RNAi inhibits viral RNA.

<p>RD cells were treated with each siRNA and then infected with strain EV71-2006-52-9 at an MOI of 0.01. At 24 h postinfection, the viral RNA was extracted and analyzed with real-time RT–PCR. The data shown represent the means ± SD of three independent experiments (**p < 0.01).</p

RNAi inhibits viral protein.

<p>RD cells transfected with siRNA were infected with strain EV71-2006-52-9 at an MOI of 0.01. At 36 h postinfection, total protein was extracted and analyzed with western blotting. β-Actin was used as the internal loading control. The protein measurements were made in three independent experiments.</p

Sequences of the synthesized siRNA molecules and their positions in the EV71 2A^pro genomic region.

<p>Sequences of the synthesized siRNA molecules and their positions in the EV71 2A^pro genomic region.</p

Transfection efficiency of siRNA in RD cells.

<p>(A) Cellular distribution of BLOCK-iT Fluorescent Oligo in transfected RD cells. RD cells were transfected with different concentrations of BLOCK-iT Fluorescent Oligo and 2 μl of Lipofectamine 2000. At 24 h after transfection, the cells were observed under a fluorescence microscope. (B) RD cells transfected with BLOCK-iT Fluorescent Oligo were quantified with flow cytometry. The cells were assayed in three independent experiments.</p

siRNAs protect cells against EV71-induced cytopathic effects.

<p>Morphological changes in RD cells were observed after infection. Cells were transfected with each siRNA at a final concentration of 60 nM and then infected with strain EV71-2006-52-9 at an MOI of 0.01. Micrographs were taken at 48 h postinfection under an inverted microscope. The tests were performed in three independent experiments.</p

RNAi inhibits the replication of different EV71 strains.

<p>RD cells transfected with siRNA were infected with strain EV71-2008-43-16 at an MOI of 0.01. Total protein was extracted at 36 h after infection, and VP1 expression was analyzed with western blotting. β-Actin was used as the internal loading control. Protein measurements were made in three independent experiments.</p

Viral titer assay.

<p>Virus titers were determined in term of TCID₅₀. RD cells pretreated with siRNAs were infected with strain EV71-2006-52-9 at an MOI of 0.01. At 48 h postinfection, the supernatants were collected to detect the progeny viral titers. Data are the means ± SD of three independent experiments (**p < 0.01).</p

Distribution of the outbreak strains and onset time.

<p>Distribution of the outbreak strains and onset time.</p

Phylogenetic analysis of the six outbreak strains.

<p>A phylogenetic tree was constructed from multiple alignment of the core genome SNPs of the 6 isolates in the present study and another 4 previous isolates from GenBank, where FPR3757 USA300 was included as an outgroup. A phylogenetic tree with 1000 bootstrap resamples of the alignment data sets was generated using the neighbor-joining method in MEGA5.0 with the contribution model of "Kimura 2-parameter". Bootstrap values are indicated at the nodes. The scale bar indicates the number of substitutions per position for a unit branch length.</p

Resistance and virulence of six outbreak strains.

<p>Resistance and virulence of six outbreak strains.</p