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.

The RT-PCR products generated with the sets of sequencing primers were purified using the NucleoSpin Extract II Kit (Macherey-Nagel, Germany). Both DNA strands were sequenced with an ABI PRISM 3100 Genetic Analyzer using a BigDye Terminator v.3.1 cycle Sequencing Kit (Applied Biosystems, USA). Overlapping fragments were aligned and assembled using SeqMan software from the DNASTAR package (DNASTAR, USA). 32 [The open reading frames were identified using the NCBI ORF Finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).] Sequence alignment was performed using the CLUSTAL W. For the phylogenetic inferences of the MAstV capsid protein region, the MAstV5 sequences that were submitted to genome amplification and 12 MAstV5 representative strains were included. MEGA6 software 29 was used for phylogeny inference calculated using "find best DNA/protein model" tool from MEGA6. The Kimura 2-parameter substitution model was selected for the MAstV5 ORF2 nucleotide inference, and the LG substitution model (frequencies +F) was used for the amino acid inference. The substitution-rate variation among sites was modeled with a gamma distribution (shape parameter = 5). Statistical support was provided by 1000 non-parametric bootstrap analyses. A nucleotide distance matrix was calculated using an alignment with ORF2 and partial genome sequences ( Table 2 ). The Mamastrovirus genotypes were distinguished based on the amino acid sequence of the full length ORF2, where the genetic distances (p-dist) 0.378-0.750 and 0.006-0.312 between and within groups, respectively, were used. 5