A molecular investigation of stem-galling Tetramesa Walker (Hymenoptera: Eurytomidae) on African grasses: applications to biological control
- Van Steenderen, Clarke Julian Mignon
- Authors: Van Steenderen, Clarke Julian Mignon
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432565 , vital:72881 , DOI 10.21504/10962/432565
- Description: South Africa is a larger donor than receiver of alien grasses, where approximately 15% (_ 165 spp.) of the country's native grass species have become naturalised elsewhere. Many of these grasses have become serious invaders, causing significant damage to native species, habitat structure, and ecosystem functioning. Biological control is a sustainable and cost-effective method for the control of invasive weeds, but its application to invasive grasses has been approached with trepidation in the past due to the fears of a lack of host-specific herbivores that may cause non-target damage to agriculturally-important crops. The Tetramesa Walker genus (Hymenoptera: Eurytomidae) is one of three genera in the family that feed exclusively on grasses, and have a record of being host-specific to a particular species, or complex of closely-related congeners. There are over 200 described Tetramesa species, but this taxonomic effort has occurred almost exclusively in the Northern Hemisphere. Only about 2% of the described species are from Africa, with none from southern Africa despite the high diversity of grasses in the region. The low morphological variability between many Tetramesa groups has made identification difficult, where there may in fact be multiple undiscovered cryptic species. This thesis generated genetic sequence data (mitochondrial COI and nuclear 28S) that revealed at least eight native southern African Tetramesa taxa that are new to science, focusing particularly on the assemblages associated with Eragrostis curvula Nees (African lovegrass) and Sporobolus pyramidalis Beauv. and S. natalensis Steud. (giant rat's tail grass) which are alien invasive pests in Australia. Approximately 200 eurytomid wasps were collected and sequenced from 19 grass species across six South African provinces. Additionally, 27 grass species were sequenced using four gene regions (rps16-trnK, rps16, rpl32-trnL, and ITS), which were added to existing sequence data to build a dataset comprising over 700 sequences. Field host ranges and the use of host grass genetic proxies were important in making inferences about the host-specificity of eurytomid wasps of interest. Nine Tetramesa groups appeared to be host-specific to a single grass species, while six Tetramesa were associated with multiple species in a single grass genus. Since S. pyramidalis, S. natalensis, S. africanus, Hyparrhenia hirta, E. trichophora, and Andropogon gayanus are weeds elsewhere, there are at least six potential Tetramesa biological control agents that have been identified. A high diversity of Tetramesa on grasses within the Eragrostis genus was reported, with at least four taxa associated with E. curvula. It is currently uncertain whether these taxa represent different cryptic species or intraspecific populations that are the result of geographic sub-structuring. No-choice host-specificity testing using Tetramesa sp. 4 on E. curvula revealed that the wasp could complete its lifecycle on two non-target African grasses; namely E. plana and E. planiculmis. The wasp was also recorded on other Eragrostis species in the field (namely E. biflora and E. capensis). Using grass genetic sequences obtained in this study, it was found that there are four native Australian Eragrostis species that are more closely related to target E. curvula than to the non-target E. plana and E. planiculmis. This suggests that Tetramesa sp. 4 may not be suitably host-specific for use as a biological control agent. Further host-specificity testing on these native Australian species is required, however, before this insect is ruled out completely. The Tetramesa on S. pyramidalis (Tetramesa sp. 1), and the unidentified Sporobolus species presumed to be S. africanus, were suitably host-specific to be used as biological control agents. Since it was unclear whether some phylogenetic clades were true species or intraspecific populations, which is essential to understand when selecting agents for biological control, a new piece of software, SPEDE-sampler", was developed. It offers users of the Generalised Mixed Yule Coalescent (GMYC) species delimitation model a means of assessing the degree to which sampling effects such as data size and parameter choice can influence species diversity estimates. When applied to the Tetramesa data set, the software assisted in identifying which groups may contain cryptic species, uncovering that the COI marker is affected more by singletons than the 28S marker (i.e. species diversity tends to be overestimated), and confirming putative Tetramesa taxa that could be useful for biological control programmes going forward. This thesis has provided evidence that South Africa contains a diverse assemblage of Tetramesa and other eurytomids that are closely associated with their grass hosts, and that many of these taxa hold promise for grass biological control. This work has also highlighted the importance of integrative taxonomy in the discovery of novel taxa, and that biological control practitioners need to be aware of the caveats of each line of evidence used in the delimitation of putative species. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Van Steenderen, Clarke Julian Mignon
- Date: 2023-10-13
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432565 , vital:72881 , DOI 10.21504/10962/432565
- Description: South Africa is a larger donor than receiver of alien grasses, where approximately 15% (_ 165 spp.) of the country's native grass species have become naturalised elsewhere. Many of these grasses have become serious invaders, causing significant damage to native species, habitat structure, and ecosystem functioning. Biological control is a sustainable and cost-effective method for the control of invasive weeds, but its application to invasive grasses has been approached with trepidation in the past due to the fears of a lack of host-specific herbivores that may cause non-target damage to agriculturally-important crops. The Tetramesa Walker genus (Hymenoptera: Eurytomidae) is one of three genera in the family that feed exclusively on grasses, and have a record of being host-specific to a particular species, or complex of closely-related congeners. There are over 200 described Tetramesa species, but this taxonomic effort has occurred almost exclusively in the Northern Hemisphere. Only about 2% of the described species are from Africa, with none from southern Africa despite the high diversity of grasses in the region. The low morphological variability between many Tetramesa groups has made identification difficult, where there may in fact be multiple undiscovered cryptic species. This thesis generated genetic sequence data (mitochondrial COI and nuclear 28S) that revealed at least eight native southern African Tetramesa taxa that are new to science, focusing particularly on the assemblages associated with Eragrostis curvula Nees (African lovegrass) and Sporobolus pyramidalis Beauv. and S. natalensis Steud. (giant rat's tail grass) which are alien invasive pests in Australia. Approximately 200 eurytomid wasps were collected and sequenced from 19 grass species across six South African provinces. Additionally, 27 grass species were sequenced using four gene regions (rps16-trnK, rps16, rpl32-trnL, and ITS), which were added to existing sequence data to build a dataset comprising over 700 sequences. Field host ranges and the use of host grass genetic proxies were important in making inferences about the host-specificity of eurytomid wasps of interest. Nine Tetramesa groups appeared to be host-specific to a single grass species, while six Tetramesa were associated with multiple species in a single grass genus. Since S. pyramidalis, S. natalensis, S. africanus, Hyparrhenia hirta, E. trichophora, and Andropogon gayanus are weeds elsewhere, there are at least six potential Tetramesa biological control agents that have been identified. A high diversity of Tetramesa on grasses within the Eragrostis genus was reported, with at least four taxa associated with E. curvula. It is currently uncertain whether these taxa represent different cryptic species or intraspecific populations that are the result of geographic sub-structuring. No-choice host-specificity testing using Tetramesa sp. 4 on E. curvula revealed that the wasp could complete its lifecycle on two non-target African grasses; namely E. plana and E. planiculmis. The wasp was also recorded on other Eragrostis species in the field (namely E. biflora and E. capensis). Using grass genetic sequences obtained in this study, it was found that there are four native Australian Eragrostis species that are more closely related to target E. curvula than to the non-target E. plana and E. planiculmis. This suggests that Tetramesa sp. 4 may not be suitably host-specific for use as a biological control agent. Further host-specificity testing on these native Australian species is required, however, before this insect is ruled out completely. The Tetramesa on S. pyramidalis (Tetramesa sp. 1), and the unidentified Sporobolus species presumed to be S. africanus, were suitably host-specific to be used as biological control agents. Since it was unclear whether some phylogenetic clades were true species or intraspecific populations, which is essential to understand when selecting agents for biological control, a new piece of software, SPEDE-sampler", was developed. It offers users of the Generalised Mixed Yule Coalescent (GMYC) species delimitation model a means of assessing the degree to which sampling effects such as data size and parameter choice can influence species diversity estimates. When applied to the Tetramesa data set, the software assisted in identifying which groups may contain cryptic species, uncovering that the COI marker is affected more by singletons than the 28S marker (i.e. species diversity tends to be overestimated), and confirming putative Tetramesa taxa that could be useful for biological control programmes going forward. This thesis has provided evidence that South Africa contains a diverse assemblage of Tetramesa and other eurytomids that are closely associated with their grass hosts, and that many of these taxa hold promise for grass biological control. This work has also highlighted the importance of integrative taxonomy in the discovery of novel taxa, and that biological control practitioners need to be aware of the caveats of each line of evidence used in the delimitation of putative species. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
Enhancing the electrocatalytic activity of phthalocyanines through finding the ideal combination of substituents in push-pull phthalocyanine-based systems
- Nkhahle, Reitumetse Precious
- Authors: Nkhahle, Reitumetse Precious
- Date: 2023-10-13
- Subjects: Phthalocyanines , Electrocatalysis , Hydrazine , Nitrites , Activating group , Deactivating group
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432578 , vital:72882 , DOI 10.21504/10962/432578
- Description: Phthalocyanines (Pcs) are a class of synthetic pigments with a similar structure to porphyrins. The work presented in this thesis is centred around these electron-rich macrocycles and their use in electrocatalysis. This body of work provides a more rigorous analysis on asymmetric Pcs, focusing on finding the “ideal” combination of substituents in the synthesis of A3B-type Pcs and how these asymmetric structures compare with their symmetric counterparts (A4) in the electrocatalysis of hydrazine and nitrite. The choice in substituents in the syntheses of the Pcs was such that there is both electron-donating and electron-withdrawing groups to induce a push-pull effect. In the studies involving the electrocatalysis of hydrazine, asymmetric cobalt Pcs (CoPcs) possessing alkyl groups as the primary substituents, with variations in the acid-containing group, along with their symmetric counterparts, probes with potential for further improvement were identified. Using voltammetric and amperometric techniques, the analyte-electrode kinetics, mechanism in which the electrochemical reaction proceeds along with the limits of detection (LoD) were determined. In the general sense, the pentadecylphenoxy-derived CoPcs performed better than those containing the tert-butyl substituent as the dominant substituent with the asymmetric CoPcs producing more favourable results than their symmetric analogues. With respect to the probes designed for nitrite, a multi-dimensional approach was undertaken in that acetaminophen was chosen as the primary substituent whilst multiple changes in the asymmetric component were made. In addition to varying the carboxylic acid-containing substituent, alkyne- and amine-based substituents were also explored in which the alkyne-containing Pc was anchored onto the electrode surface through click chemistry while the amine-bearing Pc was covalently linked (and π-stacked) to nitrogen-doped graphene quantum dots (NGQDs). Another component that was altered was the central metal where CoPcs were compared to manganese Pcs (MnPcs). The most desirable peak oxidation potential for nitrite was observed in the MnPcs as it was the lowest with adsorption sometimes being a better suited method of electrode modification relative to clicking. The inclusion of NGQDs was found to be beneficial when combined with the symmetric CoPc whilst in the presence of an asymmetric Pc complex, less desirable results were observed. Overall, there were variations in the results with the symmetric CoPc sometimes being better than some of the asymmetric CoPcs demonstrating that a blanket-approach in terms of synthesizing and applying asymmetric Pcs is not always viable. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Nkhahle, Reitumetse Precious
- Date: 2023-10-13
- Subjects: Phthalocyanines , Electrocatalysis , Hydrazine , Nitrites , Activating group , Deactivating group
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432578 , vital:72882 , DOI 10.21504/10962/432578
- Description: Phthalocyanines (Pcs) are a class of synthetic pigments with a similar structure to porphyrins. The work presented in this thesis is centred around these electron-rich macrocycles and their use in electrocatalysis. This body of work provides a more rigorous analysis on asymmetric Pcs, focusing on finding the “ideal” combination of substituents in the synthesis of A3B-type Pcs and how these asymmetric structures compare with their symmetric counterparts (A4) in the electrocatalysis of hydrazine and nitrite. The choice in substituents in the syntheses of the Pcs was such that there is both electron-donating and electron-withdrawing groups to induce a push-pull effect. In the studies involving the electrocatalysis of hydrazine, asymmetric cobalt Pcs (CoPcs) possessing alkyl groups as the primary substituents, with variations in the acid-containing group, along with their symmetric counterparts, probes with potential for further improvement were identified. Using voltammetric and amperometric techniques, the analyte-electrode kinetics, mechanism in which the electrochemical reaction proceeds along with the limits of detection (LoD) were determined. In the general sense, the pentadecylphenoxy-derived CoPcs performed better than those containing the tert-butyl substituent as the dominant substituent with the asymmetric CoPcs producing more favourable results than their symmetric analogues. With respect to the probes designed for nitrite, a multi-dimensional approach was undertaken in that acetaminophen was chosen as the primary substituent whilst multiple changes in the asymmetric component were made. In addition to varying the carboxylic acid-containing substituent, alkyne- and amine-based substituents were also explored in which the alkyne-containing Pc was anchored onto the electrode surface through click chemistry while the amine-bearing Pc was covalently linked (and π-stacked) to nitrogen-doped graphene quantum dots (NGQDs). Another component that was altered was the central metal where CoPcs were compared to manganese Pcs (MnPcs). The most desirable peak oxidation potential for nitrite was observed in the MnPcs as it was the lowest with adsorption sometimes being a better suited method of electrode modification relative to clicking. The inclusion of NGQDs was found to be beneficial when combined with the symmetric CoPc whilst in the presence of an asymmetric Pc complex, less desirable results were observed. Overall, there were variations in the results with the symmetric CoPc sometimes being better than some of the asymmetric CoPcs demonstrating that a blanket-approach in terms of synthesizing and applying asymmetric Pcs is not always viable. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-10-13
Market participation and welfare of smallholder farmers in the Eastern Cape Province South Africa
- Lesala, Mahali Elizabeth https://orcid.org/0000-0001-9921-2190
- Authors: Lesala, Mahali Elizabeth https://orcid.org/0000-0001-9921-2190
- Date: 2021-06
- Subjects: Farms, Small , Economic development -- South Africa , Agriculture -- Economic aspects -- South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/20916 , vital:46745
- Description: The low market participation of smallholder farmers in markets has received enormous attention from scholars, both in the country and the Eastern Cape Province. However, it is not clear how low their market participation is including its implications on farmer’s welfare. The purpose of the study was to determine the extent to which smallholder farmers in the homelands of the Eastern Cape participate in output markets and assess how their participation in markets has affected wellbeing of their households. This information will have important practical implications for policy regarding appropriate pathways for poverty alleviation and livelihoods improvements in the rural areas of the Eastern Cape Province. Three irrigation schemes; Qamata, Zanyokwe and Tyefu irrigation schemes were selected for this study. A sample of 210 smallholder irrigators were interviewed by means of a close-ended questionnaire. The data were analysed by means of descriptive statistical tools, the multiple-level choice models and the Propensity Score Matching (PSM) technique. SPSS and STATA computer programmes were used to carry out all the estimations. The analysis established that, although agriculture is the primary activity for rural livelihoods, it is not the main contributor to family income. Rather, remittances and social grants were the dominant sources of household income in the Qamata, Zanyokwe and Tyefu areas. From the standpoint of market, maize and potatoes are the most popular crops, but potatoes dominate the market. This result confirms that maize is the staple crop and therefore mostly grown for home consumption while production of potatoes is market-oriented. The Market Participation Index (MPI) revealed that farmers sell at least 55 percent of their farm produce, implying that farmers have made some transition from subsistence to semi-commercial farming. However, farmers’ priority still remains food self-sufficiency and market participation only takes place after satisfying their home food needs. The results revealed that the significant factors influencing the farmers’ decisions and their extent of participation in output markets were the age, gender, marital status of the household head, primary occupation of household head, size of farm cultivated, government financial support, access to extension services and farmer’s membership of cooperatives. Concerning the impact of output market participation on welfare of smallholders, the Average Treatment on the Treated (ATT) as the measure of change revealed that participation in output markets has a positive impact on welfare of the smallholder farmers through increased incomes. Farmers who participated in output market were at least R838.44 better off than those who did not participate in markets although social grants and remittances made significantly higher contribution to household welfare. The study suggests that despite some improvements in income of market participants, the standards of living of the rural households are still far from what would be considered optimal. Crop farming evidently contributes less than desired, hence the persistence of the widespread poverty. It is urgent to focus interventions on improving agricultural productivity while widening strategies for improving rural livelihoods beyond agriculture to diversify the choices open to rural dwellers. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-06
- Authors: Lesala, Mahali Elizabeth https://orcid.org/0000-0001-9921-2190
- Date: 2021-06
- Subjects: Farms, Small , Economic development -- South Africa , Agriculture -- Economic aspects -- South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/20916 , vital:46745
- Description: The low market participation of smallholder farmers in markets has received enormous attention from scholars, both in the country and the Eastern Cape Province. However, it is not clear how low their market participation is including its implications on farmer’s welfare. The purpose of the study was to determine the extent to which smallholder farmers in the homelands of the Eastern Cape participate in output markets and assess how their participation in markets has affected wellbeing of their households. This information will have important practical implications for policy regarding appropriate pathways for poverty alleviation and livelihoods improvements in the rural areas of the Eastern Cape Province. Three irrigation schemes; Qamata, Zanyokwe and Tyefu irrigation schemes were selected for this study. A sample of 210 smallholder irrigators were interviewed by means of a close-ended questionnaire. The data were analysed by means of descriptive statistical tools, the multiple-level choice models and the Propensity Score Matching (PSM) technique. SPSS and STATA computer programmes were used to carry out all the estimations. The analysis established that, although agriculture is the primary activity for rural livelihoods, it is not the main contributor to family income. Rather, remittances and social grants were the dominant sources of household income in the Qamata, Zanyokwe and Tyefu areas. From the standpoint of market, maize and potatoes are the most popular crops, but potatoes dominate the market. This result confirms that maize is the staple crop and therefore mostly grown for home consumption while production of potatoes is market-oriented. The Market Participation Index (MPI) revealed that farmers sell at least 55 percent of their farm produce, implying that farmers have made some transition from subsistence to semi-commercial farming. However, farmers’ priority still remains food self-sufficiency and market participation only takes place after satisfying their home food needs. The results revealed that the significant factors influencing the farmers’ decisions and their extent of participation in output markets were the age, gender, marital status of the household head, primary occupation of household head, size of farm cultivated, government financial support, access to extension services and farmer’s membership of cooperatives. Concerning the impact of output market participation on welfare of smallholders, the Average Treatment on the Treated (ATT) as the measure of change revealed that participation in output markets has a positive impact on welfare of the smallholder farmers through increased incomes. Farmers who participated in output market were at least R838.44 better off than those who did not participate in markets although social grants and remittances made significantly higher contribution to household welfare. The study suggests that despite some improvements in income of market participants, the standards of living of the rural households are still far from what would be considered optimal. Crop farming evidently contributes less than desired, hence the persistence of the widespread poverty. It is urgent to focus interventions on improving agricultural productivity while widening strategies for improving rural livelihoods beyond agriculture to diversify the choices open to rural dwellers. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-06
Exploring the structural integrity of a picornavirus capsid
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
- «
- ‹
- 1
- ›
- »