Introduction: Hand, foot, and mouth disease caused by enterovirus A71 (EV-A71) is more frequently associated with neurological complications and deaths compared to other enteroviruses.Areas covered: The authors discuss current understanding of the neuropathogenesis of EV-A71 based on various clinical, human, and animal model studies. The authors discuss the important advancements in virus entry, virus dissemination, and neuroinvasion. The authors highlight the role of host immune system, host genetic factors, viral quasispecies, and heparan sulfate in EV-A71 neuropathogenesis.Expert opinion: Comparison of EV-A71 with EV-D68 and PV shows similarity in primary target sites and dissemination to the central nervous system. More research is needed to understand cellular tropisms, persistence of EV-A71, and other possible invasion routes. EV-A71 infection has varied clinical manifestations which may be attributed to multiple receptors usage. Future development of antivirals and vaccines should target neurotropic enteroviruses. Repurposing drug and immunomodulators used in combination could reduce the severity of EV-A71 infection. Only a few drugs have been tested in clinical trials, and in the absence of antiviral and vaccines (except China), active virus surveillance, good hand hygiene, and physical distancing should be advocated. A better understanding of EV-A71 neuropathogenesis is critical for antiviral and multivalent vaccines development.
Enterovirus A71 (EV-A71) causes hand, foot and mouth disease (HFMD) in young children. It is associated with severe neurological complications and death. This study aims to develop a live-attenuated vaccine by codon deoptimization (CD) and codon-pair deoptimization (CPD) of EV-A71. CD is generated by introducing the least preferred codons for amino acids while CPD increases the presence of underrepresented codon pairs in the specific genes. CD and CPD chimeras were generated by synonymous mutations at the VP2, VP3, VP1 and 2A gene regions, designated as XYZ. All twelve deoptimized viruses were viable with similar replication kinetics, but the plaque sizes were inversely proportional to the level of deoptimization. All the deoptimized viruses showed attenuated growth in vitro with reduced viral protein expression at 48 h and lower viral RNA at 39 °C. Six-week-old ICR mice were immunized intraperitoneally with selected CD and CPD X and XY vaccine candidates covering the VP2-VP3 and VP2-VP3-VP1 genes, respectively. All vaccine candidates elicited high anti-EV-A71 IgG levels similar to wild-type (WT) EV-A71. The CD X and CPD X vaccines produced robust neutralizing antibodies but not the CD XY and CPD XY. On lethal challenge, offspring of mice immunized with WT, CD X and CPD X were fully protected, but the CD XY- and CPD XY-vaccinated mice had delayed symptoms and eventually died. Similarly, active immunization of 1-day-old suckling mice with CD X, CPD X and CD XY vaccine candidates provided complete immune protection but CPD XY only protected 40% of the challenged mice. Histology of the muscles from CD X- and CPD X-vaccinated mice showed minimal pathology compared to extensive inflammation in the post-challenged mock-vaccinated mice. Overall, we demonstrated that the CD X and CPD X elicited good neutralizing antibodies, conferred immune protection and are promising live-attenuated vaccine candidates for EV-A71.
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.
Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.
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.
The emergence of neurotropic Zika virus (ZIKV) raised a public health emergency of global concern. ZIKV can cross the placental barrier and infect foetal brains, resulting in microcephaly, but the pathogenesis of ZIKV is poorly understood. With recent findings reporting AXL as a type I interferon antagonist rather than an entry receptor, the exact entry mechanism remains unresolved. Here we report that cell surface sialic acid plays an important role in ZIKV infection. Removal of cell surface sialic acid by neuraminidase significantly abolished ZIKV infection in Vero cells and human induced-pluripotent stem cells-derived neural progenitor cells. Furthermore, knockout of the sialic acid biosynthesis gene encoding UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase resulted in significantly less ZIKV infection of both African and Asian lineages. Huh7 cells deficient in α2,3-linked sialic acid through knockout of ST3 β-galactoside-α2,3-sialyltransferase 4 had significantly reduced ZIKV infection. Removal of membrane-bound, un-internalized virus with pronase treatment revealed the role of sialic acid in ZIKV internalization but not attachment. Sialyllactose inhibition studies showed that there is no direct interaction between sialic acid and ZIKV, implying that sialic acid could be mediating ZIKV-receptor complex internalization. Identification of α2,3-linked sialic acid as an important host factor for ZIKV internalization provides new insight into ZIKV infection and pathogenesis.