Genetic and phylogenetic analysis
The nucleotide sequences of MuKV identified in the present study were
compared to the corresponding sequences of other kobuvirus strains
previously reported in the Genbank database using Basic Local Alignment
Search Tool (BLAST;https://www.ncbi.nlm.nih.gov).
Multiple sequences were aligned with the corresponding regions of
kobuvirus reference strains retrieved from Genbank database using the
CLUSTAL W program in Molecular Evolutionary Genetics Analysis (MEGA
version 7.0, Oxford Molecular Ltd., UK). The pairwise nucleotide (nt)
and amino acid (aa) identities among all sequences were calculated using
MegAlign program (DNASTAR, Inc. USA, Wisconsin, Madison). The open
reading frame (ORF) was predicted for the obtained genome sequences by
ORF finder
(https://www.ncbi.nlm.nih.gov/orffinder).
Similarity plot analysis of the full-length polyprotein genome was
performed using SimPlot 3.5.1 software. The RNA secondary structure
prediction for 5’ and 3’ UTR was conducted by Mfold program (Zuker,
2003). Phylogenetic analyses were generated by maximum-likelihood method
with 1000 bootstrap replicates using MEGA v7.0 and further visualized in
FigTree v1.4.0 software. The model selections were based on the results
of ’Find best DNA/Protein models’ in MEGA v7.0.