Methods
Two animals collected in 2010 and 2013, in São José de Espinharas, Paraíba State, Brazil (Figure 1A) were sent to the Animal Pathology Laboratory, Center of Health and Rural Technologic, Federal University of Campina Grande for rabies investigation, collected in 2010 and 2013, in São José de Espinharas, Paraíba State, Brazil (Figure 1A). The CNS was evaluated according to the methodology described using direct fluorescent antibody test (d-FAT) (Dean et al, 1996), mouse inoculation test described by Koprowski (1996), histologycal and immunohistochemistry tests (Araújo et al, 2014).
To characterize the genomic sequence of the virus, the total RNA was extracted from the central nervous system using the QiAmp Viral RNA kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. The RNA obtained was treated with DNAse (Sigma-Aldrich, San Luis, MO, USA), according to manufacturer’s instructions. Reverse transcription of RNA was performed with the SuperscriptTM III Reverse Transcriptase kit (Thermo Fischer Scientific, Waltham, MA, USA) and stored at -20°C.
Next, the dsDNA was obtained according as previously described (Ullmann et al, 2015). As the dsDNA had not the expected concentration for library preparation (<0.5 ng/mL), the total 50 µL reaction volume was concentrated to 5µL on the concentrator (Speed Vaccum, Eppendorf, Hamburgo, Germany). Five uL (0.2 ng/µL) of the dsDNA obtained was used as input for library preparation with the Nextera XT Library Preparation kit (Illumina, CA, USA), until clean up after PCR amplification step (3rd step of the protocol). Libraries were quantified by qPCR (Kapa, KapaBiosystems, Wilmington, MA, USA), and sequenced with MiSeq Illumina, with its commercial kit (2x300 cycles)(Illumina., San Diego, CA,USA).
The assembly of resulting contigs was performed with Geneious R6 using as reference the complete genome of RABV (Rabies virus, GenBank accession KM594039). The Phylogenetic analysis was generated with the Seaview4 software (Gouy et al, 2010), and the tree was built with the Neighbor-Joining model, with 1,000 bootstrap repetitions, and visualized using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/). Finally, the analysis of mutations in the deep-sequencing data was performed using a co-assembly of both sample reads with ViVan (Isakov et al, 2015), and the protein structure was visualized with Pymol (Version 1.2r3pre, Schrödinger, LLC.).