Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene

Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene Kok Keng Tee Tommy Tsan‐Yuk Lam Yoke Fun Chan Jon M. Bible Adeeba Kamarulzaman C. Y. William Tong Yutaka Takebe Oliver G. Pybus

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Evolutionary Genetics of Human Enterovirus 71: Origin, Population Dynamics, Natural Selection, and Seasonal Periodicity of the VP1 Gene 2010 Journal of Virology 10.1128/jvi.01019-09 tee2010-evolutionary-genetics-of-human-ent tee2010-evolutionary-genetics-of-human-ent.txt, the project's staged copy 9729b96a8954609b9c7af406ab1615cf1eba26d4 open-access evolutionary-virology

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0 spans located by searching the stored text for the quote's exact words, allowing any whitespace between them The VP1 capsid gene of human enterovirus 71, read across 628 global sequences spanning four decades, as a record of the virus's origin, spread and selection. Quotation withheld because this record is not cleared for redistribution. When did human enterovirus 71 originate, how have its subgenogroups emerged and spread, and is the VP1 capsid gene under positive selection? Quotation withheld because this record is not cleared for redistribution. existence Quotation withheld because this record is not cleared for redistribution. evolutionary dynamics of enterovirus 71 VP1 gene Quotation withheld because this record is not cleared for redistribution. phylogenetic-analysis phylogenetic-analysis Quotation withheld because this record is not cleared for redistribution. mechanisms Epidemic cycles may be driven demographically rather than antigenically: susceptible children accumulate through birth until a threshold density is crossed, which then triggers large-scale transmission. Quotation withheld because this record is not cleared for redistribution. mechanisms The trunk-versus-terminal dN/dS gap is produced by strong purifying selection acting on all lineages combined with transient deleterious mutations that accumulate on terminal branches and are then removed. Quotation withheld because this record is not cleared for redistribution. mechanisms Retaining glutamic acid at the exposed, potentially immunogenic VP1 position 98 is what allows a lineage to persist long-term; lineages that substitute it die out. Quotation withheld because this record is not cleared for redistribution. mechanisms Because VP1 is surface-exposed and targeted by host neutralizing antibodies, it is subjected to constant immune selective pressure that can drive adaptive change in the capsid. Quotation withheld because this record is not cleared for redistribution. Subgenogroups circulate at low prevalence for years before detection, so the date of common ancestry precedes the first isolation by 1 to 5 years Quotation withheld because this record is not cleared for redistribution. Rises in genetic diversity track epidemics because new subgenogroups emerging is what drives the diversity up Quotation withheld because this record is not cleared for redistribution. Purifying selection dominates the whole gene while a few exposed sites show positive selection, so the genome-wide and per-site pictures point opposite ways Quotation withheld because this record is not cleared for redistribution. Terminal branches carry a higher dN/dS than trunk branches because deleterious mutations appear at the tips and are removed before reaching the trunk Quotation withheld because this record is not cleared for redistribution. Glutamic acid at VP1 position 98 is repeatedly lost on terminal lineages and never on the trunk, suggesting its loss ends lineages rather than propagating Quotation withheld because this record is not cleared for redistribution. An alternative to antigenic escape: susceptibles accumulate by birth until a density threshold is crossed, which would produce the same periodicity without immune-driven evolution Quotation withheld because this record is not cleared for redistribution. The available sequence diversity may make the estimated date of origin too recent; more archival sequencing is needed. what this limits: the estimated date of origin: WE CANNOT AT PRESENT EXCLUDE THE POSSIBILITY THAT CURRENTLY AVAILABLE VIRUS DIVERSITY MAY UNDERESTIMATE THE DATE OF EV-71 ORIGIN Quotation withheld because this record is not cleared for redistribution. The divergence dates apply only to VP1 and may not hold for other genome regions, because recombination occurs outside VP1. what this limits: every divergence date reported: OUR DIVERGENCE DATES SHOULD BE CONSIDERED SPECIFIC TO VP1 AND MAY NOT BE APPLICABLE TO OTHER GENOME regions, because recombination is reported outside VP1 Quotation withheld because this record is not cleared for redistribution. Whether the observed diversity translates into antigenic escape in humans is not established by this work. what this limits: the vaccine and cross-protection implications: THE DEGREE OF CROSS-PROTECTION [in humans] is not established here, only in animal models Quotation withheld because this record is not cleared for redistribution. The sampled diversity may be too shallow to reach the true origin, so the dates could be too recent duplicate of: limitations[0] · what this limits: the same scope as limitations[0] - the sampled diversity may be too shallow to reach the origin Quotation withheld because this record is not cleared for redistribution. The dates apply to VP1 only, because recombination is absent here but reported elsewhere in the genome what this limits: the same scope as limitations[1] - VP1 only Quotation withheld because this record is not cleared for redistribution. Whether antigenic evolution drives the observed cycles is left open, and the competing explanation is not tested what this limits: the antigenic-evolution explanation of the epidemic cycles: WHETHER ANTIGENIC EVOLUTION IS IMPORTANT IN CAUSING THESE CYCLES IS A KEY AREA OF UNCERTAINTY Quotation withheld because this record is not cleared for redistribution. The trunk-versus-terminal selection difference rests on a p-value just above the conventional threshold what this limits: the trunk-versus-terminal selection difference, and read the number not the adjective: the paper calls it A SIGNIFICANTLY LOWER DN/DS RATIO and prints (P = 0.055) Quotation withheld because this record is not cleared for redistribution. Sequences were down-sampled by hand to keep the computation tractable, which is a judgement about what counts as redundant what this limits: every date and tree in the paper: TO REDUCE EXCESSIVE COMPUTATIONAL LOAD, CLOSELY RELATED SEQUENCES ... WERE MANUALLY REMOVED - a hand judgement about what counts as redundant, upstream of all of it Quotation withheld because this record is not cleared for redistribution. Cross-protection between subgenogroups, on which the vaccine implications depend, is unmeasured what this limits: the same scope as limitations[2] - cross-protection between subgenogroups is unmeasured in humans Quotation withheld because this record is not cleared for redistribution. Influenza phylodynamics - the reading of a ladder-like phylogeny as the signature of antigenic drift - applied to EV-71, together with an explicit statement of where the analogy may fail Quotation withheld because this record is not cleared for redistribution. Influenza vaccine strategy offered as the template for EV-71 vaccine design, conditional on EV-71 turning out to drift antigenically Quotation withheld because this record is not cleared for redistribution. all VP1 sequences screened for recombination with the Recombination Detection Program under both default and adjusted settings eliminates: recombination masquerading as phylogenetic signal - a recombinant sequence puts two histories in one alignment and the tree that results is neither · outcome (stated by the tagger, no quotation): held Quotation withheld because this record is not cleared for redistribution. Picornaviridae Picornaviridae Quotation withheld because this record is not cleared for redistribution. branch - core trunk: root to the most recent common ancestor of the latest isolates, the lineage that survived Quotation withheld because this record is not cleared for redistribution. branch - secondary trunk: lineages branching off the core trunk that persist an intermediate 4 to 9 years Quotation withheld because this record is not cleared for redistribution. branch - terminal: tip branches, whose mutations are transient and absent at later time points Quotation withheld because this record is not cleared for redistribution. emergence - estimated time of common ancestry, inferred from the molecular clock rather than observed Quotation withheld because this record is not cleared for redistribution. emergence - date of first isolation, which is when someone looked, not when the lineage began; the gap between the two senses IS the paper's central finding Quotation withheld because this record is not cleared for redistribution. selection - genome-wide purifying: mean dN/dS far below 1 across the whole VP1 gene in every subgenogroup Quotation withheld because this record is not cleared for redistribution. selection - site-specific positive: individual codons under positive selection, coexisting with purifying selection over the gene as a whole Quotation withheld because this record is not cleared for redistribution. genetic diversity - relative (g) reconstructed through time by the skyline plot, not a count of observed sequences Quotation withheld because this record is not cleared for redistribution. claim_type: existence a second reading reaching the same value. Recorded: existence — note: a second reading reaching the same value from a different sentence. Not applied - a person decides. Quotation withheld because this record is not cleared for redistribution. method: phylogenetic-analysis a second reading reaching the same value. Recorded: phylogenetic-analysis — note: a second reading reaching the same value from a different sentence. Not applied - a person decides. Quotation withheld because this record is not cleared for redistribution. phenomenon: evolutionary dynamics of enterovirus 71 VP1 gene a second reading reaching the same value. Recorded: evolutionary dynamics of enterovirus 71 VP1 gene — note: a second reading reaching the same value from a different sentence. Not applied - a person decides. Quotation withheld because this record is not cleared for redistribution. contradicts → earlier EV-71 substitution-rate estimates obtained by linear regression (reference 11) Quotation withheld because this record is not cleared for redistribution. replicates → previous analyses of recombination in human enterovirus species (reference 73) Quotation withheld because this record is not cleared for redistribution. extends → earlier EV-71 substitution rate estimates made by linear regression Quotation withheld because this record is not cleared for redistribution. supports → the comparative finding that trunk branches carry lower dN/dS than terminal branches across RNA viruses Quotation withheld because this record is not cleared for redistribution. supports → the influenza A finding that rises in genetic diversity accompany epidemics Quotation withheld because this record is not cleared for redistribution. supports → Bible and colleagues on cell culture having negligible effect on VP1 sequence Quotation withheld because this record is not cleared for redistribution. enterovirus 71 —part-of→ Picornaviridae enterovirus 71 Picornaviridae strength of the relation: 1 Quotation withheld because this record is not cleared for redistribution. subgenogroup —part-of→ genogroup subgenogroup genogroup strength of the relation: 1 Quotation withheld because this record is not cleared for redistribution. VP1 —part-of→ enterovirus 71 VP1 enterovirus 71 strength of the relation: 1 Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but the span writes 'EV-71'; endpoint abbreviated. Quotation withheld because this record is not cleared for redistribution. purifying selection —contrasted-with→ positive selection purifying selection positive selection strength of the relation: 1 Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but the span writes 'negative selection' against others' 'positive selection'; endpoint variant. Quotation withheld because this record is not cleared for redistribution. trunk —contrasted-with→ terminal branches trunk terminal branches strength of the relation: 1 Quotation withheld because this record is not cleared for redistribution. genetic diversity —operationalised-as→ Bayesian skyline plot genetic diversity Bayesian skyline plot strength of the relation: 1 Quotation withheld because this record is not cleared for redistribution. subgenogroup —causes→ genetic diversity subgenogroup genetic diversity strength of the relation: 0.8 Quotation withheld because this record is not cleared for redistribution. RETRACTED (NEITHER ENDPOINT IS NAMED IN ITS OWN SPAN) · antigenic drift —causes→ ladder-like antigenic drift ladder-like RETRACTED: NEITHER ENDPOINT IS NAMED IN ITS OWN SPAN. The edge is 'antigenic drift --causes--> ladder-like'; the span reads: Quotation withheld, with limited genetic diversity at any one time. It says this process, not antigenic drift, and the strongly temporal shape, not ladder-like. A span that names neither endpoint cannot assert a relation between them - the criterion's plainest case - and it is compounded by has been linked to, which is both relational and attributed to other work. WHAT THE SPAN DOES SAY: nothing about these two terms, so nothing is kept. would reject this edge today; it predates the rule. — strength of the relation: 0.5 — the record marks this weak Audit 2026-09-28: DOUBTFUL - read against its span and kept, but antigenic drift has been linked to temporal phylogeny shapes; causes is loose. Quotation withheld because this record is not cleared for redistribution. enterovirus 71 —contrasted-with→ influenza enterovirus 71 influenza strength of the relation: 1 Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but endpoint by anaphora: 'Both phylogenies' = subgenogroup C1 isolates and influenza virus A (previous sentence). Quotation withheld because this record is not cleared for redistribution. likelihood ratio test likelihood ratio test Quotation withheld because this record is not cleared for redistribution. Bayesian Markov chain Monte Carlo Bayesian Markov chain Monte Carlo Quotation withheld because this record is not cleared for redistribution. bootstrap bootstrap Quotation withheld because this record is not cleared for redistribution. multiple-comparison correction (Bonferroni) multiple-comparison correction (Bonferroni) Quotation withheld because this record is not cleared for redistribution. coefficient of variation coefficient of variation Quotation withheld because this record is not cleared for redistribution. posterior probability posterior probability Quotation withheld because this record is not cleared for redistribution. confidence interval confidence interval Quotation withheld because this record is not cleared for redistribution. Bayes factor Bayes factor Quotation withheld because this record is not cleared for redistribution. maximum likelihood maximum likelihood Quotation withheld because this record is not cleared for redistribution. gamma-distributed among-site rate variation gamma-distributed among-site rate variation Quotation withheld because this record is not cleared for redistribution. dN/dS dN/dS Quotation withheld because this record is not cleared for redistribution. likelihood ratio test likelihood ratio test Quotation withheld because this record is not cleared for redistribution. Bayes factor Bayes factor Quotation withheld because this record is not cleared for redistribution. Bonferroni correction Bonferroni correction Quotation withheld because this record is not cleared for redistribution. credible interval credible interval Quotation withheld because this record is not cleared for redistribution. confidence interval confidence interval Quotation withheld because this record is not cleared for redistribution. coefficient of variation coefficient of variation Quotation withheld because this record is not cleared for redistribution. posterior probability posterior probability Quotation withheld because this record is not cleared for redistribution. maximum likelihood maximum likelihood Quotation withheld because this record is not cleared for redistribution. bootstrap bootstrap Quotation withheld because this record is not cleared for redistribution. Markov chain Monte Carlo Markov chain Monte Carlo Quotation withheld because this record is not cleared for redistribution. p-value p-value Quotation withheld because this record is not cleared for redistribution. The common ancestor of human enterovirus 71 is dated to about 1941, after which the virus split into genogroups B, C and the now-extinct genogroup A. Quotation withheld because this record is not cleared for redistribution. Each subgenogroup of genogroups B and C circulated undetected in humans for up to five years before it caused a large hand, foot and mouth disease outbreak. Quotation withheld because this record is not cleared for redistribution. The VP1 gene evolves under strong purifying selection, with mean dN/dS between 0.029 and 0.075 in every subgenogroup except B3. Quotation withheld because this record is not cleared for redistribution. In subgenogroup C1 the trunk lineages have a lower dN/dS (0.030) than the terminal lineages (0.064), the opposite of the pattern expected under antigenic-drift-style positive selection along the trunk. Quotation withheld because this record is not cleared for redistribution. No homologous recombination was detectable in the capsid-encoding VP1 region, so the phylogenetic and dating analyses are not confounded by recombination. Quotation withheld because this record is not cleared for redistribution. Rises in viral genetic diversity coincide with epidemic onsets and are driven partly by the appearance of new subgenogroups. Quotation withheld because this record is not cleared for redistribution. VP1 codon 98 changed repeatedly and unidirectionally (glutamic acid to lysine or glycine) on terminal branches but never on trunk branches, so lineages that acquired the change did not persist. Quotation withheld because this record is not cleared for redistribution. A demographic model of exponential population growth fits the VP1 data far better than a constant-population-size model. Quotation withheld because this record is not cleared for redistribution. Several trunk residues toggled back and forth over time: position 145 went glutamic acid to glutamine and back, 262 isoleucine to valine and back, and 289 alanine to threonine and back. Quotation withheld because this record is not cleared for redistribution. Subgenogroups B1 to B5, C1, C2 and C4 are globally distributed, whereas C3 is confined to Korea and C5 to Vietnam. Quotation withheld because this record is not cleared for redistribution. The VP1 gene evolves at about 4.2 to 4.6 x 10^-3 substitutions per site per year, higher than earlier linear-regression estimates. Quotation withheld because this record is not cleared for redistribution. Choice of coalescent demographic model made no significant difference to the estimated divergence dates. Quotation withheld because this record is not cleared for redistribution. the common ancestor of EV-71 dates to about 1941 Quotation withheld because this record is not cleared for redistribution. each subgenogroup circulated undetected for 1 to 5 years before its first outbreak Quotation withheld because this record is not cleared for redistribution. the same subgenogroup in different countries reflects separate lineages, not one strain travelling Quotation withheld because this record is not cleared for redistribution. VP1 evolves under strong purifying selection despite substitution rates comparable to influenza and HIV-1 Quotation withheld because this record is not cleared for redistribution. trunk lineages carry a lower dN/dS than terminal lineages in subgenogroup C1 Quotation withheld because this record is not cleared for redistribution. position 98 changes repeatedly on terminal lineages and never on the trunk, and the changes are directional Quotation withheld because this record is not cleared for redistribution. amino acids toggle - changing and reverting - on the core trunk over 3 to 5 year spans Quotation withheld because this record is not cleared for redistribution. only 14 nonsynonymous substitutions occur on the trunk, at 10 sites Quotation withheld because this record is not cleared for redistribution. no recombination was detectable in VP1 Quotation withheld because this record is not cleared for redistribution. reconstructed diversity dynamics match four decades of independent surveillance data Quotation withheld because this record is not cleared for redistribution. exponential growth fits the data better than constant population size Quotation withheld because this record is not cleared for redistribution. C3 and C5 are geographically restricted while the other subgenogroups are cosmopolitan Quotation withheld because this record is not cleared for redistribution. outbreaks recur on a roughly 2 to 3 year cycle Quotation withheld because this record is not cleared for redistribution. terminal branches terminal branches Quotation withheld because this record is not cleared for redistribution. secondary trunk lineages secondary trunk lineages Quotation withheld because this record is not cleared for redistribution. ladder-like tree structure ladder-like tree structure Quotation withheld because this record is not cleared for redistribution. antigenic drift antigenic drift Quotation withheld because this record is not cleared for redistribution. core trunk core trunk Quotation withheld because this record is not cleared for redistribution. Bayesian skyline plot Bayesian skyline plot Quotation withheld because this record is not cleared for redistribution. relaxed molecular clock relaxed molecular clock Quotation withheld because this record is not cleared for redistribution. toggling toggling Quotation withheld because this record is not cleared for redistribution. enterovirus species A enterovirus species A Quotation withheld because this record is not cleared for redistribution. ratio of nonsynonymous substitutions to synonymous substitutions ratio of nonsynonymous substitutions to synonymous substitutions Quotation withheld because this record is not cleared for redistribution. relative viral genetic diversity relative viral genetic diversity Quotation withheld because this record is not cleared for redistribution. VP1 gene sequences VP1 gene sequences Quotation withheld because this record is not cleared for redistribution. substitution rate substitution rate Quotation withheld because this record is not cleared for redistribution. tMRCA tMRCA Quotation withheld because this record is not cleared for redistribution. nucleotide substitution model nucleotide substitution model Quotation withheld because this record is not cleared for redistribution. demographic model demographic model Quotation withheld because this record is not cleared for redistribution. 628 — Number of complete VP1 gene sequences with known collection dates analysed, effectively all reported EV-71 VP1 genes as of mid-2008 Quotation withheld because this record is not cleared for redistribution. 1941.0 (95% CR, 1928.8 to 1952.2) — Estimated date of origin of EV-71 as a descendant of coxsackievirus A16 Quotation withheld because this record is not cleared for redistribution. 229 and 251 — Sizes of the down-sampled genogroup B and C alignments used for the Bayesian MCMC analyses Quotation withheld because this record is not cleared for redistribution. 0.030 ± 0.009 ... 0.064 ± 0.008 ( P = 0.055) — dN/dS on trunk versus terminal lineages of subgenogroup C1, and the significance of the difference Quotation withheld because this record is not cleared for redistribution. 14 ... 10 — Number of nonsynonymous substitutions observed on subgenogroup C1 trunk branches and the number of distinct sites at which they occurred Quotation withheld because this record is not cleared for redistribution. every 2 to 3 years — Observed periodicity of EV-71 outbreaks Quotation withheld because this record is not cleared for redistribution. 145 ( P < 0.05), 98 ( P < 0.05), 237, and 241 ( P < 0.05) — VP1 codon positions detected as being under positive selection in well-sampled subgenogroups Quotation withheld because this record is not cleared for redistribution. about 3 to 5 years — Length of time a toggled residue persisted on the core trunk branches before reverting Quotation withheld because this record is not cleared for redistribution. 1 to 5 years — Lag between the date of common ancestry of a genogroup C subgenogroup and its first identification Quotation withheld because this record is not cleared for redistribution. 628 — VP1 sequences analysed, essentially every one then reported Quotation withheld because this record is not cleared for redistribution. 1941.0 (95% CR, 1928.8 to 1952.2) — the estimated origin of EV-71, decades before its first isolation in 1969 Quotation withheld because this record is not cleared for redistribution. 4.5 × 10 −3 to 4.6 × 10 −3 — substitutions per site per year in genogroup B Quotation withheld because this record is not cleared for redistribution. 0.029 to 0.075 — the range of mean dN/dS across subgenogroups, all far below 1 Quotation withheld because this record is not cleared for redistribution. 2 to 5 years — how long genogroup B subgenogroups circulated before causing large outbreaks Quotation withheld because this record is not cleared for redistribution. 20 million states — the MCMC chain length, sampled every 10,000 Quotation withheld because this record is not cleared for redistribution. 229 and 251 sequences — the down-sampled genogroup B and C data sets actually used for the clock analysis Quotation withheld because this record is not cleared for redistribution. approximately 7,400 bases — the EV-71 genome length Quotation withheld because this record is not cleared for redistribution. every 2 to 3 years — the apparent outbreak periodicity the discussion tries to explain Quotation withheld because this record is not cleared for redistribution. 1969 — first isolation of EV-71, nearly three decades after the inferred origin Quotation withheld because this record is not cleared for redistribution.

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