Evidence for Novel Hepaciviruses in Rodents

Drexler, Jan Felix; Corman, Victor Max; Müller, Marcel Alexander; Lukashev, Alexander N.; Gmyl, Anatoly; Coutard, Bruno; Adam, Alexander; Ritz, Daniel; Leijten, Lonneke M.; van Riel, Debby; Kallies, Rene; Klose, Stefan M.; Gloza-Rausch, Florian; Binger, Tabea; Annan, Augustina; Adu-Sarkodie, Yaw; Oppong, Samuel; Bourgarel, Mathieu; Rupp, Daniel; Hoffmann, Bernd; Schlegel, Mathias; Kümmerer, Beate M.; Krüger, Detlev H.; Schmidt-Chanasit, Jonas; Setién, Alvaro Aguilar; Cottontail, Veronika M.; Hemachudha, Thiravat; Wacharapluesadee, Supaporn; Osterrieder, Klaus; Bartenschlager, Ralf; Matthee, Sonja; Beer, Martin; Kuiken, Thijs; Reusken, Chantal; Leroy, Eric M.; Ulrich, Rainer G.; Drosten, Christian

No isolation attempts were made due to the small available specimen quantities and notorious difficulty of hepacivirus isolation. Instead, those rodent specimens with highest RNA concentrations were selected for full genome sequencing. Genomespanning islets were amplified by PCR using degenerate broadly reactive oligonucleotides (Supplementary Table S2 ). Bridging strain-specific oligonucleotide primers (available upon request) were then designed to perform long range PCR using the Expand High Fidelity kit (Roche) on cDNA templates generated with the SuperScriptIII kit (Invitrogen). Some cDNA templates were enriched using a Phi29-based hexamer-driven amplification using a modified protocol of the Qiagen Whole Transcriptome Amplification kit (Qiagen) as described previously [25] . Amplicons were Sanger sequenced using a primer walking strategy. The 59genome ends were determined using the Roche rapid amplifica-tion of cDNA ends (RACE) kit (Roche) generating contiguous PCR amplicons encompassing the complete 59-untranslated region (59-UTR) and the 59-terminus of the core gene. 454 junior next generation sequencing was used for confirmation of 59-UTR sequences. For determination of the 39-genome end, viral RNA was adenylated using a poly-A-polymerase (Clontech, Paris, France) followed by 39-RACE using the Invitrogen GeneRacer Kit (Invitrogen).