2.4 Whole-genome sequencing and genetic analysis of RVA
G8P[8] strains
All genes of the RVA G8P[8] strains were amplified by RT-PCR using
the specific primers for each gene.33 PCR products
were purified using the Gel/PCR DNA Fragments Extraction Kit (Geneaid)
and then sequenced using the Big-Dye Terminator Cycle Sequencing Kit
(Applied Biosystems, USA) in an automated ABI Prism 310 Genetic Analyzer
(Applied Biosystems). Similarity between G8P[8] strains in this
study and reference strains previously reported was calculated using
BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple
sequence alignments based on nucleotide sequences of each gene were
generated using Clustral X, and phylogenetic trees of partial VP1 (471
bp), VP2 (744 bp), VP3 (660 bp), VP4 (758 bp), VP6 (498 bp), VP7 (804
bp), NSP1 (708 bp), NSP2 (740 bp), NSP3 (682 bp), NSP4 (515 bp), and
NSP5 (697 bp) were constructed using the neighbour-oining method in MEGA
7.0. Statistical significance of the genetic relationship was estimated
by bootstrap resampling analysis (1000 replications). The amino acid
sequences of the VP7 antigenic epitopes of G8 strains were compared
using BioEdit 7.2. The nucleotide sequences of the G8P[8] strains
detected in this study were deposited in the GenBank database under
accession numbers OP871410-OP871473.