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.

An investigation into the role of interleukin-6 in linking systemic and local inflammatory responses in patients with colorectal cancer

Colorectal cancer is the second most common cause of cancer death in the UK. It is accepted that both tumour and host factors are important determinants of disease progression and survival. While systemic and local inflammatory responses are increasingly recognized to be of particular importance the understanding of the mechanisms linking these important inflammatory processes remains unclear. This thesis examines the prognostic importance of measures of systemic and local inflammation and proposes a hypothesis for a link between tumour necrosis, systemic and local inflammatory responses in patients with colorectal cancer. Chapter 3 reports the comparison of the prognostic value of longitudinal measurements of systemic inflammation in patients undergoing curative resection of colorectal cancer. In Chapter 3 the results demonstrate that there was no significant overall change in either mGPS or NLR from pre- to post- operatively. This study highlighted the associations between pre- and post- operative mGPS and NLR and T-stage (p<0.001), TNM stage (p<0.005) and cancer-specific survival. The relationships between pre-operative measurements were examined using multivariate analysis. For pre-operative measurement both mGPS and NLR were associated with cancer-specific survival while when post-operative measures were examined only mGPS was specifically associated with cancer-specific survival (HR 4.81, CI 2.13-10.83, P<0.001). Chapter 4 examines the prognostic value of the Klintrup-Makinen scoring method and the existing limitations with regard to its clinical utility. An automated scoring method using commercially available image analysis software was developed and compared with manual scoring of tumour inflammatory infiltrates. This study demonstrated that both manual K-M scoring (p<0.001) and automated K-M scoring (p<0.05) had prognostic value in patients who had undergone potentially curative resection of colorectal cancer, and that the novel automated method may provide an objective method of assessment of tumour inflammatory infiltrates using routinely stained haematoxylin and eosin sections of tumour samples. In chapter 5 a hypothesis was proposed that Interleukin-6 may link tumour necrosis and systemic and local inflammatory responses in patients with colorectal cancer. This chapter examined the basis for this hypothesis, which is presented in figure 5.1. In addition, in chapter 5 the importance of this potential link is examined. In chapter 6, the hypothesis outlined in chapter 5 was examined in a cohort of patients who had undergone attempted curative resection of colorectal cancer. This study examined the inter-relationships between circulating mediators, in particular IL-6, tumour necrosis and systemic and local inflammatory responses. This results of this study demonstrated that IL-6 was associated with tumour necrosis (<0.001) and mGPS (<0.001) independent of T-stage. Thus adding weight to the hypothesis that elevated circulating concentrations of IL-6 may play a role in modulating both the systemic and local inflammatory responses in patients with cancer. Chapter 7 further develops the hypothesis that IL-6 signalling may be important in modulating systemic and local inflammatory responses in patients with colorectal cancer. Further, in chapter 7 the basis for the role of trans-signalling in this signaling pathway was examined. In this study, we reported that neither expression of the soluble IL-6 receptor or soluble gp130 were associated with systemic or local inflammatory responses. As a result the possible reasons for these findings were explored and future work suggested. A prospective database of patients undergoing attempted curative resection of colorectal cancer in Glasgow Royal Infirmary was used throughout this thesis. This database was created and is maintained regularly by successive research fellows at the Royal Infirmary. The work presented in this thesis highlights the importance of the host response in the form of systemic and local inflammation in patients with colorectal cancer and proposes a link between these responses and tumour necrosis. In addition, this work adds weight to the body of evidence suggesting that assessment of these host responses may improve stratification to treatment for patients with colorectal cancer. Further, this work proposes a mechanistic link, between tumour necrosis, systemic and local inflammatory responses through Interleukin-6, that merits further investigation.

The impact and control of malignant catarrhal fever in Tanzania

Malignant Catarrhal Fever (MCF), an often-lethal infectious disease, presents as a variable complex of lesions in susceptible ungulate species. The disease is caused by a -herpesvirus following transmission from an inapparent carrier host. Two major epidemiological forms exist: wildebeest-associated MCF (WA-MCF), in which the virus is transmitted to susceptible species by wildebeest calves less than approximately four months of age, and sheepassociated MCF (SA-MCF) in which the virus is spread by sheep (primarily adolescents). Due to the lack of an in-vitro propagation system for the causative agent of the more economically significant SA-MCF, and with the expectation that cross-protective immunity may be provided, vaccine development has focused on the more easily propagated alcelaphine herpesvirus-1 (AlHV-1) that causes WA-MCF. In 2008 a direct viral challenge trial showed that a novel vaccine, employing an attenuated AlHV-1 (atAlHV-1) `C5000 virus strain, protected British Friesian-Holstein (FH) cattle against an intranasal challenge with virulent AlHV-1 `C5000 virus. For cattle keeping people living near wildebeest calving areas in sub-Saharan Africa an effective vaccine would have value as it would release them from the costly annual disease avoidance strategy of having to move their herds away from the oncoming wildebeest. On the other hand, an effective vaccine will release herd owners from the need to avoid MCF, allowing them to graze their cattle alongside wildebeest on the highly nutritious pastures of the calving areas. As such conservationists have raised concerns that the development of a vaccine might lead to detrimental grazing competition. The principle objective of this study was to test the novel vaccine on Tanzanian shorthorn zebu cross cattle (SZC).We did this firstly using a natural challenge field trial (Chapter Two) which demonstrated that immunisation with the atAlHV-1 vaccine was well tolerated and induced an oro-nasopharyngeal AlHV-1-specific and -neutralising antibody response. This resulted in an immunity in SZC cattle that was partially protective and reduced naturally transmitted infection by 56%. We also demonstrated that non-fatal infections occurred with a much higher frequency than previously thought. Because the calculated efficacy of the vaccine was less than that seen in British FH cattle we wanted to determine whether host factors, particular to SZC cattle, had impacted the outcomes of the field trial. To do this we repeated the 2008 direct viral challenge trial using SZC cattle (Chapter Four). During this trial we also investigated whether the recombinant bacterial flagellin monomer (FliC), when used as an adjuvant, might improve the vaccine’s efficacy. The findings from this trial indicated that direct challenge with pathogenic AlHV-1 is effective at inducing MCF in SZC cattle and that FliC is not an appropriate adjuvant for this vaccine. Furthermore, with less control group cattle dying of MCF than expected we speculate that SZC cattle may have a degree of resistance to MCF that affords them protection from infection and developing fatal disease. In Chapter Three we investigated aspects of the epidemiology of MCF, specifically whether wildebeest placenta, long implicated by Maasai cattle owners as a source of MCF, might play a role in viral transmission. Additionally, through comparative sequence analysis, at two specific genes (A9.5 and ORF50) of wild-type and atAlHV-1, we investigated whether the `C5000 strain, the source of which was taken from Africa more than 40 years ago, was appropriate for vaccine development. The detection of AlHV-1 virus in approximately 50% of placentae indicated that infection can occur in-utero and that this tissue might play a role in disease transmission. And, despite describing three new alleles of the A9.5 gene (supporting previous evidence that this gene is polymorphic and encodes a secretory protein with interleukin-4 as the major homologue), the observation that the most frequently detected haplotypes, in both wild-type and attenuated AlHV-1, were identical suggests that AlHV-1 has a slow molecular clock and that the attenuated strain was appropriate for vaccine development. In Chapter Five we present the first quantitative assessment of the annual MCF avoidance costs that Maasai pastoralists incur. In particular we estimated that as a result of MCF avoidance 64% of the total daily milk yield during the MCF season was not available to be used by the 81% of the family unit remaining at the permanent boma. This represents an upper-bound loss of approximately 8% of a household0s annual income. Despite these considerable losses we concluded that, given an incidence of fatal MCF in cattle living in wildebeest calving areas of 5% to 10%, if herd owners were to stop trying to avoid MCF by allowing their cattle to graze alongside wildebeest, any gains made through increased availability of milk, improved body condition and reduced energy demands would be offset by an increase in MCF-incidence. With the development of an effective vaccine, however, this alternative strategy might become optimal. The overall conclusion we draw therefore is that, despite the substantial costs incurred each year avoiding MCF, the partial protection afforded by the novel vaccine strategy is not sufficient to warrant a wholesale change in disease avoidance strategy. Nonetheless, even the partial protection provided by this vaccine could be of value to protect animals that cannot be moved, for example where some of the herd remain at the boma to provide milk or where land-use changes make traditional disease avoidance difficult. Furthermore, the vaccine may offer a feasible solution to some of the current land-use challenges and conflicts, providing a degree of protection to valuable livestock where avoidance strategies are not possible, but with less risk of precipitating the potentially damaging environmental consequences, such as overgrazing of highly nutritious seasonal pastures, that might result if herd owners decide they no longer need to avoid wildebeest.

Prevalence and associations of Trypanosoma spp. and Sodalis glossinidius with intrinsic factors of tsetse flies

Trypanosomiasis has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. Whilst assessments of the biology of trypanosomes, vectors, vertebrate hosts and the environment have provided useful information about life cycles, transmission, and pathogenesis of the parasites that could be used for treatment and control, less information is available about the effects of interactions among multiple intrinsic factors on trypanosome presence in tsetse flies from different sites. It is known that multiple species of tsetse flies can transmit trypanosomes but differences in their vector competence has normally been studied in relation to individual factors in isolation, such as: intrinsic factors of the flies (e.g. age, sex); habitat characteristics; presence of endosymbionts (e.g. Wigglesworthia glossinidia, Sodalis glossinidius); feeding pattern; host communities that the flies feed on; and which species of trypanosomes are transmitted. The purpose of this study was to take a more integrated approach to investigate trypanosome prevalence in tsetse flies. In chapter 2, techniques were optimised for using the Polymerase Chain Reaction (PCR) to identify species of trypanosomes (Trypanosoma vivax, T. congolense, T. brucei, T. simiae, and T. godfreyi) present in four species of tsetse flies (Glossina austeni, G. brevipalpis, G. longipennis and G. pallidipes) from two regions of eastern Kenya (the Shimba Hills and Nguruman). Based on universal primers targeting the internal transcribed spacer 1 region (ITS-1), T. vivax was the predominant pathogenic species detected in flies, both singly and in combination with other species of trypanosomes. Using Generalised Linear Models (GLMs) and likelihood ratio tests to choose the best-fitting models, presence of T. vivax was significantly associated with an interaction between subpopulation (a combination between collection sites and species of Glossina) and sex of the flies (X2 = 7.52, df = 21, P-value = 0.0061); prevalence in females overall was higher than in males but this was not consistent across subpopulations. Similarly, T. congolense was significantly associated only with subpopulation (X2 = 18.77, df = 1, P-value = 0.0046); prevalence was higher overall in the Shimba Hills than in Nguruman but this pattern varied by species of tsetse fly. When associations were analysed in individual species of tsetse flies, there were no consistent associations between trypanosome prevalence and any single factor (site, sex, age) and different combinations of interactions were found to be significant for each. The results thus demonstrated complex interactions between vectors and trypanosome prevalence related to both the distribution and intrinsic factors of tsetse flies. The potential influence of the presence of S. glossinidius on trypanosome presence in tsetse flies was studied in chapter 3. A high number of Sodalis positive flies was found in the Shimba Hills, while there were only two positive flies from Nguruman. Presence or absence of Sodalis was significantly associated with subpopulation while trypanosome presence showed a significant association with age (X2 = 4.65, df = 14, P-value = 0.0310) and an interaction between subpopulation and sex (X2 = 18.94, df = 10, P-value = 0.0043). However, the specific associations that were significant varied across species of trypanosomes, with T. congolense and T. brucei but not T. vivax showing significant interactions involving Sodalis. Although it has previously been concluded that presence of Sodalis increases susceptibility to trypanosomes, the results presented here suggest a more complicated relationship, which may be biased by differences in the distribution and intrinsic factors of tsetse flies, as well as which trypanosome species are considered. In chapter 4 trypanosome status was studied in relation to blood meal sources, feeding status and feeding patterns of G. pallidipes (which was the predominant fly species collected for this study) as determined by sequencing the mitochondrial cytochrome B gene using DNA extracted from abdomen samples. African buffalo and African elephants were the main sources of blood meals but antelopes, warthogs, humans, giraffes and hyenas were also identified. Feeding on multiple hosts was common in flies sampled from the Shimba Hills but most flies from Nguruman had fed on single host species. Based on Multiple Correspondence Analysis (MCA), host-feeding patterns showed a correlation with site of sample collection and Sodalis status, while trypanosome status was correlated with sex and age of the flies, suggesting that recent host-feeding patterns from blood meal analysis cannot predict trypanosome status. In conclusion, the complexity of interactions found suggests that strategies of tsetse fly control should be specific to particular epidemic areas. Future studies should include laboratory experiments that use local colonies of tsetse flies, local strains of trypanosomes and local S. glossinidius under controlled environmental conditions to tease out the factors that affect vector competence and the relative influence of external environmental factors on the dynamics of these interactions.