3.4 Whole-genome sequence analysis of G8P[8] strains
The whole-genome sequences of four representative Chiang Rai G8P[8] strains (RVA/Human-wt/THA/5CR11/2019/G8P[8], RVA/Human-wt/THA/5CR23/2019/G8P[8], RVA/Human-wt/THA/5CR33/2019/G8P[8], RVA/Human-wt/THA/5CR56/2019/G8P[8]) were analyzed to clarify the origin of these strains, whether they are derived from one of the G8P[8] reference strains, or whether they are new reassortant strains resulting from genomic reassortment. Phylogenetic trees were constructed using nucleotide sequences from the BLAST search and 51 DS-1-like G8P[8] reference strains published elsewhere, including two Chinese DS-1-like G8P[8] strains (RVA/Human-wt/CHN/GZ-0005/2021/G8P[8], RVA/Human-wt/CHN/GZ-0013/2021/G8P[8]) of the sublineage A. Analysis revealed that G8P[8] strains in this study had the genomic constellation II of the DS-1-like genetic backbone: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Surprisingly, phylogenetic analysis revealed that five genes (VP1, VP6, NSP2, NSP3, NSP4) of the Chiang Rai DS-1-like G8P[8] strains clustered in distinct lineages separated from most of the DS-1-like G8P[8] reference strains.
Phylogenetic analysis of the VP1 gene showed that the four representative G8P[8] strains in this study and the two Chinese strains, together with many G2P[4] strains belonged to lineage V and not to lineage IV, which consisted of DS-1-like G8P[8] reference strains (Figure 2C). Phylogenetic analysis of the VP6 gene revealed two points of interest (Figure 2D). First, the VP6 gene of G8P[8] strains in this study and two Chinese G8P[8] strains belonged to lineage V along with G2P[4], G3P[8], G8P[8], and G9P[8] strains, whereas the DS-1-like G8P[8] reference strains belonged to other lineages. Second, the VP6 gene of 10 DS-1-like G8P[8] strains previously reported in Thailand in 2013 belonged to a separate lineage III, which is closely related to the Vietnamese strain RVA/Human-wt/VNM/NT0082/2007/G10P[14]. Phylogenetic analysis of the NSP2 gene revealed that G8P[8] strains in this study and the two Chinese G8P[8] strains together with some of the G8P[8] strains reported after 2016 from the Czech Republic (RVA/Human-wt/CZE/H366/2017/G8P[8]) and Korea (RVA/Human-wt/KOR/CAU17L-103/2017/G8P[8]) belonged to a separate lineage IV, which composed of multiple genotypes including G1P[8], G3P[8], G8P[8], and G9P[8] strains (Figure 2E).
Phylogenetic analysis of the NSP3 gene showed that G8P[8] strains in the present study belonged to a distinct lineage IV together with G2P[4], G3P[6], G9P[4], and G12P[8] strains. The difference with the analysis of the VP1, VP6, and NSP2 genes was that the two Chinese DS-1-like G8P[8] strains did not cluster together with the G8P[8] strains in this study but were closely related to the DS -1-like G8P[8] reference strains (Figure 2F). Phylogenetic tree of the NSP4 gene revealed that G8P[8] strains detected in the current study clustered in lineage V along with G1P[4], G1P[8], G2P[4], and G9P[8] instead of lineage IV, which composed of the DS-1-like G8P[8] reference strains (Figure 2G). In addition, the phylogenetic tree also showed that the two Chinese DS-1-like G8P[8] strains formed a separate lineage V that was distinct from the G8P[8] strains in this study and the DS -1-like G8P[8] reference strains.
For the VP2, VP3, NSP1, and NSP5 genes, phylogenetic trees showed that the Chiang Rai DS-1-like G8P[8] strains identified in this study clustered in the same lineages together with the DS-1-like G8P[8] reference strains with nucleotide sequence similarity greater than 98% nucleotide sequence similarity (FIGURE S1, Supplementary materials).