In situ hybridisation for the detection of viral nucleic acids
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The technique of in situ hybridisation was optimised for the detection of viral RNA using radioactively-labelled single-stranded DNA and RNA probes, and applied to three areas of interest. Optimum hybridisation conditions were determined in vitro using cells infected with the single-stranded negative sense RNA paramyxoviruses. Transcription of RNA probes was the most rapid and efficient method of probe labelling, since electrophoretic purification was not required and large amounts of RNA were produced. However, their use for in situ hybridisation was problematic due to RNase contamination and low sensitivity. In contrast, DNA probes produced from M13 clones and oligonucleotide probes gave consistent hybridisation results and were preferred in subsequent studies for their ease of use, stability and sensitivity. The effect of virus-host interactions on the clearance of the paramyxovirus, SV5, in a mouse model was investigated by detection of viral RNA and protein in lung sections. Immunisation with purified SV5 proteins prior to infection provided protection against infection, indicated by a reduction in the level of viral RNA and protein, due to enhanced clearance of virus by primed T cells. X-irradiation of the host prior to infection resulted in prolonged or persistent infection in which RNA was detected up to 19 days post-infection. The potential of in situ hybridisation for detection of aetiological agents was demonstrated by investigation of the presence of measles virus in two chronic human diseases. Thus, measles virus RNA was detected in brain sections from a patient with subacute sclerosing panencephalitis and in the osteoclasts of bone sections from a patient with Paget's disease of bone. In situ hybridisation was used to analyse expression of the two immediate-early genes of herpesvirus saimiri, the 52K gene and the hinG gene. Differential expression was detected by hybridisation to mRNA using oligonucleotide probes, in productively-infected cells. The 52K gene was expressed asynchronously throughout the population in agreement with immunocytochemical detection of the 52K protein. In contrast, the hinG gene was expressed synchronously, with all cells showing similar levels of hybridisation, indicating a specific control mechanism for expression of the 52K gene, which differs from that of the hinG gene in requiring or being inhibited by additional factors. This may have relevance to the mechanism of establishment of latency in this virus.
Thesis, PhD Doctor of Philosophy
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