Unravelling the replication biology of Providence virus in a cell culturebased model system
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
- Full Text:
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
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Using a spatial resilience lens to understand alignments and misalignments in South Africa’s marine governance system
- Authors: Hardisty, Shannon
- Date: 2020
- Subjects: Marine parks and reserves -- South Africa , Marine parks and reserves -- South Africa -- Management , Marine parks and reserves -- Government policy -- South Africa , Table Mountain National Park (South Africa) , Spatial ecology -- South Africa , Coastal zone management -- South Africa
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164649 , vital:41152
- Description: In marine protected areas (MPAs), the radically open nature of marine ecosystems, the seemingly contradictory short-term goals of fisheries management (resource use) and conservation efforts (biodiversity protection), the variety of stakeholders, and the inherently political nature of space ha ve contributed to a disconnect between policy and practice. Many groups operate in an MPA at various spatial and socio-political scales, from national government bodies to local community groups, as such an MPA can be viewed as a complex, multi-scale and multi-level social-ecological system (SES). A greater understanding of the multi-scale processes driving the spatial resilience of SES can help address scale mismatches between continuous ecological change and the long-term governance of a seascape. South Africa has a long history of spaces and natural resources being politicised and faces high levels of unemployment and political frustration. Marine governance has been characterised as being siloed, lacking transparency regarding government processes and even between departments, and not properly addressing the needs of local communities; resulting in scale and level mismatches throughout the multi-scale processes (from national policies to local regulations)therefore, it is pertinent to understand the interactions between various national policies, their implementation, and their impacts. Using a spatial resilience lens, I set out to understand scale alignment in South Africa’s marine governance systems as it relates to MPAs. A spatial resilience lens emphasises analysis of both spatial and temporal scales, thus highlighting the multi-scale variables and interactions that occur within a system. I first sought to understand the policy and management across national and regional scales by conducting a series of semi-structured interviews with experts which were analysed using thematic analysis. I then approached the question of alignment from a local to national scale by using the Table Mountain National Park Marine Protected Area as a case study, here I carried out a series of workshops and interviews with line fishers, women’s groups, and eco-tourism operators from three different communities. Experts identified six themes when describing the structure of gove rnance in South African MPAs; they consisted of: government departments and marine legislation, management level factors, communication and information sharing, local level factors, biophysical factors, and scale. Communication and information sharing was the most mentioned referenced theme (40.26 %). The relationships between the communities and other actors (i.e. how communities are impacted, or impact on, the Department of Environmental Affairs , the Department of Agriculture Forestry and Fisheries, and Park Authorities) contributed 67.27 % to the total number of mentions, 91 % of which were from the following themes: communication and information sharing, local level factors, biophysical factors, and scale. My results indicate that weak, top-down relationships dominate the governance system in South Africa. During the Cape Town workshops I applied the spatial resilience lens in three ways. Firstly, the participants collaborated on a mapping exercise discussing the existing zone types within the MPA, as well as developing their own. There was an 87.87 % overlap of community designed zones on the existing MPA zones, however the types of zones designed by the community were different. Secondly, I carried out a document analysis and used information from the workshops to analyse the status and rights designation of important species for different user groups. The results show that the various stakeholder groups, while all active in the marine environment, interact with the ecosystem at different scales and with differing species. The differing interactions between groups result in challenges such as representation and complicated power dynamics between user groups. Finally, I asked participants to discuss factors that affect their ability to gain a livelihood from the sea. The external elements and their effects differed between the user groups, identifying important socio-political interactions. Whilst the external elements were all geographically distinct and localised, the spatial resilience lens allowed for a cross-scale, and to a certain extent cross-sector, understanding of these elements thus producing a more holistic understanding. My thesis shows that user groups operate on different spatial scales within the marine environment as a result of social, economic, and political influences leading to several scale-based challenges. The identified scale-based challenges have contributed to misalignments in marine governance in South Africa. My study shows that scale mismatches have important implications for understanding how marine policy influences user groups and identified pathways that affected policy implementation , and identifies future research application of a spatial resilience lens within social-ecological systems, for example it’s potential use in understanding the gendered nature of MPAs
- Full Text:
- Authors: Hardisty, Shannon
- Date: 2020
- Subjects: Marine parks and reserves -- South Africa , Marine parks and reserves -- South Africa -- Management , Marine parks and reserves -- Government policy -- South Africa , Table Mountain National Park (South Africa) , Spatial ecology -- South Africa , Coastal zone management -- South Africa
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164649 , vital:41152
- Description: In marine protected areas (MPAs), the radically open nature of marine ecosystems, the seemingly contradictory short-term goals of fisheries management (resource use) and conservation efforts (biodiversity protection), the variety of stakeholders, and the inherently political nature of space ha ve contributed to a disconnect between policy and practice. Many groups operate in an MPA at various spatial and socio-political scales, from national government bodies to local community groups, as such an MPA can be viewed as a complex, multi-scale and multi-level social-ecological system (SES). A greater understanding of the multi-scale processes driving the spatial resilience of SES can help address scale mismatches between continuous ecological change and the long-term governance of a seascape. South Africa has a long history of spaces and natural resources being politicised and faces high levels of unemployment and political frustration. Marine governance has been characterised as being siloed, lacking transparency regarding government processes and even between departments, and not properly addressing the needs of local communities; resulting in scale and level mismatches throughout the multi-scale processes (from national policies to local regulations)therefore, it is pertinent to understand the interactions between various national policies, their implementation, and their impacts. Using a spatial resilience lens, I set out to understand scale alignment in South Africa’s marine governance systems as it relates to MPAs. A spatial resilience lens emphasises analysis of both spatial and temporal scales, thus highlighting the multi-scale variables and interactions that occur within a system. I first sought to understand the policy and management across national and regional scales by conducting a series of semi-structured interviews with experts which were analysed using thematic analysis. I then approached the question of alignment from a local to national scale by using the Table Mountain National Park Marine Protected Area as a case study, here I carried out a series of workshops and interviews with line fishers, women’s groups, and eco-tourism operators from three different communities. Experts identified six themes when describing the structure of gove rnance in South African MPAs; they consisted of: government departments and marine legislation, management level factors, communication and information sharing, local level factors, biophysical factors, and scale. Communication and information sharing was the most mentioned referenced theme (40.26 %). The relationships between the communities and other actors (i.e. how communities are impacted, or impact on, the Department of Environmental Affairs , the Department of Agriculture Forestry and Fisheries, and Park Authorities) contributed 67.27 % to the total number of mentions, 91 % of which were from the following themes: communication and information sharing, local level factors, biophysical factors, and scale. My results indicate that weak, top-down relationships dominate the governance system in South Africa. During the Cape Town workshops I applied the spatial resilience lens in three ways. Firstly, the participants collaborated on a mapping exercise discussing the existing zone types within the MPA, as well as developing their own. There was an 87.87 % overlap of community designed zones on the existing MPA zones, however the types of zones designed by the community were different. Secondly, I carried out a document analysis and used information from the workshops to analyse the status and rights designation of important species for different user groups. The results show that the various stakeholder groups, while all active in the marine environment, interact with the ecosystem at different scales and with differing species. The differing interactions between groups result in challenges such as representation and complicated power dynamics between user groups. Finally, I asked participants to discuss factors that affect their ability to gain a livelihood from the sea. The external elements and their effects differed between the user groups, identifying important socio-political interactions. Whilst the external elements were all geographically distinct and localised, the spatial resilience lens allowed for a cross-scale, and to a certain extent cross-sector, understanding of these elements thus producing a more holistic understanding. My thesis shows that user groups operate on different spatial scales within the marine environment as a result of social, economic, and political influences leading to several scale-based challenges. The identified scale-based challenges have contributed to misalignments in marine governance in South Africa. My study shows that scale mismatches have important implications for understanding how marine policy influences user groups and identified pathways that affected policy implementation , and identifies future research application of a spatial resilience lens within social-ecological systems, for example it’s potential use in understanding the gendered nature of MPAs
- Full Text: