Partial transcriptional mapping of the fowlpox virus genome and analysis of the EcoRI L fragment

Abstract

Several fowlpox virus (FPV) DNA fragments were selected by differential hybridization using cDNA of transcripts that were strongly transcribed early and/or late after infection of QT-35 cells. The EcoRI L fragment contained three strongly transcribed FPV genes: L1L, a late 1452 bp partial (amino end) ORF; L2R, an early/late 522 bp ORF; and L3R, a late 948 bp ORF. The protein products of L1L, L2R and L3R shared homology with the products ofvaccinia virus ( W ) genes H4L (RAP94), H5R (Ag35) and H6R (topoisomerase), respectively, suggesting a conservation of gene structure and order between VV and FPV. The 5' upstream non-coding sequences of L1L and L3R were A + T rich and the sequence 5' TAAATG 3' overlapped the predicted translation start codon. Primer extension analysis of the L2R transcript mapped the transcriptional start sites of early and late mRNAs 14 nt downstream of a VV early promoter-like critical region sequence, AAAATTGAA-AAAAAAA. A VV-like TAAAT late transcriptional element was present 20 nt upstream of the L2R ATG translational start codon. A plasmid with the putative early L2R promoter cloned upstream of the Newcastle disease virus haemagglutinin-neuraminidase (HN) cDNA as a reporter gene was at least 6-fold more effective in generating HN mRNA than plasmids containing the P7.5 or P11 VV promoters in transient expression assays in FPV-infected CEF cells treated with cytosine arabinoside. The L2R promoter was also able to express an amount of HN mRNA equal to that expressed by the W promoters late in infection.

The Genome of Fowlpox Virus

“…FPV ORFs contain typical poxvirus promoter sequences upstream of their translation initiation codons. Sequences with similarity to the VV early-promoter consensus sequence (AAAAATGAAAAAA AA) have previously been noted in the 5Ј untranslated regions of known and predicted FPV early genes (90,91,191). Fifty-six FPV ORFs contain putative early promoters (Table 1).…”

“…A total of 55 putative late FPV ORFs, including many of the conserved virion-associated poxvirus genes (Table 1), contain the VV late-promoter sequence (TAAATG) at the ATG codon (131). The TAAATG late promoter has been previously described to be located upstream of FPV late genes (17,91,163,191), and it is known that early-late and late promoters can be exchanged between FPV and VV with no loss of temporal specificity (27).…”

“…FPV RNA polymerase subunits include homologues of VV RPO147 (FPV137), RPO132 (FPV189), RAP94 (FPV141), RPO35 (FPV193), RPO30 (FPV100), RPO22 (FPV135), RPO19 (FPV169), RPO18 (FPV056), and RPO7 (FPV118). Homologues of all previously described early (E), intermediate (I), and late (L) poxvirus transcription factors (TFs) are found in FPV, including the following: VETF S (FPV057), VETF L (FPV171), VITF-3 (FPV172 and FPV188), VLTF-1 (FPV126), VLTF-2 (FPV049), VLTF-3 (FPV165), and VLTF-4 (FPV142) (87,191). FPV079 and FPV183 encode elongation factors for late transcription (VV G2R and A18R) (22,44,186).…”

“…Past work on FPV genomics, much of which used highly tissue culture-passaged FPV strains, has provided genetic information on approximately one-third of the viral genome, including some viral genes with putative immune evasion and host range functions (16,18,20,57,93,127,150,163,166,191). The rational design of safer and more effective FPV vaccines and FPV-based expression vectors will require complete information on viral genes associated with viral virulence and host range and a more complete understanding of how these genes function in viral pathogenesis, immune evasion, and avian host range.…”

Development of FPV140 antigen-specific ELISA differentiating fowlpox virus isolates from all other viral pathogens of avian origin

“…The large complex avian virus has an AT-rich dsDNA genome of between 260 and 310 kb (Coupar et al, 1990;Mockett et al, 1992;Zantinge et al, 1996). Poxvirus diseases of poultry have significant economic impact worldwide with economic losses resulting from a drop in egg production in layers, reduced growth rates in broilers, blindness, and in some cases, death (Cunningham, 1984;Afonso et al, 2000).…”