Molecular comparison of virulent and non-virulent Alcelaphine herpesvirus-1 derivatives
Handley, Jennifer Ann
MetadataShow full item record
Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease affecting ruminants. The disease is caused by infection of susceptible hosts with one of two gammaherpesviruses, Alcelaphine Herpesvirus -1 (AHV -1) or Ovine Herpesvirus -2 (011V-2).On isolation AHV -1 infectivity is cell -associated and will induce MCF on inoculation into experimental animals. After serial passage cell -free infective virus is observed, but this virus cannot produce MCF experimentally. The aim of this project was to characterise the genomic alterations which occurred in one isolate, C500, associated with this altered pathogenicity.Viral DNA of virulent (PP) and cell -free attenuated (CFA) C500 derivatives was compared by restriction endonuclease profiling. Variability was observed using Sma I, as a 5kbp fragment was present in PP DNA but not CFA DNA. Conversely a 3.8kbp fragment was present in CFA DNA but absent from PP DNA. Homology was observed between these Sma I fragments. The Sma I 3.8kbp fragment was cloned (ATT -1), as were two Hind III equivalents of the PP 5kbp Sma I fragment (VIR -1 and VIR -2). These clones were mapped for several restriction endonucleases, subcloned and sequenced.The location of the C500 clones was assessed by Southern blotting and PCR. The structure of the C500 genome consisted of a central unique region of approximately 130kbp, flanked on either side by multiple copies of a 1050bp repeat unit, resulting in an overall genome size of 160kbp. The ATT -1 clone was found to be present twice in the CFA genome, located at both ends of the unique region, close to the terminal repeats, with both copies orientated in the same direction. The location of the VIR -1 and VIR -2 clones in the PP genome was not fully deteimined during this study, however, the results obtained suggested that the VIR -1 and VIR -2 clones were located approximately 1.4kbp apart, at one end of the unique region, close to the terminal repeats. The mapping data suggested that two sequential deletions occur as the C500 virus is passaged in tissue culture, resulting in a total deletion of approximately 3.8kbp. This deletion correlates with loss of virulence.The sequence of the three C500 clones was compared to other known sequences at both nucleic acid and amino acid levels, but no significant homology was observed. One open reading frame (ORF), encoding a peptide designated peptide 5, and an adjacent unsequenced region of 800óp were lost on attenuation of the C500 isolate, suggesting that this region encodes sequence essential to the development of MCF. The sequence of peptide 5 did not exhibit structural similarity to the peptides involved in the transforming potential of either of the related gammaherpesviruses Herpesvirus saimiri (HVS) or Epstein -Barr virus (EBV). However, the location of the ORF encoding peptide 5 in the C500 genome was similar to that of the transforming protein of HVS in the HVS genome.One ORF, encoding a peptide designated peptide 1, was found to be conserved in OHV -2, and is a further indication of the close relationship between these two herpesviruses. Peptide 1 also exhibited limited structural homology to the transforming proteins of HVS, however, the presence of two copies of peptide 1 in the CFA C500 genome suggested that peptide 1 did not have a similar function to the HVS peptides.