Rules governing genetic exchanges among viral types from different Enterovirus A clusters

Figures

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  Fig. 1.. The genomic comparison reveals the clustering structure of the species Enterovirus A. (a) Constructing the evolutionary relationship of 17 human-infecting Enterovirus A types using the complete prototype strain VP1 nucleotide sequences. An unrooted tree was built using the maximum-likelihood algorithm implemented in mega5, as described in Methods. Genetic distance is shown above the branches, and bootstrap values (1000 replicates) are shown below. The branch names indicate Enterovirus types and the prototype GenBank accession number. The rightmost labels indicate distinct clusters of the species Enterovirus A. (b) Similarity plot of the complete genome sequences of the selected human-infecting types from different clusters of Enterovirus A, using a sliding window of 250 nucleotides moving in 30-nucleotide steps. EVA71 BrCr strain was used as the query sequence.

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  Fig. 2.. Viruses from the conventional and unconventional Enterovirus A clusters differed in their capsid expression levels in human cells. (a) Schematic representation of the enterovirus capsid expressing vector, which contained a CMV promoter followed by EGFP reporter, VP4, VP2, VP3 and VP1 coding sequences. A viral 2A protease cleavage site (AITTL) was inserted between EGFP and VP4. (b) Image showing the capsid plasmids analysed by agarose gel electrophoresis. ‘Opti-’ stands for the codon-optimized plasmid. (c–e) HEK293T cells were transfected by capsid plasmids and EGFP signals were captured using fluorescence microscopy 2 days later. EGFP expression levels among (c) conventional Enterovirus A types, (d) unconventional Enterovirus A types and (e) those of codon-optimized. (f) Various pseudoviruses were produced by trans-encapsidation using different viral capsids and EVA71 replicon RNA. Pseudoviruses were harvested after two rounds of freeze–thaw cycle, and then added to RD cells. Then, 12 h post-infection, the luciferase activities were recorded. The GnHCl (viral replication inhibitor) treated EVA71-pseudovirus was used as the assay controls. (g) The pseudoviruses of EVA71, CVA10 and…

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  Fig. 3.. Systematic testing of trans-encapsidation between conventional and unconventional Enterovirus A clusters. (a) The cartoon showed the construct of the subgenomic enterovirus replicon carrying a luciferase reporter. (b) SalI linearized each replicon vector was in vitro-transcribed by the T7 polymerase in vitro-transcription kit to generate the replicon RNA. The quality of those RNAs was examined by agarose gel electrophoresis. (c) Luciferase activity was monitored at 12 h after introducing replicon RNAs into RD cells. GnHCL treatment was used to determine the baseline luciferase activity. (d) The schematic diagram of generating pseudoviruses by trans-encapsidation. (e) Systematic activity tests of pseudoviruses generated by cross trans-encapsidations between CVA6-, CVA10-, CVA16-, EVA89- and EVA91-replicon RNAs and capsids. The values represent relative luciferase activity normalized to signals from the replicons pseudotyped by its native capsids. Data were summarized as mean±sd (n=3). Unpaired Student’s t-test, **P<0.01; ***P<0.001.

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  Fig. 4.. Testing genome recombinations at 5′-UTR and non-structural protein regions. (a) Phylogenetic trees of the 5’UTR sequences from the various Enterovirus A types. (b) Schematic representation of the six chimeric replicons and the parental EVA71. The 5′-UTR of CVA6, CVA16, CVA10, EVA89, EVA91 and EVA125 were differentially coloured. (c) The 5′-UTR chimeric replicon RNAs were obtained by in vitro transcription and transfected into RD cells. Luciferase activity was monitored at 12 h post-transfection. GnHC1 treatment was used as the assay control. (d) The 5′-UTR chimeric replicon RNAs and EVA71-capsid plasmids were sequentially transfected into HEK293T cells to produce trans-encapsidated pseudoviruses. The infectivity of those pseudoviruses was reflected by luciferase activities at 12 h post-infection. (e) P2- or (g) P3-region of EVA71 was replaced by the counterpart of CVA6, CVA16, CVA10 and EVA89 to create the chimeric replicon constructs as indicated. (f, h) Luciferase activities in RNA transfected RD cells reflect the replication activities of those (f) P2- and (h) P3 chimeric replicons. The values represent luciferase activity in three independent experiments (mean±sd). Unpaired…

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  Fig. 5.. Generation of viable EVA71 and EVA89 recombinant by artificial recombination. (a) The cartoon showed the recreation of viral recombinants (vEVA71/89P2 and vEVA71/89P3) through artificially exchanging EVA71’s P2 or P3 by EVA89’s P2 or P3, respectively. (b) RNAs generated from infectious clones were introduced into RD cells. vEVA71/89P3 but not vEVA71/89P2 caused obvious cytopathic effect (CPE) after four blind passages in cell culture. EVA71 was used as a positive control. (c) The litres of rescued vEVA71/89P3 viral stocks were determined by plaque assay. (d) RD cells were infected by vEVA71/89P3 (m.o.i.=1) overnight, and then fixed and stained by an anti-VP1 primary antibody and AlexFlour 647-conjugated secondary antibody for fluorescence microscopy. DAPI was used to visualize the nuclei. (VP1, red; nuclei, blue). (e) RD cells were infected by vEVA71/89P3 (m.o.i.=1) for 6 h. Then the proportion of VP1-expressing cells was measured by flow cytometry. (f) RD cells were infected by EV71 or vEVA71/89P3 (m.o.i.=1) for 6 h. The cleavage of eIF4G was determined by immunoblot. (g) The infectivity of EV71 and vEV71/89P3 was examined in different cell lines. The charts depicted the viral…