An investigation into aspects of the replication of Jembrana disease virus
Stewart, Meredith (2005) An investigation into aspects of the replication of Jembrana disease virus. PhD thesis, Murdoch University.
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.
|Publication Type:||Thesis (PhD)|
|Murdoch Affiliation:||School of Veterinary and Biomedical Sciences|
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