Insights into In Vitro Adaptation of EV71 and Analysis of Reduced Virulence by In Silico Predictions.

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 occur in the EV-A71 genome. The mutations within the genome give rise to quasispecies within the viral population that could be further defined by haplotypes. In vitro virulence of EV-A71 was shown by plaque size 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 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, while only two plaque variants (small, medium) were present in 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 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.

Stability and antiviral activity of SP40 peptide in human serum.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). SP40 peptide was previously identified to inhibit EV-A71 strains from genotypes A, B and C. However, the stability and antiviral activity of SP40 peptide in human serum are yet to be established. To address this, we evaluated the stability and anti-EV-A71 activity of SP40 peptide after incubation in 25 % human serum. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC/MS) were utilized to evaluate serum stability and cleavage patterns of SP40 peptide after incubation in human serum. Cell protection assay was used to evaluate the anti-EV-A71 activity of SP40 peptide after incubation in human serum and to identify the minimal active sequence of SP40 peptide that retained antiviral activity. The results showed that the SP40 peptide was stable in human serum with 56 % of the full-length SP40 peptide being detected after 48 h incubation in human serum. The SP40 peptide was mainly cleaved by exopeptidases and no endoprotease recognition sites were identified within the SP40 peptide. Cell protection assays revealed that the SP40 peptide retained substantial activity after 24 and 48 h incubation in human serum. Furthermore, the data revealed that three amino acids at the N-terminus and one amino acid at the C-terminus of the SP40 peptide were dispensable for its antiviral activity. Importantly, the four truncated peptides displayed better potency than the full-length SP40 peptide. Overall, this study provided insights into the stability and activity of SP40 peptide in human serum and will facilitate the development of SP40 peptide as an anti-EV-A71 agent.