Both pseudorabies virus and the various strains of avian sarcoma and leukosis viruses are economically important pathogens. In Canada, pseudorabies is an exotic virus which primarily infects swine. The disease is rapidly fatal in most species although adult pigs may become carriers. Because pseudorabies virus (PrV) is exotic to Canada, imported animals are rigorously tested for the presence of antibody titers to the virus. The leukosis caused by Rous associated virus (RAV) has a major impact on the health of poultry flocks. Different strains of the avian leukosis viruses cause damage of differing severity but the general characteristics of infection include decreases in both egg and meat production. There would be significant benefits if non-infectious, antigenic fractions from both of these viruses could be produced in bacterial cells. These proteins could be used in serum antibody testing to make the detection of both viruses considerably simpler than the currently used ELISA and virus neutralization tests. As well, the production of large quantities of RAV env protein could simplify studies of the mechanisms of host specificity and provide additional information about chicken cell receptors. I have attempted to produce an antigenically recognizable protein from both pseudo-rabies virus and Rous associated virus. Attempts were made to express such a protein from pseudorabies virus by cloning either cDNA or genomic DNA fragments into plasmid vectors followed by colony immunodetection using a polyclonal antibody against PrV prepared in pig. Although several recombinants produced positive immunological results, further examination showed that none contained PrV DNA sequences. Standard screening methods were modified and improved during the course of this work. Subsequently, other laboratories identified significant difficulties in using unpurified polyclonal antibodies prepared in swine which provided an explanation for the results I observed. A cDNA library constructed from RNA prepared from RAV-1 was screened using a probe prepared from env sequences in pSR-XD2, a plasmid containing much of the RSV sequence. Transformants containing RSV homologous DNA were identified. During purification of these clones and subsequent screenings many of the plasmids identified as containing RSV homologous sequence lost their inserts. Additional studies demonstrated that plasmids which exhibited the strongest homology with the RSV probe and which contained the longest inserts, lost these inserts at a high rate. A second cDNA cloning using a different bacterial host and a different viral strain (RAV-2) produced several positive clones, with insert lengths ranging from 250 by to 950 bp. These inserts were sequenced and compared to known RSV and RAV sequences. A high degree of homology was observed. The longest RAV-2 cDNA fragments included most of the env gp37 polypeptide coding region. In order to test for expression of an antigen, inserts from two independently isolated recombinants were subcloned into lambdagt11 in each of the three reading frames. A polyclonal antibody prepared against Rous sarcoma virus was used to test for the expression of any recognizable antigenic determinants. No antigen production was detected even though nucleic acid plaque hybridizations demonstrated that the subcloning had been successful in introducing RAV-2 sequences into lambdagt11. Possible explanations for this, as well as a discussion of the composition of the RAV-2 sequences and their placement on the RSV genetic map are considered.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/10670 |
| Date | January 1992 |
| Creators | Bertrand, Stephen. |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 154 p. |
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