Browsing by Author "Dumbell, K R"

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    Characterisation of promoter sequences in a Capripoxvirus genome
    (1992) Fick, Wilhelmina Christina; Dumbell, K R; van Dijk, A A
    Capripoxviruses are of particular interest as live recombinant vectors for use in the veterinary field, since their host-range is restricted to cattle, goats and sheep. The work presented in this thesis is a preliminary study undertaken on the South African Neethling vaccine strain of lumpy skin disease virus (LSDV). As a departure point towards the eventual identification of strong promoter areas in the 143 kb genome of LSDV, a portion of its genome was cloned. Three methods for purification of LSDV DNA were compared, to determine which yielded the best quality DNA for cloning. DNA extracted directly from infected cells was excessively contaminated with bovine host-DNA, complicating the cloning of LSDV DNA. The use of pulsed field gel electrophoresis solved the contamination problem, by separating viral DNA from bovine DNA. However, insufficient amounts of viral DNA for cloning purposes, could be recovered from the gel. Sufficient amounts of good quality LSDV DNA was obtained by extraction from purified virions. Purified LSDV DNA was digested with various restriction enzymes to identify those which yielded several 4-1 0 kb fragments, for cloning into the Bluescribe plasmid transcription vector. Enrichment for large fragments (8-1 0 kb) was achieved by sucrose density centrifugation. Cloned fragments were analysed by Southern blot hybridisation to verify their viral origin. Hybridisation studies indicated that several unique regions of the LSDV genome were cloned as Pst I and Bam HI fragments respectively, i.e. the cloned fragments contained no overlapping regions. In total, 71.25 kb of the DNA of the LSDV Neethling vaccine strain has been cloned, representing approximately 50% of the viral genome. The availability of these clones now paves the way for further molecular investigations of the LSDV Neethling genome, including identification of promoter regions. A trial gene, which will be cloned and expressed in LSDV, namely the cloned VPS-gene of bluetongue virus serotype 4, was prepared and its nucleotide sequence determined. Homopolymer sequences present at the terminal ends of the gene as a result of the original cloning strategy, are known to interfere with expression and were removed by means of the polymerase chain reaction (PCR). The nucleotide sequence of the resulting PCR-tailored BTV4 VPS-genewas determined and used to deduce the amino acid sequence of the protein. The gene is 1638 bp in length and encodes a protein of 526 aa. Conserved sequences, 6 bp in length and unique to the 5'- and 3'terminal ends of all BTV genes, were detected at the termini of the tailored gene, confirming that the original clone was a full-length copy of the gene. Amplification by PCR did not mutate the open reading frame (OAF) of the gene, since it was of similar length to that reported for 5 other BTV serotypes. With a view to future investigations, including the identification of promoter sequences in the LSDV genome, a preliminary investigation of LSDV protein synthesis was undertaken, to acquire some knowledge of the growth cycle of the virus. Eighteen putative virus-specific proteins were identified by radio-labelling infected cells with [³⁵S]-methionine. By pulse-labelling infected cells with [³⁵S]methionine at various times post infection (p.i.), viral proteins were first detected at 16 hr p.i. It is, however, unlikely that the early phase of viral replication commences as late as 16 hr p.i. and these results might be attributed to various problems, such as the low multiplicity of infection used and that host protein shut-down was inefficient, thus masking the presence viral proteins. In conclusion, this investigation resulted in the cloning of 71,25 kb of the LSDV genome, the tailoring and sequencing of the BTV4 VPS gene and the identification of 18 putative LSDV proteins. This now paves the way for further research to develop LSDV as a vaccine vector.
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    Characterization of avipoxviruses for use in recombinant vaccines
    (1992) Kow, Daria Karen; Dumbell, K R
    Pox viruses have been demonstrated in over 60 types of wild and exotic birds as well as domestic birds. Avipox viruses have been isolated and characterised from fowls, quails, canaries, parrots and lovebirds. This work describes the first isolation of a poxvirus from Jackass penguins (Spheniscus dermersus) and the characterisation of the virus as a separate species of penguinpox virus.
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    Demonstration of new subtypes of adenovirus 7 in South Africa, and probing oesophageal carcinoma cell lines for evidence of adenovirus or of other oncogenic viruses
    (1987) Brooks, Louise Alexandra; Steyn, Lafras M; Renan, M J; Dumbell, K R
    This study was carried out in 2 parts: 1. Genome analysis of human adenovirus species 7; 2. Search for a possible viral aetiology in oesophageal carcinoma. Sixteen laboratory isolates of adenovirus species 7, isolated in South Africa between 1975 and 1986, were characterized by restriction endonuclease analysis of their DNA genomes. Virus was propagated in human embryo fibroblast cells; genomic DNA, extracted and purified from cellular DNA extracts, was analyzed using 9 different restriction enzymes. Results of this study have demonstrated 2 new genome types of adenovirus 7c which have not previously been identified. The 2 novel strains, designated as genome types Ad7c1 and Ad7c2, were shown to differ from prototype Ad7 c according to restriction profiles with EcoRI; 2 new EcoRI sites were demonstrated in Ad7c1 and 1 in Ad7c2. The restriction sites were mapped on the viral genomes (at 3.68kb and 5.32kb from the left terminus) by double enzyme digestions, cloning of viral DNA, and nucleic acid hybridization using a cloned Ad7 probe. Strains resembling the prototype Ad7c and Ad7p (Gomen) genome types were also identified in the 1985 and 1986 Ad7 isolates. In order to investigate the possible role of a viral co-factor in the aetiology of oesophageal carcinoma, 18 probes, derived from potentially oncogenic viruses, were used to screen 3 human oesophageal carcinoma cell lines for the possible presence of integrated viral DNA. One of these, an Ad7 recombinant plasmid probe, was developed by cloning DNA from the transforming region of the Ad7cl strain into the plasmid vector pUC19. Cellular DNA, extracted from the 3 tumor lines HCU18, HCU33 and HCU39, was tested by means of both DNA dot hybridization and Southern blot hybridization for the presence of Epstein-Barr virus, human papillomavirus (types 1, 5, 6, 8, 11, 16, 18), human adenovirus (strains 5, 7, 12, 31) and human T-lymphotropic virus type I DNA. Both assays were demonstrated to be sensitive enough to detect 1 copy of viral DNA per cell. No hybridization between HPV, EBV, HTLV-I or adenovirus DNA probes, and the cellular DNA was detected. These findings indicate that the stable integration of these tumor viruses in host chromosomes did not play a role in the maintenance of the malignant phenotype of the 3 extensively passaged cell lines. Cells of the 3 oesophageal tumor lines were further examined by transmission electron microscopy, but the presence of virus particles in these cells was not observed.
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    DNA comparisons of the two orthopoxviruses monkeypox and variola
    (1988) Pare, Nicola Jennifer; Dumbell, K R
    Although smallpox has been eradicated there are animal poxviruses which are closely related. It is desirable to measure the closeness of this relation to assess whether Variola virus could re-emerge as a complex mutant of an animal poxvirus. The most likely candidate is Monkeypox, which can produce human infection clinically resembling smallpox. The work in this thesis is the beginning of a detailed comparison of the DNA of Variola and Monkeypox. A 15.3kb section of the Variola genome was compared with a corresponding 14.4kb region of Monkeypox. This enabled both a comparison of corresponding sequences and the location of a short sequence present only in variola. Initially restriction enzyme mapping of the two stretches of DNA showed considerable homology and narrowed down the area containing any nonhomologous Variola sequences to within 2.9kb. Sequence comparisons show a level of 96% similarity. When the 2.9kb Variola fragment was compared with the corresponding 2.4kb Monkeypox fragment, a 400bp insert was found in Variola flanked by sequences common to both viruses. Analysis of the insert revealed two overlapping open reading frames present on opposite DNA strands. The DNA and putative polypeptide sequences were compared with known sequences, but no significant homology was detected. The presence or absence of this sequence in other orthopoxviruses is being established, but the expression of these open reading frames in vivo and function of the putative polypeptides is still to be investigated.
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    Intraspecies typing of orthopoxviruses
    (1988) Richardson, Madalene; Dumbell, K R
    Characterization of viruses within the Orthopox genus has, in the past, been done by evaluating a spectrum of biological properties which gave different reactions in the different species. More recently analysis of genomic DNA by restriction endonucleases has proved a valuable additional way of identifying species and revealing intra-species variations among the orthopox viruses. This thesis reports the results of investigations of poxvirus isolates in which both biological characterization and DNA analysis have been used. Specimens of scabs from sick buffaloes in 18 outbreaks of buffalopox in India were extracted and virus was isolated from 13 of them by inoculation on chick embryo chorioallantoic membranes. The lesions resembled those of vaccinia and the isolates were confirmed as orthopox viruses by their susceptibility to neutralization by anti-vaccinial serum. Purified preparations of each isolate were made and the virion DNA extracted from them, was analysed by digestion with endonuclease Hind III, followed by electrophoresis to separate the resulting DNA fragments. The profiles of fragment sizes in the Hind III digest were indistinguishable from those of vaccinia isolates, but differed from other representative orthopoxviruses. This was the first examination of any DNA from the buffalopox virus. Six isolates were characterised in more detail. In biological tests, one of the six showed the ceiling temperature, rabbit virulence, cytopathic effect in cell monolayers and sensitivity to anti-vaccinial serum which is characteristic of vaccinia, and is regarded as an isolate of vaccinia. The other five had a lower ceiling temperature, were avirulent when inoculated in rabbits and were 10 - 50 fold less sensitive than vaccinia to neutralization with anti-vaccinia serum. In these properties they differed from vaccinia and resembled the buffalopox virus described by Baxby and Hill in 1971. Examination of virion DNA, using other restriction endonucleases showed that all five isolates could be grouped together and distinguished from a group of five vaccinia strains in digests with the endonuclease Sac I, Xho I and Eco RI. The sixth isolate which was biologically like vaccinia was also in the vaccinia group in these DNA analyses. Digestion of DNA obtained from all 12 of the non-vaccinia buffalo isolates showed the same profile and this profile differed from that of any of the vaccinia strains and also from the buffalopox isolate made 15 years previously in North India and described by Baxby and Hill. In another series of experiments the viral DNA from a number of isolates of monkeypox was analysed. One of these isolates was the first to be made from a wild caught animal (a squirrel) and the question was whether this isolate could be identified with isolates made from humans who had become infected in the same area of Zaire. All the isolates had previously been characterized biologically as being monkeypox virus. DNA was prepared from the squirrel isolate and from two human isolates from about the same time and district. No difference could be detected in the profiles of fragment sizes generated by digestion with the endonucleases Hind III or Pst I. To substantiate this apparent identity, DNA was prepared from another 9 monkeypox isolates from human infections with monkeypox in Liberia, Sierra Leone, Nigeria and Zaire as well as from five outbreaks in captive monkeys in Europe or America. The isolates covered the period from 1958 to 1986. The fragment size profiles established by electrophoresis of Pst I digests of all these DNA preparations were compared and found to fall into one of three distinct patterns. All the animal isolates from outside Africa had the same pattern as the isolates from Liberia and Sierra Leone. A second pattern was given by the two isolates from Nigeria and all the seven isolates from Zaire showed a third pattern. These observations supported the idea that the squirrel had been infected with the same variant of monkeypox virus that was causing the sporadic human infections in that country. The significance of the difference between the three patterns was investigated by constructing maps showing the location of Pst I cleavage sites on the genome of one isolate from each of the three main areas. A map had been published for Hind III cleavage sites on the genome of the Denmark strain of monkeypox virus. DNA from the Denmark strain was digested with Hind III, separated by electrophoresis in agarose gels and the fragments transferred by blotting onto nylon membranes. Fragments of DNA excised from gels after electrophoresis of Pst I digests were labelled by nick translation with 32 P-nucleotide and hybridized to the Hind III blots in order to determine the part of the genome from which each fragment came. By repetitions of this technique it was possible to construct Pst I maps of the genomes of representative strains of each of the three geographical variants of the virus. The sites which were different in the various isolates were found to be located in the centre and towards the left-hand end of the genome maps.
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    Molecular and evolutionary analysis of a gene conserved in most Orthopoxviruses
    (1993) Douglass, Nicola Jennifer; Dumbell, K R
    Evidence is presented to show that variola and monkeypox viruses evolved independently from a common ancestor. An open reading frame (ORF), potentially coding for a protein of 341 amino acid residues, was found to be conserved in two strains of variola virus (Harvey and Somalia), but degenerate in the Denmark strain of monkeypox virus. Monkeypox virus had a deletion of 391 bp, two 24bp deletions and a single base pair deletion within the coding region of this single copy ORF. The ORF corresponds to the E5R ORF in the published sequence of the Copenhagen strain of vaccinia virus, and the DNA sequence was determined for an additional strain of vaccinia virus, Dairen. A number of other Orthopoxviruses were found to contain this ORF, strengthening confidence in its presence in an ancestral Orthopoxvirus. The equivalent DNA sequence was determined for a number of monkeypox virus strains from West and Central Africa. The Denmark strain was identical to one from Liberia, indicating that this virus probably originated from West Africa. A third virus from West Africa, Benin, was found to have >99% base similarity and the same pattern of deletions as the other two monkeypox viruses. The Zaire strains were identical to one another and different from the West African strains. Like the West African strains, they contained the two 24bp deletions and single base pair deletion. In place of the large deletion they had three smaller deletions of 5-, 9- and 127-bp as well as a single base pair insertion. They also had additional deletions of land 2-bp and an insertion of 3bp. The West African strains have the potential to code for a truncated gene product of 107 amino acid residues, whereas the Zaire strains have no significant ORF. This clearly shows that monkeypox virus has diverged into two geographically isolated groups (Zaire and West Africa). There was >99% base similarity between the two groups, suggesting that the divergence occurred recently. Phylogenetic analysis, by the neighbour-joining method, was undertaken on the corresponding DNA sequences from variola (2 strains), monkeypox (6 strains), vaccinia (1 strain + 2 published sequences), cowpox (2 strains), taterapox, camel pox and ectromelia viruses. For every species gerbilpox virus was the nearest neighbour, suggesting that taterapox virus may be the species most closely related to the common ancestral Orthopoxvirus. Within the variola and cowpox virus species there was >99% DNA sequence conservation. Between species, camelpox virus was the most closely related species to gerbilpox virus, with variola virus, and, more distantly, vaccinia virus, falling into the same group. Cowpox virus was the most diverged species examined. Ectromelia virus shared a branch with cowpox virus. A comparison was made of the intergenic DNA sequence between this ORF and the adjacent downstream ORF. Variation was found, both within and between species, in the form of insertions and deletions. The interrelationships between the different Orthopoxvirus species more or less parallels that of the E5R-equivalent comparison. Some of the viruses had clusters of direct repeats. A pentameric repeated unit was found in 2, 10 and 17 copies in camelpox, gerbilpox and ectromelia viruses respectively. Raccoon poxvirus had a 7bp unit in 13 adjacent copies. The two cowpox viruses had a more complex arrangement of repeated sequences. It was thought that the ESR ORF may prove to be nonessential for virus replication. This was tested by interruption of the E5R gene in vaccinia virus; this did not affect the ability of the virus to form plaques in cell culture, but appeared to reduce the pathogenicity of the 'virus for rabbits. The deduced amino acid sequences were analysed for conserved and variable regions within the gene, to which no specific function has yet been assigned.