Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing

Abstract

Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication.

Keywords: enterovirus; high-throughput nucleotide sequencing; molecular methods; oral; oral vaccines; poliovirus; poliovirus vaccine.

FIG 1

Determination of the error threshold. (A) Error events observed across the genome in low-passage-number controls. Insertions (ins), deletions (del), and substitutions (subst) are color coded. Note the short error-prone region (bp 810 to 900) in Sabin 2. The solid black line represents the overall per base error rate, 0.0033. (B) Error events plotted as a function of read depth. (C) Prediction of insertion, deletion, and substitution error rates based on sequence depth using a general linear model (Poisson) with observed error data. (D) Comparison of overall errors using Poisson and negative-binomial (gamma-Poisson mixture) models. The black curve represents the error threshold utilized in this study. The threshold was calculated from the overall per base error rate using the Poisson distribution at a P of <0.001.

FIG 2

Variant analysis of vaccine-related polioviruses. (A) Distribution of synonymous and nonsynonymous variants in the 5′ UTR and VP region of Sabin 1, Sabin 2, and Sabin 3 vaccine-related polioviruses. Horizontal black bars, medians. (B) Distribution of variant abundance in the 5′ UTR and VP region. Horizontal black bars, medians.

FIG 3

Frequency of VP1 nucleotide variants at each amino acid position in Sabin 1, Sabin 2, and Sabin 3. Dotted lines mark amino acid positions associated with phenotypic reversion. Variants include nonsynonymous and synonymous changes. AA, amino acid.