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

Additional to phylogeny and genomic properties, the novel viruses resemble HCV in important traits of the natural history of infection. The detection of non-identical virus sequences in natural groups of animals, in combination with specific antiviral antibodies, proves continuous transmission of virus among animals. Induction of controlled infections in housed animals should thus be feasible. We found clear in-vivo evidence for hepatic tropism by demonstrating histopathological signs of liver inflammation, concentrations (3.4610 8 copies/gram). Specimen 3187 shows no significantly increased inflammatory activity and no signs of fibrosis in a case with no detectable hepacivirus RNA. Due to highest tissue quality, a terminal hepatic venule instead of a portal triad is shown. D. Recognition of rodent hepacivirus clade 1 antigens by M. glareolus serum. VeroFM cells expressing complete NS3 from M. glareolus hepacivirus RMU10-3382 (GenBank, KC411777) were incubated with 1:2000-diluted rabbit-anti-NS3 3382 antiserum (control) or 1:50-diluted rodent serum (picture shows exemplary results for animal NLR 3/C12), followed by goat-anti-rabbit-Cy2 (green) and goat-anti-mouse-Cy3 (red) secondary immunoglobulins. For colocalization analysis of fluorescence signals, the 6th of 12 1-mM Z-stags is shown for every channel. Cross-reactivity with HCV antigens was analyzed by incubation of HuH7 cells, transfected with HCV replicon JFH1, with a rabbit-anti-human-HCV-NS3-49 serum, diluted 1:400 (control) or rodent serum NLR 3/C12 diluted 1:50, followed by goat-anti-rabbit-Cy2 (green) and goat-anti-mouse-Cy3 (red) secondary antibodies. Counterstaining was performed using DAPI. Bar, 25 mm. doi:10.1371/journal.ppat.1003438.g006 excessive viral RNA concentrations in the liver, as well as in-situ hybridizations demonstrating intracellular genome replication in liver cells of bank voles. A somewhat lower degree of hepatic inflammation compared to that in some HCV-infected humans might be due to the shorter life span of bank voles rarely exceeding 1-2 years in the wild, or due to a higher capacity of tissue regeneration [72, 73] . Interestingly, our serological investigations suggested bank voles might be able to clear hepacivirus infections, as antibodies did not co-occur with RNA in most, but not all animals [1] . Bank voles may therefore be more capable of clearing hepacivirus infection than humans. This would be compatible with infection patterns also observed in other Flaviviridae members, exemplified by the flavivirus West Nile virus in rhesus macaques, the pestivirus BVDV1 in cattle and the hepacivirus GBV-B in experimentally infected tamarins [61, 74, 75] . However, it would differ from equine hepaciviruses, in which RNA and antibodies cooccurred [24] . Controlled infection experiments in bank voles might yield relevant scenarios for the study of HCV persistence. Bank voles can be kept in the laboratory with comparatively little effort and have been used for virus infection studies, e.g., with herpesviruses, bornaviruses, hantaviruses, and flaviviruses [53, 54, 55, 56] . Efforts to establish bank vole infection models may benefit from the discovery of two highly divergent clades in this species. Knowledge of three full genomes in total should enable efficient rescue of virus from cDNA. Notably, the Rhabdomys-associated virus clade has a host in even closer relationship (on subfamily level) to Mus musculus commonly kept in laboratories, for which powerful tec