Computational protocol: Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil

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Protocol publication

[…] This work was conducted with samples from 73 municipalities of São Paulo State, Southern Brazil, using 305 samples of enteric content of 29 bat species of three families (Molossidae, Phyllostomidae and Vespertilionidae). These animals were submitted to Instituto Pasteur (São Paulo, Brazil) from March/2013 to July/2014, as a part of rabies surveillance program, and were stored at −20 °C. Each animal was necropsied, the entire intestine was removed from abdominal cavity and all intestinal content was extracted and stored at −20 °C.Feces suspensions (v/v, 10 %) were prepared with DEPC-treated water. Suspensions were clarified at 12,000 × g for 30 min at 4 °C and the supernatants were used in the assays. Extraction of total RNA was carried out with TRIzol Reagent™ (Life Technologies, Carlsbad, CA, USA) according to the manufacture’s instruction followed by reverse transcription with Random Primers and M-MLV™ Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) as per manufacturer’s instructions.All samples were submitted to a pancoronavirus nested RT-PCR targeting the RNA-dependent RNA-polimerase (RdRp) [], using Taq Platinum™ DNA Polymerase (Life Technologies, Carlsbad, CA, USA) as per manufacturer’s instructions. Positive samples were submitted to a RT-PCR targeting to S gene for phylogenetic inference [].Amplicons were purified using the ExoSap-IT® reagent (USB, Cleveland, OH, USA) or Illustra™ GFX™ Gel Extraction Kit (GE Healthcare, Buckinghamshire, UK) and bi-directional Sanger sequencing with the respective primers was carried out with BigDye v.3.1™ and ABI 3500 Genetic Analyzer™ (Life Technologies, Carlsbad, CA, USA).Chromatograms generated were subjected to Phred online application (http://asparagin.cenargen.embrapa.br/phph/) for assessment of their quality and the final consensus sequences were obtained with CAP Contig application in Bioedit 7.2.5 program []. Sequences obtained were aligned with homologous sequences retrieved from GenBank using CLUSTAL/W software in Bioedit 7.2.5 program [].Alignments were used for phylogenetic trees construction with distance optimization criterion with neighbor-joining algorithm and Composite Maximum Likelihood evolutionary model with 1000 “bootstrap” repetitions, using the MEGA 6 program []. […]

Pipeline specifications

Software tools BioEdit, Clustal W, MEGA
Application Phylogenetics