Detection of alpha and betacoronaviruses in multiple Iberian bat species

Falcón, Ana; Vázquez-Morón, Sonia; Casas, Inmaculada; Aznar, Carolina; Ruiz, Guillermo; Pozo, Francisco; Perez-Breña, Pilar; Juste, Javier; Ibáñez, Carlos; Garin, Inazio; Aihartza, Joxerra; Echevarría, Juan E.

Oro-pharyngeal swabs collected between 2004 and 2006 were preserved in 1 ml of lysis buffer (4 M GuSCN (Sigma), OS% N-lauroyl Sarcosine (Sigma), 1 mM dithiothreitol (DTT, Sigma), 25 mM Sodium Citrate and 20 pg/tube Glycogen (Boehringer Mannheim). Oro-pharyngeal swabs and faeces collected in 2007 were preserved in both 1 ml of lysis buffer and 1 ml of viral transport medium (VTM) (Eagle's minimal essential medium (EMEM) supplemented with 10 UI/ml of penicillin, 10 lg/ml of streptomycin, 160 lg/ml of gentamicin, 50 UI/ml of mycostatin and 1% of bovine serum albumin). All samples were frozen at -80°C before sending them to the Rabies Reference Laboratory at the Centro Nacional de Microbiología, ISCIII in Madrid. Faecal samples were clarified by centrifugation. Total nucleic acid was extracted from a 200 ll aliquot of each specimen for PCR assays and the rest were stored to -80°C in two different aliquots. Final pellets were always resuspended to 55 ll of water.