Дисертації з теми "Virus viability in droplets"

The transmission of some infectious diseases requires that pathogens can survive (i.e., remain infectious) in the environment, outside the host. The viability of pathogens that are immersed in aerosols and droplets is affected by factors such as relative humidity (RH) and the chemical composition of the liquid media, but the effects of these stressors on the viability of viruses have not been extensively studied. The overall objective of this work was to investigate the effects of RH and media composition on the viability of viruses in suspended aerosols and stationary droplets. We used a custom rotating drum to study the viability of airborne 2009 pandemic influenza A(H1N1) virus across a wide range of RHs. Viruses in culture medium supplemented with material from the apical surface of differentiated primary human airway epithelial cells remained equally infectious for 1 hour at all RH levels tested. We further investigated the viability of two model viruses, MS2 and Φ6, in suspended aerosols and stationary droplets consisting of culture media. Contrary to the results for influenza virus, we observed a U-shaped viability pattern against RH, where viruses retained their viability at low and extreme high RHs, but decayed significantly at intermediate to high RHs. By characterizing the droplet evaporation kinetics, we demonstrated that RH mediated the evaporation rate of droplets, induced changes in solute concentrations, and modulated the cumulative dose of solutes to which viruses were exposed as droplets evaporated. We proposed that the decay of viruses in droplets follows disinfection kinetics. Lastly, we manipulated the chemical composition of media to explore the stability of viruses as a function of pH and salt, protein, and surfactant concentrations. Results suggested that the effects of salt and surfactant were RH and strain-dependent. Acidic and basic media effectively inactivated enveloped virus. Protein had protective effect on both non-enveloped and enveloped viruses. Results from this work has advanced the understanding of virus viability in the environment and has significant implications for understanding infectious disease transmission.
Doctor of Philosophy
Pathogenic organisms, including bacteria, viruses, fungi, protozoa, and helminths, cause infections that are responsible for substantial morbidity and/or mortality. For example, it is estimated that influenza has caused 9 million to 45 million illnesses and 12,000 to 61,000 deaths annually since 2010 in the United States. The spread of certain diseases relies on people touching the pathogenic organism on surfaces or inhaling it from the air. Successful transmission requires that the pathogen survive, or maintain its infectivity, while it is in the environment. The survival of pathogens can be affected by temperature, humidity, composition of the respiratory fluid carrying them, and other factors. However, there is limited research investigating the effects of these factors on the survival of viruses in the environment. In this work, we studied the effect of relative humidity (RH) on the survival of viruses, including influenza virus and two other types of viruses, in inhalable aerosols and larger droplets. We found that influenza viruses survive well in aerosols across a wide range of RH levels for at least 1 h. Conversely, the two model viruses survived best at both low and very high RHs, such as found indoors in the wintertime or in tropical regions, respectively, but had a pronounced decay at intermediate RHs. By measuring how fast droplets evaporated, we found that RH affected their chemistry and determined the total amount of stress that viruses were exposed to. This explained why a "U-shaped" survival pattern was observed against RH. We also investigated the survival of viruses in droplets containing different components. Results indicated that the effects of salt, surfactant, protein, and droplet pH depended on RH and the type of virus. The outcomes of this work are meaningful in predicting the survival of viruses in aerosols and droplets of various compositions in the environment and could provide insight on developing strategies to minimize the spread of infectious diseases.

The wettability and phase change phenomena of water are ubiquitous on biological and artificial surfaces. Properties like water repellency, self-cleaning, coalescence induced condensation jumping, anti-frosting, and dew harvesting arise on surfaces with particular structures and chemistry and are of particular interest for energy and water saving. This thesis collects different studies of wettability and phase change on natural and artificial surfaces: growth and self-ejection of condensation droplets on micro and nanostructured surfaces we fabricated, their applications, the Sliding on Frost of condensation droplets observed on the Cotinus Coggygria leaf, the dew harvesting property of the Old Man of the Andes Cactus enhanced by distance coalescence through microgrooves and finally, a theoretical study of viruses viability in sessile droplets. The first chapter introduces the theoretical framework of wettability and phase changes on surfaces. In the second chapter, we present the self-ejection of condensation droplets from hydrophobic nanostructured microstructures. We modelled analytically the droplets jumping and fabricated surfaces to verify the predictions. The fabricated geometry was inspired by the modelling and the available fabrication techniques. We tested the surfaces in condensation conditions. Using a high frame rate camera coupled with a long working distance microscopy objective, we investigated the growth and ejection transient. We then compared the experimental self-ejection velocity for various structures geometry with our analytical models. In Chapter 3, we investigated the applications of the fabricated surfaces reported in Chapter 2. In Chapter 4, we explore the condensation frosting on the leaf of Cotinus Coggygria, native of our woods and with interesting hydrophobic properties. Covered by wax nanotubules, it exhibits coalescence-induced condensation jumpings that may be a useful cleaning tool. Furthermore, the frost is delayed but not only for the jumping. Surprisingly, at temperatures some degrees below zero, we observed what we called ‘droplet Sliding on Frost bridges’, that further delays frosting. We described the feasibility of this sliding in terms of dynamic contact angles of the surface and contact angles of supercooled water on ice. By capturing high temporal and spatial resolution videos we investigated the sliding on frost and droplet recalescence (fast dendrite growth that partially solidify the liquid). The responsible for the failure of sliding for temperatures from about -8 ° C down appears to be the advancing angle of water on ice that increases with the subcooling rather than the recalescence that blocks the drop in place. These results add a piece to the fundamental research on the supercooled water-ice-vapour interfaces. As it often happens, biological surfaces offer a starting point for the study of fundamental mechanisms and the development of artificial surfaces with optimized properties. In the Chapter 5, the multifunctional roles of hairs and spines in Old Man of the Andes Cactus (Oreocereus trolli) are studied. We study the morphology of the appendages, the hairs wettability, mechanical properties of both, and the dew formation on spines. The longitudinal microgrooves on the spines cause a particular phenomenon of distant coalescence (DC), in which smaller droplets flow totally or partially into larger ones through the microgrooves, with consequent accumulation of water in a few large drops. An earlier study has shown artificial micro-grooved surfaces that exhibit DC are more efficient than flat ones at collecting and sliding dew, and thus these cactus spines could act as soil dew conveyors. The agreement between our analytical model and experimental data verifies that the flow is driven by the Laplace pressure difference between the drops. This allowed us to obtain a general criterion for predicting the total or partial emptying of the smaller drops as a function of the dynamic contact angles of a surface. Based on this criterion, an hydrophilic material with small contact angle hysteresis would allow a greater number of complete drops emptying. The COVID-19 pandemic has raised the problem of contagion from airborne and deposited droplets. In the last chapter, we report the state of the art of experiments on the viability of viruses in deposited droplets. Up to date, it has been experimentally highlighted that the relative viability of some viruses (RV) depends on the material chemistry, temperature, and interestingly, on relative humidity (RH) with a U-shaped trend. One of the current hypotheses is that the cumulative dose of salt concentration (CD) affects RV. We model the RV of viruses in sessile droplets by inserting a RV-CD relation in a model of droplet evaporation. By considering a saline water droplet (one salt) as the simplest approximation of real solutions, we analytically simulate the time evolution of salt concentration, vapor pressure, and droplet volume varying contact angles, droplet sizes, and RH in the range 0–100%. The results elucidate some previously not yet well-understood dynamics, demonstrating how three main regimes—directly implicated in nontrivial experimental trends of virus RV—can be recognized as the function of RH. The proposed approach could suggest a chart of a virus fate by predicting its survival time at a given temperature as a function of RH and contact angle. We found a good agreement with experimental data for various enveloped viruses and predicted in particular for the Phi6 virus, a surrogate of coronavirus, the characteristic U-shaped dependence of RV on RH. Given the generality of the model, once experimental data are available that link the vulnerability of a certain virus (such as SARS-CoV-2) to the concentrations of salts or other substances in terms of CD, it is envisioned that this approach could be employed for antivirus strategies and protocols for the prediction/reduction of human health risks associated with SARS-CoV-2 and other viruses.

Hepatitis C virus (HCV) is remarkably capable of efficiently hijacking host cell pathways including lipid metabolism in the liver in order to create pro-viral environments for pathogenesis. It is becoming increasingly clear that identifying small molecule inhibitors that target host factors exploited by the virus will expand available HCV treatment options. As such, a thorough understanding of host-virus interactions is critical to the development of alternative therapeutic strategies. Hepatic lipid droplets (LDs) are recruited by HCV to play essential roles in the viral lifecycle. The intracellular location of LDs is modified upon interacting with viral structural core protein. This enables formation of platforms that support viral particle assembly. Because these interactions are non-static, capturing its dynamic processes in order to better understand viral assembly can be achieved with label-free molecular imaging enhanced with live-cell capabilities. Chemical biology approaches that includes CARS microscopy employed in a multi-modal imaging system was used to probe interactions between HCV and host LDs. By successfully tracking LD trajectories, we identified core protein’s ability to alter LD speed and control for LD directionality. Using protein expression model systems that allowed for simultaneous tracking of core protein and LDs, our data revealed that mutations in the core protein region that vary in hydrophobicity and LD binding strengths, are factors that control for differential modulation of LD kinetics. Furthermore, we measured bidirectional LD travels runs and velocities, and observed critical properties by which core protein induces LD migration towards regions of viral particle assembly. Given that many steps in the HCV lifecycle are directly linked to host lipid metabolism, it is not surprising that disrupting lipid biosynthetic pathways would negatively affect viral replication. From this outlook, we explored small molecule inhibitors that targeted several lipid metabolic pathways to study its antiviral properties. Using fluorescent probes covalently labeled to viral RNA, we captured the visualization of disrupted replication complexes upon antagonizing nuclear hormone receptors that are linked to regulating lipid homeostasis. Correspondingly, biochemistry and molecular imaging techniques were also employed to identify novel antiviral mechanisms of small molecule inhibitors that target additional HCV-dependent lipid metabolic pathways.

The reduction of pathogenic microorganisms is essential to minimize human health risks associated with the reuse, reclamation, and recycling of wastewater and the land application of biosolids (sewage sludge). The most advanced treatment technologies, as well as, the most representative methods and indicator organisms are necessary to ensure public safety. The goal of this dissertation is to assess advanced Bardenpho wastewater treatment technologies in regards to virus removal, suggest the most appropriate viral indicators of human fecal contamination and/or treatment process controls, and develop an updated method for enumerating Ascaris ova viability in land applied biosolids. Appendix A evaluates the incidence of 11 different virus types in sewage throughout a 12-month time period, and their subsequent reduction via advanced Bardenpho treatment processes. This study showed that wastewater treatment facilities utilizing advanced Bardenpho for secondary treatment are more effective at reducing viruses in wastewater than facilities utilizing conventional aeration basin and trickling filter processes. Appendix B develops a new method for determining the viability of Ascaris ova in land applied biosolids. In this method, early development stages prior to larval-development, are included in the estimation of potential viability. Comparisons between viability enumerations suggests that the conventional microscopy method, in which only ova containing motile larva are considered viable, underestimates the number of eggs that may progress to an infectious stage. Whereas, the method based on early-to-late stage development, considers the potential viability of all eggs, providing a more conservative approach.

Les résultats fondamentaux et biocliniques que nous présentons au travers de cette thèse illustrent la difficulté d’évaluer la part liée au virus et celle liée à des facteurs de l’hôte dans l’induction d’une stéatose hépatique chez les patients chroniquement infectés par le HCV. Les données in vitro suggèrent que le virus joue un rôle direct dans l’induction d’une stéatose, notamment par les propriétés de sa protéine de capside, et que la variabilité du virus peut avoir un impact sur l’intensité de cette stéatose. Notre étude bioclinique suggère que la variabilité du virus semble avoir un rôle beaucoup plus modéré in vivo. Ainsi, chez les patients chroniquement infectés par le HCV, les facteurs de l’hôte joueraient un rôle majeur pour moduler le degré de la stéatose associée au virus et de prochaines études seront nécessaires pour établir la nature de ces facteurs
The results presented in this thesis illustrate the difficulty of assessing the part related to the virus and that related to host factors in the induction of hepatic steatosis in patients chronically infected with HCV. In vitro data suggest that the virus plays a direct role in the induction of steatosis, due to the properties of its capsid protein, and that the variability of the virus can affect the intensity of the steatosis. Our bio-clinical study suggests that this variability seems to have a much more moderate impact in vivo. Thus, in patients chronically infected with HCV, host factors seem to play a major role to modulate the degree of steatosis associated with the virus. Further studies are needed to establish the nature of these factors

Le virus de l’hépatite C (VHC) dépend de facteurs cellulaires pour accomplir son cycle viral et persister dans l’hôte. L’une des stratégies de notre laboratoire consiste à étudier de manière approfondie le réseau d’interactions virus-hôte, afin d’identifier de nouvelles cibles thérapeutiques cellulaires et de développer des antiviraux plus efficaces pour vaincre la résistance virale. Durant ma thèse j’ai étudié deux facteurs cellulaires importants pour le VHC. Le premier est la protéine ribosomale RACK1. Nous avons montré que cette protéine est spécifiquement requise pour la traduction IRES-dépendante du VHC, et non pour la traduction coiffe-dépendante. Le deuxième facteur est une protéine de surface des gouttelettes lipidiques appelée TIP47. Nous avons montré que cette protéine est importante à la fois pour l’assemblage et pour l’export des particules virales. L’ensemble de ces travaux montre que de nouvelles cibles thérapeutiques pourraient être envisagées pour lutter contre le VHC
The hepatitis C virus (HCV) relies on cellular factors to complete its life cycle and persist in its host. One of the strategies employed by our laboratory is the in-depth study of the network of virus-host interactions to identify new therapeutic cellular targets and develop more effective antivirals to overcome viral resistance.During my PhD, I studied two cellular factors involved in the HCV life cycle. The first factor is the ribosomal protein RACK1. We have shown that this protein is specifically required for the HCV IRES-mediated translation but not for the cap-mediated translation. The second factor is the lipid droplets binding protein TIP47. We have shown that this protein is important for both assembly and export of viral particles. This work shows that new therapeutic targets could be considered in the fight against HCV

Le VHC infecte les hépatocytes, cellules polarisées du foie. Le cycle de réplication du VHC est dépendant du métabolisme lipidique de la cellule hôte. Mais la relation entre VHC, polarité cellulaire et métabolisme lipidique est mal connue. Nous avons démontré que SHIP2 joue un rȏle important dans l’établissement de la polarité apicobasale des cellules épithéliales. La protéine core du HCV induit la perte de polarité cellulaire et diminue l’expression de la phosphatase SHIP2. La réintroduction de SHIP2 dans les cellules exprimant core restitue la polarité cellulaire et diminue l’expression de core. SHIP2 agit aussi sur l’accumulation et l’organisation des gouttelettes lipidiques qui sont des éléments cellulaires nécessaires à la réplication du VHC. Ces résultats montrent le rôle de SHIP2 dans la polarité cellulaire et le désigne comme une cible intéressante pour des recherches dans la lutte contre les infections du VHC
HCV infects hepatocytes, polarized cells of the liver. HCV replication cycle is dependent on lipid metabolism of the host cell. But the relationship between HCV cell polarity and lipid metabolism is unknown. We demonstrated that SHIP2 plays an important role in establishment of the apicobasal epithelial cell polarity. The HCV core protein induces loss of cell polarity and decreases the expression of the phosphatase SHIP2. The reintroduction of SHIP2 in cells expressing core restores cell polarity and decreases the expression of core protein. SHIP2 also negatively affect lipid droplets, which are important for HCV replication. These results show the role of SHIP2 in cell polarity and designate it as an attractive target for research in the fight against HCV infection

Les septines sont une famille de protéines GTPases qui peuvent former des structures d'ordre supérieur, comme les filaments et les anneaux, et capables de se lier avec les membranes cellulaires par leur interaction avec les phosphoinositides (PIs) via un domaine polybasique en N-terminal de leur domaine de liaison au GTP. Nous avons montré par une analyse transcriptomique réalisée en utilisant les données GSE14323 que la septine 9 est significativement surexprimée dans la cirrhose induite par le virus de l'hepatite C (VHC). Nos résultats montrent, ainsi, que la septine 9 induit l’augmentation en taille des gouttelettes lipidiques (GLs) par un mécanisme dépendant le phosphatidylinositol-5-phosphate et des microtubules. Nous avons montré, également, que cette voie de régulation des GLs est exploité par le VHC. De plus, nous avons montré que la septine 9 est impliquée dans la régulation de la morphologie de l’appareil Golgi et la mise en place de la polarité cellulaire par son interaction avec les phosphoinositides via deux domaines polybasiques. Ces résultats apportent une nouvelle compréhension du mécanisme moléculaire de l’interaction des septines avec les phosphoinositides et montrent pour la première fois l’importance de cette interaction dans des fonctions cellulaires de la septine 9
Septins are a GTPases proteins family that can form high order structures such as filaments and rings, and able to bind cell membranes by interacting with phosphoinositides via a polybasic domain located at the N-terminal of their GTP binding domain. Here, We show by the transcriptomic analysis performed using the GSE14323 dataset that septin 9 is significantly upregulated in hepatitis C virus induced cirrhosis. Our findings show that septin 9 induce the lipid droplet growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV. In addition, we have shown that the septin 9 is involved in Golgi apparatus morphology regulation and cell polarity installation by interacting with phosphoinositides via two polybasic domains. These results provide new understanding of the molecular mechanism of septins interaction with the phosphoinositides and show its importance in septin 9 cellular functions shown for the first time

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
Viral hemorrhagic fever is mostly caused by dengue virus (DENV), which originates 100 million infections annually. DENV is a Flavivirus, member of the Flaviviridae family that also comprises other important human pathogens. This virus affects the host lipid metabolism, increasing intracellular lipid droplets (LDs) and imbalancing plasma lipoproteins levels and composition. Effective dengue treatments are not available yet, in part due to the poor understanding of some viral life cycle steps. The DENV capsid protein (DENV C) was recently found to interact with LDs, in a process that influences viral replication. However, the underlying molecular mechanisms of such interaction were not clear and also no function had already been attributed to its N-terminal disordered region. Moreover, human lipoproteins, similar to LDs in structure and composition, could play an important role in the dengue virus infectivity process. This study aimed at unraveling the molecular mechanism behind the DENV C protein interaction with host LDs and lipoproteins, which may explain key processes occurring in vivo. The understanding of the molecular nature and biological relevance of these interactions may also provide clues on how to inhibit them. In the present study, it was found that DENV C N-terminal region is crucial for the interaction with LDs and VLDL, requiring the presence of potassium. These interactions seem to occur mainly with LDs and VLDL intrinsic protein(s), rather than lipids. Importantly, pep14-23, a peptide based on the DENV C N-terminal conserved region, inhibits the C-LDs interaction. In contact with negative phospholipids, this peptide undergoes a structural conversion to α-helix. This lipid-driven structural conversion may thus help pep14-23 interaction with its target. These are clear breakthroughs in the understanding of DENV C-LDs interaction (and inhibition), providing a ground for the development of C protein targeted treatments for the infections by DENV and other Flavivirus.

West-Nile Virus (WNV) infection occurs through the bite of Culexspp. mosquitoes and constitutes a serious public health threat. Despite the global spread and disease severity, there is no specific and effective treatment for WNV infection, in part due to a poor understanding of the virus life cycle. A key viral life cycle step is viral assembly and encapsidation, mediated by the capsid (C) protein interaction with RNA and host lipid structures. WNVC interactionswith intracellular lipid droplets (LDs) and with very low-density lipoproteins (VLDL), crucial step for successful viral replication in related members of the Flaviviridaefamily, were studied via biophysical approaches. Zeta potential measurements showed that WNVC interaction with LDs requires K+and LDs surface proteins. WNVC interaction with lipoproteins measured via dynamic light scattering(DLS), showed that WNVC binds to VLDL but not tolow-density lipoproteins(LDL), in a potassium dependent manner. It was clear that in the presence of WNV C there is an increase of the VLDL hydrodynamic radius that correlates very well with WNV C estimated dimension. The WNVC (un)binding forcesupon interaction with LDs and VLDL werequantitatively determined by atomic force microscopy(AFM)-based force spectroscopy. WNV C specifically binds to LDs and VLDL, in a potassium-dependent manner,but not to LDL.DLS was also performed todetermine pep14-23 (an inhibitor of dengue virus–DENV –Cproteininteraction with LDs and VLDL) effect on WNVC-VLDLbinding. Data obtained indicate that pep14-23 may also be a potential inhibitor for WNVC key interactions.The results obtained in this study are in agreement with thepreviouslyobserved forDENV, showing that WNV C and DENV C proteins interact in a similar manner with LDs and VLDL, and suggesting pep14-23 asa potential inhibitor of FlavivirusC proteinsbinding tohost lipid systems.

Student Number : 9906382M MSc (Med) dissertation - Faculty of Health Sciences
The G1862T mutation in the bulge of the RNA encapsidation signal, in the precore region of hepatitis B virus, results in reduced expression of HBeAg and accumulation of the HBeAg precursor in the endoplasmic reticulum (ER)/Golgi apparatus of the cell. This accumulation can disturb the functioning of the ER and lead to the ER stress response that can affect various cellular pathways, in turn affecting cell viability. The aim of this study was to determine whether apoptosis or necrosis occurred when cultured Huh7 cells were transfected with a plasmid expressing the G1862T mutation. Plasmid constructs, with and without the G1862T mutation, were used to transfect cells. To differentiate between necrosis and apoptosis cells were stained with propidium iodide or YO-PRO-1®, respectively. These were analyzed quantitatively using flow cytometry and qualitatively using confocal microscopy. Confocal microscopy, using monoclonal anti- HBe and the Hoechst stain, was performed to ensure that apoptosis was present as a result of the accumulation of the G1862T mutant HBeAg precursor. Caspase profiling was carried out using a fluorogenic-based assay. When cells were transfected with wild-type plasmid, necrosis predominated over apoptosis. Apoptosis predominated when the cells were transfected with the G1862T mutant plasmid. The highest levels of apoptosis occurred at 72 hours post-transfection. Confocal microscopy revealed the co-localization of aggregates of mutant HBeAg precursor with apoptotic nuclei. Transfection with G1862T mutant plasmids resulted in significant differences in the expression of caspase 3, 8, and 9 relative to the wild-type, at 48 and 72 hours post-transfection. The accumulation of the G1862T mutant HBeAg precursor, in the ER/ Golgi compartment, leads to apoptosis and affects the levels of caspase expression.

Much of our understanding of the HCV life cycle and host-viral interactions has evolved from the visualisation of fixed images of infected cells. However, the recent development of live cell imaging techniques now allows viral life cycles to be visualised in live cell cultures. We have tagged the NS5A protein of the infectious Jc1 chimera (J6/JFH-1) with fluorescent tags and shown that NS5A segregates into two distinct populations: one relatively static and one highly motile, although the role and composition of these structures is not well understood. To investigate HCV RNA dynamics throughout the viral life cycle and examine whether either or both sub-classes of NS5A-positive structures are enriched with HCV RNA we developed a system to simultaneously track HCV RNA and NS5A in living cells. MS2 bacteriophage RNA stem loop sequences (6x /8x /12x /24x repeats) were inserted into the 3’UTR of the Jc1/5A-TCM virus (Jc1/5A-TCM+3’UTR:MS2) to allow indirect tracking of HCV RNA in Huh-7.5 cells via MS2.Coat-mCherry fusion protein that interacts specifically with MS2 stem loops. Jc1/5A-TCM+3’UTR:MS2 viruses replicated to significantly lower levels than the parent Jc1 as assessed by immunofluorescence analysis. However, long-term culture resulted in emergence of more efficient viral replication, with PCR and sequence analysis indicating at least partial retention of MS2 stem loops at 8 days post electroporation of HCV RNA. To further characterize and overcome the replication handicap induced by the insertion of the MS2 stem loop sequences we also generated Huh-7.5 cells that harbour the HCV subgenomic replicon featuring these MS2 stem loops insertions. Deep sequencing analysis was conducted to identify emerging adaptive mutations. However none was found to be particularly predominant. Most importantly, redistribution of the mCherry tagged-MS2 coat protein from a homogenous cytoplasmic distribution to a more punctate localisation was observed in the context of the full-length viral cultures indicating specific binding to HCV RNA. Using this approach we have simultaneously visualised HCV RNA (MS2.coatmCherry) and NS5A traffic (FlAsH) in real-time during HCV replication. Both HCV RNA-positive small motile and larger static structures were enriched with NS5A. In contrast, a subset of the trafficking NS5A-positive structures was devoid of HCV RNA. We also investigated viral RNA traffic with respect to lipid droplets (LDs) and show that two sub-types of static HCV RNA-positive structures existed: one was closely juxtaposed to LDs while the second sub-class was localised away from LDs. Moreover the system enabled visualization of putative RNA delivery at the LD surface with examples of motile HCV RNA-enriched structures dynamically interacting with LDs. Finally performing co-imaging of HCV NS5A and Rab18, an NS5A-interacting host factor located at the LD surface, we were able to illustrate the often transient nature of NS5A interaction with the LD and putative sampling of the LD that may precede interaction with core and initiation of assembly steps of the viral life cycle. These studies reveal new insights into the dynamics of HCV RNA traffic and the interactions at play in the context of the HCV life cycle.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015