Genome of horsepox virus

Abstract

Here we present the genomic sequence of horsepox virus (HSPV) isolate MNR-76, an orthopoxvirus (OPV) isolated in 1976 from diseased Mongolian horses. The 212-kbp genome contained 7.5-kbp inverted terminal repeats and lacked extensive terminal tandem repetition. HSPV contained 236 open reading frames (ORFs) with similarity to those in other OPVs, with those in the central 100-kbp region most conserved relative to other OPVs. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of vaccinia virus (VACV) and rabbitpox virus, clearly grouping together these VACV-like viruses. Fifty-four HSPV ORFs likely represented fragments of 25 orthologous OPV genes, including in the central region the only known fragmented form of an OPV ribonucleotide reductase large subunit gene. In terminal genomic regions, HSPV lacked full-length homologues of genes variably fragmented in other VACV-like viruses but was unique in fragmentation of the homologue of VACV strain Copenhagen B6R, a gene intact in other known VACV-like viruses. Notably, HSPV contained in terminal genomic regions 17 kbp of OPV-like sequence absent in known VACV-like viruses, including fragments of genes intact in other OPVs and approximately 1.4 kb of sequence present only in cowpox virus (CPXV). HSPV also contained seven full-length genes fragmented or missing in other VACV-like viruses, including intact homologues of the CPXV strain GRI-90 D2L/I4R CrmB and D13L CD30-like tumor necrosis factor receptors, D3L/I3R and C1L ankyrin repeat proteins, B19R kelch-like protein, D7L BTB/POZ domain protein, and B22R variola virus B22R-like protein. These results indicated that HSPV contains unique genomic features likely contributing to a unique virulence/host range phenotype. They also indicated that while closely related to known VACV-like viruses, HSPV contains additional, potentially ancestral sequences absent in other VACV-like viruses.

FIG. 1.

Schematic comparison of HSPV left (A) and right (B and C) terminal genomic regions to those of other orthopoxviruses. Virus names were abbreviated as follows and correspond to sequences from the following GenBank accession numbers in parentheses: VACV CPN (M35027); VACV WR (AY243312); Tian, VACV Tian Tan (AF095689); VACV MVA (U94848); m0, VACV Lister isolate LC16m0 (AY678277); RPXV, RPXV Utrecht (AY484669); GRI, CPXV GRI-90 (X94355); MPXV, MPXV Zaire-96-I-16 (AF380138); ECTV, ECTV Moscow (AF012825); CMLV, CMLV M-96 (AF438165); VARV, VARV Bangladesh-1975 (L22579); CPXV BRI (AF482758). Heavy lines indicate nucleotide sequences; boxes indicate ORFs matching those annotated in HSPV and those in genomic regions absent in HSPV. ORF names and genomic positions in kilobase pairs (K) are indicated for HSPV and CPXV Brighton Red, as are names of ORFs absent in these two species. Hatching indicates ORFs different in length (>10%) from intact orthologues from CPXV GRI-90 or Brighton Red. Red ORFs indicate HSPV ORFs intact or carried on sequences that are absent relative to VACV-like viruses. Asterisks indicate ORFs resequenced/reannotated by Upton et al. (92) as present or intact in the Tian Tan genome. Large solid-lined boxes indicate sequences matching the global alignment only at the opposite genomic terminus; dashed boxes indicate where sequences located in the opposite genomic terminus match the global alignment. Red lines indicate ITR sequence in each virus and are unaligned on the terminal side of HSPV002/HPSV206. Panel A is presented at a different scale relative to panels B and C.

FIG. 1.

Schematic comparison of HSPV left (A) and right (B and C) terminal genomic regions to those of other orthopoxviruses. Virus names were abbreviated as follows and correspond to sequences from the following GenBank accession numbers in parentheses: VACV CPN (M35027); VACV WR (AY243312); Tian, VACV Tian Tan (AF095689); VACV MVA (U94848); m0, VACV Lister isolate LC16m0 (AY678277); RPXV, RPXV Utrecht (AY484669); GRI, CPXV GRI-90 (X94355); MPXV, MPXV Zaire-96-I-16 (AF380138); ECTV, ECTV Moscow (AF012825); CMLV, CMLV M-96 (AF438165); VARV, VARV Bangladesh-1975 (L22579); CPXV BRI (AF482758). Heavy lines indicate nucleotide sequences; boxes indicate ORFs matching those annotated in HSPV and those in genomic regions absent in HSPV. ORF names and genomic positions in kilobase pairs (K) are indicated for HSPV and CPXV Brighton Red, as are names of ORFs absent in these two species. Hatching indicates ORFs different in length (>10%) from intact orthologues from CPXV GRI-90 or Brighton Red. Red ORFs indicate HSPV ORFs intact or carried on sequences that are absent relative to VACV-like viruses. Asterisks indicate ORFs resequenced/reannotated by Upton et al. (92) as present or intact in the Tian Tan genome. Large solid-lined boxes indicate sequences matching the global alignment only at the opposite genomic terminus; dashed boxes indicate where sequences located in the opposite genomic terminus match the global alignment. Red lines indicate ITR sequence in each virus and are unaligned on the terminal side of HSPV002/HPSV206. Panel A is presented at a different scale relative to panels B and C.

FIG. 2.

Phylogenetic analysis of HSPV central genomic regions. Conserved HSPV central genomic nucleotide sequences (positions 26800 to 170171) corresponding to regions used previously for OPV phylogenetic analysis (51) were aligned with homologous OPV sequences using DIALIGN, and gapped regions were realigned with CLUSTAL W and trimmed with Gblocks. The unrooted tree for 124,677 aligned characters was generated using maximum likelihood with general time reversible correction for multiple substitutions, four-category discrete gamma model, estimation for proportion of invariant residues, and 100 bootstrap replicates as implemented in PHYML. Bootstrap values greater than 70 are indicated at appropriate nodes; dots indicate values of 100. Homologous nucleotide sequences from the following viruses and accession numbers were compared: VACV strain CPN, M35027; VACV WR, AY243312; VACV Lister (Elstree) vaccine consensus (Lis), AY678276; VACV Lister-derived LC16m0 (m0), AY678277; VACV Tian Tan (Tian), AF095689; VACV MVA, U94848; RPXV Utrecht (RPXV), AY484669; CPXV strain GRI-90 (X94355); CPXV BRI, AF482758; MPXV strain Zaire-96-I-16 (MPXV ZAI), AF380138; MPXV WRAIR7-61 (MPXV W61), AY603973; MPXV USA_2003_039 (MPXV U39), DQ011157; CMLV strain M-96 (CMLV M96), AF438165; CMLV CMS, AY009089; VARV strain Bangladesh-1975 (VARV BAN), L22579; VARV India-1967 (VARV IND), X69198; VARV Garcia-1966 (VARV GAR); Y16780; ECTV strain Moscow (ECTV MOS), AF012825. The scale indicates estimated distance. Identical topologies at supported nodes were obtained using additional maximum likelihood analyses as implemented in TREE-PUZZLE, using neighbor-joining and maximum parsimony as implemented in PHYLO_WIN and PHYLIP, respectively, and using an unedited alignment (146,439 characters) (data not shown). Similar topologies were also obtained using similar analyses on whole-genomic alignments (data not shown).