Koh, Jia Xuen (2023) Insights into in vitro adaptation of enterovirus 71 and impact of spontaneous mutations by in silico analysis. Masters thesis, Sunway University.
Full text not available from this repository.Abstract
EV-A71 is a common viral pathogen that causes hand-foot and mouth disease. It is a single-stranded RNA virus that has a low fidelity RNA polymerase and as a result, spontaneous mutations frequently occurred in the EV-A71 genome. The mutations within the genome gave rise to quasispecies within the viral population that is consisted of haplotypes. The thesis aims to unravel the diversity of the quasispecies structure. In vitro virulence of EV-A71 was shown by plaque morphology in Rhabdomyosarcoma (RD) cells, which was substantiated by in vitro characterizations of growth, RNA replication, binding, attachment and host cell internalization. Viruses could exhibit different host cell adaptations in different cell lines during viral passaging. The thesis intends to verify whether the spontaneous mutations arise from low-fidelity RNA polymerase indicates adaptation-related mutations in cell cultures or mutations associated with virulence. Viral plaque characterization is included to investigate its association with plaque sizes. The impact of spontaneous mutations on in vitro virulence is analysed through in silico predictions. The EV-A71/WT (derived from EV-A71 subgenotype B4) was shown to comprise six haplotypes through next-generation sequencing, where only EV-A71/Hap2 was found to be cultivable in RD cells, while EV-A71/Hap4 was the only cultivable haplotype in Vero cells. The EV-A71/WT produced plaques of four different sizes (small, medium, big, huge) in RD cells but only two plaque variants (small, medium) were present in Vero cells. The VP3-H29Y mutation could act as a mutation critical for cell adaptation towards Vero cells. The small plaque variant isolated from RD cells displayed lower RNA replication rates, slower in vitro growth kinetics, higher TCID50 and lower attachment, binding and entry ability when compared against the EV-A71/WT due to the mutation at 3D-S228P that disrupted the active site of the RNA polymerase, resulting in low replication and growth of the variant, indicating a promising candidate to further evaluate as a live-attenuated vaccine. The medium plaque variant isolated from RD cells showed the highest in vitro virulence when compared to the other variants which could be explained by the VP3-F186L mutation that caused a loss of hydrogen bonds and salt bridges when analysed through in silico predictions, therefore resulting in a flexible GH loop that could facilitate viral infectivity. Both big and huge plaque variants showed weak in vitro infectivities similar to the small plaque variant despite their large plaque sizes, possibly affected by the VP3-H29Y and VP1-L183S mutations that indirectly impaired virion processing and receptor recognition and binding. This research provides an insight that EV-A71 exists as a quasispecies and propagation of viruses in different cell lines exhibits different outcomes. The relationship between plaque size and infectivity may not correlate positively. The mutations shall be further investigated by site-directed mutagenesis to identify possible molecular determinants of virulence. In vivo immunogenicity and challenge studies shall be performed using the small plaque variant before selecting it as a vaccine seed strain.
| Item Type: | Thesis (Masters) |
|---|---|
| Uncontrolled Keywords: | Enterovirus 71; quasispecies; in vitro virulence; structural analysis; next generation sequencing. |
| Subjects: | Q Science > QR Microbiology |
| Divisions: | Sunway University > Faculty of Medical and Life Sciences [formerly School of Medical and Life Sciences until 14 March 2025; School of Healthcare and Medical Sciences until 2020] |
| Depositing User: | Ms Yong Yee Chan |
| Date Deposited: | 20 Oct 2026 11:02 |
| Last Modified: | 20 Oct 2026 11:02 |
| URI: | http://repository.sunway.edu.my/id/eprint/3667 |
Actions (login required)
 |
View Item |