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

Including the novel rodent viruses congeneric with HCV and canine/equine viruses, the minimal distance between the genera Hepacivirus and Pegivirus would be 73.5%. While this is lower than the 85-88% between other pairs of genera, it is consistent with a separation threshold of 72.2% between all members of the genus Flavivirus and Tamana bat virus, for which a separate genus has been proposed [59] . This also corresponds to inter-generic distances within other well-studied families of plus-strand RNA viruses such as the Picornaviridae, whose twelve genera are mostly separated by 70-80% in the RdRp-encoding 3D gene [60] . The HCV 1a H77 (GenBank, NC_004102) . GenBank accession numbers of reference hepaciviruses are indicated to the right of taxon names. Tree topology was inferred using BEAST with a GTR nucleotide substitution model as described in the methods section. Rodent hepaciviruses from this study are shown in red and boldface, equine hepaciviruses from this study are shown in blue and boldface. Red squares indicate those viruses whose near full-length genomes were generated. Statistical support of grouping is shown as posterior probabilities at deep nodes. Scale bar corresponds to genetic distance. To the right, 5,000 tree replicates of the same analysis are rendered using Densitree (initial 5,000 trees discarded as burn-in). Green line color indicates low probability of all trees, line thickness corresponds to concordant topologies across tree replicates. B. Genome organization of the novel rodent hepaciviruses. Genes were annotated as described in the methods section. Black arrows on the top indicate predicted signal peptidase cleavage sites. Red arrows below indicate N-, blue arrows O-glycosylation sites. Putative gene starts and ends are numbered below polyprotein plots. HCV 1a strain H77 is depicted on top as a reference. RMU10-3382 (KC411777) also represents the highly similar virus NLR-365 ( For the Bayesian phylogeny shown to the left, the WAG amino acid substitution model was used in MrBayes as indicated in the methods section. Statistical support of grouping from Bayesian posterior probabilities is indicated at node points. Scale bar corresponds to genetic distance. The Pestivirus BVDV (NC_001461) was chosen as an outgroup and truncated for graphical reasons. Branches leading to the novel hepaciviruses from this study are in orange. GenBank accession numbers of analyzed hepaciviruses correspond to those indicated in Figure 3A . The 59-and 39-genome termini were re-drawn from published foldings for equine hepaciviruses [24] , HCV [63, 77] and GBV-B [78] and de novo for this study for the 59- The RNA-negative specimen RMU10-3187 from the same species was processed identically and is shown below as a control. Positive staining is visible as distinct red granules in the cytoplasm of hepatocytes. Magnification was 1006, the inserts shows details of single hepatocytes in 106 higher magnification. Scale bars are shown to the lower right. C. Histopathology of M. glareolus liver specimens. Liver sections were stained by Hematoxylin and Eosin (H&E) and Epson van Giesson (EvG) stains. In H&E stains, black arrows point to inflammatory lymphocytic infiltrate. In EvG stains, black arrows highlight potential signs of fibrosis. Specimen 3180 shows intermediate portal inflammatory lymphocytic activity with potential low-grade fibrosis in a case with high hepacivirus RNA concentrations (1.5610 8 copies/gram). Specimen 1602 shows low-grad