Identification of molecular determinants of cell culture growth characteristics of Enterovirus 71

Abstract

BackgroundHand, 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.ResultsIn 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.ConclusionsThe 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.

Antiviral activity of silymarin in comparison with baicalein against EV-A71

“…After 24 h, the supernatant was collected and infectious viral titers were quantified using the plaque assay [20]. RNA copy number was determined by qRT-PCR [21] [Supplementary Figure S2 Time course assay for virucidal activity RD cells (2 × 10 4 /well) were grown overnight in wells of a 96 well plate. The virus was pre-treated with 100 μg/ mL silymarin or 54 μg/mL of baicalein for 15, 30, 45 and 60 min at MOI of 1 (pfu/ml = 2 × 10 4 /0.1 mL) for 1 h. Cells were infected with the pre-treated virus-flavonoidmixture and allowed to incubate with the RD cells at 37°C for 1 h. After 1 h, the inoculum was removed, cells were washed with PBS and maintenance media was added.…”

“…The supernatant was collected after 24 h and the infectious viral titers were quantified using the plaque assay [20]. RNA copy number was determined using qRT-PCR [21] [Supplementary Figure S2 Post-infection assay RD cells (2 × 10 4 /well) were grown overnight in wells of a 96 well plate. The cells were infected with the virus at MOI of 1 (pfu/ml = 2 × 10 4 /0.1 mL) for 1 h and incubated at 37°C.…”

“…After 24 h, the supernatant was collected and infectious viral titers were quantified using the plaque assay [20]. RNA copy number was determined using qRT-PCR [21] [Supplementary Figure S2(e)].…”

Characterization of significant molecular determinants of virulence of Enterovirus 71 sub-genotype B4 in Rhabdomyosarcoma cells

“…The study concluded that the EV-A71 partial deletant (PD) and mutant 475 demonstrated the lowest RNA copy number, plaques and VP1 protein followed by EV-A71 mutants 487, 5262, 158 and 486 in the order listed. This shows that the mutant PD could serve as a potential vaccine seed strain to carry beneficial mutations that will further attenuate or stabilize it to become a good LAV candidate (Yee et al, 2016).…”

“…Compared to mutants with specific nt replacements at 475, 486 and 487, the EV-A71 PD mutant carrying the 11 base pair deletion demonstrated the lowest viral RNA copy number, plaque count and VP1 capsid protein. The PD mutant demonstrated low virulence and therefore, could possibly be a good potential seed strain for designing a LAV candidate (Yee et al, 2016).…”