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

Rodent hepacivirus immunofluorescence assay. VeroFM cells were transfected in suspension using FuGENE HD (Promega, Mannheim, Germany) with 0.75 mg plasmid expressing the complete His-tagged NS3 proteins of the rodent hepaciviruses RMU10-3382 (rNS3RMU10-3382) and NLR-AP70 (rNS3AP70) and fixed 24 hours later with acetone/methanol (80%/20%). Myodes glareolus sera were tested at screening dilutions of 1:10 and 1:40. For secondary detection, a goat-anti-mouse Ig (Dianova, 1:2000) and a donkey-anti-goat cyanine 3-labelled Ig (Dianova, 1:200) were applied. Recombinant rNS3RMU10-3382 protein including a cleavable Thioredoxin/His 6 tag was expressed in bacteria and purified under non-denaturing conditions following a standard protocol [47] . The untagged purified protein was used to produce specific rabbit polyclonal antisera at Thermo Scientific Pierce custom antibody service. Rabbit antiserum against rNS3RMU10-3382 (1:2000) was used in parallel to an rNS3RMU10-3382-reactive rodent serum (1:50) for a co-localization study by confocal laser scanning microscopy. Here, secondary detection was performed using a cyanine 2-labelled goat-anti rabbit Ig (Dianova, 1:200) and a cyanin 3-conjugated goat-anti-mouse Ig (Dianova, 1:200).