Leptospirosis in northern Tanzania: exploring the role of rodents and ruminant livestock in a neglected public health problem

Leptospirosis is an important but neglected zoonotic disease that is often overlooked in Africa. Although comprehensive data on the incidence of human disease are lacking, robust evidence of infection has been demonstrated in people and animals from all regions of the continent. However, to date, there are few examples of direct epidemiological linkages between human disease and animal infection. In East Africa, awareness of the importance of human leptospirosis as a cause of non-malarial febrile illness is growing. In northern Tanzania, acute leptospirosis has been diagnosed in 9% of patients with severe febrile illness compared to only 2% with malaria. However, little is known about the relative importance of different potential animal hosts as sources of human infection in this area. This project was established to investigate the roles of rodents and ruminant livestock, important hosts of Leptospira in other settings, in the epidemiology of leptospirosis in northern Tanzania. A cross-sectional survey of rodents living in and around human settlements was performed alongside an abattoir survey of ruminant livestock. Unusual patterns of animal infection were detected by real-time PCR detection. Renal Leptospira infection was absent from rodents but was detected in cattle from several geographic areas. Infection was demonstrated for the first time in small ruminants sub-Saharan Africa. Two major Leptospira species and a novel Leptospira genotype were detected in livestock. L. borgpetersenii was seen only in cattle but L. kirschneri infection was detected in multiple livestock species (cattle, sheep and goats), suggesting that at least two distinct patterns of Leptospira infection occur in livestock in northern Tanzania. Analysis of samples from acute leptospirosis in febrile human patients could not detect Leptospira DNA by real-time PCR but identified social and behavioural factors that may limit the utility of acute-phase diagnostic tests in this community. Analysis of serological data revealed considerable overlap between serogroups detected in cattle and human leptospirosis cases. Human disease was most commonly attributed to the serogroups Mini and Australis, which were also predominant reactive serogroups in cattle. Collectively, the results of this study led to the hypothesis that livestock are an important reservoir of Leptospira infection for people in northern Tanzania. These results also challenge our understanding of the relationship between Leptospira and common invasive rodent species, which do not appear to maintain infection in this setting. Livestock Leptospira infection has substantial potential to affect the well-being of people in East Africa, through direct transmission of infection or through indirect effects on food production and economic security. Further research is needed to quantify the impact of livestock leptospirosis in Africa and to develop effective interventions for the control of human and animal disease.

Host cellular regulatory networks in dengue virus-human interactions

Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.

Investigating the role of target cell availability in the pathogenesis of feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, which shares many similarities with its human counterpart, human immunodeficiency virus (HIV). FIV infects its main target cell, the CD4+ T lymphocyte, via interactions with its primary receptor CD134 (an activation marker expressed on activated CD4+ T lymphocytes), and, the chemokine receptor CXCR4. According to the different ways in which FIV isolates interact with CD134, FIV may be categorised into two groups. The first group contains strains that tend to dominate during the earlier phase of infection, such as GL8 and CPG41. These strains are characterized by their requirement for an additional interaction with the second cysteine rich domain (CRD2) of the CD134 molecule and are classified as “CRD2-dependent” strains. The second group, on the other hand, contains either laboratory-adapted isolates or isolates that emerge after several years of infection, such as PPR or the GL8 variants that emerged in cats 6 years post experimental infection and were studied in this thesis. These isolates are designated “CRD2-independent” as they can infect target cells without interacting with CRD2 of the CD134 molecule. This study provides the first evidence that FIV compartmentalisation is related to FIV-CD134 usage and the tissue availability of CD134+ target cells. In tissue compartments containing high levels of CD134+ cells such as peripheral blood and lymph nodes, CRD2-dependent viruses predominated, whereas CRD2-independent viruses predominated in compartments with fewer CD134+ cells, such as the thymus. The dynamics of CD4+CD134+ T lymphocytes at different stages of FIV infection were also described. The levels of CD4+CD134+ T lymphocytes, which were very high in the early phase, gradually decreased in the later phase of infection. The dynamics of CD4+CD134+ T lymphocyte numbers appeared to correlate with FIV tropism switching, as more CRD2-independent viruses were isolated from cats in the late phase of infection. Moreover, it was observed that pseudotypes bearing Envs of CRD2-dependent variants infected CD134+ target cells more efficiently than pseudotypes bearing Envs of CRD2-independent variants, confirming the selective advantage of CRD2-dependent variants in environments with high levels of CD134+ target cells. In conclusion, this study demonstrated that target cell types and numbers, as well as their dynamics, play important roles in the selection and expansion of FIV variants within the viral quasispecies. Improved understanding of the roles of target cells in FIV transmission and pathogenesis will provide important information required for the development of an improved, more successful protective FIV vaccine and will provide insight into the development of effective vaccines against other lentiviral infections such as HIV.