Identification of the Canarypox Virus Thymidine Kinase Gene and Insertion of Foreign Genes

Abstract

We mapped the canarypox virus (CaPV) thymidine kinase (TK) gene within a 5.8-kbp XbaI fragment of the genome by Southern blotting using the fowlpox virus (FPV) TK gene as a probe. Nucleotide sequence analysis of the fragment revealed seven open reading frames (ORFs) showing gene organization similar to 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. An A/T-rich region and a transcription termination signal, TTTTTAT, were found upstream and at the end of the ORF, which is consistent with poxvirus early gene regulation. The consensus sequence of the late promoter TAAAT also overlapped with the initiation codon of the ORF. The amino acid sequence similarity between the TK genes of CaPV and FPV, avipoxviruses, was 64.2%, which was lower than the similarities between vaccinia and variola orthopoxviruses (97.2%) and between Shope fibroma and myxoma leporipoxviruses (82.6%). However, the monophyly of avian clades of CaPV and FPV was supported by phylogenetic 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. The recombinant viruses obtained were unstable, although the foreign genes were expressed efficiently in the mammalian cells infected with the viruses.

The Genome of Canarypox Virus

“…CNPV contains a unique complement of genes likely involved in nucleotide metabolism. CNPV encodes, similar to FWPV and other ChPVs, thymidine kinase, dUTPase, and glutaredoxin homologues (2,5). CNPV contains two genes (CNPV199 and CNPV213) similar to both deoxycytidine and deoxyguanosine kinases, thus far avipoxvirus-specific nucleotide metabolism genes.…”

“…Improved understanding of virus-host interactions should yield improved vaccine vectors, as demonstrated by recent incorporation of vaccinia virus (VACV) host response modification genes to create a third-generation CNPV-based vaccine (28,44). Current molecular data indicate that CNPV is related to FWPV, a virus for which the genomic sequence has been determined (2,5). Features of the 288-kbp FWPV genome included (i) large genomic segments containing replicative genes conserved and syntenic with those of other ChPV, (ii) rearrangement of specific genomic regions relative to other ChPV, (iii) the presence of numerous gene families and novel homologues of cellular genes likely involved in virus-host interaction, viral virulence, and viral host range which account for the large size of the FWPV genome relative to ChPV, and (iv) the location of potential virulence and host range genes both in terminal genomic regions and discrete, variable central genomic regions near the junctions of large genomic rearrangements.…”

“…While other select avipoxvirus species closely resemble FWPV in restriction fragment patterns and estimated genomic size, the CNPV genome is larger and comparatively divergent (79,92,97). In addition, limited sequence data indicate that while CNPV and FWPV share regions of similar gene order and are likely monophyletic within the Poxviridae, amino acid identity between open reading frame (ORF) homologues (55 to 74%) is comparable to that seen between different ChPV genera (5). Thus, there are likely to be substantial differences in the genomic elements responsible for virulence and host range differences of CNPV and FWPV.…”

Avipoxviruses: infection biology and their use as vaccine vectors

“…Through use of these techniques, we now have a better understanding of the biology of vaccinia and other poxviruses, including their host range restriction. Although, some major advances have been made in genome sequencing and in vitro characterization of APVs [7,8,38,39], studies on APV host range genes are scarce. A wide array of gene homologues with likely host range functions such as NK-cell receptors, chemokines, serine protease inhibitors and homologues of genes involved in apoptosis, cell growth, tissue tropism and avian host range, have been identified in APVs, which suggests significant viral adaptation in the avian host [7].…”

Combined Cytolytic Effects of a Vaccinia Virus Encoding a Single Chain Trimer of MHC-I with a Tax-Epitope and Tax-Specific CTLs on HTLV-I-Infected Cells in a Rat Model

“…Hamster BHK cells were cultured in D-MEM supplemented with 10% FCS. Canarypox virus (a kind gift of National Institute of Animal Health) [31], mO, m8Δ [22], and LC16 m8ΔVNC110 that harbors multiple cloning site in the HA gene of m8Δ genome were described previously [32]. Viral titers were calculated on the basis of the number of plaques on RK13 cells.…”