A Selective Bottleneck Shapes the Evolutionary Mutant Spectra of Enterovirus A71 during Viral Dissemination in Humans

Abstract

RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 (VP1-31G) in viral particles disseminated into the integumentary and central nervous systems. In vitro viral growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D conferred enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to the central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this reemerging viral pathogen.IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand, foot, and mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31 helps viruses break through the bottleneck and invade the central nervous system. This substitution is also advantageous for replication in neuronal cells in vitro Considering that fatal cases contain enhanced glycine substitution at VP1-31, we suggest that the increased prevalence of VP1-31G may alter viral tropism and aid central nervous system invasion. Our findings provide new insights into a dynamic mutant spectral switch active during acute viral infection with emerging viral pathogens.

Keywords: capsid protein VP1; enterovirus A71; pathogenesis; quasispecies.

FIG 1

Divergent EV-A71 mutant spectra among various tissues in humans. (a) Pie charts displaying the proportion of each haplotype among the indicated specimens or tissues according to haplotype prediction results by using the QuasiRecomb program. Among all seven pie plots, each haplotype is represented by a unique color, which represents the distribution of each haplotype across each specimen or tissue. (b) Plot showing the frequency of distribution of each haplotype in different tissues. Each line with a unique color indicates the proportion of changes of a haplotype among specimens or tissues in the respiratory and digestive systems, integumentary system, and central nervous system. OG, orogastric.

FIG 2

Dynamic haplotype switching between the respiratory tract and the CNS. (a) The Shannon entropy value for each viral quasispecies isolated from the indicated tissues was calculated based on the frequency of distribution of all haplotypes detected in the indicated specimens or tissues. (b) Phylogeny networks and frequency of appearance of each haplotype from different tissues. The sizes of the solid circles indicate the total abundance of each haplotype among all specimens or tissues. Different colors displayed in each solid-color circle show the abundance contributed by the indicated specimens or tissues.

FIG 3

Genetic-diversity contributions of nonsynonymous and synonymous mutations of viruses isolated from various tissues. The πN (a) and πS (b) values were calculated throughout each viral genome using a 50-codon sliding window and a 1-codon step. High πN/πS values indicate cases where the SNVs evolved under positive selection, whereas low πN/πS values indicate SNVs that evolved under purifying selection. Amino acid residues for the major peaks of πN and πS are noted in the plots. The arrows indicate the πN peaks of the VP1-31, 3A-73, and 3D-423 residues and πS peaks of 2A-42 and 3C-152 residues among different specimens or tissues, respectively.

FIG 4

Higher proportion of VP1-31G viruses from fatal cases than HFMD cases. (a) The Shannon entropies of quasispecies isolated from patients with fatal disease or HFMD were compared based on the frequency of distribution of all haplotypes. The 1998-1 virus was isolated from a throat swab of an autopsy case. The center values represent the Shannon entropies among the selected fatal and HFMD cases. The proportions of VP1-31G (b) and 3A-73S (c) among virus isolates from patients with fatal disease or HFMD were determined based on the SNV calling results from deep-sequencing reads. The values represented by the horizontal lines are mean proportions among the selected fatal and HFMD cases. The Mann-Whitney U test was used to calculate P values. *, P < 0.05. (d) The πN values for EV-A71 from fatal cases and HFMD cases were calculated for each viral genome using a 50-codon sliding window and a 1-codon step.

FIG 5

Genetic diversity contributions of synonymous mutations of viruses isolated from patients with different disease severities. The values of πS of EV-A71 from fatal cases and HFMD cases were calculated throughout each viral genome using a 50-codon sliding window and 1-codon step. The 1998-1 virus is the virus isolated from the throat swab of the autopsy case.

FIG 6

Effects of VP1-31 substitution on thermostability, viral replication, and viral fitness. Panels a and b display, respectively, the thermostability and viral replication of a virus with VP1-31 substitutions. (a) A total of 10⁷ PFU of the VP1-31G (green) or VP1-31D (blue) virus was incubated at different temperatures. Viruses were harvested after the indicated number of days and titrated in plaque assays (n = 6). The values and error bars are the mean and ±1 standard deviation, respectively. A paired Student's t test was used to calculate P values. (b) DLD-1, RD, and SK-N-SH cells were infected with VP1-31G (red) or VP1-31D (blue) at an MOI of 0.1, as indicated. Viral titers were determined at the indicated days postinfection. The values and error bars are the mean ±1 standard deviation, respectively. A 2-tailed paired Student's t test was used to calculated P values. (c) DLD-1, RD, and SK-N-SH cells were individually inoculated or coinoculated with the VP1-31G and VP1-31D viruses, using the same amount of each virus. Viruses were harvested once obvious cytopathic effects were observed. Harvested viruses were sequentially passaged in the same cell line. The dominant haplotype appearing in various passages of viruses were defined by Sanger sequencing of the VP1-31 residue.

FIG 7

Effects of VP1-31 substitution on virus binding and RNA release. (a) VP1-31G and VP1-31D viruses were absorbed to DLD-1, RD, and SK-N-SH cells, as indicated. Bound viruses were quantified by ELISA. Values and error bars are the mean ±1 standard deviation, respectively. (b) DLD-1, RD, and SK-N-SH cells were infected with neutral red-labeled VP1-31G and VP1-31D viruses, as indicated. Based on the characteristics of a neutral red-labeled virus, which can be inactivated under light exposure unless the virus releases its RNA, the numbers of viruses that released their RNA genome into the cells were determined using the infectious center assay. Values and error bars are the mean ±1 standard deviation, as indicated. A paired Student's t test was used to calculate P values. The error bars represent 1 standard deviation from triplicate experimental results.

FIG 8

Localization of the VP1-31 residue and potential protein structure changes at the VP1-31 substitution site. (a) The external and internal surfaces of the EV-A71 capsid pentamer are shown, as generated using the UCSF Chimera program, version 1.9. The red residue indicates the location of the VP1-31 residue in the outer (left panel) or internal (right panel) surface of the pentamer structure (PDB code 4AED). (b) The VP1-31 residue (red) is displayed on the VP1 protein ribbon structure. N and C indicate the N- and C-terminal ends of the VP1 protein. The red box region is enlarged in panel c of this figure. (c) Diagram displaying protein modeling of the structural consequences of the VP1-31 D-to-G mutation. Blue and red sticks expanding from the backbone structure represent the side chains of VP1-71D (in both panels), VP1-31D (left panel), and VP1-31G (right panel) residues. Solid yellow lines indicate potential interactions between atoms of VP1-71D and VP1-31D (left panel) or VP1-31G (right panel).

FIG 9

Model of EV-A71 haplotype evolution under bottleneck selection in humans. (a) A broad mutant spectrum of EV-A71, which has major VP1-31D (blue) and minor VP1-31G (red) haplotypes and can infect humans through oral-oral or oral-fecal routes through the respiratory and digestive tract. (b) After entry into the human body, these haplotypes infect epithelial cells in the pharynges (upper respiratory tract) and intestines (digestive tract) and colonize at the initial infection sites. (c) Once the virus colonizes, it next invades other tissues, including the integumentary system and CNS. The mutant spectrum is shaped by a selection bottleneck such as tissue tropism, which changes the haplotype composition in these tissues. (d) The haplotype with VP1-31G becomes dominant in skin, spinal, and brain tissues, which causes HFMD and severe neurological diseases. (e) These evolutionary changes may depend on the effects of the VP1-31 substitution on virus property. The VP1-31D virus showed high thermostability, viral growth, and fitness in colorectal cells, whereas the VP1-31G virus showed better replication fitness in neuronal cells.