High throughput AS LNA qPCR method for the detection of a specific mutation in poliovirus vaccine strains.

Sabin Inactivated Poliovirus Vaccine (sIPV) has become one of the preferred vaccination options for the last step in the Poliovirus eradication program. Sequencing of poliovirus samples is needed during the manufacturing of poliovirus vaccines to assure the safety and immunogenicity of these vaccines. Next-generation sequencing analysis is the current costly and time-consuming gold standard for monitoring the manufacturing processes. We developed a low-cost and quick, highly sensitive, and allele-specific locked nucleic acid-probe-based reverse transcription quantitative PCR alternative that can accurately detect mutations in poliovirus vaccine samples during process development, scaling up, and release. Using the frequently in vitro occurring and viral replication-impacting VP1-E295K mutation as a showcase, we show that this technology can accurately detect E295K mutations in poliovirus 2 samples to similar levels as NGS. The qPCR technology was developed employing a synthetic dsDNA fragment-based standard curve containing mixes of E295K-WT (wildtype) and Mut (mutant) synthetic dsDNA fragments ranging from 1 × 107 copies/µL to 1 × 102 copies/µL to achieve a linear correlation with R2 > 0.999, and PCR efficiencies of 95-105 %. Individual standard concentration levels achieved accuracies of ≥92 % (average 96 %) and precisions of ≤17 % (average 3.3 %) RSD. Specificity of locked nucleic acid (LNA)-probes was confirmed in the presence and absence of co-mutations in the probe-binding region. Application of the developed assay to Sabin Poliovirus type 2 production run samples, illustrated a linear relationship with an R2 of 0.994, and an average accuracy of 97.2 % of the variant (allele)-specific AS LNA qPCR result, compared to NGS. The assay showed good sensitivity for poliovirus samples, containing E295K mutation levels between 0 % and 95 % (quantification range). In conclusion, the developed AS LNA qPCR presents a valuable low-cost, and fast tool, suitable for the process development and quality control of polio vaccines.

Genetic characterization and molecular evolution of type 3 vaccine-derived polioviruses from an immunodeficient patient in China.

In 2013, a case of immunodeficiency vaccine-derived poliovirus (iVDPV) was identified in Jiangxi Province, China. In this study, we purified 14 type 3 original viral isolates from this case and characterized the molecular evolution of these iVDPVs for 298 days. Genetic variants were found in most of the original viral isolates, with complex genetic and evolutionary relationships among the variants. A phylogenetic tree constructed based on the P1 region showed that these iVDPVs were classified into lineage A and B. The dominant lineage B represents a major trend in virus evolution. The nucleotide substitution rate at the third codon position (3CP) estimated by the BEAST program was 1.76 × 10-2 substitutions/site/year (95% HPD: 1.23-2.39 × 10-2). The initial OPV dose was given dating back to March 2013, which was close to the time of the last OPV vaccination, suggesting that OPV infection may have originated with the last dose of vaccine. Recombinant analysis showed that these iVDPVs were inter-vaccine recombinants with two recombination patterns, S3/S2/S1 and S3/S2/S3/S2/S1. Whole genome sequence analysis revealed that key nucleotide sites (C472U, C2034U, U2493C) associated with the attenuated phenotype of Sabin 3 have been replaced. Temperature sensitivity test showed that all tested strains were temperature-sensitive, except for the variant Day11-5. Interestingly, we observed that the variant Day11-5 temperature resistance properties may be associated with the Lys to Met substitution at the VP2-162 site. Serological test and whole genome sequence analysis showed that the seropositivity rate remained high, and mutations in the antigenic sites did not significantly alter neutralization ability.

Genetic stabilization of attenuated oral vaccines against poliovirus types 1 and 3.

Vaccination with Sabin, a live attenuated oral polio vaccine (OPV), results in robust intestinal and humoral immunity and has been key to controlling poliomyelitis. As with any RNA virus, OPV evolves rapidly to lose attenuating determinants critical to the reacquisition of virulence1-3 resulting in vaccine-derived, virulent poliovirus variants. Circulation of these variants within underimmunized populations leads to further evolution of circulating, vaccine-derived poliovirus with higher transmission capacity, representing a significant risk of polio re-emergence. A new type 2 OPV (nOPV2), with promising clinical data on genetic stability and immunogenicity, recently received authorization from the World Health Organization for use in response to circulating, vaccine-derived poliovirus outbreaks. Here we report the development of two additional live attenuated vaccine candidates against type 1 and 3 polioviruses. The candidates were generated by replacing the capsid coding region of nOPV2 with that from Sabin 1 or 3. These chimeric viruses show growth phenotypes similar to nOPV2 and immunogenicity comparable to their parental Sabin strains, but are more attenuated. Our experiments in mice and deep sequencing analysis confirmed that the candidates remain attenuated and preserve all the documented nOPV2 characteristics concerning genetic stability following accelerated virus evolution. Importantly, these vaccine candidates are highly immunogenic in mice as monovalent and multivalent formulations and may contribute to poliovirus eradication.

Epidemiological survey and genetic characterization of type 3 vaccine-derived poliovirus isolated from a patient with four doses of inactivated polio vaccine in Henan Province, China.

BACKGROUND: Vaccine-derived poliovirus (VDPV) is a potential threat to polio eradication because they can reintroduce into the general population and cause paralytic polio outbreaks, a phenomenon that has recently emerged as a prominent public health concern at the end of global polio eradication. This study aimed to describe the epidemiology and genetic characteristics of the first VDPV identified from a patient with acute flaccid paralysis (AFP), with four doses of inactivated polio vaccine immunization in Henan Province, China in 2017. METHODS: The patient was diagnosed with type 3 VDPV. Subsequently, a series of epidemiological approaches was implemented, including a retrospective search of AFP cases, rate of vaccination assessment, study of contacts, and supplementary immunization activities. Fecal samples were collected, viral isolation was performed, and the viral isolates were characterized using full-length genomic sequencing and bioinformatic analysis. RESULTS: Phylogenetic analysis showed that the viral isolates from the patient were different from other reported genetic clusters of type 3 VDPV worldwide. They were identified as a Sabin 3/Sabin 1 recombinant VDPV with a crossover site in the P2 region. Nucleotide substitutions, including U → C (472) and C → U (2493), have been identified, both of which are frequently observed as reversion mutations in neurovirulent type 3 poliovirus. A unique aspect of this case is that the patient had been vaccinated with four doses of inactive polio vaccine, and the serum neutralizing antibody for Sabin types 1 and 3 were 1∶16 and 1∶512, respectively. Thus, the patient was speculated to have been infected with type 3 VDPV, and the virus continued to replicate and be excreted for at least 41 d. CONCLUSIONS: The existence of this kind of virus in human population is a serious risk and poses a severe challenge in maintaining a polio-free status in China. To the best of our knowledge, this is the first report of VDPV identified in the Henan province of China. Our results highlight the importance of maintaining a high-level vaccination rate and highly sensitive AFP case surveillance system in intercepting VDPV transmission.

Genetic Characterization of Novel Oral Polio Vaccine Type 2 Viruses During Initial Use Phase Under Emergency Use Listing - Worldwide, March-October 2021.

The emergence and international spread of neurovirulent circulating vaccine-derived polioviruses (cVDPVs) across multiple countries in Africa and Asia in recent years pose a major challenge to the goal of eradicating all forms of polioviruses. Approximately 90% of all cVDPV outbreaks are caused by the type 2 strain of the Sabin vaccine, an oral live, attenuated vaccine; cVDPV outbreaks typically occur in areas of persistently low immunization coverage (1). A novel type 2 oral poliovirus vaccine (nOPV2), produced by genetic modification of the type 2 Sabin vaccine virus genome (2), was developed and evaluated through phase I and phase II clinical trials during 2017-2019. nOPV2 was demonstrated to be safe and well-tolerated, have noninferior immunogenicity, and have superior genetic stability compared with Sabin monovalent type 2 (as measured by preservation of the primary attenuation site [domain V in the 5' noncoding region] and significantly lower neurovirulence of fecally shed vaccine virus in transgenic mice) (3-5). These findings indicate that nOPV2 could be an important tool in reducing the risk for generating vaccine-derived polioviruses (VDPVs) and the risk for vaccine-associated paralytic poliomyelitis cases. Based on the favorable preclinical and clinical data, and the public health emergency of international concern generated by ongoing endemic wild poliovirus transmission and cVDPV type 2 outbreaks, the World Health Organization authorized nOPV2 for use under the Emergency Use Listing (EUL) pathway in November 2020, allowing for its first use for outbreak response in March 2021 (6). As required by the EUL process, among other EUL obligations, an extensive plan was developed and deployed for obtaining and monitoring nOPV2 isolates detected during acute flaccid paralysis (AFP) surveillance, environmental surveillance, adverse events after immunization surveillance, and targeted surveillance for adverse events of special interest (i.e., prespecified events that have the potential to be causally associated with the vaccine product), during outbreak response, as well as through planned field studies. Under this monitoring framework, data generated from whole-genome sequencing of nOPV2 isolates, alongside other virologic data for isolates from AFP and environmental surveillance systems, are reviewed by the genetic characterization subgroup of an nOPV working group of the Global Polio Eradication Initiative. Global nOPV2 genomic surveillance during March-October 2021 confirmed genetic stability of the primary attenuating site. Sequence data generated through this unprecedented global effort confirm the genetic stability of nOPV2 relative to Sabin 2 and suggest that nOPV2 will be an important tool in the eradication of poliomyelitis. nOPV2 surveillance should continue for the duration of the EUL.

Africa battles out-of-control polio outbreaks.

Cases tumble in Pakistan and Afghanistan but African outbreaks now threaten eradication.

The Early Evolution of Oral Poliovirus Vaccine Is Shaped by Strong Positive Selection and Tight Transmission Bottlenecks.

The emergence of circulating vaccine-derived polioviruses through evolution of the oral polio vaccine (OPV) poses a significant obstacle to polio eradication. Understanding the early genetic changes that occur as OPV evolves and transmits is important for preventing future outbreaks. Here, we use deep sequencing to define the evolutionary trajectories of type 2 OPV in a vaccine trial. By sequencing 497 longitudinal stool samples from 271 OPV2 recipients and household contacts, we were able to examine the extent of convergent evolution in vaccinated individuals and the amount of viral diversity that is transmitted. In addition to rapid reversion of key attenuating mutations, we identify strong selection at 19 sites across the genome. We find that a tight transmission bottleneck limits the onward transmission of these early adaptive mutations. Our results highlight the distinct evolutionary dynamics of live attenuated virus vaccines and have important implications for the success of next-generation OPV.

Case of Poliomyelitis Caused by Significantly Diverged Derivative of the Poliovirus Type 3 Vaccine Sabin Strain Circulating in the Orphanage.

Significantly divergent polioviruses (VDPV) derived from the oral poliovirus vaccine (OPV) from Sabin strains, like wild polioviruses, are capable of prolonged transmission and neuropathology. This is mainly shown for VDPV type 2. Here we describe a molecular-epidemiological investigation of a case of VDPV type 3 circulation leading to paralytic poliomyelitis in a child in an orphanage, where OPV has not been used. Samples of feces and blood serum from the patient and 52 contacts from the same orphanage were collected twice and investigated. The complete genome sequencing was performed for five polioviruses isolated from the patient and three contact children. The level of divergence of the genomes of the isolates corresponded to approximately 9-10 months of evolution. The presence of 61 common substitutions in all isolates indicated a common intermediate progenitor. The possibility of VDPV3 transmission from the excretor to susceptible recipients (unvaccinated against polio or vaccinated with inactivated poliovirus vaccine, IPV) with subsequent circulation in a closed children's group was demonstrated. The study of the blood sera of orphanage residents at least twice vaccinated with IPV revealed the absence of neutralizing antibodies against at least two poliovirus serotypes in almost 20% of children. Therefore, a complete rejection of OPV vaccination can lead to a critical decrease in collective immunity level. The development of new poliovirus vaccines that create mucosal immunity for the adequate replacement of OPV from Sabin strains is necessary.

Expression of immunogenic poliovirus Sabin type 1 VP proteins in transgenic tobacco.

One of the milestones of vaccinology is the depletion of the global impact of Poliomyelitis. The current vaccines to deal with Polio comprise the Sabin and Salk formulations. The main limitation of the former is the use of attenuated viruses that can revert into pathogenic forms, whereas the latter is more expensive and induces no protection in the intestinal tract; the site of virus replication. Genetically engineered plants cope with such limitations. In addition, they offer a low-cost alternative for production, storage and delivery of vaccines. This technology has been narrowly applied in the development of Polio vaccines. Herein, we explored the ability of tobacco cells to express the immunogenic VP1, VP2, VP3, and VP4 Polio antigens, which are relevant for vaccine development. Evidence on the expression of the plant-made Polio VPs is presented and an immunogenicity assessment proved their capacity to induce local and systemic humoral responses when administered by subcutaneous and oral routes. The plant-made VPs will be useful in the development of low-cost vaccine formulations able to induce effective mucosal immunity without the risks associated to the use of attenuated viruses; therefore there is a potential for this technology to contribute toward Polio eradication.

Engineering the Live-Attenuated Polio Vaccine to Prevent Reversion to Virulence.

The live-attenuated oral poliovirus vaccine (OPV or Sabin vaccine) replicates in gut-associated tissues, eliciting mucosa and systemic immunity. OPV protects from disease and limits poliovirus spread. Accordingly, vaccination with OPV is the primary strategy used to end the circulation of all polioviruses. However, the ability of OPV to regain replication fitness and establish new epidemics represents a significant risk of polio re-emergence should immunization cease. Here, we report the development of a poliovirus type 2 vaccine strain (nOPV2) that is genetically more stable and less likely to regain virulence than the original Sabin2 strain. We introduced modifications within at the 5' untranslated region of the Sabin2 genome to stabilize attenuation determinants, 2C coding region to prevent recombination, and 3D polymerase to limit viral adaptability. Prior work established that nOPV2 is immunogenic in preclinical and clinical studies, and thus may enable complete poliovirus eradication.

Multiplex PCR-based titration (MPBT) assay for determination of infectious titers of the three Sabin strains of live poliovirus vaccine.

BACKGROUND: Conventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge. RESULTS: A new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1-5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay. CONCLUSIONS: The multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.

Evaluation of the genetic stability of Sabin strains and the consistency of inactivated poliomyelitis vaccine made from Sabin strains using direct deep-sequencing.

Inactivated poliomyelitis vaccine made from Sabin strains (sIPV) has been encouraged to introduce in the "Global Polio Eradication & Endgame Strategic Plan" and increasingly used worldwide. Attenuated Sabin strains used in manufacture of oral poliovirus vaccine (OPV) and sIPV may regain full or partial neurovirulence during growth in vaccine recipients and the vaccine manufacturing processes. Ensuring the molecular consistency of sIPV batches and that no mutation accumulates beyond the level present in past batches are important for quality control of vaccine manufacture process. Direct deep-sequencing allows the construction of a library of virus RNA and the detection of genetic mutations throughout the viral genome. In the present study, direct deep-sequencing was conducted to detect molecular mutations in virus passages, multiple sIPV monovalent lots, and virus monovalent lots from different polio type III strains. The results indicated that direct deep-sequencing can be used to identify and quantify small amounts of mutant viruses in vaccine preparations, trace the source of a specific virus seed, and monitor the batch-to-batch consistency of vaccines, suggesting that this technique could be suitable for the quality control and consistency monitoring of sIPV production.

Molecular mechanism of poliovirus Sabin vaccine strain attenuation.

Recruitment of poliovirus (PV) RNA to the human ribosome requires the coordinated interaction of the viral internal ribosome entry site (IRES) and several host cellular initiation factors and IRES trans-acting factors (ITAFs). Attenuated PV Sabin strains contain point mutations in the PV IRES domain V (dV) that inhibit viral translation. Remarkably, attenuation is most apparent in cells of the central nervous system, but the molecular basis to explain this is poorly understood. The dV contains binding sites for eukaryotic initiation factor 4G (eIF4G) and polypyrimidine tract-binding protein (PTB). Impaired binding of these proteins to the mutant IRESs has been observed, but these effects have not been quantitated. We used a fluorescence anisotropy assay to reveal that the Sabin mutants reduce the equilibrium dissociation constants of eIF4G and PTB to the PV IRES by up to 6-fold. Using the most inhibitory Sabin 3 mutant, we used a real-time fluorescence helicase assay to show that the apparent affinity of an active eIF4G/4A/4B helicase complex for the IRES is reduced by 2.5-fold. The Sabin 3 mutant did not alter the maximum rate of eIF4A-dependent helicase activity, suggesting that this mutant primarily reduces the affinity, rather than activity, of the unwinding complex. To confirm this affinity model of attenuation, we show that eIF4G overexpression in HeLa cells overcomes the attenuation of a Sabin 3 mutant PV-luciferase replicon. Our study provides a quantitative framework for understanding the mechanism of PV Sabin attenuation and provides an explanation for the previously observed cell type-specific translational attenuation.

Diagnostic Assay Development for Poliovirus Eradication.

With poliovirus eradication nearing, few pockets of active wild poliovirus (WPV) transmission remain in the world. Intratypic differentiation (ITD) plays a crucial part in laboratory surveillance as the molecular detection method that can identify and distinguish wild and vaccine-like polioviruses isolated from acute flaccid paralysis cases or environmental sources. The need to detect new variants of WPV serotype 1 (WPV1) and the containment of all serotype 2 polioviruses (PV2) in 2015 required changes to the previous version of the method. The ITD version 5.0 is a set of six real-time reverse transcription-PCR (rRT-PCR) assays that serve as accurate diagnostic tools to easily detect and differentiate PV serotypes and genotypes. We describe the creation and properties of quantitation standards, including 16 control RNA transcripts and nine plaque-isolated viruses. All ITD rRT-PCR assays were validated using these standards, and the limits of detection were determined for each assay. We designed and pilot tested two new assays targeting recently circulating WPV1 genotypes and all PV2 viruses. The WPV1 assay had 99.1% specificity and 100% sensitivity, and the PV2 assay had 97.7% specificity and 92% sensitivity. Before proceeding to the next step in the global poliovirus eradication program, we needed to gain a better understanding of the performance of the ITD 5.0 suite of molecular assays and their limits of detection and specificities. The findings and conclusions in this evaluation serve as building blocks for future development work.

Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses: Evolutionary Implications.

Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing) of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These "weak" segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

MicroRNA-134 regulates poliovirus replication by IRES targeting.

Global poliovirus eradication efforts include high vaccination coverage with live oral polio vaccine (OPV), surveillance for acute flaccid paralysis, and OPV "mop-up" campaigns. An important objective involves host-directed strategies to reduce PV replication to diminish viral shedding in OPV recipients. In this study, we show that microRNA-134-5p (miR-134) can regulate Sabin-1 replication but not Sabin-2 or Sabin-3 via direct interaction with the PV 5'UTR. Hypochromicity data showed miR-134 binding to Sabin-1 and 3 but not Sabin-2 IRES. Transfection of a miR-134 mimic repressed translation of Sabin-1 5'UTR driven luciferase validating the mechanism of miR-134-mediated repression of Sabin-1. Further, site directed mutagenesis of the miR-134 binding site in Sabin-1 IRES relieved miR-134-mediated repression indicating that these regulatory molecules have an important role in regulating the host gene response to PV. Binding of miR-134 to Sabin-1 IRES caused degradation of the IRES transcript in a miR-134 and sequence specific manner. The miR-134 binding site was found to be highly conserved in wild type PV-1 as well as EV71 strains indicating that miR-134 may regulate function of these IRES sequences in circulation.

Quantifying low-frequency revertants in oral poliovirus vaccine using next generation sequencing.

Spontaneous reversion to neurovirulence of live attenuated oral poliovirus vaccine (OPV) serotype 3 (chiefly involving the n.472U>C mutation), must be monitored during production to ensure vaccine safety and consistency. Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has long been endorsed by the World Health Organization as the preferred in vitro test for this purpose; however, it requires radiolabeling, which is no longer supported by many laboratories. We evaluated the performance and suitability of next generation sequencing (NGS) as an alternative to MAPREC. The linearity of NGS was demonstrated at revertant concentrations equivalent to the study range of 0.25%-1.5%. NGS repeatability and intermediate precision were comparable across all tested samples, and NGS was highly reproducible, irrespective of sequencing platform or analysis software used. NGS was performed on OPV serotype 3 working seed lots and monovalent bulks (n=21) that were previously tested using MAPREC, and which covered the representative range of vaccine production. Percentages of 472-C revertants identified by NGS and MAPREC were comparable and highly correlated (r≥0.80), with a Pearson correlation coefficient of 0.95585 (p<0.0001). NGS demonstrated statistically equivalent performance to that of MAPREC for quantifying low-frequency OPV serotype 3 revertants, and offers a valid alternative to MAPREC.

The Evolutionary Pathway to Virulence of an RNA Virus.

Paralytic polio once afflicted almost half a million children each year. The attenuated oral polio vaccine (OPV) has enabled world-wide vaccination efforts, which resulted in nearly complete control of the disease. However, poliovirus eradication is hampered globally by epidemics of vaccine-derived polio. Here, we describe a combined theoretical and experimental strategy that describes the molecular events leading from OPV to virulent strains. We discover that similar evolutionary events occur in most epidemics. The mutations and the evolutionary trajectories driving these epidemics are replicated using a simple cell-based experimental setup where the rate of evolution is intentionally accelerated. Furthermore, mutations accumulating during epidemics increase the replication fitness of the virus in cell culture and increase virulence in an animal model. Our study uncovers the evolutionary strategies by which vaccine strains become pathogenic and provides a powerful framework for rational design of safer vaccine strains and for forecasting virulence of viruses. VIDEO ABSTRACT.

A Cluster of Paralytic Poliomyelitis Cases Due to Transmission of Slightly Diverged Sabin 2 Vaccine Poliovirus.

UNLABELLED: Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July to August 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features. (i) Until this outbreak, Sabin-like viruses (in distinction to more markedly evolved vaccine-derived polioviruses [VDPVs]) were reported to cause only sporadic cases of VAPP. Consequently, VAPP cases were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPVs in epidemiological terms. (ii) The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity. (iii) The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events. (iv) The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking, and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems. IMPORTANCE: The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: those caused by slightly diverged (Sabin-like) viruses on the one hand and those caused by significantly diverged VDPVs on the other. This classification is based on the number of mutations in the viral genome region encoding a viral structural protein. Until now, only sporadic poliomyelitis cases due to Sabin-like polioviruses had been described, and in distinction from the VDPV-triggered outbreaks, they did not require broad-scale epidemiological responses. Here, an unusual outbreak of poliomyelitis caused by a Sabin-like virus is reported, which had an exceptionally high disease/infection ratio. This outbreak blurred the borderline between Sabin-like polioviruses and VDPVs both in pathogenicity and in the kind of responses required, as well as underscoring important gaps in understanding the pathogenicity, epidemiology, and evolution of vaccine-derived polioviruses.