Reverse genetics technology has facilitated the genetic manipulation of many nonsegmented negative strand RNA viruses (NNSV), including Sendai virus (SeV). It has provided the means to exploit SeV as a vector for vaccines or gene therapy, via insertion of extra-numeral transcription units (ENTU) encoding heterologous genes. SeV contains six contiguous genes flanked by the leader (Ld) and trailer (Tr) promoter sequences (3 'Ld-N-P-M-F-HN-L-Tr 5 '). Characterisation of an infectious clone containing the entire antigenome of SeV confirmed that it contained all elements necessary for recombinant (r)SeV rescue. We inserted a heterologous gene encoding respiratory syncytial virus (RSV) fusion (F) protein between the Nand P genes of SeV. A recombinant virus (rSeV/RSV F) was successfully rescued and expressed functional RSV F protein. Importantly, it induced protective immunity in a BALB/c mouse model, demonstrating its vaccine potential against RSV. A common consequence of ENTU insertion is growth attenuation. Therefore, we hypothesised that a rSey containing a bicistronic gene, in which the second cistron encodes a heterologous gene, would circumvent this limitation. To address this, we used a 9nucleotide sequence with known Internal Ribosome Entry Site (IRES) activity. Furthermore, . multiple linked repeats of this IRES increased the expression efficiency of the second cistron. We inserted the Renilla luciferase (rLlle) ORF, preceded by 1, 3 or 7 synthetic IRES copies within the SeV N gene 5' untranslated region. We successfully rescued the corresponding rSeVs, thereby confirming the feasibility of generating bicistronic NNSVs. We confirmed luciferase expression in infected cells and that the number of IRES copies influenced expression efficiency. However, luciferase activity was invariably lower than that from rSeV expressing rLuc from an ENTU. Importantly, and in contrast to rSeV ENTU constructs, our data demonstrated that bicistronic rSeVs were not growth attenuated. Therefore, our approach offers a novel way to express heterologous genes from NNSVs.

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This thesis begins with a comprehensive review of bacterial indicator systems and. by drawing on published material related to the transmission of enteric viral disease in the marine environment. shows that bacterial indicators are inadequate for assessing risks from viruses. Literature in respect of coliphages has also been reviewed as an introduction to the experimental section of this present study. which looks at some of the factors influencing bacteria, viruses and phage in sewage and the marine environment. This study has thrown new light on the survival of coliphage and bacteria and has assisted in showing for the first time the interrelationship between factors thought to contribute to viral inactivation. It is evident that adsorption is the major factor involved and that this applies to bacteria and coliphage as well as viruses. Other factors such as salinity, temperature and pH may exert an influence by increaSing or decreasing adsorption processes. It is concluded that there are potentially serious limitations in thB USB of DNA T-type phages as virus models and that. while coliphages clearly have useful applications. the use of the single-stranded RNA coliphages is to be preferred. As indicators of risk from viral pathogens coliphage do not conform reliably to the criteria demanded of an indicator. However, they may be usefully applied in conjunction with the continued use of an accepted bacterial indicator such as E. coli. Ultimately the best indicators for viruses may be the viruses themselves. It is apparent from the review of literature and experimental work undertaken for this present study that many questions remain unanswered. Suggestions have been made as to some priorities for future studies which it is envisaged would make a significant contribution to our knowledge of viruses in the marine environment.

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Hepatitis B virus (HBV) is of universal concern: currently, around one-third of the global population (ca. 2 billion people) is, or has been, infected by HBY, it is estimated that there are 350-400 million chronic carriers and that half a million people die from HBV associated disease a year. This thesis investigates Hepatitis B Virus evolutionary dynamics, molecular epidemiology and molecular variants. First, Hepatitis B virus has presented a considerable challenge for evolutionary rate estimation. Here, this challenge is re-addressed using a novel analysis of newly acquired serial samples from the indigenous peoples of the South Pacific, in combination with previously published data. Second, using probabilistic Bayesian models to estimate evolutionary rates from noncontemporaneous sequences, as well as, phylogenetic methods for detecting recombination, the evolutionary history of hepatitis B virus was examined in the geographical region of Oceania; evolutionary rates, dates of divergence, as well as, genotype distributions are investigated. Finally, to investigate if Hepatitis B virus is a reemerging disease in the developing nations of the South Pacific the epidemiological status in the region was examined in two overlapping surveys. In the first survey the efficacy of the Hepatitis B virus vaccination programme was examined in three Pacific Island Countries: Papua New Guinea, the Republic of Fiji Islands and the Republic of Kiribati. e In the second survey 562 randomly selected human serum samples from Madagascar, Indonesia and Oceania are screened for naturally occurring surface gene variants of Hepatitis B virus. A combination of serological and nucleic acid testing techniques are used to determine both the apparent and hidden, historical as well as contemporary, incidence of HBV in the region.

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Plasmacytoid dendritic cells (pDCs) are thought to be specialized for early viral recognition and secrete large amounts of the anti-viral cytokines type I interferon (lFNa/f3). This thesis studied viral recognition by pDCs and aimed to identify novel viral sensing receptors for viral glycoproteins. Murine pDCs were found to express the predominantly myeloid restricted lectins (mannose receptor, SIGNRI, Dectin-I and Dectin-2); a scavenger receptor, immunomodulatory receptor-ligand pairs (CD200R, CD200, SIRPa and CD47) and myeloid differentiation antigens (F4/80 and 7/4). This extends the known phenotype of pDCs and identified candidate receptors for further study. A protocol based on flow cytometric intracellular staining was developed to measure IFNa/f3 production by pDCs. This, together with an IFNa ELISA and IFN bioassay, was used to investigate viral recognition by pDCs. HI and H5 subtypes of influenza virus haemagglutinin (HA) and gp 120 from human immunodeficiency virus (HIVICN54) stimulated IFN production by splenocytes (containing pDCs). Mannan inhibited inactivated influenza virus (A/Guangdong/25/93), HA and gpl20 stimulated IFN production which implied a role for a mannose specific receptor in viral sensing, however, no requirement for mannose receptor or SIGNRI was observed. IFN induction by inactivated influenza virus, HA and gpl20 was also dependent on MyD88, but influenza virus recognition was not dependent on Toll-like receptor (TLR)2 or TLR4, raising the possibility of glycoprotein recognition through another TLR. The ability of CD200 to modulate IFNa induction was also tested. Consistent with the known inhibitory functions of CD200 through CD200R, CD200 knock-out macrophages produced more IFNa in response to the MyD88-independent stimulus polyinosinic-polycytidylic acid. In contrast, the blocking anti-CD200 monoclonal antibody OX90 inhibited MyD88-dependent IFNa production by spleen cells stimulated with influenza virus and CpG ODN suggesting that C0200 could promote IFNa production. C0200 may therefore differentially regulate IFNa induction pathways.