MHV-68 is a gammaherpesvirus that is related to Epstein-Barr virus, Kaposi’s sarcoma associated herpesvirus, and Herpesvirus saimiri. The aim of this thesis was to characterize the role of the putative plasmid maintenance protein encoded by ORF73 of MHV-68 in viral latency and pathogenesis. In order to investigate the role of this gene, MHV-68 mutant viruses deficient for ORF73 were constructed and the recombinant viruses were analysed <i>in vitro</i> and <i>in vivo</i>. It was determined that ORF73 was not required for lytic replication of MHV-68 <i>in vitro</i> or<i> in vivo.</i> In contrast, a severe deficit in viral latency <i>in vivo</i> was observed in the ORF73-deficientviruses suggesting that the virus was unable to persist in the host in the absence of ORF73. Furthermore it was shown that this gene product is necessary for the maintenance of latent viral episomes in an <i>in vitro</i> assay utilizing a mouse myeloma B-cell line, NSO, indicating that ORF73 is a critical determinant in gammaherpesvirus latency. In this project I also attempted to rescue the phenotype of the ORF73-deficient virus by insertion of an ORF73-EGFP expression cassette at an ectopic genomic locus. While expression of ORF73 at this site enabled maintenance of viral episomes in NSO cells <i>in vitro, </i>the <i>in vivo</i> latency deficit was not restored. Further <i>in vitro</i> characterization of the ORF73-deficient and ORF73-EGFP rescuant viruses suggests that ORF73 has a role in the regulation of lytic replication. A final aspect of this project involved the potential use of an ORF73-deficient virus as a live-attenuated gammaherpesvirus vaccine. Analysis of vaccinated animals challenged with wild type MHV-68 showed that exposure to the ORF73-deficient virus afforded significant protection against wild type infection and reduced viral latency to a level below the limit of detection. These results indicate that a live-attenuated gammaherpesvirus that cannot persist is an effective vaccine.

The major part of the dissertation concerns the isolation and genetic characterisation of temperature sensitive mutants of an avian influenza virus, Fowl Plague Virus (FPV). Thirty four mutants of the Rostock strain of FPV have been studied by genetic recombination and shown to fall into six high-frequency recombination groups. The hypothesis that each of these groups corresponds to a single genome segment (single gene) of FPV, led to the work reported in the second part of the dissertation. Each recombination group has been assigned to a particular segment of the FPV genome in the following way. Non-t.s. interstrain recombinants have been constructed from a t.s. Rostock parent and another FPV strain (Dobson) whose RNA segments can be distinguished on the basis of electrophoretic migration in polyacrylamide gels. Determination of the parental origin of each gene in a number of recombinants has led to the identification of the gene carrying the t.s. lesion in the mutant parent. A similar analysis of virus specified polypeptides has also allowed the identification of the defective protein. As a consequence of these analyses the coding function of each segment of the FPV genome has been determined. A separate chapter of the dissertation has been devoted to an investigation of the genetic basis of host range of FPV Dobson. Its relevance to the question of virulence in influenza viruses is discussed. The dissertation concludes with a discussion on several interesting features of certain mutants which may give some indication of their functional defect.

A full length endoE gene construct has been generated from the partial clones used to sequence the gene. Recombinant endoE has been expressed and purified as a fusion protein that is catalytically active and possesses the same kinetic characteristics as the bacteriophage enzyme. The mature, active endoE is 76 kDa in size and is probably produced by the post-translational cleavage of a 90 kDa primary translation product. Recombinant endoE has been purified to homogeneity, in milligram quantities, with the fusion protein partner removed for further studies of the enzyme. The role of the C-terminus of endoE was studied by expression of C-terminally truncated proteins. These were expressed from 3' truncated endoE gene constructs generated either by exonuclease III-mediated nested deletion or by PCR. The site of proteolytic processing has been located to within 18 amino acids and further truncation beyond this cleavage site renders the enzyme inactive and more susceptible to proteolytic degradation. The endoE sequence contains two copies of the sialidase motif and one P-loop motif. The importance of these motifs in enzyme activity was studied by site-directed mutagenesis, replacing part or all of the motifs with alanine residues. These indicated that the P-loop motif was not involved either in binding or hydrolysis of polysialic acid and that the sialidase motifs appear to be necessary to allow production of correctly folded, soluble protein but are not involved directly in catalysis. The kinetic characteristics of the binding interaction of the endoE fusion protein and polysialic acid have been studied and compared to those of C-terminally truncated endoE fusion proteins. As a first step in investigating the use of endoE as a reagent in cell biological studies, the subcellular localisation of polysialic acid and the effects of chloroquine and brefeldin A treatment on its localisation in cultured MCF-7 breast tumour cells have been studied.

The role of GAGs in HSV-1 SC16 adsorption and entry, was examined here in the context of three different cell types using infection as a readout. These experiments demonstrate that the importance of GAGs in infection varies from one cell type to another, reinforcing the view that a role for GAGs as entry receptors is not universal for all virus - cell type combinations. As well as participating in receptor-mediated attachment and playing a crucial role in virion envelope-plasma membrane fusion, HSV-1 gD is also essential for cell-associated spread. On the basis that some of the essential functions of the gD of other related herpes viruses can become dispensable under certain conditions, a study was carried out to determine whether a variant of HSV-1, capable of gD-independent cell-associated spread could be isolated. However, these attempts were unsuccessful in the context of HSV-1, implying that the function of gD in cell-associated spread cannot be compensated for in other parts of the virus. Finally, the issue of secretion and release of HSV-1 progeny virus particles was examined. Electron microscopy studies established that large numbers of virus particles are present on the infected cell surface, and that the bottle-neck to production of virus in the tissue culture medium is release from the infected cell surface rather than secretion through the plasma membrane. Since it is known that components of virus particles interact with GAGs, the role of GAGs in release of progeny virions from the cell surface was investigated. In this way it was confirmed that in some cell types, the presence of GAGs is important in retaining progeny virus in a cell-associated state.

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Using fluorescent antibody virus neutralisation tests, optimised for each virus, I have measured the neutralisation titres of rabbit sera against a global panel of divergent lyssaviruses. Those neutralisation titres were then used to generate antigenic maps, allowing visualisation of antigenic distances to within 1.3 two-fold dilutions in titre. Antigenic distances were compared with glycoprotein ectodomain sequence data, of which 20/30 sequences were obtained by direct sequencing as part of this study. These comparisons revealed clinically important differences between genetic and antigenic relationships. Vaccine strains and wild-type genotype 1 rabies viruses were antigenically distinct, and three of the Eurasian bat viruses were antigenically similar to known genotypes. To evaluate the validity of extrapolating animal model data to other species, I have compared the serological responses of mice, dogs, monkeys, foxes and raccoon dogs using antigenic cartography. The responses of rabbits, mice, monkeys and dogs correspond but orally vaccinated foxes and racoon dogs do not. Passive immunoglobulin treatment is a crucial part of rabies post exposure treatment, and here I have also shown that it is possible to predict the titre of polyclonal passive immunoglobulin treatments to divergent lyssaviruses. Together the results of this study show a measurable spectrum of antigenic distances between lyssaviruses that can be used for rational design of active and passive preventative treatments.

The working hypothesis tested in this thesis that there was no strain variation between louping-ill virus isolates was found wanting. Diversity of antigenic subtypes and biotypes among louping-ill virus isolates was detected and the identified types appeared to be confined to distinct geographical location. A panel of 21 mouose monoclonal antibodies raised against the standard isolate of louping-ill virus (li.31) were selected on the basis of reaction to the virus in indirect immunofluorescence (IIF) tests. The monoclonal antibodies, further characterised on their ability to neutralise virus, inhibit haemagglutination, influence the course of infection in mice and their reactivity in an ELISA, fell into five groups based on their established characterisitics. Five monoclonal antibodies neutralized the virus. Only one antibody also possessed haemagglutination-inhibition (HAI) activity, and two protected mice very significantly against subcutaneous challenge with 2 x 10<SUP>4</SUP> plaque-forming units of louping-ill virus, where as for non-neutralizing antibodies enhances the viral pathogenicity as determined by the number and time when mice became affected. A two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed and a combination of 170 monoclonal antibody pairs were evaluated for detection of soluble viral antigens in this system. Competitive assay with seven selected monoclonal antibodies showed that epitopes defined by five antibodies were either close together on the virus particle or over lapped whereas the epitopes defined by the other two antibodies appear to be spatially distant from the other epitopes. The application of monoclonal antibodies in functional assays (neutralization and HAI) as well as in binding assays (IIF and ELISA) detected antigenic variation among 21 louping-ill virus isolates from 4 countries in the British Isles and 3 countries in mainland Europe. According to their reactivity in these tests isolates could be placed in five distinct groups three of which are divisible into two subgroups. Isolates from the same geographical location tended to be of the same group as did isolates from the same location from different species isolated over a 50 year period. Further characterisation of the isolates indicated that additional differences could be detected on the basis of plaque morphology and pathogenicity for mice which correspond to the groupings identified with the monoclonal antibodies.

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