Survival of Escherichia coli has been examined under the effect of various environmental factors, nutrient and stress conditions in natural lake water or sterile microcosms. E.coli could survive for at least 200 days at temperatures below 25 °C under starvation stress in sterile lake water. Whilst E.coli survived for the longest period of time at 4°C, the shortest survival time was always at 37°C in both natural and sterile lake water microcosms. Predation by protozoa was not found to be a significant factor affecting the survival of E.coli in natural lake water microcosms. The addition of sources of nutrients to lake water enhanced the survival of E.coli in both natural and sterile lake water. E.coli could grow in the lake water with the addition of a single carbon source or single amino acid addition but ultimately there was an accelerated decline in numbers. On the other hand the addition of ammonium sulphate and casein enhanced survival and allowed growth. The measurement of metabolic enzyme activities such as succinate and NADH dehydrogenase activity showed that E.coli reduced its metabolic activity very quickly upon starvation especially at 37°C. The reduction in activity was again temperature dependent. E.coli cells also reduced in size under starvation stress in lake water and the rate at the size reduced was temperature dependent. Measurements of the respiratory activity of the cells showed that although the viable count declined below detection limits under starvation stress, at least 50% of the cells could still respire. This indicates that the cells enter a viable but non-culturable phase in lake water. Alkaline phosphatase activity increased in cells which were incubated under starvation conditions. Pretreatment of the cells with alcohol, hydrogen peroxide or salt to induce stress proteins also lead to an increase in alkaline phosphatase activity. The addition of nutrient sources, particularly amino acids, to sterile lake water microcosms increased the activity of alkaline phosphatase and fi-galactosidase enzymes Two-dimensional gel electrophoresis was used to show changes in the protein patterns of E.coli affected by starvation and other stress conditions. Some proteins were induced under starvation conditions, others were repressed. The changes in the relative concentration of the outer membrane proteins, OmpC, OmpF and OmpA were quantified. All were affected by starvation stress with OmpA disappearing from the outer membrane. A 45 kDa protein was induced under all stress conditions. Other stresses resulted in the induction of a 70 kDa protein (oxidation stress by hydrogen peroxide) or the complete disappearance of the major outer membrane proteins (sodium dodecyl sulphate treatment). The survival of E.coli under starvation and other stress conditions was correlated with the induction of specific proteins.

The work described in this thesis identifies the structural polypeptide profile of human astrovirus type 1, grown in tissue culture in the presence of trypsin. Four major polypeptides were observed, of molecular weights: 34,000, 33.000, 26,500 and 6-8,000. A fifth of molecular weight 37,000 observed when the virus is prepared by PEG precipitation is considered to represent a precursor polypeptide. Of the four structural polypeptides, designated VP1, VP2, VP3 and VP4 respectively, VP1 and VP2 were found to be immunologically reactive with anti-astrovirus type 1 polyclonal antiserum. Astrovirus type 1 particles were found to have a buoyant density of 1.33-1.34 gm/ral in caesium chloride and a genome approximately 7,200 bases in length, corresponding to a molecular weight of 2.43 x 10° which is assumed to be RNA. This data suggests a relationship between astroviruses and picornaviruses, most especially the enterovirus subgroup, which have similar structural polypeptide and physico-chemical properties to those described above. However, the use of trypsin for the propagation of astrovirus in tissue culture means that further analysis of the structural polypeptides is required to determine whether those observed in this analysis represent four individual proteins, similar to the profile of picornaviruses, or whether some are cleavage products of the larger proteins due to the action of trypsin. The 3' terminal sequence of the astrovirus genome has been cloned and sequenced. It was found to have a poly (A) tail, stop codons in all three reading frames within the last 95 nucleotides and no polyadenylation signals. Several other astrovirus specific clones were obtained, but there is no suggestion as to which regions of the genome these represent. Comparisons between the astrovirus specific clones and the microgenie sequence databank has shown no significant homologies. The highest homology obtained for the 3' terminal sequence when compared to those of other picornaviruses was 47Z with coxsackie A21, this is not taken to confirm a relationship between the viruses. Assay systems were developed for astrovirus during the course of this study. An immunofluorescent end point titration, using type specific antisera, allowed the determination of the infectivity of a virus stock within 48 hours of infection. An immuno dot blot system was developed to assay for amounts of virus protein in samples, such as gradient fractions, and was used to determine the peak banding of virus in sucrose.

Promyelocytic Leukaemia proteins (PML), the core component of PML nuclear bodies (NB), are implicated in many critical cellular functions. One of those is confronting virus infection: DNA and RNA viruses target PML-NB and interact with PML proteins and this is thought to facilitate efficient replication by interfering with their antiviral functions. One of those viruses is human adenovirus type 5 (Ad5). Its early E4Orf3 protein interacts specifically with PML isoform II (PML-II), disrupting PML-NB into track-like structures to inhibit PML- antiviral responses. PML-II has been shown to be a positive regulator of type I interferon responses, as are typically induced by virus infection, through regulating the transcription factors that control this innate immune response. Given this role of PML-II and its interaction with Ad5 E4Orf3, the first question that addressed here is how does PML-II affect the progress of Ad5 infection under normal conditions? PML-NBs are also involved in cellular stress responses, being disrupted by heat shock and other stresses. Thermo-tolerance due to mild heat conditioning protects cells from more aggressive stresses such as higher temperatures through triggering the heat shock response, which helps in re-folding the affected cellular proteins. Interferon and other cellular cytokines are also induced in response to heat shock, suggesting a mechanism whereby heat stress might affect infection, possibly dependent on PML-NBs. The second question addressed here was whether cells became more resistant to infection by pre- stressing them and if so would PML-II have a role in that resistance? To address these questions, PML-II-depleted cells were first engineered by lentiviral vector delivery of specific shRNA. Physical and functional knock-down was confirmed by measuring mRNA levels for PML-II and for genes normally induced by NF-kB activation. The biology of Ad5 infection in these cells was then assessed in comparison with control cells. Ad5 gene expression displayed substantial increases with the transient or permanent depletion of PML-II in HeLa and MRC5 cells. This effect was particularly marked for late gene expression; hexon mRNA and all other late proteins showed substantial increase in PML-II depleted cells compared to several distinct control cell lines. This increase reached up to 100% with PML-II removal, quantified by flow cytometry. Virus yield also showed a 3-fold increase in PML-II depleted cells. This effect was only partly due to the impaired interferon pathway in these cells, which would be expected to augment Ad5 gene expression. The more significant factor in that increase was the overexpression of HSP70 chaperone, a specific member of the heat shock protein family, in PML-II depleted cells. siRNA-mediated HSP70 reduction caused a drastic decrease in Ad5 gene expression in PML-II Kd cells to more or less the same level seen in control cells . This effect was not NF-kB-dependent but HSP70 depletion did increase expression of interferon response genes such as ISG56. Pre-exposure of cells to mild heat stress made them significantly more resistant to Ad5 infection: such treatment reduced viral gene expression, in particular hexon and other late mRNA and protein expression, in HeLa cells and MRC5 cells. Ad5 genome replication was also reduced in pre-stressed cells compared to the control conditions. Consistent with previous studies of more extreme heat stress, mild heat stress affected the morphology of PML-NB. It also affected expression of PML protein and specifically showed an increase in PML-II mRNA expression after 3 hours of exposure to 40 ̊C compared to the control conditions. In light of the enhanced infection seen when PML-II was depleted, this increased expression of PML-II following heat stress might play a role in the reduced infection efficiency in such cells. Indeed, PML-II removal reversed the negative effect of the mild heat stress on Ad5 infection: such cells showed more late gene expression after heat stress than control cells.

Foot-and-mouth disease virus (FMDV) (Family: Picornaviridae, Genus: Aphthovirus) is a significant global pathogen with extensive economic impact. FMDV has a low fidelity RNA-dependent RNA polymerase and lacks proof reading capability. This coupled with its relatively short generation time and large population sizes means it exists in a swarm of genetically closely related variants. The reservoir of diversity contained within this mutant spectrum allows the virus to adapt rapidly to new environments. Much of the previous work looking at virus evolution has focused on the consensus level genetic sequence. The advent of next generation sequencing (NGS) technologies enables evolutionary studies of the entire viral swarm. This PhD project uses NGS technologies to interrogate the swarm structure by investigating factors affecting the viral swarm and the dynamics of variants within it. Furthermore, this work shows how analysis of the swarm can reveal fundamental information about virus biology. A PCR-free NGS methodology was developed to create deep sequencing data sets of all genomes present within an FMDV viral swarm. The elimination of the PCR step results in less errors being introduced in the sequencing process thereby improving the resolution and reliability of the identification of low level variants. This optimised method was then used to define and compare the FMDV swarms of several wildtype isolates. This revealed differences in swarm structure from isolate to isolate and produced evidence of within swarm selection. Not all proteins known to be under selection at the consensus level were also under selection within the swarm. The diversity of viruses within the swarm was found to be dependent upon the host from which a virus was sampled, with African buffalo potentially able to maintain multiple infections. Subconsensus variants in these mixed samples had mutations at positions previously associated with immune escape. Investigation of the evolution of swarm structure when adapting to new cell type in vitro indicated that two distinct population structures can exist relative to the existence of adaptive pressure. These two population structures have different distributions of variable nucleotides but comparative total levels of variation (as measured by Shannon's entropy). Deep sequencing of the virus swarm enabled the discovery of conserved novel stem loop structures, which were hypothesized to be required for packaging of the virus genome. Mutating these sites produced a virus with decreased packaging efficiency. This thesis includes novel analysis techniques for considering the viral swarm. It demonstrates how investigating the diversity in the swarm can help us to understand virus molecular biology, its evolution and the limits upon this. Understanding viral evolution at this scale has the capacity to improve our fundamental understanding of the biology and evolution of FMDV which can in turn inform vaccine design and disease control strategies.

This thesis explores the ability of RUNX1 and its fusion oncoprotein derivatives to induce senescence-like growth arrest (SLGA) in primary cell cultures. While this phenomenon resembles replicative senescence seen in normal diploid human fibroblasts after extensive passage, it does not involve telomere attrition. From previous studies in the host laboratory it can also be distinguished from Ras oncogene-induced senescence (OIS) as it does not appear to depend on a DNA damage response secondary to hyper-proliferation. Despite these differences, this study supports the hypothesis that RUNX SLGA is an anti-cancer fail-safe which protects cells against oncogenic transformation. In favour of the fail-safe model, two out of the three RUNX1 fusion oncoproteins examined fail to induce SLGA in a well characterised human fibroblast cell system (Hs68). In the case of TEL-RUNX1 (TR), a fusion which is associated with around 25% of childhood B-cell acute lymphoblastic leukaemias, loss of SLGA activity is due to N—terminal fusion to TEL. SLGA activity is regained by deletion of the HLH dimerisation domain in TEL or by introduction of a single amino acid mutation (K99R). The other major RUNX1 fusion oncoprotein, RUNX1-ETO (RE) which is commonly observed in M2 subtype acute myeloid leukaemia (AML), induces a potent SLGA. However, the RE phenotype differs from that of RUNX1, as RE strongly induces reactive oxygen species (ROS) and a pronounced senescence-associated secretory phenotype (SASP). In human AML, RE is co-expressed with a truncated variant generated by alternative splicing (RE9a), which has been shown to be much more potently leukaemogenic in animal models. This study demonstrates that RE9a also fails to induce SLGA and induces a markedly attenuated SASP. RE is generated by fusion of the N-terminal moiety of RUNX1 to the ETO C-terminus that encompasses a series of repressive nervy homology regions (NHRs), three of which are missing from RE9a. The effect of deleting or mutating NHRs from RE was also examined and show that loss of both NHR3 and NHR4 is necessary for complete ablation of RE SLGA. It was also demonstrated that aspects of RUNX1-induced SLGA are dissociable as, for example, the RUNX1P2 isoform induces profound growth arrest but only a minimal SASP. This study supports the hypothesis that RUNX1-induced SLGA must be overcome to allow oncogenic transformation, either by intrinsic inactivation (TR, RE9a) or by co-operating mutations (RUNX1, RE). Notably, loss of sensitivity to RUNX1-induced SLGA appears to correlate with mutational loss of p16INK4A, and it was shown that growth of the human leukaemia cell line REH is stimulated rather than repressed by the TR K99R mutant. The possibility that the RE-induced SASP has pro-oncogenic effects through effects on cell survival or on bystander cells is also discussed.

Serosurveillance is a powerful tool fundamental to understanding infectious disease dynamics. The presence of virus neutralising antibody (VNAb) in sera is considered the best evidence of infection, or indeed vaccination, and the gold standard serological assay for their detection is the virus neutralisation test (VNT). However, VNTs are labour intensive, costly and time consuming. In addition, VNTs for the detection of antibodies to highly pathogenic viruses require the use of high containment facilities, restricting the application of these assays to the few laboratories with adequate facilities. As a result, robust serological data on such viruses are limited. In this thesis I develop novel VNTs for the detection of VNAb to two important, highly pathogenic, zoonotic viruses; rabies and Rift Valley fever virus (RVFV). The pseudotype-based neutralisation test developed in this study allows for the detection of rabies VNAb without the requirement for high containment facilities. This assay was utilised to investigate the presence of rabies VNAb in animals from a variety of ecological settings. In this thesis I present evidence of natural rabies infection in both domestic dogs and lions from rabies endemic settings. The assay was further used to investigate the kinetics of VNAb response to rabies vaccination in a cohort of free-roaming dogs. The RVFV neutralisation assay developed herein utilises a recombinant luciferase expressing RVFV, which allows for rapid, high-throughput serosurveillance of this important neglected pathogen. In this thesis I present evidence of RVFV infection in a variety of domestic and wildlife species from Northern Tanzania, in addition to the detection of low-level transmission of RVFV during interepidemic periods. Additionally, the investigation of a longitudinal cohort of domestic livestock also provided evidence of rapid waning of RVF VNAb following natural infection. Collectively, the serological data presented in this thesis are consistent with existing data in the literature generated using the gold standard VNTs. Increasing the availability of serological assays will allow the generation of robust serological data, which are imperative to enhancing our understanding of the complex, multi-host ecology of these two viruses.

Ovine pulmonary adenocarcinoma (OPA) is a respiratory disease caused by jaagsiekte sheep retrovirus (JSRV). This virus induces the growth of large lung tumours in affected sheep and is a significant problem for the sheep industry. An interesting feature of OPA is that it occurs only in sheep. Goats may also be infected by JSRV but disease progression is limited to the early stages so that clinical signs do not develop. The ability of a virus to replicate in its host is dependent on a wide range of cellular proteins, including essential, required (‘dependency’) factors, and proteins that act to inhibit replication, referred to as restriction factors. Greater understanding of the roles of dependency and restriction factors can provide insights into pathogenesis and the species-specificity of infection. The aim of this study was to investigate the potential role of previously identified restriction factors on the replication of JSRV, and specifically whether APOBEC3 or TRIM5 proteins are responsible for the specificity of OPA for sheep. To examine this question, ruminant genes for APOBEC3 were cloned and their activity against JSRV was tested using a replication-defective reporter virus that expresses GFP. This system allows the activity of putative restriction factors to be measured quantitatively by flow cytometry. These experiments revealed that ruminant APOBEC3 proteins, including those from sheep, inhibit JSRV infection in vitro. Further analysis of the mechanism of restriction of JSRV by sheep APOBEC3 provided evidence for cytidine deaminase-dependent and independent mechanisms against this virus. In order to extend the studies on the species-specificity of APOBEC, several human and mouse APOBEC proteins were analysed for their activity against JSRV. Murine APOBEC3 and human APOBEC3F were both able to restrict JSRV in vitro, while other human APOBECs tested were not. These results have impact for the development of murine model of OPA and for the development of JSRV as a gene delivery vector. To assess the impact of TRIM5 on JSRV replication, derivatives of the permissive cell line CRFK were created that stably express TRIM5 from a range of ruminant and primate species. Infection studies performed in cell culture indicated that none of the TRIM5 proteins tested restrict JSRV, at least during the early stages of virus infection. Further studies are needed to examine other potential mechanisms of activity of TRIM5 against JSRV. This thesis has revealed new insights into host-pathogen interactions in OPA that may contribute to the development of control strategies against this disease. In addition, these data provide a background for the future development of JSRV as a gene delivery vector.

Mosquito-borne arboviruses are a considerable threat to human and animal health across the world. Many of them are classed as emerging or remerging pathogens and the incidence of disease for a number of serious viral infections has increased as they expand their geographical and host ranges. As with other invertebrates, mosquitoes lack the adaptive immune response present in vertebrates and instead rely on their innate immune defences to modulate viral infections. Nevertheless, in contrast to vertebrates, arboviral infections in their arthropod vector are non-pathogenic and have no cytopathic effect or detrimental impact on their survival. The response considered to be the most important for antiviral defence in mosquitoes is RNA interference (RNAi) which is a sequence-specific, RNA silencing mechanism. Most of what is known about antiviral RNAi in arthropods has been established in Drosophila as the model insect organism. These studies have benefited from an extensive range of genetic mutants, molecular tools, reporter assays and genetic profiling. The absence of these tools for use in mosquito research is a substantial deficit for arboviral studies in their natural vector system and must be rectified in order to fully understand the influence vector immunity has on virus transmission. This thesis discusses the development of a ‘molecular tool-box’ for advancing the acquisition of knowledge in this area. Efficient RNAi gene silencing and its effect on the antiviral RNAi response was established in vitro using Semliki Forest virus (SFV) as model arbovirus. This assay determined that knock-down of Argonaute-2 had the most substantial impact on virus replication compared to the knockdown of other RNAi proteins. In addition, the limited detection of virus-derived small RNAs, key molecules of the antiviral RNAi response by Northern blot analysis provides further support to previous evidence that SFV may circumvent the antiviral response by sequestering its genomic RNA, resulting in restricted access by the RNAi machinery and preventing the generation of large quantities of virus-derived small RNAs. However, some SFV-derived small RNAs are known to be produced and these have been shown to generate a pattern of ‘hot’ and ‘cold’ spots along the full-length coding sequences. This thesis has determined that this pattern is not exclusive to viral-derived dsRNA trigger molecules but is also exhibited following the treatment of mosquito cells in culture with non-viral dsRNA. This implies that all exogenous dsRNA is processed by RNAi in a similar manner. This study has also characterised the presence of an RNA-dependent RNA polymerase (RdRP) encoded by Aedes aegypti mosquitoes. RdRPs are important for the amplification and spread of the RNAi signal in other organisms such as plants and worms; however, only one study suggested the existence of one in Drosophila. Although, this project proposed the presence and transcription of a homologue of the Drosophila RdRP in the Aedes aegypti-derived Aag2 cell line, protein knockdown assays revealed that it has no effect on virus replication in vitro; suggesting that it does not function as an RdRP. Due to the lack of antibodies against the major RNAi proteins Dicer-1, Dicer-2, Argonaute-1 and Argonaute-2 in mosquitoes, these were designed and screened which allowed the identification of several candidates for the detection of the proteins in mosquito cells in culture. Further to this, recombinant forms of the RNAi initiator protein Dicer-2 and the slicer protein Argonaute-2 were successfully generated and tested in vitro using different promoters to establish their use for future temporal and spatial kinetic studies. It was concluded that of the promoters tested the most successful for the expression of these reporter constructs was the subgenomic promoter of SFV. On the other hand a second promoter, the PUb promoter, may prove more suitable in the future. Finally, this project studied the antiviral capabilities of a non-haematophagous mosquito cell line which would not come across an arboviral infection by traditional blood- feeding routes. Instead the mosquito larvae sustain their adult life stages by feeding on the larvae of other species which may be vertically infected. A cell line derived from Toxorhynchites amboinensis was characterised and was shown to carry out RNAi if induced by dsRNA suggesting that they are able to mount an antiviral response to acquired infections. This study also determined that the cell line contains an endogenous insect specific virus and, although the source of this is unknown, it adds an interesting new dimension to mosquito antiviral immunity. This thesis enhances RNAi research in Aedes mosquitoes by presenting novel molecular tools and reporter assays which will be highly valuable for facilitating future investigations. The studies performed also add to what is already understood regarding the interaction between SFV and mosquito antiviral immunity through the RNAi response.

Human papillomaviruses (HPVs) are the causative agent in cervical cancer and have been implicated in a rising number of head and neck cancers in both men and women. There are two licensed prophylactic vaccines, both target HPV 16 and 18, the two most common, oncogenic types. However, there are no antiviral drugs for the treatment of HPV infection and disease. Papillomaviruses encode two DNA binding proteins, E1 and E2, which interact with host cell proteins to induce replication. Two essential cellular factors for viral transcription and replication are TopBP1 and Brd4. TopBP1 is a known interactor of HPV16 E2, and is essential for the initiation of DNA replication in eukaryotic cells. Previous studies with E2 mutants have shown that Brd4 is essential for transactivation properties of E2. Brd4 has also been implicated in the regulation of E1-E2 mediated viral DNA replication. However, the role of E2 in regulating the host genome has been less well studied. As attempts at developing a therapeutic vaccine have failed, and current small molecule inhibitors which block the interaction of replication factors, E1 and E2, are not effective across all HPV types due to slight differences in E1-E2 interactions, Brd4 and TopBP1 may present themselves as pan-type specific targets. Blocking the interaction of the host proteins Brd4 and TopBP1 with viral E2 is a viable target for HPV related cancers. This thesis set out to understand how E2 interacts with TopBP1 and Brd4 to regulate the cellular genome as well as the viral genome, to better understand how to terminate the viral life cycle. Two E2 mutants, E2-TopBP1 and E2-Brd4, which fail to bind TopBP1 and Brd4 respectively, were made to address this question. Functional assays with E2 wildtype and the two E2 mutants have enhanced our understanding of viral replication. TopBP1 and Brd4 are present at the viral origin of replication in an E1-E2 dependent manner, and optimal initiation of DNA replication is dependent on the interaction with E2. TopBP1 and Brd4 locate into E1-E2 foci and shRNA targeting these proteins destroys these replication foci. However, this has no effect on E1-E2 mediated levels of DNA replication. The results suggest a role for TopBP1 and Brd4 in the initiation of HPV16 E1-E2 DNA replication but not continuing replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. To address the question of how E2 regulates the host, we stably expressed HPV16 and 18E2 wildtype, E2-TopBP1 and E2-Brd4, in U2OS cells, a p53 positive cell line that tolerates E2 expression. These cells were used in human exon array analysis, to determine which host cellular genes E2 regulates. We determined that HPV16 and 18E2 can regulate cellular genes and a failure to bind either TopBP1 or Brd4 increases the number of cellular genes altered. Overall the results suggest that the levels of TopBP1 and Brd4, which can interact, regulate E2 function and therefore could regulate viral infection.

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.