Ovine Herpesvirus-2 (OvHV-2) is a gamma-herpesvirus that belongs to genus macavirus, is endemic in sheep worldwide. The virus infects sheep subclinically but when it is transmitted to cattle; it induces malignant catarrhal fever (MCF), a frequently fatal lymphoproliferative disease. The pathogenesis and site of OvHV-2 is unknown in both species. In this study, we tried to: first detect the virus presence and measure its DNA loads; secondly, localise the precise cellular location of the virus in the tissues of sheep and domestic cattle. For the first purpose, we optimised and validated a sensitive quantitative polymerase chain reaction (qPCR) technique using Taqman® probe system that can detect and measure the virus’s DNA as low as one viral DNA copy in a qPCR reaction. Secondly, we applied RNA in situ hybridisation (RNA-ISH) technique to detect viral transcripts (Ov2.5 a latent gene and ORF65 a lytic gene), and in addition we used immunohistology to stain the viral Ov8 antigen (glycoprotein) by specific polyclonal antibodies. For these purposes we have used a variety of organs and tissues, namely: respiratory tract, tongue, muzzle, lymphoid and reproductive organs as well as nasal swabs and peripheral blood leukocytes from randomly selected sheep (n=28), cows without MCF (n=50) and cattle with MCF (n=12). The results in sheep have shown that 88 % of them harbour viral DNA in most of their organs at very low amounts. The viral mRNA and antigen were also detected in a wide range of organs including epithelial cells of respiratory tract, tongue and muzzle, macrophages and lymphocytes (B cells) in bronchial associated lymphoid associated lymphocytes (BALT), lymph nodes in spleen as well as vascular endothelial cells of many of these tissues. Interestingly in cattle without MCF, results were very similar as in sheep i.e.; viral DNA was found in a large population of cattle (67 %); and viral transcripts and antigen detected in a large proportion of tested organs, similar as seen in sheep. In the MCF-affected cattle, similar types of cells were found infected as in cattle without MCF, but with significantly higher viral loads (more than three logs). This study shows for the first time OvHV-2 location and cell types they infect in sheep, and in cattle that do not show any evidence of MCF. The new question is what triggers inducing of MCF in the subclinically infected healthy cattle?. That can be addressed by further investigations.

Across the developing world peste des petits ruminants virus (PPRV) places a huge disease burden on small ruminant agriculture. PPR is mainly controlled by vaccinating animals with live attenuated vaccines. However, the current PPR vaccines and companion serological tests do not enable serological differentiation between naturally infected and vaccinated animals (DIVA), therefore a meaningful serological assessment of vaccine coverage and epidemiological surveillance is not possible. Therefore, the main objective of this PhD study was to establish a reverse genetics system for PPRV, so that a marker vaccine could be developed to enable the serological differentiation between vaccination and infection, alongside developing proof of concept for increasing the valency of the existing vaccines. Initially, as a prerequisite to full genome synthesis the full genome sequence for a PPRV vaccine strain was confirmed. An efficient reverse genetics system for the PPRV Nigeria75/1 vaccine strain was established in this study and 3 recombinant PPRVs were rescued including a faithful clone of the vaccine strain (rPPRV Nigeria75/1), a clone expressing GFP as a heterologous protein (rPPRV+GFP Nigeria75/1) and a negatively marked vaccine containing mutations to the haemagglutinin (H) gene (rPPRV-C77 Nigeria75/1). All 3 rescued viruses showed similar growth characteristics in vitro when compared to the parental vaccine strain and, following in vivo assessment the H mutant provided full protection in goats upon virulent virus challenge. Although the mutations made to H abrogated in vitro binding of C77, the mutations made were not sufficient to enable DIVA in vivo. Finally proof of concept was developed for the segmentation of PPRV and expression of heterologous proteins in an effort to generate a multivalent vaccine. A recombinant two-segmented version of PPRV was successfully rescued that expressed GFP from one segment and the bluetongue virus VP2 from the other. This virus was partially characterised in vitro and demonstrates the potential for this approach in the development of multivalent vaccines for small ruminants.

Orthobunyaviruses are the largest genus within the Bunyaviridae family, with over 170 named viruses classified into 18 serogroups (Elliott and Blakqori, 2001; Plyusnin et al., 2012). Orthobunyaviruses are transmitted by arthropods and have a tripartite negative sense RNA genome, which encodes 4 structural proteins and 2 non-structural proteins. The non-structural protein NSs is the primary virulence factor of orthobunyaviruses and potent antagonist of the type I interferon (IFN) response. However, sequencing studies have identified pathogenic viruses that lack the NSs protein (Mohamed et al., 2009; Gauci et al., 2010). The work presented in this thesis describes the molecular and biological characterisation of divergent orthobunyaviruses. Data on plaque morphology, growth kinetics, protein profiles, sensitivity to IFN and activation of the type I IFN system are presented for viruses in the Anopheles A, Anopheles B, Capim, Gamboa, Guama, Minatitlan, Nyando, Tete and Turlock serogroups. These are complemented with complete genome sequencing and phylogenetic analysis. Low activation of IFN by Tete serogroup viruses, which naturally lack an NSs protein, was also further investigated by the development of a reverse genetics system for Batama virus (BMAV). Recombinant viruses with mutations in the virus nucleocapsid protein amino terminus showed higher activation of type I IFN in vitro and data suggests that low levels of IFN are due to lower activation rather than active antagonism. The anti-orthobunyavirus activity of IFN-stimulated genes IFI44, IFITMs and human and ovine BST2 were also studied, revealing that activity varies not only within the orthobunyavirus genus and virus serogroups but also within virus species. Furthermore, there was evidence of active antagonism of the type I IFN response and ISGs by non-NSs viruses. In summary, the results show that pathogenicity in man and antagonism of the type I IFN response in vitro cannot be predicted by the presence, or absence, of an NSs ORF. They also highlight problems in orthobunyavirus classification with discordance between classical antigen based data and phylogenetic analysis.

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Protection against viruses is provided by the host innate, adaptive and cellular intrinsic immunities. Cellular intrinsic immunity, or intrinsic defence, against herpesviruses is in part enacted by components that constitute nuclear substructures known as ND10. ND10 (also called PML nuclear bodies after its major organising component) are dynamic nuclear domains that contain various cellular proteins, including PML itself, Sp100, hDaxx and ATRX. In the early stages of infection, herpesvirus genomes and sites of immediate early transcription become associated with ND10 and their components. Representative herpesviruses such as an alphaherpesvirus HSV-1 and a betaherpesvirus HCMV encode for strong transcriptional activators, namely the Immediate Early protein ICP0 and the tegument protein pp71, respectively. These proteins are known to counteract cellular intrinsic defence mechanisms. In the context of HSV-1 and HCMV infections, the major ND10 component PML has been identified as an important constituent of cellular intrinsic defence. In addition, a number of research studies have demonstrated that Sp100 and hDaxx contribute to these processes during infection with HSV-1 and HCMV, respectively. The general hypothesis of the present study implies that cellular chromatin-associated factors within ND10 may act to repress viral gene expression. The two representative ND10 components present a particular interest for the current investigation based on the facts that: (i) hDaxx is a transcriptional co-repressor; (ii) ATRX is a chromatin-remodelling enzyme; and (iii) ATRX and hDaxx interact with each other to form a chromatin-remodelling complex with repressive properties. The purpose of the present study was therefore to investigate the roles of these two proteins in HSV-1 and HCMV infection. By using virus mutants incapable of efficient stimulation of Immediate Early gene expression (ICP0-null HSV-1 and pp71-null HCMV) it was possible to analyse the contribution of ATRX and hDaxx to the repression mechanism that occurs in the absence of these viral transactivators. An RNA interference approach was utilised for generating cell lines depleted of ATRX or hDaxx in order to assess their roles in viral infectivity. In addition, cell lines reconstituted with wt hDaxx, and ATRX-interaction or SUMO-interaction deficient hDaxx mutants were constructed in order to study the contribution of these functional elements to the role of hDaxx in the repression of ICP0-null mutant HSV-1. The key findings presented in the current study can be summarised as follows: (1) ATRX contributes to the intrinsic resistance against HCMV infection, and this mechanism is strongly counteracted by viral pp71; (2) A chromatin-remodelling complex formed between ATRX and hDaxx contributes to the efficient repression of ICP0-null HSV-1 genomes, thereby constituting a part of anti-HSV-1 intrinsic cellular defence. ICP0 counteracts this process, and the possible mechanisms of ICP0 action are proposed. These data provide the first evidence for the role of ATRX in viral infection and in addition demonstrate a role for hDaxx in the regulation of HSV-1 infection. The strong indication that ATRX and hDaxx act as a complex opens a possibility of chromatin-dependent repression of ICP0-null HSV-1 genomes. Whether ATRX and hDaxx contribute to the repression of pp71-null HCMV genomes as a complex is yet to be established. In summary, the conclusion of the studies presented in this thesis suggests regulatory roles of ND10-localised chromatin remodelling proteins ATRX and hDaxx in cellular anti-herpesvirus intrinsic resistance mechanisms.

Analysis of primary sequence data revealed a subset of open reading frames that were predicted to encode HSV-I dUTPases based on five areas of local primary sequence conservation. The differences in the primary sequence organisation of these motif regions allowed the description of two distinct dUTPase classes. The class I dUTPases are encoded by a diverse range of organisms and are characterised by a trimeric arrangement with subunit protein lengths approximating 150 amino acids. The class II dUTPases are specific to the herpesviruses and are characterised by a monomeric arrangement with a protein chain length approximately double that of their class I counterparts. It has been proposed that the class II dUTPases arose by the intragenic duplication of the class I open reading frame. In this thesis the class I structures were used as a basis to investigate the HSV-1 class II dUTPase in terms of structural and evolutionary relationships. To allow a defined approach to functional analysis of the HSV-1 dUTPase a tertiary structural model was generated for the class II enzymes. Following intensive primary sequence analysis a method was devised for comparing class I and class II sequences directly. Secondary structure prediction programs were utilised to judge the basic structural similarities between the two classes allowing the proposition of several defined hypotheses. The available class I structural information was utilised in order to characterise highly conserved structural elements within the class I group. In was then possible to relate this data set to class I primary sequences and subsequently to the generation of a class II model. Various modelling techniques were used based on the constraints on the structural organisation that could achieve a functionally active monomer plus the set of hypotheses defined in the earlier work. Mutagenic analysis of the HSV-1 dUTPase was then possible using the class II model as a reference. Several targets were investigated based on predicated functionally important regions. Analysis of these mutant enzymes was performed using purified recombinant HSV-1 dUTPase expressed from the T7 E.coli expression system.

The aim of this thesis was to further our knowledge about the contribution of silent alleles on megabase chromosomes to the late stages of trypanosome infection and test the hypothesis that this contribution takes shape in a hierarchy of expression due to differences between alleles in terms both of flanking regions and coding sequence. This was achieved through a combination of bioinformatics and molecular studies. The initial approach was to undertake an extensive manual curation of the available VSG archive; this endeavour resulted in establishment of a fertile collaboration with the Trypanosoma brucei genome sequencing project, and in creation, with the aid of P. Ward and S. Menon, of a dedicated web-based tool to handle and query curated VSG genes. Out of an updated estimate of ~1600 VSG genes, 940 (between half and three quarters) were annotated and shown to be arranged in subtelomeric arrays and to be largely present as pseudogenes (~90%). By considering separately the hypervariable N-terminal domain (three types, A, B and C) and the more conserved C-terminal domain (types 1 to 4, with two additional types identified in this study), it appeared that most of the degeneracy lay in the C-terminal domain. This suggested that N-terminal domains (one third of them being intact) would be utilised by a process of segmental gene conversion yielding hybrid genes, by recombination with functional C-terminal ends resident at the expression site. Under the assumption that “order” within the genome (the presence of patterns within the VSG archive) helps inform “order” in VSG expression (a hierarchy based on different activation probabilities), it was somewhat surprising to detect little evidence of clear substructuring within the archive: no “classes” of VSGs could be identified, based on coding sequence and flanking sequence features. In keeping with the observed high level of divergence within the VSG archive, clear orthologue groups (here defined as alleles sharing >60% amino acid sequence identity) were found not to include more than three to four members and to be scattered at random across the arrays. Putative functional genes could not be separated into groups based on expected differences in activation probabilities, such as a different number of upstream 70-bp repeats, shown to be involved in copying silent alleles to the expression site.

Human and bovine respiratory syncytial viruses (HRSV and BRSV), along with pneumonia virus of mice (PVM) are the members of the genus Pneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae. Although both HRSV and BRSV have been associated with important diseases in human and livestock, there is no clearcut description of the molecular aspects of their pathogenesis. For HRSV, the lack of a suitable study model is one of the main reasons hampering the study of aspects of pathogenesis of the virus. HRSV infects a wide range of animal models, however most of the common laboratory animal models are not sufficiently permissive to study the infectivity of the virus. PVM naturally infects mice and causes a disease indistinguishable from that of HRSV in humans. Two strains of PVM have been described: strain 15 (Warwick) which is not pathogenic and strain J3666 which is highly pathogenic. The main difference between these two strains lies in the organisation of the gene encoding the attachment (G) glycoprotein. The G gene in strain J3666 has two ORFs. The larger second ORF codes for the G glycoprotein and is located downstream of the first ORF which has no known function. The strain 15 G gene also contains two ORFs but in this case both the first and main ORFs overlap each other. The aim of the project was to investigate the molecular basis for pathogenesis of PVM as a model for pneumoviruses. As a first step, the pathogenesis of PVM strain J3666 was revaluated and the effect of consecutive tissue culture passages on the pathogenicity of the virus was examined. It was shown that consecutive passages of PVM strain J3666 caused attenuation of the virus. To investigate the possible mutations causing the attenuation the majority of the virus genome was sequenced from three passage stocks where the transition from pathogenic to non-pathogenic occurred. No differences in the genome sequences for the three passage stocks were found. However, sequence analysis of individual clones of the SH and G genes of the viruses showed evidence that the stocks contained a mixed population of sequences. A robust reverse genetics system was established to rescue recombinant PVM from cDNA using co-transfection of plasmids coding for the ribonucleoprotein complex of the virus (N, L, M2-1 and P proteins) and a cDNA copy of the virus genome cloned under the control of the bacteriophage T7 RNA polymerase. Using this system, four viruses differing in their G gene organisation were generated and used to infect mice to study the effect of mutations on pathogenicity. It was shown that the viruses with the G gene of strain 15 (Warwick) lacking the first ORF manifest a modest increase in their pathogenicity compared to the non-pathogenic PVM strain 15(Warwick) parent. The recombinant viruses containing the G gene organisation of strain J3666 showed the highest level of pathogenicity. The reverse genetics system was used to study the role of the first ORF in G glycoprotein expression. Using a dicistronic minigenome construct, the effect of the presence or absence of the first ORF in both the strain 15 and strain J3666 G gene organisation was studied. It was shown that the presence of the first ORF of the G gene in the strain 15 (Warwick) suppressed the expression of the G protein, while the first ORF in the strain J3666 did not have any significant effect on G protein expression.