Whilst the volume of literature concerning rubella virus has increased markedly during recent years, few authors have compared the properties of strains of varied origin and history. In the main, interest has centred on the biological characteristics of the attenuated strains of rubella and the modification of such properties during loss of virulence for man. A study of the plaque morphology in GL-RK[1]3 cells, of some 36 strains from pre and post-natally acquired infection was undertaken. The strains included laboratory adapted, highly passaged and freshly isolated material with little or no history of culture in vitro. It was found that in the main, low pass strains showed a typical small plaque form but two freshly isolated strains with large plaque type were also encountered. The laboratory passaged strains showed a range in plaque morphology from small to large which did not appear to be dependant on the tissue selected for propagation of the virus. A brief study of the influence of tissue culture passage on the plaque morphology of selected strains yielded evidence of two systems of change; the first being a gradual transition from a small to large plaque, whilst the second appeared to be closer to a selection process, the new plaque type emerging and becoming predominant. Six representative strains of rubella were selected for further study. Growth of virus and production of haemagglutinating antigen in several cell culture systems was compared and the results suggested that the previous passage history of a strain could influence its potential for growth in cell culture. The thermal stability of these strains was also investigated and the two isolates from congenitally infected infants were shown to differ in their pattern of inactivation. All of the strains were shown to be immunogenic in rabbits, and the antisera thus produced were used in cross haemagglutination inhibition and neutralization studies. Apparent anomalies were encountered with the antisera produced in response to the Cendehill strain. It was not possible to demonstrate haemagglutination inhibiting antibodies against some of the strains in spite of the fact that these were present to high titres against other strains of rubella. Examination of the kinetics of homologous and heterologous neutralization showed some difference between the strains and the existence of two particularly antibody sensitive strains.

Assessment of the relative potencies of compounds which inhibit the multiplication of herpes virus is rendered difficult by the fact that they have been investigated by different workers who were not using comparable test systems. The aim of this work was therefore to evaluate such test systems as could be used for studying the chemotherapy of herpes virus infections with particular reference to their suitability for determining relative potencies. Both in vitro and in vivo systems were investigated. Tissue culture methods included parallel observations of the time of appearance of cytopathic effect in the presence and absence of the test compound, carried out in tubes and microtitre plates. The compounds were also examined in plaque inhibition and plaque reduction tests, by the gradient plate technique, and by determining the reduction in virus yield produced by the test compound in single-cycle experiments. The gradient plate technique gave a visual indication of the minimal inhibitory concentration. With the other methods dose-response lines were obtained, the most satisfactory being given by the plaque reduction method. Cytarabine was the most active compound, the ID50 with a standard strain of type 1 herpes being 0. 23 muM. The values for the other compounds were 0. 76 muM for trifluorothymidine, 0. 89 muM for idoxuridine, 14. 96 muM for vidarabine and 20. 89 muM for diaminopurine arabinoside. The compounds fell in the same order in tests with type 2 strains, except that 2 strains were found to be insensitive to trifluorothymidine and diaminopurine arabinoside. The in vivo models used were herpetic encephalitis in mice and herpetic keratitis in rabbits. Dose-response lines could be obtained in mice by taking the mean reciprocal survival time as the response, but they were not as satisfactory as those obtained by plaque reduction. The keratitis model could not be used for determinations of relative potency, but it was satisfactory for assessing the therapeutic effect of different concentrations of the test compound, and thus for ranking comparable solutions of the different compounds in order of activity. Satisfactory clinical cure was obtained with all compounds regardless of their relative potencies as determined in tissue culture. From the results of both in vitro and in vivo test systems it was possible to demonstrate variations in sensitivity between types and between strains of the same type. There are several reports in the literature that type 2 strains of herpes virus are less sensitive than type 1. In the present work this was found to be not the case, since there was a wide overlap in the sensitivities of the strains of the two types, with some type 2 strains being considerably more sensitive than those of type 1. The present studies have thus established satisfactory test methods for the quantitation of the relative potency of the standard anti-herpes compounds, which will be valuable in the assessment of the antiviral potential of compounds to be discovered in the future.

The evolution of ON/OFF switching phase variable loci is presumed to have arisen due to the need for bacterial populations to cope with uncertain environments where selection fluctuates between opposing pressures. One such selection is believed to be the presence of bacteriophage. Bacteriophage can influence bacterial evolution by forcing the development of bacteriophage resistance mechanisms within bacterial populations. However, resistance mechanisms can often come at a cost, such as reduced survival against the immune responses. As such, phase variation may circumnavigate this cost by generating heterogeneous populations containing both resistant and sensitive phenotypes. This study aimed to investigate the two known phase variable bacteriophage resistance genes in Haemophilus influenzae hsdM and lic2A, which encodes the methyltransferase of a type I restriction modification system and a lipooligosacharide biosynthesis associated glycosyltransferase respectively. Analysis of the diversity of repeat tract lengths and ON/OFF states of these genes across H. influenza genomes within GenBank revealed that while lic2A was ON in the majority of strains, hsdM was OFF. Thus lic2A may be consistently beneficial while the hsdM benefit is transient. Analysis of samples from patients with COPD, showed that lic2A was ON in the majority of samples, while hsdM may also be ON state in a number of samples. Although a resistance phenotype could be observed for lic2A, no resistance could be observed for hsdM. Therefore, the dynamics of bacteriophage spread through populations heterogeneous for lic2A was investigated. The heterogeneous populations generated by phase variation reduced bacteriophage dispersal through bacterial populations. This mechanism may also allow bacterial populations to adjust their heterogeneity levels to control bacteriophage densities. The heterogeneity may further create diverse bacteriophage densities across the bacterial macropopulation through resistant populations acting as a barrier. The results demonstrate the potential for phase variation to aid in the survival of bacterial populations against bacteriophage predation.

In the last two decades a surge in antibiotic resistance has limited antibiotic effectiveness increasing the risk of uncontrolled epidemics especially for biofilm-related infections. The National Institute of Health reports that 80 % of human infections are biofilm related. The Proteus mirabilis bacteria were focused on in this study as they are significant biofilm formers in chronic infections such as biofilm-related urinary tract infections for which there are currently no completely effective treatment strategies. As biofilms can increase antibiotic resistance by up to 1000-fold, there is an urgent need for the development of novel antimicrobials. Thus, bacteriophages which are viruses that target and kill bacteria have been proposed as suitable alternatives, but factors like storage stability and re-isolation pose limitations. Towards investigating the development of new antimicrobial strategies, the aims of this thesis were to assess: (i) the therapeutic potential of bacteriophages against Proteus mirabilis biofilms and (ii) the development of a novel antimicrobial strategy based on a synthetic biology approach for improvement of bacteriophage-based biofilm control. The work presented in this thesis led to or may lead to four areas of development, which have the potential to contribute to fields of biofilm research, bioengineering and materials science. Firstly, novel bacteriophages against clinical strains of Proteus mirabilis were isolated with physicochemical and genomic characterisation. Unlike other studies, the effect of temperature was included in the selection of favourable bacteriophages for anti-biofilm use. Secondly, towards improving bacteriophage-based treatment, dendrimeric nanoparticles known as dendron were posed as alternatives, these were synthesised and characterised, and demonstrated improved biofilm reduction and eradication by 35 % and 100 % respectively compared with the most effective bacteriophage. Thirdly, this study developed insight into the dendron’s mechanism of action, which was previously unreported, and was proposed to be through disruption of Proteus mirabilis DNA systems. Fourthly, in a unique approach, the dendron was bioengineered with bacteriophage DNA using electrostatic interactions. The results suggested that the dendron has potential to be used as a carrier for bacteriophage DNA, and presents the first attempt in published literature at combining the anti-biofilm properties of bacteriophages and dendrons towards futuristic development of synthetic bacteriophages. The results also provide a promising antimicrobial strategy for use of dendrons as therapeutic agents, alone or in combination with antibiotics and bacteriophages for treatment of biofilm-related infections.

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This thesis studies the evolution, influence, and proliferation of endogenous retroviruses (ERVs) within animal genomes. First, a simple mathematical model is constructed to address the question of whether retroviral endogenizations occur most often in male or female hosts. The result of applying the model to a diversity of genomes suggests that there may be female risk factors to endogenization, or that selection may be acting on full-length ERVs. Second, a study of the divergence of orthologous full-length ERVs from human and chimpanzee is performed. It is found that highly transcribed members of the HERV-H family have been under directional selection in the last six million years. Third, the insertion and deletion activity of the largest ERV families in five primate species is studied. Using a phylogenetic model it is demonstrated that ERVs are likely to be deleted early if they are to be deleted at all. Notably, it is also shown that HERV-H is an outlier family that is unusually slowly deleted. Fourth, the HERV-H loci in the human genome are studied on an individual basis. It is found that the long terminal repeats of HERV-H affect the magnitude and specificity of its transcription. Surprisingly, a region of the retroviral gag gene is positively associated with transcription and it is argued that this association is a partial explanation for the preferential maintenance of HERV-H in a full-length form. In conclusion, it is argued that researchers should take seriously the notion that many ERVs have not drifted to fixation. It is also argued that taking account of solo-LTR formation is important to accurately assessing the historical activity of ERVs. Finally, it is hypothesised that the application of bioinformatics techniques like those developed in this thesis may be sufficient to identify exaptation events in species quite distant from the primates that are studied here.

This thesis analyzes the evolutionary trajectories that drive the evolution of several RNA viruses. These viruses have been identified to be the leading causes of viral outbreaks and deaths in humans. Studying the mechanisms influencing their evolution could therefore produce vital information for controlling the spread of these viruses or for their eradication. The availability of huge sequence repositories and advancement in computing and sequencing technologies allows for the development of novel methods for understanding the evolution of viruses even during an on-going outbreak, epidemic or pandemic. In this study, I developed a method that incorporates phylogenetic and structural based techniques to study the evolution of drug resistance in (A) HIV-1 Pol proteins, (B) the evolutionary dynamics of the 2013 - 2016 EBOV outbreak and (C) the evolution of the A(H1N1) influenza virus amongst human, avian and swine species. Findings from this thesis show that though HIV-1 evolves differently in the presence and absence of drug selection pressure, the virus is generally constrained by the need to maintain viral protein structure stability. The virus achieves this by accumulating enabling mutations early in its evolutionary history in order to accommodate the emergence of drug resistance associated mutations, which are mostly destabilizing. I also show that although the 2013 - 2016 EBOV was evolving rapidly, early data indicated that it was not changing at the functional level and not adapting to the human host. This is because most of the mutations occur in either inter genic or intrinsically disordered regions, which are less constrained, while the structured bits are characterized by neutral impact mutations. This again suggests that the virus needs to maintain a stable protein structure in order to remain functional. I show that EBOV is relatively stably evolving and the major force driving its evolution is more of an epidemiologic rather than a molecular factor whereas HIV-1 is evolving adaptively and its evolution is driven by molecular processes. However, one residue change, A82V seems to have altered the ability of the virus to bind its human receptor. This suggests that adaptive or functional mutations (which are mostly destabilizing in nature) work hand in hand with enabling mutations in such a way that a virus can acquire a mutation that confers drug resistance or leads to a gain of function without compromising its fitness while also retaining its functions such as infectivity and transmissibility. This indicates that the mechanisms described above may be a general way through which viruses evolve. The methods developed in the study can easily be applied to studying the evolution of other viruses and other systems e.g. microorganisms and cancer cells. Even if selection analysis does not show positive selection or any mutations in functional site, my thesis has demonstrated that structural analysis will be very useful for identifying and also predicting mutations that could facilitate adaptation of viruses. Also the influenza study shows that though the A(H1N1) is evolving somewhat differently in the humans, avian and swine species, one thing they seem to have in common is that stability constrains their evolution. I also show that my findings based on the human A(H1N1) influenza virus is consistent with the other human viruses (HIV and EBOV) analyzed in this project work.

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No description available.

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