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

The near full genomes of five representative hepaciviruses from all rodent clades were determined, including two viruses from R. pumilio, two from M. glareolus clade 1 and one from M. glareolus clade 2 (identified by red squares in Figure 3A ). The polyprotein genes were of different sizes including 2,781; 2,887; and 3,007 amino acid residues, respectively, compared to 3,008-3,033 in HCV. All genomes shared the typical hepacivirus polyprotein organization, encoding putative proteins in the sequence C-E1-E2-p7-NS2-NS3-NS4A/4B-NS5A-NS5B ( Figure 3B ). The putative structural C, E1, E2 and p7 proteins were predicted by signal peptidase cleavage site analysis (Supplementary Table S3 ) to be comparable in their sizes to that of known hepacivirus proteins. Placentalia (Eutheria) evolutionary lineages according to [76] . Major mammalian clades are identified at basal nodes of the Placentalia phylogeny: Afrotheria (e.g., elephants), Xenarthra, (e.g., anteaters) and Boreoeutheria, divided into the two superorders Euarchontoglires, (e.g., primates, rodents) and Laurasiatheria (e.g., dogs, bats). Sampled mammalian orders are shown in boldface type. Orders containing novel hepaciviruses identified in this study are shown in red and boldface. Orders with known hepaciviruses (perissodactyla, primates, carnivora) are given in red. Numbers of extant families and species per order adapted from [33] All rodent viruses had considerably fewer predicted glycosylation sites in their structural proteins, in particular their putative E2 proteins, as opposed to HCV. A detailed genome analysis is provided in Figure 3B . The 59-terminus of the core gene of the R. pumilio hepacivirus clade contained a putative adenosine-rich slippery sequence at codons 10-14 (AAAAAAAACAAAAA, Supplementary Figure 3B) . In HCV, a very similar sequence (AAAAAAAAAACAAA), located at nearly the same positions (codons 8-12) of the core gene induces production of a protein termed F in vitro due to ribosomal frameshift event [49] . Depending on the HCV genotype, the size of the F protein ranges from 126 to 162 amino acid residues which vary considerably in sequence composition [50] . The size of a putative F protein in SAR46 would be 65 amino acid residues and no homology to the HCV F proteins was observed.