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

hnologies such as gene knock out and in-vivo imaging exist. Also for this virus clade, two different full genomes have been determined. In the present study focusing on viral ecology, however, we have not conducted infection or virus rescue trials in cell cultures or animals. Due to the strict liver tropism those viruses can be expected to be as difficult to cultivate as HCV, and we currently lack any possibilities to generate primary Myodes hepatocytes. The housing of those animals is in preparation, as are attempts to rescue fully sequenced viruses by reverse genetics. Additionally, our finding of presence of hepaciviruses in the Murinae subfamily have triggered more targeted ecological investigations to potentially identify viruses from hosts in even closer relationship to Mus musculus. The availability of rodent surrogate models of HCV infection may obviate one of the most critical obstacles to HCV vaccine development by obviating the need for primate experiments in early stages of experimentation [12, 34] . Figure S1 BEAST polyprotein phylogeny including the novel rodent hepaciviruses. The complete polyprotein sequence of all hepaciviruses was analyzed in BEAST [37] using the FLU amino acid substitution matrix and a strict clock over 10,000,000 trees sampled every 1,000 generations. After exclusion of 2,500 trees as burn-in, all trees are depicted using Densitree [39] . Blue color corresponds to most probable topologies, red to second-best, green to third-best and dark green to remaining topologies. First round RT-PCR reactions were used a touchdown protocol with reverse transcription at 48u for 30 minutes, denaturation at 95u for 3 minutes, followed by PCR 10 cycles of 15 sec at 94uC, 20 sec at 60uC with a decrease of 1uC per cycle, and extension at 72uC for 45 seconds, followed by another 40 cycles at 50uC annealing temperature. Second round reactions used the same cycling protocol without the RT step. RNA quantification was performed in 25 mL reaction volumes using the SSIII One-Step RT-PCR system (Invitrogen) as described above with 300 nmol/L of respective forward and reverse primers and 200 nmol/L of respective probes. Amplification involved 15 min at 55uC; 3 min at 95uC; 45 cycles of 15 sec at 94uC, and 25 sec at 58uC. Fluorescence was measured at the 58uC annealing/extension step. Published assays from which oligonucleotide primers were used in this study included [35, 79, 80, 81, 82] . (DOC) Table S3 Putative cleavage sites for cellular signal peptidases within the N-terminal half of hepacivirus polyproteins. NN: neural networks; HMM: hidden Markov models (the values represent probabilities for putative SP cleavage sites). Only SP cleavage sites predicted by both NN and HMM were considered. All scores were re-calculated upon putting a suggested cleavage site at amino acid position 20 of a query polypeptide. *Y-scores were zero for these sites, however they were supported by uncorrected S-scores (not shown). Hepaciviruses included were SAR46 (KC411807) and SAR3 (KC411806) from Rhabdomys pumilio, RMU10-3382 (KC411777), NLR-365