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dc.contributor.authorWan, Floraen
dc.date.accessioned2018-05-14T10:16:56Z
dc.date.available2018-05-14T10:16:56Z
dc.date.issued2002en
dc.identifier.urihttp://hdl.handle.net/1842/30039
dc.description.abstracten
dc.description.abstractMurine gammaherpesvirus 68 (MHV-68) is a lymphotropic virus which infects wild murid rodents and can readily infect experimental mice. Thus, MHV-68 is an invaluable small animal model for elucidating gammaherpesvirus pathogenesis and immune evasion strategies. The MHV-68 genome has been fully sequenced (Virgin et al., 1997) and comparative sequence analysis has shown that it is more closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) and herpesvirus saimiri (HVS) than Epstein-Barr virus (EBV). There are at least 80 open reading frames (ORFs) within the genome, many of which are present in other gammaherpesviruses. There are, however, 4 ORFs, termed M1-M4, which are unique to MHV-68. Gammaherpesviruses have the propensity to abduct immunomodulatory elements, therefore it is likely that such elements will be encoded within the MHV-68 genome. One such candidate is the M4 gene. The aim of the project was to functionally characterise the M4 gene and elucidate its role in the pathogenesis of MHV-68.en
dc.description.abstractHalf of the M4 protein was expressed in a bacterial expression system and the purified protein was used to generate anti-sera. Immunoprecipitation studies demonstrated the M4 sera bound to a protein of ~44kDa, the predicted size of the M4 product. Analysis of M4 transcription in vitro showed that the gene was transcribed both early, prior to DNA synthesis, and late in the virus lifecycle.en
dc.description.abstractThe M4 gene was cloned into a mammalian expression construct and transfected into COS-7 cells. Cells containing the construct were selected under G418 (neomycin derivative) antibiotic selection. However, we were unable to demonstrate that the COS7 cells expressed the M4 protein.en
dc.description.abstractThe M4 gene was inserted into MHV-76, a virus which lacks part of the left hand end of the genome including the M4 gene, generating a M4 knock-in (M4KI) recombinant virus. Apart from this deletion, MHV-76 harbours the full contigency of MHV-68 genes. Polymerase chain reaction (PCR) and Southern analysis were used to demonstrate the presence of the M4 gene in the M4KI virus. Reverse trancription-PCR (RT-PCR) and northern analysis were used to show that the M4KI virus was expressing M4 RNA in vitro. The M4KI virus was used for comparative studies with MHV-76, which lacks the M4 gene, and MHV-68, which contains the M4 gene. The growth kinetics of MHV-76 are similar to MHV-68 in vitro but in contrast the virus appears to be cleared more efficiently in vivo in the lungs of mice. The establishment of latency is also impaired in the spleens of MHV-76-infected mice (Macrae et al., 2001). In vitro studies revealed the M4KI virus growth kinetics were similar to MHV-68 and MHV-76. Infection of BALB/c mice with the M4KI virus revealed that it replicates in the lung with the same kinetics as MHV-76. Thus, compared to MHV-68, MHV-76 and M4KI virus titres rise slightly faster, and levels of virus are reduced or cleared quicker. M4KI virus infective centres were much reduced in the mediastinal lymph nodes in comparison to MHV-76 and MHV-68, and were not detectable in the spleen. Thus, the M4 gene product, in the context of the MHV-76 genome prevented the establishment of detectable latent infection in the spleen.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2018 Block 18en
dc.relation.isreferencedbyAlready catalogueden
dc.titleCharacterisation of the Murine Gammaherpesvirus-68 M4 geneen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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