Type I interferon is a cytokine induced upon virus infection that generates an antiviral state in neighbouring cells. Historically, research into avian interferon has lagged behind that into the mammalian system and hence reagents and assays are poorly developed. The avipoxvirus Fowlpox virus is naturally host restricted to avian cells. Modified Vaccinia virus Ankara (MVA) is an attenuated Vaccinia virus derived by serial passage in chicken embryonic fibroblasts (CEF), so that it too is now host restricted. In CEF pretreated with chicken interferon (ChIFN), plaque formation by Fowlpox virus (FP9 strain) was approximately 500-times less inhibited than MVA. A lack of bioinformatic clues within the FP9 genome sequence justified a broad-scale screen to identify any molecular determinants that contributed to this interferon resistance. The 266 kbp FP9 genome was divided into 61 overlapping fragments, each approximately 8 kbp long, to include each unique open reading frame (ORF) intact in at least one fragment. Splice-overlap-extension PCR protocols were optimised to assemble each genomic fragment with a guanine phosphoribosyl transferase (GPT) gene under the control of a poxvirus promoter, between two MVA flanks. These linear constructs were transfected into cells co-infected with MVA, for homologous recombination to insert the FP9 fragment and GPT cassette into the MVA genome. GPT-positive virus was enriched by culture with mycophenolic acid. In total, 65 recombinant viruses were recovered, representing 85% of the FP9 ORFs. Fourteen fragments were independently duplicated to control for mutations that may have been introduced during PCR. Recombinant viruses were screened for an increased ability to plaque in ChIFN-treated cells identifying 2 FP9 fragments, Fowlpox Interferon Resistance locus (FIR) 1 and FIR2. Further recombinant MVA viruses each containing a single gene from FIR1 were constructed and assessed for interferon resistance using the same techniques, identifying FP9.014 as contributing to FP9 ChIFN resistance.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:506461 |
| Date | January 2008 |
| Creators | Buttigieg, Karen Rachel |
| Contributors | Skinner, Michael |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/4415 |
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