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

Tan CW, Tee HK, Lee MH, Sam IC, Chan YF

PLoS One, 2016;11(9):e0162771.
PMID: 27617744 DOI: 10.1371/journal.pone.0162771

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.

Matched MeSH terms: Enterovirus A, Human/growth & development
Identification of molecular determinants of cell culture growth characteristics of Enterovirus 71

Yee PT, Tan KO, Othman I, Poh CL

Virol J, 2016 11 28;13(1):194.
PMID: 27894305

BACKGROUND: Hand, foot and mouth disease is caused by Enterovirus 71 (EV-A71) and Coxsackieviruses. EV-A71 infection is associated with high fever, rashes and ulcers but more severe symptoms such as cardiopulmonary failure and death have been reported. The lack of vaccines highlighted the urgency of developing preventive agents against EV-A71. The molecular determinants of virulent phenotypes of EV-A71 is unclear. It remains to be investigated if specific molecular determinants would affect the cell culture growth characteristics of the EV-A71 fatal strain in Rhabdomyosarcoma (RD) cells.
RESULTS: In this study, several genetically modified sub-genotype B4 EV-A71 mutants were constructed by site-directed mutations at positions 158, 475, 486, 487 and 5262 or through partial deletion of the 5'-NTR region (∆ 11 bp from nt 475 to 486) to generate a deletion mutant (PD). EV-A71 mutants 475 and PD caused minimal cytopathic effects, produced lowest viral RNA copy number, viral particles as well as minimal amount of viral protein (VP1) in RD cells when compared to mutants 158, 486, 487 and 5262.
CONCLUSIONS: The molecular determinants of virulent phenotypes of EV-A71 sub-genotype B4 strain 41 (5865/Sin/000009) were found to differ from the C158 molecular determinant reported for the fatal EV-A71 sub-genotype B1 strain (clinical isolate 237). The site-directed mutations (SDM) introduced at various sites of the cDNA affected growth of the various mutants when compared to the wild type. Lowest viral RNA copy number, minimal number of plaques formed, higher infectious doses required for 50% lethality of RD cells and much reduced VP1 of the EV-A71 sub-genotype B4 strain 41 genome was attained in mutants carrying SDM at position 475 and through partial deletion of 11 bp at the 5'-NTR region.

Matched MeSH terms: Enterovirus A, Human/growth & development*
Characterization of significant molecular determinants of virulence of Enterovirus 71 sub-genotype B4 in Rhabdomyosarcoma cells

Yee PTI, Mohamed RAH, Ong SK, Tan KO, Poh CL

Virus Res, 2017 06 15;238:243-252.
PMID: 28705680 DOI: 10.1016/j.virusres.2017.07.010

One of the leading causes of the hand, foot and mouth disease (HFMD) is Enterovirus 71 (EV-A71), displaying symptoms such as fever and ulcers in children but some strains can produce cardiopulmonary oedema which leads to death. There is no FDA-approved vaccine for prevention of severe HFMD. The molecular determinants of virulence for EV-A71 are unclear. It could be a single or a combination of amino acids that determines virulence in different EV-A71 genotype/sub-genotypes. Several EV-A71 strains bearing single nucleotide (nt) mutations were constructed and the contribution of each mutation to virulence was evaluated. The nt(s) that contributed to significant reduction in virulence in vitro were selected and each mutation was introduced separately into the genome to construct the multiply mutated EV-A71 strain (MMS) which carried six substitutions of nt(s) at the 5'-NTR (U700C), VP1-145 (E to G), VP1-98E, VP1-244K and G64R in the vaccine seed strain that had a partial deletion within the 5'-NTR region (nt. 475-485) of Δ11bp. In comparison to the wild type strain, the MMS showed low virulence as it produced very low RNA copy number, plaque count, VP1 and had 105-fold higher TCID50, indicative of a promising LAV candidate that should be further evaluated in vivo.

Matched MeSH terms: Enterovirus A, Human/growth & development*
Fulltext Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1

Tan CW, Chan YF, Sim KM, Tan EL, Poh CL

PLoS One, 2012;7(5):e34589.
PMID: 22563456 DOI: 10.1371/journal.pone.0034589

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.

Matched MeSH terms: Enterovirus A, Human/growth & development