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Strong adaptive evolutionary forces shape the interactions between pathogens and their hosts and typically lead to a stable co-existence. In this process of co-evolution, mammals have developed restriction factors that limit retrovirus infectivity, replication or assembly and narrow the spectrum of potential host species. These restriction factors are either constitutively expressed, such as APOBEC3 proteins, cytidine deaminases that interfere with reverse transcription, or form part of the type I interferon-induced innate immunity, such as TRIM5, a member of the tripartite motif protein family that induces degradation of retroviral capsid, blocks reverse transcription, or tetherin (BST-2, CD317), which inhibits release of nascent viral particles from infected cells. Conversely, viruses have evolved antagonists of restriction factors or proteins that limit IFN-induced gene expression, thus evading immune surveillance. The interaction between host and viral components is delicately balanced and has a significant impact on disease outcome. Feline immunodeficiency virus (FIV), a lentivirus closely related to human immunodeficiency virus (HIV), is a recent introduction into domestic cats and causes an immunodeficiency syndrome analogous to human AIDS. Interestingly, non-domestic cats such as lion or pumas have co-existed with lentiviruses for prolonged periods of time and FIV infections are largely benign. Although plasma viral and proviral loads are high in both domestic and non-domestic cats, in vitro studies have shown that FIV infection of non-domestic cat T lymphocytes is significantly less efficient than that of domestic cat T cells. Thus, this thesis tests the hypothesis that the differential disease outcome of FIV infections in felids is caused by differences in lentiviral restriction factor activities or their sensitivities to FIV restriction factor antagonists. Data presented in this study show for the first time that feline APOBEC3 proteins are expressed in tissues and cell types relevant for FIV infection. The APOBEC3 proteins A3H and A3CH exhibited a high antiviral activity against FIV lacking the APOBEC3 antagonist Vif in single-cycle replication assays, with no difference in activity being detected between domestic and non-domestic cat proteins. However, domestic cat A3CH was significantly more sensitive to antagonism by FIV Vif than lion or puma A3CH, which would allow efficient viral replication in domestic cat T lymphocytes and subsequently lead to T cell loss and immunodeficiency. Furthermore, this thesis provides evidence that felid tetherins can prevent FIV particle release from producer cells in single-cycle replication assays; however, stable expression of domestic and non-domestic cat tetherins in feline cell lines did not abrogate FIV replication. Indeed, syncytium formation indicative of viral cell-to-cell spread was significantly enhanced in type I interferon-treated feline cells infected with CD134-independent strains of FIV which often arise in chronic (late) stages of FIV infections in vivo. Finally, this work reports the generation of a synthetic domestic cat TRIM5α-cyclophilin A fusion protein which was highly efficient at preventing FIV pseudotype and productive infection. This novel feline restriction factor represents a potent antiviral defence agent with very low potential for toxicity and could in future be used in gene therapy approaches to treat FIV-infected cats.

Retroviruses possess several biological features that differentiate them from all other infectious agents. The obligatory integration step of the retrovirus genome into the host genome has allowed these viruses to associate, modulate and alter the biology of the cell with a variety of unique mechanisms. Integration of retroviruses into the germ line of the host results in the formation of vertically transmitted “endogenous” retroviruses (ERVs). It is now becoming apparent that ERVs have often been selected as they provided evolutionary advantages to the host. Sheep Betaretroviruses provide a unique biological system to study the complex interaction between retroviruses and their hosts. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a naturally occurring lung cancer of sheep. The JSRV Env glycoprotein is a dominant oncoprotein and its expression is sufficient to induce cell transformation in vitro and in vivo. Thus, OPA is a unique large animal model for the study of lung carcinogenesis. The sheep genome harbours at least 27 copies of ERVs highly related to JSRV (enJSRVs). Studies on enJSRVs have provided evidence supporting the idea that ERVs, exogenous retroviruses and the host have coevolved through a dynamic process throughout evolution. enJSRVs play a critical role in conceptus development and placental morphogenesis, and can block JSRV replication in vitro at both early and late stages of the replication cycle. The work presented here focuses on the study of the exogenous and endogenous JSRV Envs and their role in cell transformation and trophoblast differentiation respectively. We were able to show that: I) the JSRV Env transforms epithelial cells in vitro independently from its cellular receptor; II) both the exogenous and endogenous JSRV Envs interact with the receptor tyrosine kinase RON and that the cytoplasmic tail of the Env is the major determinant modulating the biological effects of the Env-RON interaction; III) the molecular chaperone Hsp90 regulates JSRV Env induced cell transformation, in part by downregulating Akt; and IV) OPA is a useful large animal model for the evaluation of new anti-cancer therapeutic agents. Moreover, we characterized the transforming properties, receptor usage and fusogenic activity of enJSRVs Envs to gain insight into their role in placental morphogenesis. The studies described in this thesis contributed to the understanding of JSRV induced cell transformation and the biology of enJSRVs.

To determine whether recurrent genomic imbalances are a feature of HL, CD30-positive HRS cells were laser microdissected from 20 cHL cases and 4 HL-derived cell lines and subjected to analyses by CGH. In primary tumours, the most frequently involved chromosomal gains were 17q (70%), 2p (40%), 12q (40%), 17p (40%), 22q (35%), 9p (30%), 14q (30%), 16p (30%), with minimal overlapping regions at 17q21, 2p23-13, 12q24, 17p13, 22q13, 9p24-33, 14q32, 16p13.3 and 16p11.2. The most frequent losses involved 13q (35%), 6q (30%), 11q (25%) and 4q (25%), with corresponding minimal overlapping regions at 13q21, 6q22, 11q22 and 4q32. Statistical analysis revealed significantly more gains of 2p and 14q in the older adult cases; loss of 13q was associated with a poor outcome. The results suggest that there is a set of recurrent chromosomal abnormalities associated with cHL and provide further evidence that cHL is genetically distinct from nodular lymphocyte predominance Hodgkin lymphoma (NLPHL). Combined immunophenotype and interphase cytogenetic (FICTION) studies were used as techniques for follow-up studies. High expression of both STAT3 and STAT5a has been described in cHL and their genes are located in 17q21.2. Constitutive activation of NF-?B has been found to be a feature of the HRS cells in cHL and has been shown to facilitate escape from apoptosis. The c-rel gene encodes for a subunit of NF-?B and is located on chromosome 2p16. REL amplification has been shown in some cHL cases. Non-functional inhibitor proteins of NF-?B, such as I?Ba, have been described as an alternative mechanism for the aberrant activation of NF-?B. As part of the follow-up studies, IKBa gene mutation status and loss of heterozygosity (LOH) in the HRS cells were determined by sequence analysis and SNP assays. FICTION confirmed that gains of 2p involved the REL gene but gains on 17q were not due to amplification of STAT3/5a. Frequent IKBa mutations were detected but many are not considered to be of functional importance. REL gain, EBV status, IKBa mutation or LOH were not mutually exclusive mechanisms in the pathogenesis of cHL.

There is a delicate evolutionary balance between viruses and their hosts. The host has evolved the intrinsic, innate and adaptive immunity to fight viral infections. However, viruses have acquired several counteracting measures to evade host defences. Ovine Betaretroviruses, including the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) and the highly related endogenous enJSRVs are a unique model system to investigate virus-host interaction over long evolutionary periods. Sheep have co-opted some defective enJSRV loci to (i) counteract infection by exogenous viruses and likely (ii) to cope with the continuous retroviral invasion of their genome. In addition, various genes of the innate and intrinsic immunity of the host have evolved to block viral replication. The work presented in this thesis focuses on the ovine bone marrow stromal cell antigen 2 (Bst-2)/ tetherin, a recently identified cellular restriction factor with a broad antiviral activity, and its interaction with sheep Betaretroviruses. In sheep, the BST-2 gene is duplicated into two paralogs termed oBST-2A and -2B. Studies presented in this thesis show that oBST-2B possesses several biological properties distinct from the paralog oBST-2A and from all the other BST-2 orthologs. oBST-2A prevents the release of JSRV/enJSRV viral particles by ‘tethering’ them at the cell membrane similarly to what observed by human BST-2. On the other hand, oBST-2B, does not reach the cell membrane but remains within the Golgi stacks and the trans-Golgi network. Several lines of evidence obtained in this thesis suggest that oBST-2B reduces significantly Env incorporation into viral particles. Therefore, oBST-2B possesses a unique antiviral activity that complements the classical tethering restriction provided by oBST-2A.

Feline immunodeficiency virus (FIV) is an important veterinary pathogen with comparative significance because of its similarities to its human counterpart HIV. Since FIV is the only non-primate lentivirus which induces AIDS-like symptoms in its natural host, it serves as a valuable animal model for both prophylactic and therapeutic studies of HIV. It is accepted that the induction of neutralising antibodies (NAbs) is a key element in the control of lentiviral infection, since T-cell based vaccines alone failed to prevent infection in most experimental animal model systems. In this project a robust and reproducible in vitro neutralisation assay was developed and optimised, permitting the assessment of the NAb response in naturally infected cats and with the potential to evaluate candidate vaccines. It was demonstrated that, in general, primary FIV strains in the UK belong to subtype A, and therefore the development of a regional, subtype A-specific, FIV vaccine could be considered for use in the UK. The identification of a neutralisation resistant isolate of FIV led to the finding that a linear neutralisation determinant was located within the V5 region of Env and mutations in this region may lead to immune evasion in vivo. In addition, a second neutralisation determinant was identified in the C3/V4 region of Env. Finally, it was observed that a small proportion of naturally infected cats generated NAbs against FIV. Of these, only a very small proportion of the cats had antibodies with the potential to cross neutralise strains within the same subtype as the homologous isolate. Nonetheless, a plasma sample from a single cat was identified that neutralised all strains tested, including strains from different subtypes and geographical regions. It is likely that studies of the homologous isolate that induced the broad NAb response may be capable of inducing a similar broad response in vaccinated cats. Such a finding would have important implications for the design of potential novel lentiviral immunogens.

The work presented in this thesis is concerned with the characterisation of the UL32 gene of herpes simplex virus type 1 (HSV-1). UL32 encodes an essential 596 amino acid cysteine-rich, zinc-binding protein that is highly conserved throughout the herpesviruses. The UL32 protein is essential for the cleavage of concatemeric viral DNA into monomeric genomes and their packaging into preformed capsids. Conservation is highest at the C-terminus and three CxxC motifs are present in almost all known herpesvirus UL32 sequences The UL32 antibodies available in the laboratory at the beginning of the project were incapable of detecting small amounts of UL32 protein and so new rabbit antisera were created. Soluble extracts from insect cells infected with a UL32-expressing baculovirus (AcUL32) were fractionated by anion exchange chromatography and the UL32-containing fractions used to immunise rabbits. The resultant antisera successfully recognised UL32 from transfected, HSV-1 infected and baculovirus infected cells on western blots, and UL32 in transfected cells by immunofluorescence. I performed random mutagenesis of the UL32 gene in an effort to examine structure-function relationships within this protein, and generated a panel of 37 mutants containing 5 amino acid insertions at distinct positions. The abilities of these mutants to complement the DNA packaging and growth defects of a virus lacking a functional copy of UL32 (the null mutant hr64) were examined and 15 of the mutants retained functionality in both assays. A complete correlation was found between the ability of mutants to support growth and DNA packaging, suggesting that the key functions of UL32 are confined to the DNA packaging pathway. There was also good correlation with the degree of amino acid conservation within UL32, with most of the mutants which abolished functionality being located in the highly conserved regions, and the functional mutants in less conserved regions. A number of site-specific mutants were also created, in which the paired cysteine residues were replaced with serines (i.e. CxxC to SxxS). Mutation of the first and third cysteine pairs (from the N-terminus) completely abrogated growth and packaging, whereas significant functionality was maintained following mutagenesis of the central pair. Finally, removal of the C-terminal 4 amino acids also resulted in generation of a non-functional protein. Generation of an HA-tagged UL32 construct and the introduction of this into HSV-1 allowed the localisation of UL32 in infected cells to be studied. In contrast to previous reports, I detected UL32 predominantly in the nuclei of infected cells, co-localising with ICP8 in replication compartments. DNA packaging has previously been shown to occur within the replication compartments and a number of the other packaging proteins also localise to these sites. It was previously reported that UL32 played a role in the localisation of capsids to the replication compartments. However, work presented in this thesis shows this not to be the case, and that capsid proteins VP5 and VP19C were correctly localised in replication compartments during infections with the UL32 mutant hr64. I found no evidence of UL32 interaction with the UL6, UL25 or UL17 DNA packaging proteins in HSV-1 infected or transfected cells, or using immunoprecipitation from baculovirus-expressed cells. Immunofluorescence studies of co-transfected cells showed that UL15 could direct the partial re-localisation of UL32 from the cytoplasm to the nucleus. The addition of the other terminase subunits UL28 and UL33 caused the complete re-localisation of UL32 to the nucleus, suggesting that UL32 might interact with the terminase complex. Fifteen of the insertional mutants were completely re-localised to the nucleus in the presence of UL15, UL33 and UL28, with eleven further mutants showing an intermediate phenotype of partial nuclear localisation. The ability to at least partially co-localise with the terminase complex appeared necessary for the ability of the mutants to support virus growth and DNA packaging, suggesting that this interaction may be essential for the function of UL32. However, no interaction could be demonstrated between UL32 and any of the individual terminase subunits using immunoprecipitation from insect cells. A series of experiments was undertaken to further characterise the UL32 protein. A new UL32 mutant virus (Δ32EP) was generated by insertion of a kanamycin resistance cassette in place of a large portion of the UL32 gene. This mutant had an indistinguishable phenotype from hr64, confirming that the main function of UL32 is within DNA packaging. The functional conservation between HSV-1 UL32 and the homologues from HCMV and VZV was examined, but neither protein could support the growth of Δ32EP. DNA fragments from replicated concatemeric DNA from Δ32EP infected cells behaved similarly to wt HSV-1 fragments in PFGE, suggesting that UL32 is not involved in the resolution of branched structures within the genome prior to packaging. UL32 had previously been reported to bind zinc, and this was confirmed using a zinc-release colourimetric assay. The amount of zinc bound to soluble baculovirus-expressed UL32 was quantified, showing that UL32 bound zinc in a 1:1 molar ratio. UL32 does not share all of the characteristics of a zinc finger motif, but the results of the mutagenesis experiments suggest that the outer CxxC/CxxxC motifs may be important for zinc binding. Because of its zinc-binding properties and potential interaction with the terminase complex, the DNA binding properties of UL32 were also investigated. It was found that UL32 did not bind to dsDNA containing either the minimal packaging sequence (Uc-DR1-Ub) or an unrelated non-HSV-1 sequence using an electrophoretic mobility shift assay (EMSA).

Outbreaks and sporadic cases of viral Infectious Intestinal Disease (IID) are a major public health issue resulting in significant morbidity and sometimes mortality each year. The economic costs associated are substantial. Laboratory diagnosis of viral IID is important as the many infectious and non-infectious causes cannot be reliably differentiated using clinical or epidemiological characteristics alone. An accurate diagnosis can aid patient management, infection control procedures and reduce health care costs by preventing unnecessary treatments, testing for alternative causes and hospital stay. It also aids public health surveillance. At the start of the research described in this thesis the West of Scotland Specialist Virology Centre (WOSSVC) used Electron Microscopy (EM) as the frontline test for outbreaks and sporadic cases of IID. However, although rapid on a small number of samples, this technique has been shown to be insensitive, laborious and is not suited to testing large numbers of samples. The research presented in this thesis sought to examine whether molecular diagnostic techniques such as conventional gel-based or real-time Polymerase Chain Reaction (PCR) assays could be a viable replacement for EM as the frontline test(s) for viral IID in a routine laboratory service of this type, and whether their implementation could bring benefits to the laboratory service in terms of improved rapidity, sensitivity and throughput. The aim was to adapt published PCR methods for use in routine diagnostic work rather than for research purposes, an approach that distinguishes this research from previous work in this area. In order to achieve this aim, the appropriate PCR techniques were first selected from the literature, based on a combination of clinical and laboratory requirements, and were adapted for use in the laboratory service. A series of laboratory experiments was then carried out in order to compare the sensitivity of the adapted methods to existing techniques such as EM and antigen detection assays (EIAs) and to other methods that emerged during the period of study including alternative PCR assays. Where found to be suitable, the selected PCR tests were implemented in the routine diagnostic service for viral IID. The effects of these changes on the laboratory service were then examined. The results show that since the introduction of molecular tests at WOSSVC for the detection of viral pathogens in cases of gastroenteritis the number of samples tested has risen steadily, as have the detection rates for each of the main viral causes of IID. Furthermore, this has been achieved at the same time as a substantial reduction in sample turn-around-times. Such improvements will have a positive impact in several areas of public health relating to viral IID and are discussed fully, including patient management, infection control and national surveillance.

Typical Rett syndrome (RTT) is a paediatric neurological disorder caused in >95 % of cases by loss-of-function mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2). The gene product, MeCP2, is a widely expressed nuclear protein that is especially abundant in postmitotic neurons of the central nervous system (CNS). Knocking out Mecp2 function in mice recapitulates many of the overt neurological features seen in RTT patients and provides a very useful model for testing potential therapeutic applications. The absence of a curative therapy together with the monogenecity of the disorder and established reversibility of the phenotype in mice suggest that replacement of the MECP2 gene is a potential therapeutic option worthy of exploration. In this study I used several viral vectors to test the potential of gene therapy in RTT mice. First, I generated different viral vectors which can express tagged-MeCP2 under the control of ubiquitous and cell-type specific promoters. Secondly, I assessed the ability of these vectors to deliver the Mecp2 transgene into Mecp2 knockout mice at both neonatal and adult stages of development. I then aimed to investigate the effect of exogenously delivered Mecp2 on RTT-like phenotypes. The results demonstrated that lentiviral vectors were able to effectively deliver an RFP-tagged Mecp2 minigene into neurons of Mecp2-null mice both in vitro and in vivo. Exogenous Mecp2 was targeted to the nucleus and displayed heterochromatin localization with no evidence of ectopic expression. Use of the synapsin1 (syn1, neuron-specific) promoter resulted in cellular levels of Mecp2 equal to 85 ± 0.1% of endogenous protein levels, whereas the phosphoglycerate kinase (PGK) promoter produced cellular levels of exogenous Mecp2 at a relatively high levels (210 ± 0.1 % of endogenous levels). Direct brain injection of Lentiviral vector was able to deliver exogenous Mecp2 into the CA1 region of the hippocampus and to produce high transduction efficiency around the injection sites but with limited spread. The early mortality of the injected mice precluded assessment of the functional consequences of exogenous Mecp2 expression. However, assessment of the cellular morphology was possible and this analysis revealed delivery of exogenous Mecp2 to normalise neuronal nuclear volume deficits seen in the Mecp2stop/y mice from 86 ± 0.1 % of WT values to 100 ± 0.04 % of WT levels. At the molecular level, I showed that exogenous Mecp2 3 becomes phosphorylated at serine 421 under basal conditions and that the level of phosphorylation of exogenous Mecp2 is disproportionately higher (5.5 ± 0.4 times) than that seen for endogenous Mecp2. I also showed the Mecp2 overexpression in WT neurons is associated with a reduction in the cellular levels of total histone 4 (78 ± 0.01 % of the endogenous level) and a parallel reduction in cellular levels of acetylated histone 4 (79 ± 0.01 % of the endogenous levels). In second phase of experiments, I showed that the single stranded Adeno-associated virus (ssAAV)-based vector with chicken beta actin (CBA) promoter and encapsulated with capsid of AAV serotype 9, was able to efficiently deliver exogenous MECP2 into the brain of Mecp2-null as well as WT neonatal mice after intracranial (IC) injection. In contrast to lentiviral vectors, there was widespread transduction of cells throughout the nervous system with transduction efficiency varying between 6.8 ± 2.3 % and 41.5 ± 11.3% of all cells dependent on the brain region. The transgene was mostly expressed in neurons which represented 67 ± 11.8 % to 98 ± 0.8 % of all transduced cells. ssAAV9 vector expressed exogenous MeCP2 at near-physiological levels (100-125 % of endogenous levels). At the cellular level, exogenous MeCP2 was able to rescue the neuronal nuclear volume of Mecp2-null mice (69 ± 0.02 % of WT values) to WT comparable values (97 ± 0.03 % of WT values). At the organismal levels Mecp2-null mice treated with ssAAV9/MECP2 showed extended survival (median survival of 16.7 weeks compared to 9.3 weeks for the GFP-treated control) and also displayed a modest, but significant, reduction in the RTT-like phenotype severity score compared to the GFP-treated control group. The most robust improvement reported in this study was in the locomotion activity (velocity and total distance moved in the open field test and in performance on a forced motor task). Interestingly, WT mice receiving neonatal injections of ssAAV9/CBA-MECP2 did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. In a further experiment, I showed that the self-complementary AAV 9 (scAAV9) vector with an Mecp2-endogenous core promoter fragment was able to deliver exogenous MECP2 into the brain of neonatal mice after intravenous (IV) or (IC) injection. Brain transduction efficiency was 8 - 12 % after IV injection and 48 - 68 % after IC injection in neonatal mice. Cellular levels of exogenous MeCP2 were between 1.4-1.8 times the endogenous levels. At the organismal level, 4 scAAV9/MECP2-injected mice displayed an overt hindlimb motor dysfunction which was observed 3 and 5 weeks post-injection after IV and IC injection respectively. The stereotyped hindlimb dysfunction suggested a toxicity issues with this vector and examination of the lumbar segment of the spinal cord confirmed evidence of axonal degeneration in the dorsal columns. IV injection of scAAV9/MECP2 into RAG-/- knockout mice (immunocompromised) displayed hindlimb motor dysfunction similar to that observed with Mecp2stop/y mice suggesting that the adaptive immune response is not likely to be involved in the pathogenesis of this phenotype. MECP2stop/y mice treated with scAAV9/MECP2 displayed higher RTT-like phenotype severity score than GFP-treated controls which is probably due to the effect produced by the hindlimb motor dysfunction on regular RTT-like phenotype (gait, mobility and hindlimb clasping). In summary, I have demonstrated the successful application of lentiviral and AAV2/9 vectors to deliver exogenous MECP2 both in vitro and in vivo. I showed that lentiviral vectors are unlikely to be useful for global brain delivery of MECP2 due to limited spread of the virus. However the lentiviral vectors I developed are potentially useful where localized brain injection is desirable. The main translational finding is the first, at the proof of concept level, demonstration of therapeutic benefits (including enhanced survival) of exogenously delivered MECP2 using the ssAAV9/CBA-MECP2 vector. I also however, identify potential toxicity issues of exogenous MECP2 delivery whereby a scAAV9 vector was found to produce overt neuromotor deficits. Overall, my data supports the potential of gene therapy in RTT but also emphasises the importance of issues including careful vector design, choice of delivery methods and the timing of treatment in any future clinical translation.

Parasite life history theory predicts that lifetime reproductive success evolves through differential allocation of energy to life history traits constrained by trade-offs. These life history traits govern the characteristics of parasites such as their virulence, transmission and infection phenotypes, so understanding their evolution is a key concern for infectious disease prediction and management. This thesis uses the powerful tool of experimental evolution to gain a fuller understanding of the factors and constraints involved in parasite life history evolution, using bacteriophage Φ2 as a model. I found that the evolution of life history in this phage is sensitive to spatial structure, UV-C exposure and coparasitism with plasmids, and evolution can be mediated by co-evolution with the host. The high levels of variance I observed here suggest that evolution of parasite life history is more complex than a single trajectory towards a predicted optimum, and likely involves some degree of epistasis or pleiotropy with genes elsewhere on the genome. There was some degree of independent evolution of individual life-history traits, indicating that simple direct trade-offs were not in operation. I demonstrated that co-evolution with the host provided additional mutational input, resulting in a greater degree of evolution in co-evolved populations than those evolved to a static host. Furthermore, I note that co-parasitism with phage and plasmid may provide the necessary conditions for plasmid persistence under fluctuating selection for plasmid-encoded traits, and that the efficacy and suitability of phage as therapeutic agents against plasmid-encoded antibiotic resistance is complicated. No direct link between mutation and phenotype could be elucidated in this study, suggesting that evolution in life history is either governed by genes not examined in this thesis, or involves epistasis and pleiotropy with genes elsewhere on the genome. I concluded that it is important to consider the specific ecology of the focal parasite, its host and any co-occuring symbionts in order to make informed predictions of life history evolution, and general predictions may not be achievable.