Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool.

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3' ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71

Replication kinetics of clone-derived EV-A71 in RD cells.

<p>RD cells were infected with various clone-derived EV-A71 at an MOI of 0.1. The viral titers in log₁₀ PFU/ml were determined at 0, 6, 12, 24, 48 and 72 hours post-infection using plaque assay. Error bars indicate standard deviations around the means.</p

Schematic illustration and characterization of pCMV-EV71-Nluc.

<p>(A) Nluc was cloned downstream of the EV-A71 5’UTR and upstream of the EV-A71 VP4 gene. Nluc was inserted through <i>Age</i>I and <i>Hin</i>dIII restriction enzyme sites. A 2A cleavage site was inserted after the Nluc gene. (B) RD cells were infected with EV-A71-Nluc at an MOI of 1, and the luciferase activity was determined upon 48 hours post-infection. (C) RD cells were infected with EV-A71-Nluc at an MOI of 0.1, and the luciferase activity was determined at 12, 24, 48, and 72 hours post-infection. (D) Plaque morphologies of clone-derived EV-A71 and EV-A71-Nluc are shown.</p

Characterization of EV-A71 infectious clones by virus titers and plaque size.

<p>Characterization of EV-A71 infectious clones by virus titers and plaque size.</p

Schematic illustration and characterization of pCMV-EV71-EGFP.

<p>(A) EGFP was cloned downstream of the EV-A71 5’UTR and upstream of the EV-A71 VP4 gene. EGFP was inserted through <i>Age</i>I and <i>Hin</i>dIII restriction enzyme sites. A 2A cleavage site was inserted after the EGFP gene. (B) The virus titers in log₁₀ PFU/ml of pCMV-EV71-EGFP at P₀ and P₁. (C) RD cells were infected with EV-A71-EGFP at an MOI of 0.1, and green fluorescence was captured 24 and 48 hours post-infection. (D) Plaque morphologies of clone-derived EV-A71 and EV-A71-EGFP are shown.</p

Schematic illustration of the mechanism of EV-A71 DNA-launched infectious clone.

<p>The viral RNA requires exportation to the cytosol for cap-independent translation by mRNA export complexes. The HH ribozyme removes the m⁷G cap and thus ensures precise 5’ ends of the EV-A71 transcripts. The HDV ribozyme removes the SV40 termination signal and ensures precise 3’ ends of viral RNA. Viral RNA is exported into the cytosol by RNA export complexes. The m⁷G cap is required for nuclear RNA export. Removal of the cap diminishes the RNA exportation efficiency and therefore, reduces overall virus production. <i>In vitro</i>-transcribed infectious RNA is transfected into the cytosol for IRES-dependent translation. All newly synthesized positive-strand viral RNA are VPg-linked. RE indicates restriction enzyme; and IVT indicates <i>in vitro</i> transcription.</p

Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive enterovirus A71 virulence in mice.

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71