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

Figures

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  Fig. 1. Predicted RNA secondary structure of EV-A71 strain 41 from nt. 474 to 541. Due to slight differences in the length of the 5′-NTR, nt. 480 in Sabin 1 is at the equivalent position 486 in this figure. Nt. 481 in Sabin 2 is at the equivalent position 487, and nt. 472 in Sabin 3 is at the equivalent position 475 of the EV-A71 genome

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  Fig. 2. Cytopathic effects (CPE) caused by mutants a EV-A71 (A158T) b C475T c A486G d G487A e A5262G and f PD (Partial Deletant, 5′-NTR) in Rhabdomyosarcoma (RD) cells in comparison with g uninfected RD cells (negative control using Opti-MEM) and h EV-A71 wild type strain 41 infected RD cells (positive control). Transfection of infectious RNA into RD cells was performed with the use of Lipofectamine 2000 reagent using EV-A71 mutants at a MOI of 0.1. CPE was seen as round and shrunken cells that eventually dislodged from the surface. Magnification used (X 100)

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  Fig. 3. Quantification of Viral RNA Copy Number. Transfection of infectious RNA into RD cells was performed with the use of Lipofectamine 2000 reagent and EV-A71 mutants at a MOI of 0.1. The viral RNA copy number was determined at 24 h post-infection by TaqMan Real-Time PCR. Viral RNA copy numbers are the average of three biological replicates. Error bars represent the standard deviation of the mean

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  Fig. 4. Quantification of plaque forming units by EV-A71 mutants 158, 475, 486, 487, 5262, partial deletant (PD) and the wild type EV-A71 strain 41. The negative control represents uninfected RD cells. EV-A71 mutants were transfected at a MOI of 0.1. The plaque assays were performed on monolayer RD cells incubated at 37 °C and were repeated at least two separate times

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  Fig. 5. Plaque Forming Units by EV-A71 mutants and the wild type EV-A71 strain 41. RD cells were transfected with EV-A71 mutants and the wild type EV-A71 strain 41 at a MOI of 0.1. Plaque formation was observed 72 h post-infection. PFU numbers are the average of two biological replicates; Error bars represent the standard deviation of the mean

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  Fig. 6. Western blot analysis of VP1 formed by the various EV-A71 mutants. The amount of EV-A71 VP1 protein and β-actin loaded in each lane was 5 μg. The lanes are labelled as follows: lane 1, EV-A71 Mutant 158; lane 2, EV-A71 Mutant 475; lane 3, EV-A71 Mutant 486; lane 4, EV-A71 Mutant 487; lane 5, EV-A71 Mutant 5262; lane 6, EV-A71 Mutant Partial Deletant (PD) 5′-NTR; lane 7, Positive control (EV-A71 Virus); lane 8, Negative control (uninfected RD cells). The molecular weight of the EV-A71 VP1 protein is estimated to be 32.7 kDa with reference to molecular weight markers (from 10–250 kDa) (data not shown)