Adherence to prenatal screening guidelines varied by type of infection, age of women and area of residence. Overall, Rubella, HBV and syphilis prenatal screening were requested more frequently than HIV, CT and GC. From year to year, a significant improvement of HIV prenatal screening uptake was observed. Rubella, HBV and syphilis screening declined while GC and CT screening remained stable. Among screened women, HIV testing was requested significantly less frequently in the youngest <15 and oldest >45 age groups versus other age groups. Women >45 years old also received less GC and CT screening. A year- II to-year increase in HIV and GC screening was observed in pregnant women aged 15-25, 26-35, and 36-45 years old. Although HIV screening uptake increased over time among residents of Brandon and rural areas, the overall HIV screening test was still higher among residents of Winnipeg versus other areas. Similarly, residents of Brandon and rural areas were tested less frequently for CT infection. A significant improvement in GC screening among residents of Winnipeg and rural areas was observed. The results described in this thesis demonstrates inconsistent adherence to provincial guidelines – creates higher risk areas and population subsets for congenital infections.. The results also demonstrate the importance of promoting testing of this type among pregnant women. Improvement and enhancement of current practice is required to reach standard, satisfactory and appropriate adherence to screening guidelines. Ongoing periodic assessments are suggested to continually document and monitor uptake and adherence to recommended prenatal screening in Manitoba.

Thesis (M.S. in zoology)--Washington State University, August 2008. / Includes bibliographical references.

Introduction This study describes the disease burden, clinical characteristics, antibiotic management, impact of multidrug resistance and outcome of Pseudomonas aeruginosa bloodstream infection (PABSI) among children admitted to a tertiary referral hospital for children in Cape Town, South Africa. Methods A retrospective descriptive study was conducted at a paediatric referral hospital in Cape Town, South Africa. Demographic and clinical details, antibiotic management and patient outcome information were extracted from medical and laboratory records. Antibiotic susceptibility results of identified organisms were obtained from the National Health Laboratory Service database. Results The overall incidence risk of PABSI was 5.4 PABSI episodes / 10,000 hospital admissions and the most common presenting feature was respiratory distress, 34/91 (37%). Overall, 69/91 (76%) of the PA isolates were susceptible to all antipseudomonal antibiotic classes evaluated. Fifty (55%) of the PABSI episodes were treated with appropriate empiric antibiotic therapy. The mortality rate was 24% and in multivariable analysis, empiric antibiotic therapy to which PA isolate was not susceptible to, infections present on admission, and not being in the intensive care unit at the time that PABSI was diagnosed were significantly associated with 14-day mortality. Conclusion The study provided insight into factors associated with PABSI in a tertiary hospital in SubSaharan Africa. Empiric antipseudomonal antibiotic therapy was associated with a decrease in 14-day mortality.