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.

Direct pyrosequencing of RNA extracted from a blood plasma sample of a wild adult male red colobus from Kibale National Park, Uganda, generated 77,891 reads of 280 bp average length, consisting of 19,285 host reads (24.8%) and 58,606 non-host reads (75.2%). De novo assembly of singleton non-host reads resulted in a 15.5 kb contiguous sequence (contig) that aligned to the SHFV type strain LVR 42-0/M6941 (GenBank accession number NC_003092.1). A reference assembly against the 15.5 kb contig yielded 35,120 sequence reads with full coverage across the entire contig. Two additional contigs with lengths of 13 kb and 1.9 kb also aligned to the SHFV genome but were highly divergent from the initial 15.5 kb contig. The positions of these additional aligned contigs relative to the SHFV genome suggested that the two fragments might belong to the same viral variant. Using one step RT-PCR and 39 RACE, respectively, we were able to fill the gap between these two contigs and extend the 39 end of the contig to complete the terminal open reading frame (ORF9), yielding a new complete contig of 15.2 kb. A reference assembly against this 15.2 kb contig yielded 1,068 sequence reads with full coverage across the entire contig. Initial alignments indicated that both contigs covered all ORFs of the SHFV type strain and extended partially into the 39 and 59 untranslated regions. The 15.5 kb and the 15.2 kb contigs were designated SHFV-krc1 and SHFV-krc2, respectively, to indicate their origins in Kibale red colobus and to reflect nomenclature previously used to describe novel simian retroviruses in this same population of red colobus [20] .