Esteban and Hutchinson BMC Genomics 2011, 12:261 http://www.biomedcentral.com/1471-2164/12/261 Analysis of the content and organization of the ortho- poxvirus genome implicates gene gain and loss as major mechanisms in their evolution [8]. Cowpox virus strain Brighton Red (CPXV-BR) has a “master set” of genes; all other orthopoxviruses have a smaller subset of those genes. Thus it is likely that as the orthopoxviruses evolved and diversified into different hosts, some genes were lost while those that were retained adapted to the specific host. The sequence conservation of the core genes may be the result of stringent structural or func- tional constraints on these core proteins. Host-response modifier genes in the terminal regions, however, may be more able to change and thus show greater sequence divergence. As such, we were interested in understand- ing the role of adaptive molecular evolution in poxvirus genes and the selective pressures that act on genes in different regions of the genome. Adaptive molecular evolution, or diversifying selection, is a key mechanism for species divergence and identifying proteins or speci- fic residues experiencing diversifying selection may be important in understanding gene function. Diversifying selection can be detected by measuring the ratio of non-synonymous/synonymous mutation fixation rates (ω = dN/dS) [9-11]. Codons experience continual nucleotide substitutions as a result of errors in replication. Substitutions that result in synonymous mutations are selectively neutral. However, non-synon- ymous mutations can occur and become fixed as a result of selection. In purifying selection (ω < 1), a higher rate of synonymous than non-synonymous muta- tions occurs, suggesting stringent structural or func- tional constraints on the amino acid. In neutral selection (ω = 1) a change to a different amino acid has neither a positive nor negative effect on the protein. In diversifying selection (ω > 1) the rate of fixation of non- synonymous mutations is greater than the rate of fixa- tion of synonymous mutations, indicating adaptive molecular evolution. In this study we analyzed a set of 175 orthopoxvirus gene families using CPXV-BR as a reference genome to deter- mine the selective pressures acting upon them. We show that diversifying selection occurs most strongly in the terminal regions of the genome. The results of this study may help identify important functional regions in genes of known or unknown function, and may help categorize genes of unknown function as host-interacting genes. Results Gene families The data set collected from the Viral Bioinformatics Resource Centre database contained 19,874 chordopox- virus gene sequences. After the removal of identical sequences, 7758 unique sequences from 103 viruses remained (Additional file 1). Sequences were parsed into Page 2 of 12 380 gene families of which 214 were present in the CPXV-BR genome. The CPXV-BR genome encodes 235 genes belonging to 214 gene families. Of the 214 gene families, 38 were excluded from the analysis (Additional file 2). 16 gene families (containing a total of 37 genes) were excluded because they were duplicated in the genome and there- fore have the potential for differential evolutionary pres- sures on each paralog. 17 families with fewer than 6 sequences were excluded, since too few taxa in the data- set results in poor power to identify amino acid sites under diversifying selection [12]. Gene family size in the final dataset ranged from 6-69 sequences. 6 gene families were removed because of difficulty in generating a reli- able alignment, fragmentation of the gene in CPXV, or excessive computational time required for analysis due to large family size and length of genes. The final dataset had a total of 175 gene families, comprising a total of 6058 sequences analyzed (Additional file 3). CPXV-BR is used as the reference strain for the purposes of referring to gene names, codon positions and genome organiza- tion, however, because the analysis is performed on gene families, not single gene sequences, the results apply to homologous genes of all poxviruses used in this study. Treelength (S), measured as the number of substitu- tions per codon in the tree, can be used as a measure of sequence divergence in the gene family. Genes with tree- lengths that are too short (S < 0.11) are insufficiently divergent for this analysis [12]. All genes included in this analysis had S > 0.11. S values ranged from 0.225 to 43.02, with only three above 30 (Additional file 3). Genes found to be under diversifying selection (see below) were distributed throughout the range of treelengths. The length of CPXV genes ranged from 42 to 1286 codons, with the majority having a length of less than 400 codons, and only 5 greater than 800 codons. Genes found to be under diversifying selection were distributed throughout the range (data not shown). Diversifying selection We wanted to determine which genes in poxvirus gen- omes showed adaptive molecular evolution (diversifying selection). To do this, we used site models of codon evolu- tion to determine the rate of fixation of synonymous and non-synonymous mutations that can identify the presence of an amino acid site (codon) class under diversifying selection. Codon models use a statistical distribution to describe the variation in ω among sites, and make no assumptions about which sites are under diversifying selection. Site models permit differing dN/dS ratios at each codon, and because selective pressures are expected to vary across amino acids, this approach has high power to detect diversifying selection in genes [11]. Each gene family was analyzed for evidence of diversifying selection