Viruses are biological agents that infect cellular organisms. Most viruses are bacteriophages, these are the most abundant biological entities on earth. Not much is known about virus diversity in the human mouth, including dental plaque, compared to other environments. A culture-independent based approach was tried using metagenomic analysis to characterize uncultured virus gene fragments in human dental plaque. The isolated viral genomes were amplified using a multiple displacement amplification method. Eighty, eleven and ten clones were sequenced from three volunteers, respectively. TBLASTX analysis showed that 44% of the sequences had significant identities to the GenBank databases. Of these 66% were viral; 12% human; 10% bacterial; 6% mobile and 6% eukarya. These sequences were sorted into six contigs and forty five single sequences. Four contigs and one single sequence were found to have a significant identity to a small region of a putative prophage in the Corynebcterium diphtheria genome. The gaps between these were filled by primer walking and PCR to give a continuous contig of 11554 bp. Two viruses A1 and A2 and their bacterial host were isolated from the human mouth. The 16S rRNA gene sequence of the host had a 99% identity to several Neisseria sp. The A1 virus was found to appear spontaneously on soft top agar plates, and might be a lysogenic virus. The A2 virus was a lytic virus. The two viruses have different morphological shapes. A1 has a varied isometric head size that ranges from 32 to 58 nm and no tail; it may belong to the Tectiviridae family. It has a linear dsDNA genome with a size between 12 kb and 23kb. A limited amount of the genome of the A1 virus was sequenced. The A2 virus has an icosohedral head with size of 60±3 nm and a sheathed rigid tail about 175 nm long with no detectable base plate or tail fibres. It can be classified into the order Caudovirales family Siphoviridae. The size of the A2 virus genome is estimated to be 35 to 40 kb. 31703 bp of unique sequence has been determined and sorted into three contigs and 14 single sequences. Further attempts at gap filling using primer walking and PCR were unsuccessful. It has a linear dsDNA genome, with a GC content of 49 mol%. A latent period of 25 min and a burst size of 25±2 particles were determined by a single step growth curve. Bioinformatic approaches were used to identify ORFs in the genome. A2 virion associated proteins were analysed by SDS–PAGE gel electrophoresis, and some proteins sequences were directly related to the translated genomic sequence.

The RNA genomes of neurovirulent and attenuated type 3 polioviruses have been cloned in E. cola using an efficient RNA.cDNA hybrid technique. The complete nucleotide sequence of the vaccine-associated neurovirulent revertant P3/119 and, in collaboration with others, the attenuated vaccine strain P3/Leon 12 a1b, have been determined. These have been compared with that of the neurovirulent parent strain P3/Leon/3 7. Ten nucleotide sequence differences were observed between the parent P3/Leon/37 and the vaccine P3/Leon 12 a1b, three of which resulted in amino acid substitutions. Between the vaccine and the revertant P3/119, seven nucleotide sequence differences were observed. Three of these resulted in amino acid substitutions. The possible significance of individual nucleotide sequence differences to the attenuation of and reversion to neurovirulence in poliovirus type 3 is discussed. The nucleotide sequence of P3/Leon 12 a1b was the first to be determined for a type 3 poliovirus. Comparison of this sequence with published type 1 sequences has demonstrated the extent of the molecular homology between them.

No description available.

This work is concerned with the ability of poxviruses to overcome the antiviral state induced by interferons. A major part of the antiviral state induced by interferons is mediated by PKR, a serine/threonine protein kinase activated by dsRNA which phosphorylates eIF-2a, thereby inhibiting translation initiation. Many viruses interfere with the PKR response. Vaccinia virus (VV), like all poxviruses, replicates in the cytoplasm where it is directly exposed to the action of PKR (and 2'-5'A synthetase). In order to replicate it must overcome the action of interferons and PKR (and 2,-5,A synthetase). VV encodes proteins which bind interferons and encodes two proteins known to interfere with PKR: E3L, encodes a dsRNA-binding protein and K3L, encodes a homologue of eIF-2cx. The investigation has shown that an avian poxvirus, fowlpox virus (FPV), is resistant to more than 8 U/ml chicken IFN. FPV is capable of rescuing an IFN-sensitive virus, Semliki Forest virus (SFV) from the effects of chicken IFN. FPV is also capable of promoting the replication of SFV in the presence or absence of IFN. These results suggest that FPV does make antagonists of IFN. Biochemical assays were used to test IFN-treated avian cell extracts for the ability to phosphorylate known substrates of PKR. Major changes occur in the phosphorylation profile of CEF treated with IFN. Poly IC-binding proteins from IFN-treated CEF are capable of phosphorylation of histone proteins and phosphorylation of mammalian eIF-2oc peptides. These data suggest the presence of a protein(s) with properties similar to PKR. Attempts to clone an avian homologue of PKR from a commercial chicken cDNA library, proved unsuccessful. A FPV homologue of E3L does not lie between the homologues of E2L and E4L. Southern blot analysis of FPV DNA suggested that homologues of E3L and K3L exist in a 5.3 kbp restriction fragment. The appropriate fragment has been cloned and sequenced. The sequence shows that FPV has potential ORFs to encode homologues of VV E10R, E11L, OIL, I1L, I2L, and I3L but no ORF for E3L, K3L or 02L could be found.

The object of the work described in this thesis was to investigate the attachment, and penetration of the host cell walls and membranes by the minute bacteriophage andPhi;X 174. In an attempt to simplify the investigation, the interaction between viral particles and cell walls isolated from the bacterial host, Escherichia coli strain C, was examined. Conditions for maximum, irreversible adsorption to cell walls were shown to be in buffered solutions at pH 6-9, at 35-45°, in the presence of calcium or magnesium ions. It was established that these conditions gave efficient attachment to intact bacteria. When phage particles labelled with ³²P-phosphate became attached to isolated bacterial cell walls, up to 30% of the viral ³²P was converted to a form accessible to the enzyme, pancreatic deoxyribonuclease (DNase). This indicated that some of the phage deoxyribonucleic acid (DNA) was released on attachment of virus to cell walls. At the same time, some of the adsorbed andPhi;X 174 virus were converted to particles which could be eluted from the cell walls by a solution of sodium borate, containing ethylene diamine tetra acetate (EDTA). The particles were resolved into two components on fractionation by sedimentation through sucrose density gradients. One component consisted of particles with a sedimentation coefficient of 45-50S (50S particles). When viral preparations, doubly labelled with ³²P- and ³⁵S- were used, comparison of the value of the ³⁵S/³²P ratio in the original phage and in the 50S particles, showed that the latter contained the same quantity of DNA as the original virus. In contrast to intact virus, however, the 50S particles were infectious towards bacterial spheroplasts, but not towards whole bacteria. Examination in the electron microscope of 50S particles, isolated by sedimentation through sucrose gradients, failed to reveal any gross contamination with cell wall or other material, which might have caused anomalous sedimentation behaviour. It was concluded that a conformational change in the arrangement of the viral protein sub-units occurred on attachment to the cell walls and was responsible for the changes in infectivity and sedimentation coefficient. Similar investigations on the second component, showed that it had a sedimentation coefficient of 70, was not infectious towards whole bacteria or spheroplasts, and handd a reduced DNA complement. The particles appeared to be identical with the "top component" previously identified by others in preparations of andPhi;X 174 (e.g. Sinsheirner R.L., (1959) J. Mol. Biol. <strong>1</strong>:37). Attachment of andPhi;X 174 to intact, starved bacteria suspended in buffer also resulted in the formation of 50S and 70S particles, which were eluted from the bacteria by sodium borate and EDTA. After prolonged incubation (30 minutes) of virus with either cell walls, or intact bacteria, only the 70S component could be detected in eluates made with borate-EDTA solution. When the viral material eluted from cell walls was treated with DNase there was no loss in infectivity detectable in the bacterial spheroplast assay. It was concluded that all of the infectivity resided in the 50S component, and none in the viral DNA released on attachment of the phage particles. The results show that changes occur in andPhi;X 174 particles when these attach to isolated cell walls or to non-metabolising bacteria. The possible role of the 50S component in the natural infectious process could not be determined from the experiments described here. Its properties suggest that it contains a hitherto unrecognised particle which can be formed by conformational changes in whole intact andPhi;X 174.

Insect pathogenic viruses can be divided into five groups. Nuclear polyhedrosis viruses, cytoplasmic polyhedrosis viruses, granulosis viruses, and 'pox-like' viruses are viruses of the occluded type in which the virus particles are embedded in a large crystalline inclusion which may be either polyhedral or capsular in shape. Other viruses do not occur in such 'inclusion-bodies' and are often referred to as non-occluded viruses. Nuclear polyhedrosis viruses have been extensively investigated but knowledge of many of their properties is ill-defined or lacking and assumptions have been made about their structure and multiplication which are contrary to accepted concepts of virus assembly. In this work two nuclear polyhedrosis viruses have been investigated, largely by electron microscopic methods, in order to determine how the structural and dynamic properties of the viruses can be correlated. Consideration has also been given to the compliance of these properties with current thinking on virus structure and replication. The viruses employed were those of the small tortoise-shell butterfly, Aglais urticae, and the gipsy moth, Lymantria dispar. Polyhedra were extracted from infected cadavers and purified by density gradient centrifugation. Suspensions of polyhedra were subjected to various alkaline treatments to solubilize the crystalline matrix of protein and to liberate the occluded virus particles. These virus particle and an aggregated form of the polyhedron protein were separated and purified most effectively by density gradient centrifugation. Samples of virus particles and aggregated polyhedron protein were examined electron microscopically and used for the production of antisera. Antisera were also produced to polyhedron protein solutions obtained, by precipitation, from dissolutions of polyhedra from which virus particles and aggregated polyhedron protein had been removed by centrifugation. Serological tests in gel and amine acid analysis showed that the different types of polyhedron protein were related, but some probably lacked certain antigens. Serological tests showed no relationship between virus particles and polyhedron protein but the polyhedron proteins from the two viruses were related but not identical. Aggregated polyhedron proteins often exhibited a lattice-like arrangement when examined electron microscopically in negatively stained preparations and from a comparison of such lattices and the lattice structure of the polyhedron protein seen in ultra-thin sections of polyhedra a cubic arrangement has been proposed which is built from inter-connecting six-armed nodal units. The polyhedron is bounded by a membrane and preparations containing such membranes could be harvested from solutions of dissolved polyhedra by centrifugation. When examined electron microscopically the membranes appeared to possess a cubic lattice structure of similar dimensions to polyhedron protein. Closer examination however showed them to be composed of holes or hollows arranged hexagonally each hole having a central core. Polyhedron membranes did not have the structure of a unit membrane when seen in section. Virus particles extracted from polyhedra were either enveloped, naked, or empty. In A. urticae polyhedra the virus envelope (or outer membrane) surrounds a single virus particle whereas in L. dispar polyhedra bundles of virus particles are surrounded by the envelope. The envelope appears to be a three-layered structure with an outer surface layer in which no detailed substructure can be resolved, a layer of hexagonally packed subunits or peplomers 20 mandmu; in diameter arid a flexible membrane composed of 40andAring; subunits, hexagonally packed, which resembles a unit membrane when seen in section. It is likely that the surface layer and the peplomers are virus-coded structures which become attached to a host cell membrane. The virus particle consists of an internal component which is probably coiled, surrounded by a virus capsid (or inner membrane) which is apparently composed of 30andAring; subunits arranged in a diamond-shaped lattice network. The virus capsid appears to connect those virus particles (or nucleocapsids) enveloped in bundles one to another as a result of 'pinching' of the capsid. However it is likely that each individual virus particle is capable of establishing virus replication as the number of virus particles within an envelope varies considerably. The dynamic properties of the viruses were investigated by dissecting larvae at intervals after infection and embedding certain tissues for ultra-thin sectioning and electron microscopy. In A. urticae larvae enveloped virus particles were found adjacent to the microvilli of midgut columnar cells and naked virus particles were found within the microvilli. The nuclei of these cells were usually virus-infected but although some crystalline polyhedron protein was present in the nuclei no polyhedra were formed. Enveloped virus particles were found both in the cell cytoplasm and in the underlying basal lamina. The tracheal epithelium cells beneath the basal lamina were normally virus-infected and polyhedron formation occurred in the usual manner. Such infected columnar cells were not observed in L. dispar larvae but enveloped virus particles were found in the basal lamina. It is suggested that the pathway of infection of the virus is initiated by infection of the gut columnar cells as a result of attachment to the plasma membrane by the virus envelope. Many enveloped virus particles are then produced in the nuclei of these cells without the formation of polyhedra. The enveloped virus particles are therefore available for the infection of cells and tissues in the host larva and the virus spreads largely as a result of infection of the epithelial cells of the tracheal system. Typical development of the viruses was studied in fat body cells. A large densely staining network, the virogenic stroma, is clearly visible in the enlarged nuclei of the cells and naked rod-shaped virus particles appear to be produced in association with it. The virus particles acquire an envelope in spaces within the virogenic stroma or in the area between the virogenic stroma and the nuclear membrane. In L. dispar virus-infected cells the virus particles are often criss-crossed within the envelope. Polyhedron protein crystallines in a lattice arrangement between the enveloped virus particles and compression of the envelope against the virus particles takes place as a result. The criss-cross formation of the virus particles within the envelope in L. dispar virus-infected cells becomes compressed into a side-by-side arrangement because of this process. The polyhedron membrane is visible as a densely stained periphery around mature polyhedra. Similar infected tissue was embedded in water-soluble glycol methacrylate and the sections were treated with enzyme solutions. The virus particles were digested both by DNase and pronase but the virogenic stroma was mostly affected by pronase. Polyhedron protein was somewhat resistant to digestion with pronase. Phospholipase D appeared to cause partial digestion of the virus envelope. Nuclear polyhedrosis viruses are extremely complex structurally but conform to the general concepts of virus assembly. The dynamic properties of the viruses cause gross disturbance of the architecture of the normal host cell nucleus and many of their structural components can be observed during the replicative process. Nuclear polyhedrosis viruses raise problems of nomenclature and classification which can only be resolved by further work on their properties. Such work should help to establish these viruses as useful and stimulating entities for study.

The work described in this thesis set out to elucidate the heterogeneity of lymphocytes from rats by investigating methods for the separation and analysis of lymphocyte sub-populations. In order to separate lymphocytes on the basis of differences in their size, the technique of velocity sedimentation under the influence of gravity in a shallow albumin gradient was applied to rat thoracic duct lymphocytes. Tractions containing small lymphocytes, contaminated by fewer than 0.3% large, dividing cells could be obtained and were tested for their immunological function. In addition, large lymphocytes could be enriched from the 5% or so found in normal lymph to 50 to 90% in fractions from the second sedimentation. The separation was checked by sedimenting thoracic duct lymphocytes in which the large cells were specifically labelled by tritiated thymidine (CHAPTER III). The tests to which the fractions were subjected included (a) their ability to restore a primary response to Salmonella adelaide flagella in irradiated rats (b) the adoptive transfer of the secondary response to tetanus toxoid (c) their graft-versus-host activity across a strong histocompatibility barrier. In each ease, small lymphocytes purified by velocity sedimentation performed equally as well as unfractionated cells, confirming the results of earlier authors using other purification techniques, while in assays (b) and (c) fractions containing large lymphocytes were less active; such activity as there was could have been explained by small lymphocyte contamination. No positive immunological function of large lymphocytes could be checked, however, so that it was possible that their poor performance resulted from handling the cells and the conditions of sedimentation. With this reservation, it was concluded that small and not large lymphocytes are the cells responsible for the induction of the immune responses investigated. (CHAPTER III). Using the same technique, it was shown that in the spleen of rats depleted of lymphocytes through a thoracic duct fistula, the immunological memory that the rats still possessed was carried by small lymphocytes and probably not large, dividing cells. The performance of these non-recirculating small cells was, surprisingly, as good as that of recirculating small lymphocytes from the thoracic duet (CHAPTER III). Possible explanations are discussed in CHAPTER VI. The ability of the technique of velocity sedimentation to discriminate another heterogeneity was then established. Thymus-dependent (T) and thymus-independent (B) lymphocytes were distinguished and partially separated according to the following criteria. Rapidly sedimenting (approximately 4.5 to 5.0 mm/hr) small lymphocytes from the thoracic duct took up considerably more uridine on incubation in culture than more slowly sedimenting smell lymphocytes: the peak of greatest incorporation coincided with the position to which T lymphocytes from radiation chimaeras sedimented. In addition, the rapidly sedimenting cells migrated in the manner characteristic of T lymphocytes when intravenously transfused to syngeneic recipients; they were to be found, after 24 hours, in the peri-arteriolar regions of spleen and in the deep cortex of lymph nodes. In the sedimented fractions they also coincided with the peak of graft-versus-host activity, which is probably initiated by T lymphocytes. These studies (CHAPTER IV) therefore showed the existence of T and B lymphocytes in normal rat lymph, and indicated a way in which they might be partially separated. Columns of fine siliconed glass beads have been used in the past to filter out large lymphocytes and thus to collect small lymphocytes in the filtrate. In CHAPTER IV it was suggested that such columns could also discriminate T and B lymphocytes, B lymphocytes being preferentially retained: the argument in support of this depended on the enrichment in the filtrate of cells from thoracic duct lymph able to take up relatively great quantities of uridine, shown earlier to correspond to T lymphocytes. The depletion of B lymphocytes by the columns could explain the results of certain experiments where small lymphocytes purified by this technique were found to be defective in their ability to induce humoral antibody formation (CHAPTER III). These experiments also allowed the derivation of a minimum estimate of 30 to 35% for the proportion of B lymphocytes in the rat thoracic duet (CHAPTER IV). The possibility of developing a rapid and simple plaque assay to enumerate the frequency of lymphocytes in a test population bearing a receptor for a particular antigenic determinant was explored in CHAPTER V. The assay involved the binding of dinitrophenylated bacteriophage to thoracic duct lymphocytes from normal and immune donors and counting the numbers of phage eluted from the cells by competing free hapten, after their immobilisation on an agar plate containing indicator bacteria sensitive to the phage. Various factors influencing the measured frequency of antigen-binding cells were investigated, including temperature of the incubation, alterations in the cell:phage ratio and methods of washing the cells, A model system using antibody-coated Sephadex beads was also studied, which revealed the complication that binding of phage to the surface was very firm (possibly due to polyvalent attachment) and was not readily reversed by free hapten. The chief factor preventing the useful application of the assay was the variability of the results, for which the causes were investigated: suggestions were made for improving the assay (CHAPTER V). To accompany the studies with dinitrophenylated bacteriophage the responses of rats to immunisation with dinitrophenylated bovine gamma globulin, and the performance of their thoracic duct lymphocytes when transferred to irradiated recipients were studied (CHAPTER V). A passive haemagglutination assay was developed to measure the responses, which used rat erythrocytes coated with a dinitrophenylated, non-agglutinating fragment of rabbit immunoglobulin whose antibody activity was directed against rat erythrocytes (APPENDIX I). It turned out to be a sensitive assay, and substantial titres of anti-DNP antibody could be neaaured following primary or secondary immunisation of rats with hapten-protein conjugate.

Natural and induced factors inhibiting foot and mouth disease virus were investigated in bovine secretions, especially in those from the upper respiratory and oro pharyngeal areas. Techniques were devised to collect lachrymal, nasal, buccal and pharyngeal fluids from normal, convalescent and passively or actively immunised steers. The pH and total protein content of secretions were established in normal cattle. Immunoglobulin types IgA and IgGl predominated. Interferon was not detected. Normal tears exhibited no antiviral activity but nasal secretion, oral saliva and p~al fluid were inhibitory due to their alkaline pH and, in the case of salivary fluids, to the presence of an additional anti viral factor which was partially characterised. Virus lost infectivity in vitro due to natural, non specific factors at rates which varied with the strain of virus to a maximum of 1.25 log units per hour. Clinical disease, viral excretion, interferon and antibody were studied following infection with virus of types 0, A and C. Interferon was detected for up to 4 days at the period of maximum viral excretion. Secretory neutralising antibody developed , - 5 days after exposure, reached a peak within 21 - 28 days and was associated principally with IgA and IgGl. Serum levels were consistently greater than those in secretions. Passive immunisation studies showed that some secretory antibody was derived from serum. years after infection. Antibody persisted for at least ~ Secretory antibody levels increased with successive subcutaneous doses of inactivated vaccine. A single dose elicited good humoral but poor secretory responses. Levels in serum and secretions rose after a second dose and approached convalescent levels after a third. Secretory antibody was principally IgGl but after a third vaccination IgA was also detected. The relation between secretory antibody and the outcome of exposure to infection was studied. In steers vaccinated once or twice and exposed 14 days after the last vaccination, no lesions were observed and little virus was recovered from their secretions excepting pharyngeal fluid. After three vaccinations much less pharyngeal virus was recovered following exposure. Convalescent animals reexposed 5 and a half years after primary infection were immune and did not become carriers, pharyngeal samples remaining negative. These observations correlated well with the presence of neutralising IgA in the secretions.

No description available.

No description available.