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.).