Jembrana disease virus (JDV) is an acutely pathogenic lentivirus affecting Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute disease process. Reported for the first time are 2 techniques that enable quantification of the virus, and the use of these techniques to quantify the virus load in plasma of cattle during the acute disease process. The 2 techniques were a qualitative one-step JDV real-time reverse-transcription polymerase chain reaction (qRT-PCR) assay for the detection and quantification of JDV RNA, and a JDV p26 capture ELISA for the detection and quantification of JDV capsid protein. The limit of detection of the qRT-PCR was 9.8 x 102 JDV viral RNA copies over 35 cycles, equivalent to 4.2 x 104 JDV genome copies/ml, and a peak virus load of 1.6 x 1012 JDV genome copies /ml during the acute febrile period. Viral RNA and JDV p26 levels were correlated in 48 plasma samples obtained from experimentally infected cattle. A significant positive correlation (R = 0.835 and r2 = 0.697) was observed between the 2 techniques within the range of their detection limits, providing a solid basis for the use of the economical capture ELISA to quantify JDV load when real-time PCR capability is not available. The detection of JDV p26 by capture ELISA was, however, much less sensitive than the real-time RTPCR with a detection limit equating to approximately 1 x 108 JDV genome copies/ml. The transcriptional pattern of JDV during the acute phase of infection was studied by RT-PCR, sequencing and northern blot analysis. Analysis revealed a complex pattern of transcription with the identification of 14 transcripts, which confirmed 6 predicted splice sites and the identified 7 splice sites not reported previously. A small 78 bp putative non-coding exon was identified that shared the same splice acceptor as vif and was associated with the alternative transcripts of tat, rev and env. Four tat, 3 rev and 2 env transcripts were identified. The rev and env transcripts were demonstrated to use the same splice site. The study confirmed that the production of a tmx transcript, a unique gene identified in the two bovine lentiviruses JDV and Bovine immunodeficiency virus (BIV). Northern blot analysis identified 11 of the 14 transcripts identified by RT-PCR, including a 7.8 kb gag/pol primary transcript and singly spliced transcripts. The complexity of the transcript map produced suggested that JDV replication is a highly regulated process. One of the aims of this thesis was to determine the functional role of the Tmx and Vif accessory proteins of the bovine lentiviruses. Although this aim was not achieved, molecular reagents were produced that will allow these investigations to proceed. The Vif and Tmx proteins of both JDV and BIV were successfully expressed as C-terminal fusions with glutathione S-transferase (GST) using the pGEX-6P-1 bacterial expression system. The recombinant proteins were purified and were recognised by both BIV and JDV antisera from Bos taurus and Bos javanicus respectively, and by antibody in sera from cattle that had been vaccinated with a tissue-derived JDV vaccine and also those that had been naturally infected with JDV. The Vif, Tmx and Rev proteins of JDV and vif BIV were successfully expressed in a Rev-independent manner in COS7 and bovine macrophage cells using a pcDNA3.1® mammalian expression system. Cellular localisation of the recombinant viral proteins varied in the 2 cell types: in COS7 cells, both JDV and BIV Vif were detected predominantly in the nucleus, whereas in bovine macrophage cells BIV Vif localised in the cytoplasm and JDV Vif localised in the cytoplasm and nuclear membrane. JDV Tmx localised in the cytoplasm of COS7 cells but the nuclear membrane of bovine macrophage cells, and BIV Tmx localised in the nucleus and nuclear membrane in both cell types and appeared to affect the morphology of the nucleus. Mutations of vif and tmx were also successfully engineered into an infectious clone of BIV and these mutated clones will provide a valuable resource for further investigation of the role of Vif and Tmx in replication of the bovine lentiviruses.

The anaphase promoting complex (APC/C) is an ubiquitin ligase that is an essential regulator of multiple steps in the cell cycle. The complex consists of at least 12 subunits with a catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. The Parapoxvirus, Orf virus (OV), encodes a RING-H2 protein, B5L, with clear sequence similarities to APC11. The disruption of APC/C function leads to pre-mature entry into S phase and a delayed M phase exit and, potentially, apoptosis. This investigation explored the functional significance of the similarity between B5L and APC11 and specifically sought to determine if B5L manipulates cell cycle regulation by targeting APC/C function. Co-immunoprecipitation experiments from lysates of cells expressing a range of constructs revealed an interaction between B5L and APC2 in the same manner as seen with APC11. Furthermore, B5L was found to associate with endogenous APC/C. However, although APC11 promoted the formation of polyubiquitin chains in substrate-independent in vitro assays, B5L was inactive in this assay. Bioinformatics comparisons of APC11 and other known RING ubiquitin ligases with B5L and its poxviral homologues revealed some subtle differences. In particular a domain of APC11 (amino acids 61-74), that is essential for its ubiquitin ligase activity is not conserved in B5L or its homologues. When this APC11 domain was incorporated in place of the corresponding region of B5L (amino acids 59-67), the mutated B5L acquired ubiquitin ligase activity. On the other hand, APC11 protein in which the domain was replaced with that of B5L lost ubiquitin ligase activity. Stable cell lines expressing B5L showed an increased number of cells in G2/M phase (30�4%) compared with cell lines expressing APC11 (11�2%, n=3, p<0.05, ANOVA, Tukey�s), consistent with impaired APC/C function. APC/C substrates such as cyclin A, cyclin B and the thymidine kinase were stablized in B5L-expressing cells compared with control cells. Furthermore, transient hyper-expression of B5L induced apoptosis in 25�2% (n=3, p<0.05) of the cell population compared with only 6�1% apoptotic cells when APC11 was hyper-expressed. Analysis of the DNA content of OV-infected cells revealed enhanced DNA synthesis compared with cells infected with a B5L knockout OV. These observations indicate that B5L is a non-functional mimic of APC11. It associates with APC/C, but lacks ubiquitin ligase activity, and hence disrupts APC/C function. These abilities may enable OV to induce a cellular environment that enhances viral replication.

Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: Job van Woensel. Met lit. opg. - Met samenvatting in het Nederlands.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Entomology, Waite Agricultural Research Institute, 1973.

Thesis (Ph.D. 1976) from the Department of Plant Pathology, University of Adelaide.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1978.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Applied and Molecular Ecology, 2001. / Includes corrigendum attached to back leaf. Bibliography: leaves 114-132.

Thesis (M.Phil.)--Murdoch University, 2004. / Thesis submitted to the Division of Veterinary and Biomedical Sciences. "A dissertation submitted to Murdoch University in fulfilment of the requirements for the degree of Masters in Philosophy in Zoo and Wildlife Medicine" Bibliography: leaves 134-143.

Thesis (Ph.D.) -- University of Adelaide, 1957. / Typewritten copy. "The embryoculture investigations and the results of the dissection of tomato spotted wilt virus infected seeds described in Section B were presented in my M.Sc. thesis in 1953. They are again included here for the sake of completion".