Malignant catarrhal fever (MCF) is a lethal lymphoproliferative disorder of cattle and deer caused by infection with either Alcelaphine Herpesvirus-1 (AHV-1) or Ovine Herpesvirus - 2 (OHV-2). It was proposed that the viruses induce an interleukin-2 (IL-2) hyperproduction which is responsible for the lymphoid cell hyperplasia observed. The hyperplasia of lymph node cells was investigated by examining the growth characteristics of freshly explanted lymph node cells from AHV-1 infected rabbits. The lymph node cells had a higher thymidine uptake respective to control cultures in the first 24 hours. IL-2 increased viability and thymidine uptake and Con A did not stimulate these cells as expected. Very little IL-2 activity could be detected in supernatants from short term cultures using the CTLL-based bioassay. To establish a TR-PCR/immunoblot for the detection of rabbit IL-2 mRNA, IL-2 cDNA from Con A stimulated rabbit lymphocytes was cloned and partially sequenced. IL-2 transcripts could be detected in lymphoid cells from pyrexic rabbits with AHV-1 and in cells from control rabbits. Furthermore, it was shown that lymphoblastoid cell lines (LCL) derived from MCF-affected cattle did not transcribe IL-2. These data lead to the conclusion that IL-2 is involved in the acute state of MCF, but does not have a central role in the pathogenesis. Further characterisation of IL-2 dependent LCL showed that they responded weakly to Con A, were inhibited by Cyclosporin A and transcribed constitutively IL-4, IL-10, INFγ and TNFα, whereas no IL-1β mRNA could be detected. These data together with the results derived from the short term cultures of lymph node cells from AHV-1 infected rabbits clearly show that MCF inducing viruses alter the behaviour of lymphoid cells. The possible interference of OHV-2 and AHV-1 with transductional pathways, the expression of IL-2R and the activation of self-reacting lymphocytes are discussed.

The main aim of this thesis is to investigate the molecular evolution of human and non-human primate HBV to gain further insights into the origin of HBV in these species. This investigation was carried out in three main sections. The first comprised an extensive and detailed genetic analysis of the distribution of human HBV genotypes in HBV endemic areas in sub-Saharan Africa and South East Asia. In the second section complete genome sequences of HBV variants were analysed for recombination between different HBV genotypes. This analysis included the use of a novel method based on the calculation of association scores for phylogenetic groups, an approach that helps resolve much of the uncertainties and difficulties of interpretation of results arising from conventional methods, such as SimPlot. The third section investigated the frequencies of BHV infection in non-human primates, and the relationship between HBV genotype, primate species and geographical range. In my survey, HBV infection was confined to African and Asian apes, and uniformly absent from a wide range of African monkey species. Phylogenetic analysis of chimpanzee-, gibbon- and orang-utan-derived HBV variants indicated that a geographical rather than a species correlation with genotypes, implying the co-circulation and cross-species transmission of HBV between species of overlapping habitats. However, in no cases were primate-associated HBV variants found in humans, nor human genotypes in non-human primates. These findings and the interspersed nature of human and non-human primate HBV genotypes deepens the mystery of HBV origins and evolution in humans. The findings, however, provide a context for ongoing studies of HBV biological variability and genotype-associated differences in pathogenicity and outcomes of infection.

Intranasal infection of gamma interferon receptor knockout (IFNγR^-/-) mice with murine gammaherpesvirus - 68 (MHV-68) causes unique pathological changes in the spleen characterised by development and resolution of fibrosis. Delineation of virological, cellular and molecular events taking place in the spleen during these pathological changes was the main objective of this study. Sequential histopathological and cellular characterisation studies showed early invasion of lymphoid follicles (harbouring latently infected B-lymphocytes) with CD4+ and CD8+ T-lymphocytes. Concomitantly, invasion of alternatively activated macrophages characterised by arginase-1 expression was observed. These events were followed by a reduction in the number of B-lymphocytes and development of a ‘fibrotic cage’ around the shrinking lymphoid follicles. In spite of the decrease in the number of B-lymphocytes, the number of latently infected cells showed a progressive increase until the development of fibrosis. During fibrosis, a dramatic reduction in the number of latently infected cells was observed followed by repopulation of lymphoid follicles with B-lymphocytes indicating a recovery phase of the fibrotic response. These observations suggest that: 1) The progressive reduction and re-population of B-lymphocytes in the lymphoid follicles of the spleen constituted the primary events during the development and resolution of fibrosis respectively. 2) Fibrosis as a host response mechanism is able to contain the expansion of latently infected cells in the spleen. 3) Presence of latently infected cells might be the trigger for fibrosis development by preventing the re-population of lymphoid follicles. The role of IFNγ responsiveness of bone marrow derived cells in the pathogenesis of splenic fibrosis was investigated by generating radiation bone marrow chimera mice and subsequent infection with MHV-68. The study showed that, replacing the bone marrow derived cells in IFNγR^-/- mice with bone marrow derived cells from wild type mice prevented the development of splenic fibrosis following MHV-68 infection. Other pathological changes occurred in the lung, liver, aorta and spleen of IFNγR^-/- mice infected with MI-IV-68. Focal areas of interstitial fibrosis were observed in the lung during day 12-16 post infection. At the same time, cuffing of pulmonary vessels with primarily mononuclear cells was observed. A few of the infiltrating cells were productively infected with MHV-68 which coincided with clearing of productive infection from alveolar epithelial cells. The change in the tropism of viral infection may be related to the development of fibrosis.

Murine gamma herpesvirus-68 (MHV-68) is able to undergo lytic replication in a range of cell types <i>in vitro</i> and can infect inbred strains of mice. These properties make MHV-68 an excellent model for the study of gammaherpesvirus pathogenesis. MHV-68 allows investigation of gammaherpesvirus infection of and persistence in the lung – following intranasal inoculation the virus establishes a life-long infection in this organ, with virus persisting in epithelial cells and/or B cells. Identification of key viral genes required for persistence may allow for development of vaccination and/or treatment strategies. Using real-time PCR the long-term viral load in the lungs was reduced following the deletion of key genes from the viral genome. Genes identified are the Thymidine kinase gene, previously shown to play a role during acute infection of the lung and ORF73, a homologue of the HSKV LANA-1 gene. Initial data also suggests that the ORF72 and M11 genes, both involved in reactivation from latency, may play a role in maintaining viral load at late time points post-infection. <i>In vivo</i> investigation of the M1 gene of MHV-68 has demonstrated a potential role in control of viral reactivation from latency in the spleen. A novel MHV-68 mutant, M1Δ, lacking 1171 bp of the M1 ORF, was used to study the role of M1 in pathogenesis. Initial data suggests that <i>in vivo</i> infection with M1Δ results in increased viral titres during acute infection of the lung, indicating a potential role in control of initial infection. The major role of M1 appears to be during acute phase latency in the spleen.

To compare the mode of action of NVV Rev with that of HIV-1 Rev, a series of functional assays were planned. The <I>rev </I>gene of a British isolate of MVV (EV-1) was cloned and sequenced. Recombinant Rev was expressed as a fusion protein in yeast and bacterial systems. Expression in yeast was characterised by a low product yield, due to the highly toxic nature of the Rev fusion protein. Alternative expression and purification protocols were unable to greatly improve the yield and purity of product. The bacterial pGEX system, in which Rev was fused to glutathione S-transferase (GSTRev), was employed as an alternative. Purification by affinity chromatography resulted in an improved yield and purity of product. Partial instability of the fusion protein may have resulted in observed contamination. Binding of GSTRev to RNA corresponding to the predicted RRE was assayed by filter binding experiments. A specific vector context and low temperature were required to generate high quality RNA. GSTRev bound with high affinity to RRE-RNA, but not to RNA corresponding to antisense RRE. Addition of a non-specific competitor RNA reduced binding to antisense, but not sense, RNA. Rev is the least well conserved protein amongst sequenced isolates of MVV. To test for the functional conservation of the Rev/RRE axis, the cross reactivity of Rev function on heterologous RREs was examined by transient transfection assay. Whilst cross-strain functional reciprocity was observed, both the EV-1 and 1514 isolate Rev proteins demonstrated greatest activity on cognate RRE. Co-divergence of the <I>rev</I> gene and RRE structure of each strain has therefore occurred. MVV Rev was able to function through the RRE of the closely related caprine arthritis encephalitis virus. These results may have implications for the possible development of anti-lentiviral gene therapy based on <I>trans-</I>dominant, inhibitory Rev molecules.

The local and systemic immune response to rotavirus was investigated, both in previously exposed adult sheep and gnotobiotic lambs. Vaccines composed of rotavirus or recombinant VP6 mixed with or incorporated into ISCOMs were used to examine if one oral dose could induce a mucosal immune response and protection against subsequent challenge. Gnotobiotic lambs were vaccinated orally either with PBS/ISC, inactivated rotavirus (IRV)/ISC, recombinant VP6/ISC, or IRV alone, challenged 3 weeks later with a live virulent lamb strain, and killed 8-9 days after challenge. The immune response was measured as described above. The rotavirus-vaccinated groups had RV IgG ASC and antibodies in blood from 7 and 11 days respectively. After challenge, rotavirus-vaccinated groups cleared the virus in a reduced period (6-7 days vs 8-9 days), however this was only significant (p<0.05) in the IRV/ISC group. RV IgA antibodies were observed in serum and nasal secretions from 4 days after challenge. In intestinal scrapings, these were significantly (p<0.05) higher in the IRV/ISC and IRV groups. RV IgG antibodies were significantly (p<0.05) increased in nasal secretions and intestinal scrapings in the IRV/ISC and IRV groups. Lymphocytes proliferated significantly in JPPs against rotavirus in the IRV/ISC and IRV groups. CD45R<SUP>+</SUP> cells were significantly increased in blood in the IRV/ISC group before challenge, however no differences were found in other lymphocyte subpopulations either in blood or GALT. A downregulation of IFNγ transcripts was observed in JPPs and MLNs in the IRV/ISC group while the VP6/ISC group had a higher expression compared with the PBS/ISC and IRV groups. IL-4 transcripts were low in MLNs in all groups but in JPPs the rotavirus-vaccinated groups had a higher expression. IL-6 transcripts in JPPs were higher in the IRV/ISC and VP6/ISC groups but in MLNs all rotavirus-vaccinated groups had a higher level of expression. One oral dose of inactivated rotavirus alone, mixed with ISCOMs, or recombinant VP6 incorporated into ISCOMs, can induce priming and partial protection. These results suggest also that different immunological mechanisms take place when different vaccination protocols are used.

Twenty-five monoclonal antibodies (mabs) against orf virus, a parapoxvirus (ppv), were produced following the immunisation of mice with a lysate of cells infected with orf-11 virus. These mabs, together with 2 others recloned from an earlier fusion, were identified by ELISA and IFT and characterised. No neutralising activity was shown by any of the mabs. The size of orf proteins detected by the mabs was measured using western blotting and radioimmunoprecipitation (RIP). Western blotting was conducted with two types of orf-11 antigen preparations - gradient purified virus or a lysate of orf-11 infected cells. Five mabs detected a protein of approximately 40 kDa with both purified virus and infected lysate antigens. These mabs detected a protein approximately 65 kDa in size, but only with infected cell lysate antigen. In RIP studies, 21 mabs produced bands 13 of which were against the 65 kDa protein, 7 against that 40 kDa protein while one was against a 50 kDa protein. Twenty-one of the 27 mabs reacted with at least two of 18 vaccinia virus orf virus (VVOV) recombinants expressing a library of orf genome fragments of the NZ-2 virus strain. Four of the mabs which had identified the native 40kDa protein reacted with 2 overlapping recombinants (245 and 247). Seventeen of the mabs, 16 of which had identified the native 65 kDa protein recognised three recombinants 79, 285 and 286 all of which contain different inserts from the same region of the orf virus genome. Subsequent sequencing of the overlapping site between recombinants 245 and 247 by New Zealand collaborators has identified a new orf gene, designated FIL which has been shown to be analogous to the H3L vaccinia virus gene which codes for an immunodominant 35 kDa envelope protein. Cells infected with a new VVOV recombinant expressing only the FIL orf gene showed positive fluorescence with 3 or the 4 mabs which reacted with the 245 and 246 recombinants, confirming the target of these mabs is the product of the FIL gene.

The aim of the project was to functionally characterise the M4 gene and elucidate its role in the pathogenesis of MHV-68. Half of the M4 protein was expressed in a bacterial expression system and the purified protein was used to generate anti-sera. Immunoprecipitation studies demonstrated the M4 sera bound to a protein of ~44kDa, the predicted size of the M4 product. Analysis of M4 transcription <i>in vitro</i> showed that the gene was transcribed both early, prior to DNA synthesis, and late in the virus lifecycle. The M4 gene was cloned into a mammalian expression construct and transfected into COS-7 cells. Cells containing the construct were selected under G418 (neomycin derivative) antibiotic selection. However, we were unable to demonstrate that the COS-7 cells expressed the M4 protein. The M4 gene was inserted into MHV-76, a virus which lacks part of the left hand end of the genome including the M4 gene, generating a M4 knock-in (M4KI) recombinant virus. Apart from this deletion, MHV-76 harbours the full contingency of MHV-68 genes. Polymerase chain reaction (PCR) and Southern analysis were used to demonstrate the presence of the M4 gene in the M4KI virus. Reverse transcription-PCR (RT-PCR) and northern analysis were used to show that the M4KI virus was expressing M4 RNA <i>in vitro. </i>The M4KI virus was used for comparative studies with MHV-76, which lacks the M4 gene, and MHV-68, which contains the M4 gene. The growth kinetics of MHV-76 are similar to MHV-68 <i>in vitro</i> but in contrast the virus appears to be cleared more efficiently <i>in vivo</i> in the lungs of mice. The establishment of latency is also impaired in the spleens of MHV-76-infected mice (Macrae <i>et al.,</i> 2001). <i>In vitro</i> studies revealed that M4KI virus growth kinetics were similar to MHV-68 and MHV-76. Infection of BALB/c mice with the M4KI virus revealed that it replicates in the lung with the same kinetics as MHV-76. Thus, compared to MHV-68, MHV-76 and M4KI virus titres rise slightly faster, and levels of virus are reduced or cleared quicker. M4KI virus infective centres were much reduced in the mediastinal lymph nodes in comparison to MHV-76 and MHV-68, and were not detectable in the spleen. Thus, the M4 gene product, in the context of the MHV-76 genome prevented the establishment of detectable latent infection in the spleen.

No description available.

No description available.