Genetic variation, recombinant characteristics, and seroprevalence analysis of echovirus 3 causing severe and mild cases of hand, foot, and mouth disease in Guizhou Province

Figures

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  Fig 1. The phylogenetic tree based on the VP1 coding sequences of E-3 obtained from GenBank. The eight E-3 strains in this study were indicated by blue triangles (the four strains from mild cases) and red solid circles (the four strains from severe cases). Bootstrap values >75 were shown and inferred by bootstrapping with 1,000 replicates.

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  Fig 2. The phylogenetic trees based on the VP1, P1, P2, and P3 sequences of EV-B. The four strains from severe cases were indicated by red solid circles, the four strains from mild cases were indicated by blue triangles, and the E-3 prototype was indicated by a brown diamond. Eight Guizhou E-3 strains and other EV-B strains were analyzed through nucleotide alignment using the neighbor-joining method and the Kimura 2-parameter model conducted in the MEGA 11.0 program. Bootstrap values >75 were shown and inferred by bootstrapping with 1,000 replicates. (A) VP1 coding sequences. (B) P1 coding sequences. (C) P2 coding sequences. (D) P3 coding sequences.

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  Fig 3. Analysis of amino acid sites under positive selection pressure identified by MEME analysis and sites with high variation identified by entropy value analysis in E-3 strains isolated from mild and severe cases. Green boxes indicate sites under significant positive selection (MEME test, P < 0.1). Red boxes highlight sites with high entropy (Shannon entropy > 0.6). Gray dots indicate the remaining sites (shown as background reference). Line height represents the Shannon entropy value (a measure of amino acid diversity). Circles indicate sites under positive selection pressure (P < 0.1). All sites are displayed consecutively along the x-axis. (A) P1 region. (B) P2 region. (C) P3 region.

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  Fig 4. 3D structural mapping of key amino acid variations in E-3 virus P1, P2, and P3 polyproteins. (A) 3D structure of the P1 capsid polyprotein. VP1 (yellow), VP2 (pink), VP3 (blue), and VP4 (green). (B) 3D structure of the P2 non-structural polyprotein. 2A (pink), 2B (green), and 2C (blue). (C) 3D structure of the P3 non-structural polyprotein. 3A (blue), 3B (yellow), 3C (green), and 3D (pink). Amino acid sites VP4-26, VP2-156, VP2-228, 2A-952, 2C-1180, 2C-1217, 2C-1389, 3C-1594, and 3D-1883 were under positive selection pressure (MEME test, P < 0.1) and highly variable (Shannon entropy > 0.6), and sites 3A-1451 and 3D-2079 were highly variable sites (Shannon entropy > 0.6). Red boxes indicate shared amino acid substitutions between the severe and mild case groups, as well as high-variation or positively selected sites.

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  Fig 5. Recombination analyses of the Guizhou E-3 strains with other EV-B strains. Bootstrap values >75 were shown and inferred by bootstrapping with 1,000 replicates. All the tree branches were colored according to the results of the similarity plots and bootscanning analysis. (A) The analysis involved similarity and bootscanning among Guizhou E-3, E-21, EV-B80, E-30, Jiangsu E-3 strains, and the E-3 prototype strain to identify potential parental relationships. The GZ group was utilized as the query sequences. (B) The phylogenetic tree of the P1 region. (C) The phylogenetic tree of the P2 region. (D) The phylogenetic tree of the P3 region.

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  Fig 6. RD cells were infected with the E-3 strains isolated from both mild and severe case groups (MOI = 1). (A) AO/EB staining of RD cells at 48 h post-infection with E-3 strains from mild and severe case groups. The P30, P37, P40, and P47 strains were isolated from the mild case group, and the P0, P17, P22, and P28 strains from the severe case group. The nuclei of infected cells exhibited membrane blebbing, condensed chromatin, and fragmented nuclei (indicated by white arrows). Bar = 10 μm. AO penetrates all cells and stains nuclei green, while EB selectively enters cells with compromised membrane integrity, causing red or orange nuclear fluorescence. Consequently, live cells show uniformly green nuclei; early apoptotic cells display nuclear condensation or fragmentation with yellowish-green to yellow fluorescence; late apoptotic as well as necrotic cells exhibit red fluorescence. (B) Cell viability of RD cells infected with E-3 strains from mild and severe case groups was assessed by CCK-8. (C) Relative RNA expression of E-3 strains from mild and severe case groups was measured by RT-qPCR using GAPDH as the internal reference gene. (D) Viral titers of the E-3 strains isolated from…

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  Fig 7. The temperature sensitivity results for the eight Guizhou E-3 strains. The growth trends of the viruses on RD cells at 36°C (blue line) and 39.5°C (orange line) are illustrated in the graph. (A) P0/GZ/CHN/E-3 strain. (B) P17/GZ/CHN/E-3 strain. (C) P22/GZ/CHN/E-3 strain. (D) P28/GZ/CHN/E-3 strain. (E) P30/GZ/CHN/E-3 strain. (F) P37/GZ/CHN/E-3 strain. (G) P40/GZ/CHN/E-3 strain. (H) P47/GZ/CHN/E-3 strain.