Magister Scientiae - MSc / Nevirapine (NVP) is a cheap anti-retroviral drug used in poor countries worldwide, administered to pregnant women at the onset of labour to inhibit HIV enzyme reverse transcriptase. Viruses which may get transmitted to newborns are deficient in this enzyme, and HIV-1 infection cannot be established, thereby preventing mother to child transmission (MTCT). In some cases, babies get infected and positive selection for viruses resistant to nevirapine may be inferred. Positive selection can be inferred from sequence data, when the rate of nonsynonymous substitutions is significantly greater than the rate of synonymous substitutions. Unfortunately, it is found that available positive selection methods should not be used to analyse before- and after- NVP treatment sequence pairs associated with MTCT. Methods which use phylogenetic trees to infer positive selection trace synonymous and nonsynonymous substitutions further back in time than the short time duration during which selection for NVP occurred. The other group of methods for inferring positive selection, the pairwise methods, do not have appreciable power, because they average susbtituion rates over all codons in a sequence pair and not just at single codons. We introduce a simple counting method which we call the Pairwise Homologous Codons (PHoCs) method with which we have inferred positive selection resulting from NVP treatment in longitudinal HIV-1 reverse transcriptase sequences. The PHoCs method estimates rates of substitutions between before- and after- NVP treatment codons, using a simple pairwise method. / South Africa

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.

Wang Hui. / "December 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 256-280) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

Zhu Wen. / "May 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 217-249). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Ma Shuang-Cheng. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 289-324). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Deng Xue-Long. / "January 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 196-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Ho, Wing Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 116-137). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.v / Table of Contents --- p.vii / List of Figures --- p.x / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter One: --- General Introduction / Chapter 1.1 --- Respiratory Syncytial Virus (RSV) --- p.1 / Chapter 1.2 --- RSV biology --- p.2 / Chapter 1.3 --- RSV strains --- p.10 / Chapter 1.4 --- RSV pathogenesis and host antiviral responses --- p.11 / Chapter 1.5 --- Prevention of RSV infection --- p.13 / Chapter 1.5.1 --- Vaccines --- p.13 / Chapter 1.5.2 --- Passive anti-RSV antibodies --- p.17 / Chapter 1.6 --- Treatment for RSV infections --- p.20 / Chapter 1.6.1 --- Ribavirin (Virasole®) --- p.20 / Chapter 1.6.2 --- Other antiviral strategies --- p.21 / Chapter 1.6.2.1 --- Attachment inhibitors --- p.22 / Chapter 1.6.2.2 --- Fusion inhibitors --- p.23 / Chapter 1.6.2.3 --- Replication inhibitors --- p.25 / Chapter 1.6.2.4 --- Ethnobotanic medicines --- p.28 / Chapter 1.6.2.4.1 --- Anti-RSV medicinal plant components --- p.31 / Chapter 1.6.2.4.1.1 --- Phenolics and polyphenols --- p.31 / Chapter 1.6.2.4.1.2 --- Flavonoids --- p.32 / Chapter 1.6.2.4.1.3 --- Terpenoids and essential oils --- p.34 / Chapter 1.6.2.4.1.4 --- Lectins --- p.34 / Chapter 1.6.2.4.1.4.1 --- General introduction to lectins --- p.34 / Chapter 1.6.2.4.1.4.2 --- Historical aspects of lectins --- p.35 / Chapter 1.6.2.4.1.4.3 --- Applications of lectins --- p.36 / Chapter 1.7 --- Objectives of the project --- p.37 / Chapter Chapter Two: --- Screening of medicinal plants and phytochemicals for antiviral activity against RSV / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.2 --- Materials and methods --- p.47 / Chapter 2.2.1 --- Medicinal plants and phytochemicals --- p.47 / Chapter 2.2.2 --- Plant extracts preparation --- p.48 / Chapter 2.2.2.1 --- Aqueous extracts --- p.48 / Chapter 2.2.2.2 --- Ethanol extracts --- p.48 / Chapter 2.2.3 --- Cell and virus --- p.49 / Chapter 2.2.4 --- Endpoint titration of RSV infectivity --- p.50 / Chapter 2.2.5 --- Cytotoxicity test --- p.50 / Chapter 2.2.6 --- Antiviral assay --- p.52 / Chapter 2.3 --- Results --- p.53 / Chapter 2.4 --- Discussion --- p.58 / Chapter Chapter Three: --- "Mechanistic studies of anti-RSV actions of various fractions of Adina pilulifera, and daphnoretin, a purified compound from Wikstroemia indica" / Chapter 3.1 --- Introduction --- p.60 / Chapter 3.2 --- Materials and methods --- p.65 / Chapter 3.2.1 --- Fractionation of A. pilulifera ethanol extract --- p.65 / Chapter 3.2.2 --- Cell and virus --- p.65 / Chapter 3.2.3 --- Cytotoxicity test --- p.65 / Chapter 3.2.4 --- Endpoint titration of RSV by TCID50 method --- p.66 / Chapter 3.2.5 --- Antiviral study by CPE reduction assay --- p.66 / Chapter 3.2.6 --- Endpoint titration of RSV by plaque assay --- p.66 / Chapter 3.2.7 --- Antiviral study by plaque reduction assay --- p.67 / Chapter 3.2.8 --- Mode of antiviral action study --- p.68 / Chapter 3.3 --- Results --- p.70 / Chapter 3.4 --- Discussion --- p.76 / Chapter Chapter Four: --- Antiviral activity of Narcissus tazetta proteins / Chapter 4.1 --- Introduction --- p.81 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Crude proteins extraction from Narcissus tazetta cultivar --- p.88 / Chapter 4.2.2 --- Separation of proteins with affinity column --- p.88 / Chapter 4.2.3 --- Gel filtration of protein fractions on Superose column --- p.89 / Chapter 4.2.4 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.89 / Chapter 4.2.5 --- Electroblotting and N-terminal amino acid sequence analysis --- p.90 / Chapter 4.2.6 --- Protein concentration determination --- p.90 / Chapter 4.2.7 --- Isolation and purification of N. tazetta lectin (NTL) --- p.91 / Chapter 4.2.8 --- Antiviral activities of N. tazetta proteins and NTL --- p.92 / Chapter 4.2.8.1 --- Cell and virus --- p.92 / Chapter 4.2.8.2 --- Cytotoxicity test --- p.92 / Chapter 4.2.8.3 --- Endpoint titration of RSV by TCID50 method --- p.92 / Chapter 4.2.8.4 --- Antiviral study by CPE reduction assay --- p.92 / Chapter 4.2.8.5 --- Endpoint titration of RSV by plaque assay --- p.92 / Chapter 4.2.8.6 --- Antiviral study by plaque reduction assay --- p.93 / Chapter 4.2.8.7 --- Mode of antiviral action study --- p.93 / Chapter 4.3 --- Results --- p.94 / Chapter 4.4 --- Discussion --- p.107 / Chapter Chapter Five: --- General Discussion and Conclusions --- p.111 / References --- p.116

The emergence of viruses with zoonotic potential, i.e. with the potential ability to cross species barriers to infect unnatural hosts, poses a huge threat to humans. It is therefore essential to develop new methodologies to rapidly and efficiently generate attenuated virus vaccine candidates to attempt to control the threat. Viruses need to be able to at least partially inhibit the host’s innate defence mechanism, known as the interferon (IFN) system, to replicate efficiently in vivo and establish a productive infection. It has been previously reported that viruses that have lost their ability to circumvent the host’s IFN response, or IFN-sensitive viruses, are promising candidates for live attenuated virus vaccines. Here we report on the development of a cell-based method to attempt to rapidly select IFN-sensitive viruses that can not block IFN signalling, from wild-type virus populations. Lentivirus vectors containing selection markers (HSV-tk – Herpes Simplex virus thymidine kinase gene and pac – puromycin resistance gene) under the control of a tight IFN-inducible promoter (the murine Mx1 promoter) were generated and used to specifically engineer HEp2 cell lines, termed Mx GIPSE and Mx TIPSE, for this purpose. The developed methodology relies on the engineered cell lines and a selection procedure using exogenous IFN-α and puromycin: if a cell is infected with IFN-resistant virus, it will die in the presence of IFN-α and puromycin because IFN signalling will be blocked, thereby blocking the activation of the Mx1 promoter and consequent expression of pac; if a cell is infected with an IFN-sensitive virus, it will survive in the presence of IFN-α and puromycin because the Mx1 promoter will become activated through the IFN signalling pathway, leading to the expression of pac. IFN-sensitive viruses can then be rescued from the surviving cells, and amplified using IFN-permissive cell lines expressing viral IFN antagonist proteins (proteins that block the host’s IFN response). When tested on PIV5 strains CPI- (an IFN-sensitive virus) and CPI+ (an IFN-resistant virus), the developed method allowed the survival and amplification of cells infected with CPI-, whilst cell death was observed for cells infected with CPI+. Whilst the developed methodology seems promising, further developments of the system are required. The possibilities of using the developed methodology in combination with other techniques, such as FACS sorting and immune selection, to rapidly select IFN-sensitive mutant viruses from wild-type and mutagenised virus populations are discussed. The potential to use Mx TIPSE cells to select IFN-resistant revertant viruses from IFN-sensitive virus populations is also discussed. In addition, a high throughput screening assay has been developed using the engineered Mx GIPSE and Mx TIPSE cell lines to search for compounds that block IFN signalling or that block the action of viral IFN antagonist proteins. Compounds that block IFN signalling would potentially be useful as anti-inflammatory drugs whilst compounds that block the action of viral IFN antagonist proteins would be valuable as antiviral drugs.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences