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.

One ml of plasma was centrifuged at 5,0006g at 4uC for 5 min with subsequent filtration of the supernatant through a 0.45-mm filter (Millipore, Billerica, MA, USA) to remove residual host cells. Viral RNA was isolated using the Qiagen QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, except that carrier RNA was omitted. The eluted RNA was treated with DNase I (DNA-free, Ambion, Austin, TX, USA), and double stranded DNA was generated using the Superscript double-stranded cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA) primed with random hexamers. DNA was purified using the Agencourt Ampure XP system (Beckman Coulter, Brea, CA, USA) and approximately 1 ng of DNA was subjected to simultaneous fragmentation and adaptor ligation (''tagmentation'') with the Nextera DNA Sample Prep Kit (Roche Titanium-compatible, Epicentre Biotechnologies, Madison, WI, USA). DNA was subsequently cleaned using the Agencourt Ampure XP system, briefly PCR amplified with primers to add Roche/454 titanium-based adaptors onto each fragment (15 cycles) , and cleaned again with the Agencourt Ampure XP system. DNA fragments were then sequenced using the GS Junior pyrosequencing system (Roche 454 Life Sciences, Branford, CT, USA).