Figure 1.
The 5′-UTR correlates with the distinct virulence of EV71 in mice.
Three-day-old ICR mice were i.p. inoculated with unadapted EV71 clinical isolate 4643 or 237 at 5×104 pfu/mouse (A, B), and 5′-UTR-replaced recombinant virus 4643 M or 237 M and backbone MP4 at 5×104 pfu/mouse (C, D). The survivals (A, C) and clinical scores (B, D) of mice were monitored for two weeks. Mock control mice were given viral medium.
Figure 2.
Molecular determinant of EV71 virulence in mice was mapped to SL II.
Genomic structure of EV71 shown in the upper part and schematic presentation of the compositions of 5′-UTRs of infectious clones are shown in the lower panel. Open, closed, and slash box represent sequence of isolates 4643, 237, and MP4 respectively. Number and average survival time of mice infected with these clone-derived viruses were also indicated. † Survival time of mice alive on Day 14 post-inoculation was calculated as 14.
Figure 3.
Multiple genetic variations concentrate in the SL II upper domain.
Secondary structures of SL II of isolate 237 and 4643 predicted using RNA Dyalign of RNAstructure Version 5.3 are shown in the upper part. Sequence alignment of the two SL IIs revealed 20 nucleotide variations, indicated as bold in predicted secondary structure of 237 SL II, 10 concentrated from nucleotide 142 to 162 which form the SL II upper domain as boxed.
Figure 4.
Short stem in the upper domain of SL II determines EV71 virulence in mice.
The RNA secondary structure of 4643 SL II predicted, as described in Materials and Methods, is shown in the left panel. Composition of chimeric SL IIs of infectious clones is shown in the middle panel. Open and closed boxes represent sequence of isolates 4643 and 237, respectively. USLII and LSLII were generated by replacing nucleotides in the upper or lower half of 4643 SL II with corresponding sequences of isolate 237. Top loop of USLII was further replaced with corresponding sequence of isolate 4643 to generate USLII-LOOP. The average survival time of mice infected with recombinant viruses generated from these clones is shown in the right panel. Base numbers are from EV71 isolate 4643. † Survival time of mice alive on Day 14 post- inoculation was calculated as 14.
Figure 5.
Short fragment of SL II regulated viral translation and replication in vitro.
(A) Construction of dual reporter plasmid to determine translational activity mediated by EV71 5′-UTR. (B) Relative translational activities mediated by various 5′-UTRs. Dual reporter plasmids with various 5′-UTR were transfected into L929 cells, firefly luciferase activity determined 24 hours post-transfection and normalized with β-galactosidase activity. Relative translational activity to isolate 237 is shown as fold increase. (C) Effect of SL II on virus translational activity. Infectious RNAs USLIIM and 4643SLII-237M were transfected into L929 cells, lysates harvested at 2, 4, 6 and 8 hours post-transfection. Western blot was performed by MAb979 to detect viral translated products. Collective band density of each time-point was rated with ImageJ and expressed as fold increase relative to band density of USLIIM at four hours post-transfection. Western blot analysis of actin was included as control for normalization. (D) Effect of SL II on viral replication. Replicon RNAs of USLII and 4643SLII-237 were transfected into L929 cells and the cell lysates were harvested at 4, 6 and 12 hours post-transfection; luciferase activities at 4 hours reflect transfection efficiency. Their replication rates were expressed as fold increase of firefly luciferase activity at 6 or 12 hours versus 4 hours post-transfection.
Figure 6.
Short stem of SL II affected virus replication in mice.
(A) Effect of 237 SL II upper domain on virus replication in vivo. Three-day-old ICR mice were i.p. inoculated with USLIIM or 4643SLII-237M at 5×104 pfu/mouse. Brain, spinal cord and posterior limb muscle were recovered at Day 3 post-inoculation and virus titer determined with plaque assay. a: no virus detected in brain of mice infected with 4643SLII-237M. b: virus detected in only one spinal cord sample from 4643SLII-237M infected mice. (B) Effect of 237 SL II upper domain on viral replication in brain. Three-day-old ICR mice were given 2,500 pfu/dose of USLIIM or 4643SLII-237M recombinant viruses via i.c. route. Brains of infected mice were recovered post-inoculation. a: virus was detected only in one brain of 4643SLII-237M infected mice. (C) Correlation between tissue viral load and paralysis in 4643SLII-237M-infected mice. Mice were i.p. inoculated with 4643SLII-237M at 5×104 pfu/mouse. Brain, spinal cord and muscle were recovered after posterior limb paralysis was observed. a: virus was detected in one brain of 10 paralyzed mice. b: virus was detected in two spinal cord samples from 10 paralyzed mice. Results represent virus titer (log10 pfu) per milligram of tissue.
Figure 7.
Single nucleotide substitution attenuated EV71 virulence.
(A) Diagram of nucleotide substitution in SL II. Dashed rectangle represents the identified domain determining EV71 virulence. Substituted nucleotides appear in bold type; C142, A146 and C158 of USLIIM 5′-UTR were substituted with corresponding U, G and U of isolate 4643 to generate USLIIM-C142U, USLIIM-A146G and USLIIM-C158U, respectively. Clone USLIIM-142/159 contains two nucleotide substitutions of C142U and G159A. Average survival time is shown in the right panel. Recombinant viruses USLIIM, USLIIM-C142U, USLIIM-A146G, USLIIM-C158U and USLIIM-142/159 were i.p. inoculated to three-day-old ICR mice at 5×104 pfu/mouse. Survival (B) and disease severity (C) were monitored for two weeks. (D) Effect of nucleotide substitution on translational activity mediated by EV71 5′-UTR. Point mutated bicistronic plasmids were transfected into L929 cells as described above, reporter activity determined 24 hours post-transfection. Relative translational activity is shown as fold increase of normalized luciferase activity to USLII.