The development and op timisation of a Theiler’s murine encephalomyelitis virus antiviral assay
- Authors: Naidoo, Urisha Tirah
- Date: 2023-10-13
- Subjects: Theiler's encephalomyelitis virus , Picornaviruses , Antiviral agents , Immunofluorescence , Western immunoblotting , Drug development
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424677 , vital:72174
- Description: Picornaviruses belong to the Picornaviridae family which are one of the largest and most diverse family of RNA viruses that cause a broad spectrum of infections in both humans and animals. These diseases range from severe infections such as poliomyelitis, meningitis, myocarditis to mild illnesses such as the common cold. Picornavirus outbreaks are a worldwide threat as they are continuously occurring. A recent outbreak of foot-and-mouth disease caused by a picornavirus occurred in South Africa, resulting in a temporary ban on the movement of cattle. Currently, the FDA has not approved any antiviral drugs against this virus, increasing the urgency for identifying effective antivirals. Picornaviruses have similar genomes and capsid organisation as such, those that are non-hazardous to humans can be used as a model system. A Theiler’s murine encephalomyelitis virus (TMEV) strain GDVII and Baby Hamster Kidney fibroblasts (BHK-21 cells) was used as a replication system to develop and optimise a medium-throughput antiviral screening assay. The TMEV GDVII replication system in BHK-21 cells was validated, and preliminary experiments were performed that were necessary for the development of the TMEV GDVII antiviral assay. This was achieved by conducting a CPE assay to visually monitor the onset and development of CPE induced by TMEV GDVII. Plaque assays accurately quantified the number of infectious virus particles required for calculating the MOI in downstream experiments. Lastly, indirect immunofluorescence and Western blot analysis detected the expression of viral proteins using previously generated antibodies against the TMEV GDVII VP1 capsid and 2C protein, thereby confirming infection in BHK-21 cells. The development of robust and reproducible assays is an essential component in antiviral drug discovery. Therefore, the confirmed replication system was then used as a foundation to develop a medium-throughput CPE-based TMEV GDVII antiviral assay whereby the parameters were optimised to produce one of high quality. Firstly, the quantitation of viral-induced CPE was examined and confirmed in a 96-well plate using resazurin as a cell viability indicator. Each parameter was tested at varying conditions, and the optimal was concluded as 2 % FBS in the assay media, a 15 000 cells/well seeding density, infecting the cells with TMEV GDVII at an MOI of 0.00625 and measuring resazurin at an endpoint of 72 hpi. Furthermore, the parameters were ii validated by calculating the Z’- factor, which consistently produced scores above 0.5, indicative of a reliable, robust, reproducible antiviral assay. Currently, there are no inhibitors against TMEV GDVII that have been reported or confirmed in cell lines, animal models or clinical trials. Therefore, once the optimal assay parameters were selected, it presented an opportunity to assess whether potential compounds, including itraconazole (ITZ) and dipyridamole (DIP), possessed antiviral activity that could firstly, be utilised as a control inhibitor when screening compounds against TMEV GDVII and secondly, contribute to research on this virus. Additionally, the previously produced anti-TMEV GDVII capsid antibody was shown to neutralise viral infection and was also included as a potential control. The sensitivity of the cells towards DMSO, a solution in which the compounds were solubilised, was first investigated. It was found that concentrations above 1 % are toxic to the cells; as such, the final DMSO concentrations were always kept below 1 % when screening compounds. Lastly, the generation of dose-response curves aided in the conclusion that the antibody was the most suitable control inhibitor as it displayed potent antiviral activity and no cytotoxicity towards the cells. In contrast, ITZ and DIP did not possess effective antiviral action and were toxic to cells at high concentrations. Finally, after all the components of the medium-throughput TMEV GDVII antiviral assay were identified, it was possible to screen 24 compounds from a coumarin and marine natural product library for cell cytotoxicity and antiviral activity. After generating dose-response curves, it was concluded that no compound effectively inhibited virus-induced CPE, and most were toxic to cells at relatively high concentrations. In conclusion, this is the first study that describes the development and optimisation of a robust medium-throughput CPE-based antiviral assay that has immense potential to screen other libraries of compounds for antiviral activity against TMEV GDVII. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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- Date Issued: 2023-10-13
The development of techniques for the identification of novel viruses associated with acute infantile gastroenteritis in South Africa
- Authors: Jaquet, Brittany J
- Date: 2017
- Subjects: Gastroenteritis in children -- Treatment , Gastroenteritis in children -- Treatment -- South Africa , Antiviral agents , Viral vaccines , Rotaviruses , Virus diseases in children
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/38013 , vital:24725
- Description: Gastroenteritis is a serious disease affecting both children and adults globally, but is more predominant in children with over half a million deaths reported each year. The leading cause of this disease is rotavirus, which accounts for 38% of all hospitalised cases. There has, however, been a significant decrease in the number of deaths associated with rotavirus worldwide since the introduction of the two vaccines, Rotarix® and RotaTeq®. A large number of cases are therefore either associated with other viruses, such as norovirus, Aichi virus (AiV) or Saffold virus (SAFV), or are of unknown aetiology. This study thus aims to develop techniques for the identification of viruses associated with gastroenteritis. Theiler’s murine encephalomyelitis virus (TMEV) was used to develop the sample preparation, transmission electron microscopy and RT-PCR techniques used in this study. This virus was chosen as a replication system using baby hamster kidney cells can be used to create high concentrations of viral particles from which RNA can be extracted preventing the waste of the limited samples. The virus particles are also similar in size and morphology to the viruses to be identified in this study, as it belongs to the same family. After sample preparation, TEM analysis showed the presence of small, round, non-enveloped virus particles in the TMEV sample. Due to the low concentration of virus particles, PEG precipitation was performed using both 0.15 M and 0.25 M NaCl and 8% (w/v) PEG 6000. TEM analysis then showed an increase in viral particle concentration, with the highest concentration observed at 0.25 M NaCl and 8% PEG 6000. RNA was successfully extracted and RT-PCR assays were performed for both the VP1 and 2B coding regions of TMEV. A method for creating a positive control for the RT-PCR assay was developed by the in vitro transcription of RNA from pTMEV, which contains the cDNA of TMEV. The RNA was then used as the template for the 2B two-step RT-PCR assay. A product of 412 bp was successfully amplified from the in vitro transcribed RNA and the sensitivity of the RT-PCR assay was determined. Using a Norovirus GII positive stool sample provided by Maureen Taylor, a nested RT-PCR assay was developed for the NoV GII N/S domain using a previously-published primer set and cycling parameters. A 342 bp product was successfully amplified from the RNA extracted from the stool sample and cloned into pGEM®-T Easy to produce pNoVGII. Using the plasmid containing the AiV 5’UTR and the PCR amplicon for AiV 3CD, RT-PCR assays were developed for AiV 5’UTR and partial 3CD. The RT-PCR assays produced a 1008 bp product for AiV 5’UTR and 266 bp for AiV 3CD, which were cloned into pGEM®-T Easy to produce pAiV5’UTR and pAiV3CD, respectively. Using in vitro transcribed RNA from pNoVGII, pAiV5’UTR, pAiV3CD and pSAFV, which contains the SAFV cDNA, positive controls were developed for the RT-PCR assays for NoV GII, AiV 5’UTR, AiV 3CD and SAFV 2C. The sensitivity of these assays was determined. The samples chosen for this study include wastewater collected from the Belmont Valley water treatment plant, oysters suspected to be infected with viruses collected from Port Elizabeth, South Africa and 30 stool samples from symptomatic patients. With the methods developed using TMEV, the wastewater, oysters and 30 stool samples were filter-sterilised, concentrated and screened by TEM. All samples showed the presence of virus particles. RNA was successfully extracted and the wastewater, oyster and 30 stool samples were screened for NoV GII using the NoV GII RT-PCR assay. The wastewater, oysters and 11 of the stool samples produced the 342 bp NoV GII PCR product and BLAST analysis determined the nucleotide sequences to be NoV GII.4. This shows that this study was able to develop sample preparation techniques and TEM analysis for selected samples and RT-PCR assays for NoV GII, AiV and SAFV. The NoV GII RT-PCR assay was successfully used for the screening of the wastewater, oysters and 30 stool samples for NoV GII. Due to the high number of gastroenteritis cases with unknown aetiology in South Africa, the development of techniques for the identification of NoV, AiV, SAFV and other viruses is very important. The identification of these viruses will allow for better surveillance, treatment and prevention of gastroenteritis in South Africa.
- Full Text:
- Date Issued: 2017
Prediction of interacting motifs within the protein subunits of Picornavirus capsids
- Authors: Ross, Caroline Jane
- Date: 2015
- Subjects: Picornaviruses , Antiviral agents , Poliovirus , Coxsackieviruses , Hepatitis A virus , Foot-and-mouth disease virus , Viral proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4151 , http://hdl.handle.net/10962/d1017912
- Description: The Picornaviridae family contains a number of pathogens which are economically important including Poliovirus, Coxsakievirus, Hepatitis A Virus, and Foot-and-Mouth-Disease-Virus. Recently the emergence of novel picornaviruses associated with gastrointestinal, neurological and respiratory diseases in humans has been reported. Although effective vaccines for viruses such as FMDV, PV and HAV have been developed there are currently no antivirals available for the treatment of picornavirus infections. Picornaviruses proteins are classified as: the structural proteins VP1, VP2, VP3 and VP4 which form the subunits of the viral capsid and the replication proteins which function as proteases, RNA-polymerases, primers and membrane binding proteins. Although the host specificity and viral pathogenicity varies across members of the family, the icosahedral capsid is highly conserved. The capsid consists of 60 protomers, each containing a single copy of VP1, VP2 and VP3. A fourth capsid protein, VP4, resides on the internal side of the capsid. Capsid assembly is integral to life-cycle of picornaviruses; however the process is complex and not fully-understood. The overall aim of the study was to broaden the understanding of the evolution and function of the structural proteins across the Picornaviridae family. Firstly a comprehensive analysis of the phylogenetic relationships amongst the individual structural proteins was performed. The functions of the structural proteins were further investigated by an exhaustive motif analysis. A subsequent structural analysis of highly conserved motifs was performed with respect to representative enteroviruses, Foot-and-Mouth-Disease-Virus and Theiler’s Virus. This was supplemented by the in silico prediction of interacting residues within the crystal structures of these protomers. Findings in this study suggest that the capsid proteins may be evolving independently from the replication proteins through possible inter-typic recombination of functional protein regions. Moreover the study predicts that protomer assembly may be facilitated through a network of multiple subunit-subunit interactions. Multiple conserved motifs and principle residues predicted to facilitate capsid subunit-subunit interactions were identified. It was also concluded that motif conservation may support the theory of inter-typic recombination between closely related virus sub-types. As capsid assembly is critical to the viral life-cycle, the principle interacting motifs may serve as novel drug targets for the antiviral treatment of picornavirus infections. Thus the findings in the study may be fundamental to the development of treatments which are more economically feasible or clinically effective than current vaccinations.
- Full Text:
- Date Issued: 2015
Development, assessment and optimisation of oral famciclovir formulations for paediatric use
- Authors: Magnus, Laura
- Date: 2012
- Subjects: Drugs -- Dosage forms , Drugs -- Analysis , Capsules (Pharmacy) , Antiviral agents , Pediatrics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3870 , http://hdl.handle.net/10962/d1018244
- Description: Many Active Pharmaceutical Ingredients (API) such as the antiviral agent famciclovir (FCV) are required for paediatric treatment but are not commercially available in age-appropriate dosage forms. It is common practice to prepare oral liquid dosage forms using commercially available tablets, capsules or powdered API and then dispersing or dissolving the crushed and/or powdered materials in a vehicle that the patient can swallow. Vehicles that are commonly used for this purpose include methylcellulose, syrup or combinations of these carriers where possible or commercially available suspending agents such as Ora-Sweet®, if available, can be used. However, several critical factors are overlooked when manufacturing extemporaneous formulations including, but not limited to, physical and chemical properties of the API, excipients, compatibility, stability and bioavailability issues. A stability-indicating High Performance Liquid Chromatography (HPLC) method for the analysis of FCV was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The method is sensitive, selective, precise, accurate and linear over the concentration range 2-120 μg/ml. The stability of 25 mg/ml FCV formulations was assessed in vehicles manufactured from syrup simplex, hydroxypropyl methylcellulose (HPMC), Ora-Sweet® and an aqueous buffer (pH 6) following storage at 25 °C/60% RH and 40 °C/75% RH over six (6) to eight (8) weeks. The shelf life of the products was calculated as the longest period of storage for approximately 90% of the added FCV to be recovered. Formulations were manufactured using syrup simplex or HPMC with methylparaben and propylparaben individually or in combination and with sodium metabisulphite, ascorbic acid or citric acid as antioxidants. The resultant products were subject to quality control analysis for API content, viscosity, pH and appearance and the resultant data were subject to statistical analysis. The degradation rates were calculated for each product and a degradation profile plotted. The degradation rates of FCV in extemporaneous formulations were compared to those of FCV manufactured using a commercially available suspending agent and a buffered vehicle. FCV undergoes major degradation in the presence of sucrose, as observed for formulations in which the vehicle was syrup and Ora-Sweet®. FCV was found to be most stable when dissolved/dispersed in an HPMC vehicle incorporating sodium metabisulphite and a combination of parabens. The formulation that exhibited the maximum stability was manufactured using an aqueous solution buffered to pH 6. Due to the enhanced stability of FCV when added to a buffered vehicle a formulation in which an HPMC vehicle buffered to pH 6 with sodium metabisulphite, methylparaben and propylparaben was selected for optimisation using a Central Composite Design approach (CCD). In this way it was possible to establish a relationship between input variables such as pH, % w/v HPMC, % w/v antioxidant and % w/v preservative and the responses selected for monitoring by means of response surface modelling. A quadratic model was found to be the most appropriate to describe the relationship between input and output variables. Thirty batches of product were randomly manufactured according to the CCD and analysed to establish the stability in respect of viscosity, pH and the amount of FCV remaining following storage and the data were fitted to models using Design-Expert® software. A correlation between input variables and the responses was best described by a quadratic polynomial model. Analysis of Variance indicated that the response surface models were significant (P-value < 0.0001). The pH to which a FCV formulation was buffered was the most significant factor to effect the % drug content and the ultimate pH of the formulation, while the % w/v HPMC had the most significant effect on the viscosity of the product. The optimum composition for the manufacture of an oral liquid FCV formulation was predicted using the optimisation function of the Design-Expert® software. A low % error of prediction was established, indicating that the model is robust and that RSM is an appropriate formulation optimisation tool as it has a high prognostic ability. A liquid FCV formulation was developed, optimised and found to be suitable for its intended purpose. However further optimisation is required in respect of colourants, sweeteners and/or flavourants. The approach followed is useful in ensuring the development of quality products and can be applied in future.
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- Date Issued: 2012