Bats host major mammalian paramyxoviruses

Drexler, Jan Felix; Corman, Victor Max; Müller, Marcel Alexander; Maganga, Gael Darren; Vallo, Peter; Binger, Tabea; Gloza-Rausch, Florian; Rasche, Andrea; Yordanov, Stoian; Seebens, Antje; Oppong, Samuel; Sarkodie, Yaw Adu; Pongombo, Célestin; Lukashev, Alexander N.; Schmidt-Chanasit, Jonas; Stöcker, Andreas; Carneiro, Aroldo José Borges; Erbar, Stephanie; Maisner, Andrea; Fronhoffs, Florian; Buettner, Reinhard; Kalko, Elisabeth K.V.; Kruppa, Thomas; Franke, Carlos Roberto; Kallies, René; Yandoko, Emmanuel R.N.; Herrler, Georg; Reusken, Chantal; Hassanin, Alexandre; Krüger, Detlev H.; Matthee, Sonja; Ulrich, Rainer G.; Leroy, Eric M.; Drosten, Christian

Sampling and specimen preparation. For all capturing, sampling and exportation of small mammal specimens, permission was obtained from the respective countries' authorities. Bats and rodents were identified by trained field biologists. Fresh bat droppings were collected on plastic film below roost sites 12 . Additionally, bats were caught with mist nets at roost or foraging sites, kept separately in bags until individual examination. Sampling relied on faecal pellets produced in bags, vein puncture for serum samples and mouth swabs. For organ samples, bats were euthanised with ketamine and dissected immediately. Rodents were caught with live traps or snap traps, euthanised and dissected. For faecal specimens, ca. 100 mg of faeces was suspended in 500 µl of RNAlater solution (Qiagen, Hilden, Germany) immediately after collection. Suspensions were homogenised by vortexing, and 50 µl were suspended into 560 µl of buffer AVL from the Qiagen Viral RNA Mini kit (Qiagen) and processed further according to the instructions of the manufacturer. For blood or serum samples, up to 140 µl (depending on the available quantity) were extracted. For solid organs, approximately 30 mg of tissue were homogenised in a TissueLyser (Qiagen) or a ball-mill tissue grinder (Genogrinder 2000, Spex Centripep), followed by extraction of RNA using the RNeasy Kit (Qiagen) or the ABI PRISM 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA). Elution volumes were generally 50 µl for serum/blood and faecal specimens, and 100 µl for tissue specimens. RNA specimens were subjected to molecular screening for PVs using a panel of oligonucleotides and RT-PCR assays listed in Supplementary Tables S9 and S10. Supplementary Tables S9 and S10 . Virus quantification was done as described previously 55 . Briefly, amplicons from initial nested RT-PCR screening assays were TA-cloned (Invitrogen), plasmids purified and re-amplified with vector-specific oligonucleotides, and finally in vitro transcribed using the T7 promotor-based Megascript kit (Applied Biosystems, Darmstadt, Germany). Further details are available upon request from the authors.