Identification of the canarypox virus thymidine kinase gene and insertion of foreign genes

We mapped the canarypox virus (CaPV) thymidine kinase TK gene within a 5.8- kbp Xbal fragment of the genome by Southern blotting using the fowlpox Viru s (FPV) TK gene as a probe. Nucleotide sequence analysis of the fragment re vealed seven open reading frames (ORFs) showing gene organization similar t o that of FPV. The TK gene contained in this region had an ORF of 179 amino acids encoding a polypeptide with a putative molecular mass of 20.0 kDa. A n A/T-rich region and a transcription termination signal, TTTTTAT,were foun d upstream and at the end of the ORF which is consistent with poxvirus earl y gene regulation. The consensus sequence of the late promoter TAAAT also o verlapped with the initiation codon of the ORF. The amino acid sequence sim ilarity between the TK genes of CaPV and FPV, avipoxviruses, was 64.2%, whi ch was lower than the similarities between vaccinia and variola orthopoxvir uses (97.2%) and between Shope fibroma and myxoma leporipoxviruses (82.6%). However, the monophyly of avian clades of CaPV and FPV was supported by ph ylogenetic analysis. We then inserted the genes encoding lacZ, luciferase ( luci), and envelope of human T-lymphotropic virus type 1 (HTLV-1 env) into the TK gene of CaPV to evaluate its suitability as an expression vector. Th e recombinant viruses obtained were unstable, although the foreign genes we re expressed efficiently in the mammalian cells infected with the viruses. 1999 Academic Press.