Novel, Divergent Simian Hemorrhagic Fever Viruses in a Wild Ugandan Red Colobus Monkey Discovered Using Direct Pyrosequencing

Lauck, Michael; Hyeroba, David; Tumukunde, Alex; Weny, Geoffrey; Lank, Simon M.; Chapman, Colin A.; O'Connor, David H.; Friedrich, Thomas C.; Goldberg, Tony L.

To estimate the similarity of newly discovered SHFV variants to the type strain and to prototypical arteriviruses, nucleic and amino acid sequences of individual ORFs were aligned using the MAFFT method [13] , alignments were concatenated, and uncorrected percent sequence identities for the resulting full-length coding genomes were calculated using the computer program MEGA4 [14] . To determine the phylogenetic relationships of newly discovered SHFV variants to known arteriviruses, nucleotide sequences of homologous ORFs in SHFV, EAV, LDV, and PRRSV (1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7, with reference to the EAV genome) were compiled from the literature, with strains chosen from available full-length genome sequences to represent the known within-species diversity of each virus. Non-overlapping coding regions were aligned using a codon-guided version of the MAFFT method [13] with poorly aligned sites removed using the Gblocks alignment cleaning method [15] implemented in the computer program TranslatorX [16] . Phylogenetic analyses were then conducted on concatenated, non-overlapping regions of aligned, cleaned ORFs, using first and second codon positions only, to avoid error associated with third-codon position saturation. Phylogenetic trees were constructed using the maximum likelihood method implemented in the computer program PAUP* 4.0 [17] . The substitution model used in the analysis was estimated using jModelTest [18] and was of the form GTR+I+C with the following parameters: nucleotide frequencies of A = 0.2386; C = 0.2413; G = 0.2633; T = 0.2568; substitution rates of AC = 2.5732, AG = 2.7382, AT = 1.4815, CG = 1.6751, CT = 2.1065, and GT = 1; I (proportion of invariant sites) = 0.1330; and C (gamma distribution of among-site rate variation) = 2.144, with 4 rate categories. Robustness of phylogenetic groupings was assessed in PAUP* using 1000 bootstrap replicates of the data [19] . New SHFV sequences were deposited in GenBank under accession numbers HQ845737 (SHFV-krc1) and HQ845738 (SHFV-krc2).