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Nucleic Acid Technology (NAT), introduced in the late 90s, is a molecular amplification method that can be used for the diagnosis and management of patients with infectious diseases. NAT test results are obtained quicker and are quantified, providing great.er information than the positive/negative results available from traditional techniques. However, NATs arc technically demanding, susceptible to contamination and hence results from associated diagnostic tests may be inaccurate. External Quality Assessment (EQA) services are programmes developed to assess and advance the quality performance of laboratories that use NAT kits to diagnose, manage and control human diseases. Quality Control for Molecular Diagnostics (QCMD) , an organisation that provides EQA, uses proficiency panels designed with samples containing no , weak, medium and strong microbial loads. The panels are distributed to participating laboratories who analyse them knowing the pathogen but blind to the microbial load.

The development of protective immune responses against respiratory viral infections requires the coordinated actions of components of innate and adaptive immunity. Key to the induction of adaptive immune responses is the acquisition of viral antigen in peripheral tissues by antigen presenting cells, such as dendritic cells (Des) and the transport of the antigen to regional lymphoid tissue for presentation to T lymphocytes. Accumulating evidence suggests that chemokines play an important role in the development of protective Immune responses. Although primarily recognised for their ability to regulate the migration of cells of the immune system, it has become clear that chemokines exert a variety of effects on immune cells. We, and others, have demonstrated that the chemokine eeL3 is required for the development of protective antiviral immunity against respiratory viruses. Surprisingly this effect is not due to a requirement for CCL3 in regulating the migration of effector T cells to the infected tissue. In the current study I have examined the effect of eeL3 on the maturation and function of Des using two De cell lines. Des are the most potent antigen presenting cells in the body. In their immature state they infiltrate infected tissues and pick up foreign antigens for presentation to T cells. The interaction between the De and the antigen, in concert with cytokines released at the site of infection, triggers a pathway of differentiation in the Des resulting in their phenotypic and functional conversion into mature Des. These mature Des are poor at taking up antigen but, by virtue of their enhanced surface expression of eo stimulatory molecules together with their ability to secrete immunomodulatory cytokines, they are highly effective at stimulating T cell responses. The data obtained in this study indicate that eeL3 does not promote complete or partial maturation of DCs indicating that the mechanism whereby CCL3 promotes the development of protective immunity is independent of DC maturation. In contrast, these data indicate that CCL3 enhances the ability of DCs to take up soluble antigen. The uptake of FITC-conjugated HSA was increased eight-fold by treatment of the lA WSII dendritic cell line with CCL3 and this increased uptake was inhibited by mannan, an inhibitor of the mannose receptor-dependent pathway of uptake. The ability of DCs to acquire particulate antigen was not augmented by CCL3, suggesting the differential regulation of these two pathways of antigen uptake. CCL3 treatment also enhanced the ability of influenza A-infected lA WSII cells to present the MHC class I-restricted influenza NP366 epitope to an NP366-specific T cell hybridoma. CCL3-dependent enhancement of antigen presentation was not restricted to the lA WSII cell line. Using an independent approach, it was found that presentation of an epitope contained in influenza A haemagglutinin, HA258 (defined in this research), is also augmented by CCL3 treatment of another dendritic cell line, tsDC. We propose that the enhancement of DC-mediated antigen uptake and presentation by CCL3 contributes to the CCL3-dependent development of protective antiviral immunity in vivo.

SUMMARY (To be printed on this form) Replication of the -30-kb positive-sense RNA genomes of the Coronaviridae and transcription of a set of subgenomic mRNAs is mediated by a large protein complex of nonstructural proteins (nsps) involving two RNA-dependent RNA polymerase activities that, in coronaviruses, are associated with nonstructural proteins (nsp) 8 and 12. To further characterise the RNA polymerase (primase) activity suggested to be associated with coronavirus nsp8, homologs from Human coronavirus 229E (HCoV- 229E) and the bafinivirus White bream virus (WBV), along with a set of mutant proteins containing alanine substitutions of conserved residues, were expressed in E. coli and the polymerase activities of the purified proteins were characterised in vitro. The data revealed robust metal ion-dependent polymerase activities for both HCoV-229E nsp8 and the WBV homologue. The mutant proteins retained varying degrees of activity, with single or double substitutions of three conserved Asn and Lys residues (ppla/ppJab N3672A, N3672A/K37 I lA, K3687A/K371IA) displaying the most detrimental effects on HCo V -229E nsp8 activity. The polymerase activities were further characterised using a range of homopolymeric and virus-specific template RNAs, revealing insight into substrate preferences of the coronavirus primase. HCo V -229E nsp8 substitutions were subsequently studied in the context of HCoV-229E replication in cell culture using a reverse genetics system. The data show that nsp8 activity is essential for coronavirus RNA synthesis, with substitutions of functionally important residues causing lethal phenotypes or defects in viral RNA synthesis and production of infectious virus progeny. The data provides interesting insight into the enzymatic and functional properties of two as yet poorly characterized polymerase/primase activities from distantly related viruses from the Alphacoronavirus and Bafinivirus genera. Furthermore, the study identified a number of WBV main protease cleavage sites, providing information on the substrate specificity of the WBV main protease and the proteolytic processing events used to release the putative WBV primase from larger polyprotein precursors.