Detection and genetic characterization of Mamastrovirus 5 from Brazilian dogs

Alves, Christian D.B.T.; Budaszewski, Renata F.; Torikachvili, Marcela; Streck, André F.; Weber, Matheus N.; Cibulski, Samuel P.; Ravazzolo, Ana P.; Lunge, Vagner R.; Canal, Cláudio W.

An initial screening using RT-PCR to detect a larger number of Mamastrovirus species was achieved by amplifying 422 bp of the ORF1b fragment using oligonucleotides, as previously described. 28 For the specific detection of MAstV5, 92 nucleotide sequences of this species were retrieved from GenBank database (http://www.ncbi.nlm.nih.gov/nucleotide), and aligned using CLUSTAL W within Molecular Evolutionary Genetics Analysis version 6 (MEGA6). 29 The MAstV5 specific RT-PCR was designed with a primer pair targeting the region of ORF2 that amplified a 250 bp fragment selected using Primer3 software. 30 In addition, the 16S rRNA gene from Escherichia coli was amplified using the primer pair FC27 and R530 as an endogenous internal control in each fecal sample evaluated for the specific presence of MAstV5. 31 For partial genome amplification, sets of 12 pairs of sequencing primers were selected to amplify overlapping fragments of ORF1 (ORF1a and ORF1b) and capsid protein (ORF2) segment representing a consensus sequence of approximately 5000 nucleotides. The primer sequences are shown in Table 1 .