Different strains of scrapie can be distinguished by the characteristic pattern of vacuolar degeneration which develops in the murine brain, and by the length of the incubation period. The causal agent has not been identified, and the presence of infection within tissues can only be demonstrated by bioassay. In this study, the intraocular route of infection was exploited to examine the development of the pathology produced by six different scrapie strains, and to compare the pattern of lesion targeting within this well-studied anatomical system with the spread of infectivity. Scrapie incubation period is controlled by the murine <i>Sinc</i> gene, which has two alleles; lesion development was recorded in both mouse genotypes following infection with several of the scrapie strains. The pattern of lesion development was found to correspond closely with the neuroanatomical pathways from retina via the optic nerve to the visual projection areas in the brain. The earliest lesions in all models appeared in the second half of the incubation period, and were confined to the visual projection areas on the contralateral side of the brain to the injected eye (there is almost complete decussation at the murine chiasma). The first lesions were seen either in the superior colliculus or the dorsal lateral geniculate nucleus, depending on the scrapie model; these are both major projections of the optic nerve. The pattern of subsequent lesions within the visual system also varied, gradually affecting both sides of the brain, until in the terminal mouse, the lesions were usually similar to those resulting from an intracerebral route of infection. The spread of the infectivity following intraocular infection was studied by bioassay of tissues throughout the incubation period. The rise in infectivity levels of ME7 scrapie correlated closely with the sequence of lesion targeting, although infectivity was detected much earlier in the incubation period. Several questions relating to the sites of replication, and the spread of infectivity were investigated by bioassay of appropriate tissues. The lower levels of infectivity in retinas from mice with an inherited retinal defect, compared to normal mice, suggested that the photoreceptor cells support scrapie replication; these cells are destroyed following infection with 79A scrapie. The <i>Sinc</i> gene was shown to delay replication in the single neuronal relay to the superior colliculus by 50 - 60 days, suggesting that this gene acts on the transport and/or replication of infectivity. High levels of infectivity were found in the spleen following intraocular infection, indicating that inoculum escaping from the eye initiates replication in the lymphoreticular system. Similarly. disease could also be produced by conjunctival instillation of inoculum with no evidence of contralateral targeting of lesions. The way in which infectivity spreads within neurons was studied using serial enucleation after infection, alteration of retinal ganglion cell number in mice, and attempted modification of initial infection. Following serial enucleation of the infected eye, the lesion pattern and incubation periods with ME7 scrapie indicated that infectivity spread at a similar rate to that of slow axoplasmic transport. Preliminary results with two other scrapie strains show similar timings, suggesting that spread within axons is a passive process associated with normal cellular metabolism. This means that <i>Sinc</i> gene control is exerted through its effect on replication rather than transport.

No description available.

The cellular prion protein (PrPC) is a glycoprotein with unknown function constitutively expressed in mammalian neurons. PrPC converts to a pathogenic misfolded isomer (PrPSc) through a poorly understood process, resulting in a group of fatal neurodegenerative diseases collectively known as transmissible spongiform encephalopathy or prion disease. Elucidating the molecular mechanisms behind prion conversion requires production of PrPC in recombinant systems. This study was designed to generate transgenic tobacco plants expressing recombinant mouse prion protein (mPrP). Using a synthetic gene encoding the mouse prion protein, plant expression vectors were constructed for constitutive mPrP expression in the apoplast (pGreen35SmPrP-Apo), cytosol (pGreen35SmPrP-Cyto) and endoplasmic reticulum (pGreen35SmPrP-ER). Putative transgenic plants transformed with either pGreen35SmPrP-Cyto or pGreen35SmPrP-ER were analysed by PCR, ELISA and immunoblot for transgene integration and expression. However, no viable plants were obtained from the pGreen35SmPrP-Apo transformants. ELISA analysis showed that recombinant mPrP accumulated up to 0.0024% of total soluble leaf protein in transgenic tobacco leaves transformed with the pGreen35SmPrP-Cyto construct and 0.0016% of total soluble leaf protein in plants designed to sequester recombinant mPrP to the ER. Furthermore, immunoblot analysis showed that ER-targeted recombinant mPrP was mainly unglycosylated, although a glycosylated mPrP isoform was observed indicating that transgenic tobacco plants process ER-targeted recombinant mPrP in a manner analogous to mammalian systems. The nutrient composition of several transgenic plants were analysed to determine the phenotypic effect of expressing recombinant mPrP in tobacco plants. The analysis revealed that transgenic lines expressing cytosolic-mPrP had elevated average levels of Mn2+ and Fe2+. In addition, kanamycin-treated transgenic plants expressing cytosolic-mPrP developed a non-rooting phenotype. Conversely, the average Cu2+ level was increased in analysed transgenic plants designed to sequester recombinant mPrP in the ER. Furthermore, the plants developed no visible phenotype upon kanamycin treatment. This result support studies that suggest that the PrPC has functional role in metal homeostasis and loss of its thermodynamic structure leads to metal dyshomeostasis which in turn has been linked to prion disease associated neurotoxicity. Finally, the recombinant mPrP was purified via affinity chromatography facilitated by the presence of a C-terminal polyhistidine tag on the synthetic gene constructs.

The capsid plays many important roles in the virus life cycle, including host cell recognition, cell entry, uncoating and protection of viral RNA. In this thesis, artificial selection, use of inhibitory compounds and structural analysis have been combined to further understand aspects of these multiple roles. The VP1 pocket is a hydrophobic cavity in the capsid that harbours a fatty acid, known as the pocket factor. The presence of this fatty acid can increase capsid stability and its release is required for the virus to uncoat. Certain compounds are able to bind to the pocket and displace the natural fatty acid (causing a further increase in capsid stability) and can inhibit infection by making the virus so stable that it is unable to uncoat. Novel compounds, termed NLD and ALD, have been designed in silico based upon the previous best EV71 pocket binding inhibitor GPP3. These were shown to inhibit EV71 infection in cells, with NLD being more than one order of magnitude more potent than the previous best EV71 pocket-binding inhibitor. Resistance towards these compounds was studied to reveal a double mutation in VP1 (I113M and V123L). Mapping the mutations to the EV71 crystal structure revealed that they were located in the VP1 pocket, and modelling predicted that they would prevent NLD and the natural pocket factor from binding. Further characterisation of the resistant isolate revealed that the mutations were thermally- and genetically-unstable. Further investigation on thermal stability involved selection of a thermally-stable virus. This generated viruses with a double mutation in the VP1 pocket (V179A and L183V). These mutations were predicted to increase the size of the VP1 pocket, and were shown to affect the way the virus interacts with the natural pocket factor. The effects of a variety of different pocket factors on the heat stability of WT EV71, the thermostable mutant, and the inhibitor-resistant mutant were analysed. In addition, work was conducted to assess the effect of pH on uncoating. For uncoating it is known that EV71 must be incubated at a low pH in the presence of its major receptor SCARB2. To investigate the effects of this, EV71 was incubated at a low pH during infection. This was shown to reduce the virus titre, and repeated exposure to this condition selected for a VP1 mutation (N104S). This residue is predicted to be involved in binding to SCARB2, and the mutant virus was shown to differ in susceptibility to compounds that affect entry and uncoating.

No description available.

Studies essentially involved examination of the structural details of the pathogenicity of single and mixed infections of mycoplasma and CELO virus in tracheal explants and infected chickens. The first part of the work concerned study of the growth response of the parasites in explants, alone or together. This was followed by study by light and electron microscopy of their effects, and interaction with the explants. Tracheal specimens from infected birds were also examined for any evidence of pathogenicity similar to that found in explants. In the explants there was evidence for interference between the growth of the parasites but only with Mycoplasma gallisepticum were extensive cytopathological effects detected. Mycoplasma gallinarum also caused some damage to the tissues but by a mechanism which appeared to be different to that associated with M. gallisepticum, which early on developed an intimate relationship with the cells. Structural differences between the two mycoplasmas were also detected in the scanning and transmission electron microscope which probably related to their functional activities. CELO virus was virtually without effects on the explants although viral material was seen in many epithelial cells, and there was little evidence for a synergistic relationship between virus and mycoplasma. No morphological evidence of mycoplasma or virus was found in the trachea of infected birds and the pathogenic effects, which were detected, seemed to reflect more on the treatment the birds experienced at infection than on the activities of the parasites.

A virus isolate, strain B1209, was shown by morphological, physicochemical and biological studies to be an adenovirus. The virus grew and produced plaques in both chicken kidney and chick embryo cell cultures, the higher infective titres were obtained in the former cell system. B1209 also grew to a high titre in embryonated chicken eggs, causing stunting and curling of the embryos. The virus was shown to agglutinate rat RBCs and the effect of various parameters on the reaction were examined. Substances present in the chicken sera, inhibiting the HA activity of the virus with a pattern differing from that of "true" inhibition, were considered to be non-specific inhibitors. B1209 was serologically indistinguishable from chicken embryo lethal orphan (CELO) virus. Adenovirus infection was found to. be widespread in chicken flocks the incidence increasing with age to about 70-80% in birds over 60 week old. In experimentally infected chickens, neutralizing and HI antibodies appeared almost at the same time and followed the same pattern of persistence, while precipitin antibodies were detectable slightly earlier but showed a cyclic appearance in many cases. B1209 and GAL 3 strains induced a generalized infection which was not prevented by maternal antibodies in the latter experiment. No significant difference was found in the susceptibility of the very young chicks and older birds to infection but there was an earlier decline in the faecal virus titre in the latter. A delayed antibody response was evident in young chicks. The pathology of CELO virus infection by its own or in conjunctio with Mycoplasma gallisepticum was studied in a series of experiments, using various routes. Clinical signs in birds infected with virus alone consisted of depression, sneezing, coughing and gasping, This was more severe in adult birds. In gross examination, the most striking lesions were severe hyperaemia and exudation in the lungs, and varying degrees of air sacculitis, produced in aerosol experiments. In histological examinations, these lesions in the lungs corresponded to consolidation and pneumonia, and in the air sacs to exudation, oedema and epithelial hyperplasia. More severe air sacculitis, and also epithelial metaplasia in the air sacs, seen in dual infections, were considered to be the features of the synergistic effect of the virus and mycoplasma. This was also supported by the results of serological tests and mycoplasma isolation. Further studies are needed to elucidate the pathogenicity of the other strains or types of virus, and to relate the experimental data to the disease problems in the field.

The purpose of the research was to investigate the biological features of human cytomegalovirus replication. At the onset of the project five years ago, the amount of information available concerning this subject was limited. During this time results of some comparable studies have been published and the results presented here confirm and complement such studies and contribute to the understanding of virus-directed events in the infected cell. Evidence for virus replication was obtained from (a) microscopic and macroscopic cytopathology (b) cytopathology using acridine orange (c) immunofluorescence (d) quantitative analysis of protein and nucleic acid synthesis using the pulse labelling procedure and (e) titration of infectivity. Virus replication was inhibited by physical and chemical factors including (a) temperature (b) inhibitors of protein synthesis and (c) inhibitors of nucleic acid synthesis. Each inhibitor was added or removed at selected times and the effects were monitored by one or more of the methods mentioned above. It is concluded from the results that viral transcription and translation were essential for the initiation of cell rounding and the early immunofluorescence. The viral protein or oligopeptide responsible may be rich in particular amino acids, for example arginine. An early consequence of infection was the reduction in host-cell macro-molecular synthesis. Cytomegaly developed after the initial cell rounding provided protein synthesis was not interrupted. At 48 hours the infected cells, termed single cell foci (SCF), were easily enumerated. By 84 hours pi infective virus was detected in the cell-free medium and reached a maximum level about 144 hours pi; the level of infectivity in the cell-free medium was maintained for long periods. The onset of DNA synthesis in cells infected with the Rawles virus was 37 hours pi. Protein synthesis was required to initiate and maintain DNA synthesis and uninterrupted DNA synthesis was required for continual virus production. Virus growth was blocked when an inhibitor of host-cell polymerase type II was added to infected cells at 24 hours pi but this inhibitor had no effect at 48 hours pi or later. Virus production and DNA synthesis were blocked at a temperature of 40&deg;C or higher. Two temperature sensitive events were highlighted: one event was completely blocked and one event was partially blocked and the kinetics of the latter event were affected by small changes in supraoptimal temperature. Both events were required for viral DNA synthesis. However, even when viral DMA synthesis had been allowed to occur, production of infective virus was still prevented by shift-up to supraoptimal temperature.

The relevant published literature concerning the nature of herpes simplex virus and its growth in fertile hens' eggs was reviewed. Laboratory strains and fresh isolates of types 1 and 2 were grown on the chorioallantoic membrane of fertile hens' eggs and examined by biological, histological and ultra-structural techniques. The type 1 strains induced small discreet pocks, gave no haemorrhage of the chorioallantoic membrane, or embryo, embryos did not die and virus was recovered only from the inoculated membrane. Similar inoculation with the type 2 strains induced large necrotic pocks and haemorrhage of the chorioallantoic membrane as well as haemorrhage and death of the embryo; virus was recovered from the inoculation site, allantoic fluid, amniotic fluid and various selected organs of the embryo. Inoculation of either virus type into the allantoic cavity did not result in spread to the embryo. The effects of adaptation to growth in eggs were examined. Temperature marker tests in eggs showed that fresh isolates of type 1 grew less readily on the chorioallantoic membrane at elevated temperatures than those of type 2. There was no difference in the capacity of laboratory strains of either type to grow in eggs at these temperatures. Primary chick embryo fibroblasts and other egg-derived cell cultures were used to examine the growth characteristics of a strain of each virus type; the results obtained in vitro could not be entirely predicted from those in ovo. The structure of the chorioallantoic membrane and the lesions produced by each virus type following inoculation were examined by optical and electron microscopy. The type 1 induced lesion was basically proliferative and confined primarily to the chorion with some inflammatory cell infiltration into the mesoderm, particularly following prolonged incubation. With the type 2 lesion, reaction occurred throughout the entire thickness of the membrane and haemorrhages, necroses, ulceration and cellular infiltration of the mesoderm were the most prominent features. The fine structure of the herpesvirus lesions and of viral morphogenesis was examined. Inoculation with type 2 virus resulted in many more infected chorion cells compared with type 1, whilst the cells of the mesoderm and the blood vessels also became infected with type 2 virus but not type 1. Features specific to type 2 virus infected cells were the presence of two types of intra-nuclear granules and lattice structures in both nuclei and cytoplasm. Cores with various structured forms were also found in type 2 capsids but not in those of type 1. The results of this study were discussed in relation to other published work.

The behaviour of f2 coliphage and poliovirus I in activated sludge treatment was studied under different operating conditions of flow through time, mixed liquor suspended solids, temperature and virus loading in a bench scale model plant whose performance was similar to that of a full scale treatment plant. The liquid and solids fractions of mixed liquor samples containing virus were assayed separately, with the solids fraction receiving ultrasonic treatment. The recovery of poliovirus from mixed liquor by this method was about 83%, while that of f2 coliphage was in the range of about 54-85%. The average removal of f2 coliphage across the model plant was about 84 %, and was not significantly altered by altering the flow through time, mixed liquor suspended solids and temperature in the plant. The removal was, however, decreased from about 96 % to about 70% with higher virus loads. The association of f2 with the mixed liquor solids showed an inverse relation with increased flow, a direct relation with increased mixed liquor solids, and apparently direct relation (with an optimum) with increased temperature and a clear direct relation with increased virus load. The removal of poliovirus across the plant over the range of conditions studied was generally high and reached up to about 99.7 %. The behaviour and removal of both viruses in the model plant correlated with the association of these viruses with the suspended solids. The degree of association, which appeared to depend upon the nature of each virus and was achieved by physical adsorption, was strikingly contrasting with about 18 and 85 % of f2 and poliovirus respectively detected on the solids. The striking differences between the behaviour of f2 coliphage and poliovirus I imposed interesting implications on the concept of indicator virus from the public health viewpoint.