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No description available.

Gammaretroviruses are known to cause leukaemia, lymphoma and immunosuppression in many species and the original isolation of KoRV (koala retrovirus) (Hanger et al 2000) was made as part of a study into the cause of the high rate of leukaemia and lymphoma in koalas. The virus however displayed some unusual features. While classified as an endogenous (inherited) gammaretrovirus it was unusually active. While endogenous viruses are very common with up to 8% of the human genome consisting of retroviral material they are usually mutated and inactive (Gifford and Tristem 2002). KoRV possessed a full length replication competent genome, was actively transcribed and produced viral particles. KoRV was also very closely related to the exogenous (horizontally transmitted) retrovirus of Gibbons (Gibbon ape leukaemia virus or GaLV). Normally closely related viruses are found in closely related species. This unusual similarity between KoRV and GaLV raised the possibility of a recent host species jump (Hanger et al 2000). This thesis makes a case for the recent introduction of KoRV into the koala genome and ongoing endogenisation into the koala genome. KoRV is demonstrated definitively as an endogenous virus but present at a mixed prevalence within the Australian koala population. The thesis also further characterizes KoRV’s activity and establishes an association between KoRV and disease in koalas. It is difficult to prove that a virus present in all animals in a population (as is the case for most endogenous viruses) is the cause of a particular disease syndrome. Here real time PCR was used to quantify the level of viraemia and proviral DNA load in the blood of individuals. A significant association between high viral load and leukaemia and lymphoma was demonstrated. This was further confirmed in a follow up study of a group of animals where those with a high viral RNA level were at a greater risk of dying from neoplasia. The real time PCR study also indicated that animals suffering from clinical chlamydiosis had higher viral RNA levels than their healthy counterparts though this was not significant. This disease association was further strengthened when KoRV free populations were identified and were shown to have lower incidences of these diseases. The real time PCR studies demonstrated a considerable variation in the proviral copy number in individual animals. With most endogenous retroviruses the proviral copy number is fixed within the species or population (Boeke and Stoye 1997). Southern blotting was used to confirm this variation in copy number and also demonstrated variation in the pattern of these proviral inserts between unrelated animals. The random insertion pattern of KoRV provirus loci was further confirmed using cytogenetics. Sequencing of the KoRV envelope gene revealed marked variation in sequence within individual animals. These sequences despite considerable mutation in some cases were all potentially functional and indicate positive selection pressure for active virus within the individual animals. Inverse PCR was used to identify the koala genomic sequence interrupted by the proviral loci. While koala genomic sequence was successfully amplified no potential oncogenes were found. This study did however demonstrate the presence of truncated KoRV sequences missing a large part of the gagpro- pol gene. Electron microscopy also demonstrated KoRV viral particles in the bone marrow of a leukaemic animal. KoRV had originally been classified as an endogenous virus based on the fact that it was present in all animals tested and in all tissues of individual animals. However the accepted definition of an endogenous virus is one that is present in the germ line DNA and is inherited over several generations (Boeke and Stoye 1997). Single cell PCR of koala sperm was used to demonstrate that KoRV is present in germ line DNA. This was confirmed using fluorescent in situ hybridization (FISH) techniques. Southern blotting of blood obtained from a group of related captive animals showed the inheritance of proviral loci over several generations. To determine whether all koalas carried KoRV loci, samples from geographically diverse populations were obtained. As expected all animals in Queensland were positive for KoRV using PCR techniques. Unexpectedly animals from Kangaroo Island in South Australia were free of KoRV. Samples obtained from Victorian animals demonstrated a mixed KoRV status. The animals on Kangaroo Island have been isolated from other populations since the 1920’s and this raises the possibility that KoRV has integrated into the mainland koala population during the last 100 years. This is unprecedented for an endogenous virus with the most recent estimate for integration in other species being the type C viruses of pigs 5000 years ago (Mang et al 2001). The mixed prevalence of KoRV and its high activity indicate that this virus is still undergoing the process of endogenisation and provides a unique opportunity to study this process in a wild animal population.

No description available.

No description available.

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No description available.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / 1. The most appropriate methods were investigated for producing Rous sarcoma virus of suitable quantity and quality for use in the study of the viral RNA by electron microscopy. The roller bottle method of Smith and Bernstein (1973) which was adopted, produced virus yields of up to 5mg per litre of transformed cell culture supernatant after 24 hour incubations, and 0.2mg per litre of culture supernatant after 4 hour incubations. 2. The method of purifying RNA tumour viruses which resulted in the least damage to the virions was found to be isopyncic and velocity sedimentation in Ficoll density gradients containing 5mM tris-HCl and 1mM EDTA pH 8.5. The use of solutions of sucrose or >0.1M salt resulted in both osmotic changes in the virus and viral aggregation. 3. The lipoprotein coat of the Rous sarcoma virus was shown by freeze-fracturing and electron microscopy to have properties similar to those of plasma membranes, except that the number of intramembranous particles was smaller. The hydrated diameter of Rous sarcoma virus was estimated from freeze-fracture replicas of purified virions to be 140nm. 4. Vesicular contaminants, derived from serum, were present in Rous sarcoma virus preparations that had been purified from transformed cell culture supernatants. The isolated contaminants resembled virus when examined by both freeze-fracturing and negative-staining, but were readily distinguished from virus in thin sections. The virus-like serum vesicles were present in sera from several different sources. When treated with detergent and subjected to polyacylamide gel electrophoresis, the vesicles were found to contain polypeptides that possessed similar electrophoretic mobilities to those of Rous sarcoma virus polypeptides. It is probable that extraneous nucleic acid molecules, observed in preparations of Rous sarcoma virus RNA were the result of VLSV contamination of virus suspensions. 5. Contamination of purified virus suspensions by virus-like material derived from serum was reduced by centrifugation of the serum prior to its addition to cell culture medium. Virus suspensions, purified from cell supernatants from which the contaminating vesicles had been removed, were resolved in sharp bands at p = 1.07 g/ml in Ficoll density gradients; in the analytical ultracentrifuge they sedimented as homogenous populations with a sedimentation value of 740s20,w and were observed by electron microscopy to be relatively free of contaminants. 6. The maximum length of molecules from preparations of both 60-70s and 30-40s viral RNA prepared in 80% and 50% formamide respectively was 2.5μm, but both preparations were not homogeneous since they contained other, smaller molecules. 7. A model is proposed in which the difference in physical properties between the native (60-70S) form and the denatured (30-40S) form of the viral RNA is suggested to be the result of two possible conformations of a single RNA molecule. This model is an alternative to the prevailing model in which the RNA tumour virus genome is proposed to contain a number of RNA molecules of equivalent size.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / 1. The most appropriate methods were investigated for producing Rous sarcoma virus of suitable quantity and quality for use in the study of the viral RNA by electron microscopy. The roller bottle method of Smith and Bernstein (1973) which was adopted, produced virus yields of up to 5mg per litre of transformed cell culture supernatant after 24 hour incubations, and 0.2mg per litre of culture supernatant after 4 hour incubations. 2. The method of purifying RNA tumour viruses which resulted in the least damage to the virions was found to be isopyncic and velocity sedimentation in Ficoll density gradients containing 5mM tris-HCl and 1mM EDTA pH 8.5. The use of solutions of sucrose or >0.1M salt resulted in both osmotic changes in the virus and viral aggregation. 3. The lipoprotein coat of the Rous sarcoma virus was shown by freeze-fracturing and electron microscopy to have properties similar to those of plasma membranes, except that the number of intramembranous particles was smaller. The hydrated diameter of Rous sarcoma virus was estimated from freeze-fracture replicas of purified virions to be 140nm. 4. Vesicular contaminants, derived from serum, were present in Rous sarcoma virus preparations that had been purified from transformed cell culture supernatants. The isolated contaminants resembled virus when examined by both freeze-fracturing and negative-staining, but were readily distinguished from virus in thin sections. The virus-like serum vesicles were present in sera from several different sources. When treated with detergent and subjected to polyacylamide gel electrophoresis, the vesicles were found to contain polypeptides that possessed similar electrophoretic mobilities to those of Rous sarcoma virus polypeptides. It is probable that extraneous nucleic acid molecules, observed in preparations of Rous sarcoma virus RNA were the result of VLSV contamination of virus suspensions. 5. Contamination of purified virus suspensions by virus-like material derived from serum was reduced by centrifugation of the serum prior to its addition to cell culture medium. Virus suspensions, purified from cell supernatants from which the contaminating vesicles had been removed, were resolved in sharp bands at p = 1.07 g/ml in Ficoll density gradients; in the analytical ultracentrifuge they sedimented as homogenous populations with a sedimentation value of 740s20,w and were observed by electron microscopy to be relatively free of contaminants. 6. The maximum length of molecules from preparations of both 60-70s and 30-40s viral RNA prepared in 80% and 50% formamide respectively was 2.5μm, but both preparations were not homogeneous since they contained other, smaller molecules. 7. A model is proposed in which the difference in physical properties between the native (60-70S) form and the denatured (30-40S) form of the viral RNA is suggested to be the result of two possible conformations of a single RNA molecule. This model is an alternative to the prevailing model in which the RNA tumour virus genome is proposed to contain a number of RNA molecules of equivalent size.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / 1. The most appropriate methods were investigated for producing Rous sarcoma virus of suitable quantity and quality for use in the study of the viral RNA by electron microscopy. The roller bottle method of Smith and Bernstein (1973) which was adopted, produced virus yields of up to 5mg per litre of transformed cell culture supernatant after 24 hour incubations, and 0.2mg per litre of culture supernatant after 4 hour incubations. 2. The method of purifying RNA tumour viruses which resulted in the least damage to the virions was found to be isopyncic and velocity sedimentation in Ficoll density gradients containing 5mM tris-HCl and 1mM EDTA pH 8.5. The use of solutions of sucrose or >0.1M salt resulted in both osmotic changes in the virus and viral aggregation. 3. The lipoprotein coat of the Rous sarcoma virus was shown by freeze-fracturing and electron microscopy to have properties similar to those of plasma membranes, except that the number of intramembranous particles was smaller. The hydrated diameter of Rous sarcoma virus was estimated from freeze-fracture replicas of purified virions to be 140nm. 4. Vesicular contaminants, derived from serum, were present in Rous sarcoma virus preparations that had been purified from transformed cell culture supernatants. The isolated contaminants resembled virus when examined by both freeze-fracturing and negative-staining, but were readily distinguished from virus in thin sections. The virus-like serum vesicles were present in sera from several different sources. When treated with detergent and subjected to polyacylamide gel electrophoresis, the vesicles were found to contain polypeptides that possessed similar electrophoretic mobilities to those of Rous sarcoma virus polypeptides. It is probable that extraneous nucleic acid molecules, observed in preparations of Rous sarcoma virus RNA were the result of VLSV contamination of virus suspensions. 5. Contamination of purified virus suspensions by virus-like material derived from serum was reduced by centrifugation of the serum prior to its addition to cell culture medium. Virus suspensions, purified from cell supernatants from which the contaminating vesicles had been removed, were resolved in sharp bands at p = 1.07 g/ml in Ficoll density gradients; in the analytical ultracentrifuge they sedimented as homogenous populations with a sedimentation value of 740s20,w and were observed by electron microscopy to be relatively free of contaminants. 6. The maximum length of molecules from preparations of both 60-70s and 30-40s viral RNA prepared in 80% and 50% formamide respectively was 2.5μm, but both preparations were not homogeneous since they contained other, smaller molecules. 7. A model is proposed in which the difference in physical properties between the native (60-70S) form and the denatured (30-40S) form of the viral RNA is suggested to be the result of two possible conformations of a single RNA molecule. This model is an alternative to the prevailing model in which the RNA tumour virus genome is proposed to contain a number of RNA molecules of equivalent size.