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.