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.

A pan-coronavirus nested PCR was designed in the RdRp gene. A total of 5 ll of extracted RNA was added to 45 ll of reaction mixture of OneStep RT-PCR kit (QIAGEN, Valencia, CA, USA) containing 200 lM dNTPs and 60 pmol of generic CoV-specific degenerated primers (forward 5 0 -CARATGAATYTIAARTAYGC-3 0 and reverse 5 0 -TGYTGWGARCAAAAYTCRTG-3 0 ) and following manufacturer indications. Amplifications were carried out into thin-walled reaction tubes (Sorenson, BioScience, UT) in a PTC-200 (Peltier Thermal Cycler, MJ Research, Watertown, MA). Nested PCR amplifications were performed using 2 ll of first amplification product and 48 ll of reaction mixture containing 60 mM Tris-HCl (pH 8.5), 15 mM (NH4) 2 SO 4 , 200 lM dNTPs (Amersham Pharmacia Biotech, Piscataway, NJ), 3 mM MgCl 2 , 35 pmol of generic CoV-specific degenerated primers (forward 5 0 -ATGGGWTGGGAYTAYCCIAARTG-3 0 and reverse 5 0 -ACRTTRTTYTGRWARTA-3 0 ) and 1.25 U AmpliTaq DNA Polymerase (Perkin-Elmer Cetus, Norwalk). Amplification product size of 512 nt was visualized by agarose gel electrophoresis and sequenced directly in both directions using an automated ABI PRISM 377 model sequencer. For phylogeny reconstruction, consensus sequences were aligned together with others obtained from public genomic databases using the program CLUSTAL X (version 1.83) ( Table 2) . A Bayesian phylogenetic inference was obtained using Mr Bayes version 3.1 [24] with random starting trees without constraints. For the analyses GTR substitution model, gamma estimation and two simultaneous runs of 10 7 generations were done, each with four Markov chains, and the trees were sampled every 100 generations. Amino acid identity was calculated with MEGA 4 using the pairwise deletion option. The alignment comprised the same 396 bp of the RdRp gene used for the phylogenetic reconstruction.