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Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18-22 weeks.In two similarly designed clinical trials (NCT02521974 and NCT03554798) conducted in Panama, infants aged 18-22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model.Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5' untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 logThe data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses.BillMelinda Gates Foundation.

Acute flaccid paralysis (AFP) surveillance has been used to identify polio cases and target vaccination campaigns since the inception of the Global Poliovirus Eradication Initiative (GPEI) in 1988. To date, only Afghanistan and Pakistan have failed to interrupt wild poliovirus transmission. Circulation of vaccine-derived polioviruses (VDPV) continues to be a problem in high-risk areas of the Eastern Mediterranean, African, and Southeast Asian regions. Environmental surveillance (ES) is an important adjunct to AFP surveillance, helping to identify circulating polioviruses in problematic areas. Stools from AFP cases and contacts (>200,000 specimens/year) and ES samples (>642 sites) are referred to 146 laboratories in the Global Polio Laboratory Network (GPLN) for testing. Although most World Health Organization supported laboratories use the two-phase separation method due to its simplicity and effectiveness, alternative simple, widely available, and cost-effective methods are needed. The CAFÉ (Concentration and Filtration Elution) method was developed from existing filtration methods to handle any type of sewage or residual waters. At $10–20 US per sample for consumable materials, CAFÉ is cost effective, and all equipment and reagents are readily available from markets and suppliers globally. The report describes the results from a parallel study of CAFÉ method with the standard two-phase separation method. The study was performed with samples collected from five countries (Guatemala, Haïti, Thailand, Papua New Guinea, and the Philippines), run in three laboratories–(United States, Thailand and in the Philippines) to account for regional and sample-to-sample variability. Samples from each site were divided into two 500 ml aliquots and processed by both methods, with no other additional concentration or manipulation. The results of 338 parallel-tested samples show that the CAFÉ method is more sensitive than the two-phase separation method for detection of non-polio enteroviruses (p-value < 0.0001) and performed as well as the two-phase separation method for polioviruses detection with no significant difference (p-value > 0.05). The CAFÉ method is a robust, sensitive, and cost-effective method for isolating enteroviruses from residual waters.

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.

In 1988, the World Health Assembly established the Global Polio Eradication Initiative (GPEI). Since then, wild poliovirus (WPV) cases have decreased approximately 99.99%, and WPV types 2 and 3 have been declared eradicated. Only Afghanistan and Pakistan have never interrupted WPV type 1 (WPV1) transmission. This report describes global progress toward polio eradication during January 1, 2020-April 30, 2022, and updates previous reports (1,2). This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.* Five WPV1 cases were reported from Afghanistan and Pakistan in 2021, compared with 140 in 2020. In 2022 (as of May 5), three WPV1 cases had been reported: one from Afghanistan and two from Pakistan. WPV1 genetically linked to virus circulating in Pakistan was identified in Malawi in a child with paralysis onset in November 2021. Circulating vaccine-derived polioviruses (cVDPVs), with neurovirulence and transmissibility similar to that of WPV, emerge in populations with low immunity following prolonged circulation of Sabin strain oral poliovirus vaccine (OPV) (3). During January 2020-April 30, 2022, a total of 1,856 paralytic cVDPV cases were reported globally: 1,113 in 2020 and 688 in 2021, including cases in Afghanistan and Pakistan. In 2022 (as of May 5), 55 cVDPV cases had been reported. Intensified programmatic actions leading to more effective outbreak responses are needed to stop cVDPV transmission. The 2022-2026 GPEI Strategic Plan objective of ending WPV1 transmission by the end of 2023 is attainable (4). However, the risk for children being paralyzed by polio remains until all polioviruses, including WPV and cVDPV, are eradicated.

We sequenced 109 type 2 Sabin-like poliovirus isolates that had been collected from acute flaccid paralysis patients or healthy children in Nigeria. Understanding the genetic makeup of these viruses may contribute to polio eradication efforts.

In July 2022, a case of paralytic poliomyelitis resulting from infection with vaccine-derived poliovirus (VDPV) type 2 (VDPV2)

Background: The polio eradication endgame continues to increase in complexity. With polio cases caused by wild poliovirus type 1 and circulating vaccine-derived polioviruses of all three types (1, 2 and 3) reported in 2022, the number, formulation, and use of poliovirus vaccines poses challenges for national immunization programs and vaccine suppliers. Prior poliovirus transmission modeling of globally-coordinated type-specific cessation of oral poliovirus vaccine (OPV) assumed creation of Sabin monovalent OPV (mOPV) stockpiles for emergencies and explored the potential need to restart OPV if the world reached a specified cumulative threshold number of cases after OPV cessation. Methods: We document the actual experience of type 2 OPV (OPV2) cessation and reconsider prior modeling assumptions related to OPV restart. We develop updated decision trees of national immunization options for poliovirus vaccines considering different possibilities for OPV restart. Results: While OPV restart represented a hypothetical situation for risk management and contingency planning to support the 2013-2018 Global Polio Eradication Initiative (GPEI) Strategic Plan, the actual epidemiological experience since OPV2 cessation raises questions about what, if any, trigger(s) could lead to restarting the use of OPV2 in routine immunization and/or plans for potential future restart of type 1 and 3 OPV after their respective cessation. The emergency use listing of a genetically stabilized novel type 2 OPV (nOPV2) and continued evaluation of nOPV for types 1 and/or 3 add further complexity by increasing the combinations of possible OPV formulations for OPV restart. Conclusions: Expanding on a 2019 discussion of the logistical challenges and implications of restarting OPV, we find a complex structure of the many options and many issues related to OPV restart decisions and policies as of early 2023. We anticipate many challenges for forecasting prospective vaccine supply needs during the polio endgame due to increasing potential combinations of poliovirus vaccine choices.

Despite exhaustive and fully-financed plans to manage the risks of globally coordinated cessation of oral poliovirus vaccine (OPV) containing type 2 (OPV2) prior to 2016, as of 2022, extensive, continued transmission of circulating vaccine-derived polioviruses (cVDPVs) type 2 (cVDPV2) remains. Notably, cumulative cases caused by cVDPV2 since 2016 now exceed 2,500. Earlier analyses explored the implications of using different vaccine formulations to respond to cVDPV2 outbreaks and demonstrated how different properties of novel OPV2 (nOPV2) might affect its performance compared to Sabin monovalent OPV2 (mOPV2). These prior analyses used fixed assumptions for how outbreak response would occur, but outbreak response implementation can change. We update an existing global poliovirus transmission model to explore different options for responding with different vaccines and assumptions about scope, delays, immunization intensity, target age groups, and number of rounds. Our findings suggest that in order to successfully stop all cVDPV2 transmission globally, countries and the Global Polio Eradication Initiative need to address the deficiencies in emergency outbreak response policy and implementation. The polio program must urgently act to substantially reduce response time, target larger populations - particularly in high transmission areas - and achieve high coverage with improved access to under-vaccinated subpopulations. Given the limited supplies of nOPV2 at the present, using mOPV2 intensively immediately, followed by nOPV2 intensively if needed and when sufficient quantities become available, substantially increases the probability of ending cVDPV2 transmission globally.

Aims This study compared the bag‐mediated filtration system (BMFS) and standard WHO two‐phase separation methods for poliovirus (PV) environmental surveillance, examined factors impacting PV detection and monitored Sabin‐like (SL) PV type 2 presence with withdrawal of oral polio vaccine type 2 (OPV2) in April 2016. Methods and Results Environmental samples were collected in Nairobi, Kenya (Sept 2015–Feb 2017), concentrated via BMFS and two‐phase separation methods, then assayed using the WHO PV isolation algorithm and intratypic differentiation diagnostic screening kit. SL1, SL2 and SL3 were detected at higher rates in BMFS than two‐phase samples (P 0·05), while SL3 was detected less frequently with higher shipment temperatures (P = 0·027). SL2 was detected more frequently before OPV2 withdrawal in BMFS and two‐phase samples (P

Since the Global Polio Eradication Initiative (GPEI) was established in 1988, two of the three wild poliovirus (WPV) serotypes (types 2 and 3) have been eradicated.* Transmission of WPV type 1 (WPV1) remains uninterrupted only in Afghanistan and Pakistan. This report summarizes progress toward global polio eradication during January 1, 2018-March 31, 2020 and updates previous reports (1,2). In 2019, Afghanistan and Pakistan reported the highest number of WPV1 cases (176) since 2014. During January 1-March 31, 2020 (as of June 19), 54 WPV1 cases were reported, an approximate fourfold increase from 12 cases during the corresponding period in 2019. Paralytic poliomyelitis can also be caused by circulating vaccine-derived poliovirus (cVDPV), which emerges when attenuated oral poliovirus vaccine (OPV) virus reverts to neurovirulence following prolonged circulation in underimmunized populations (3). Since the global withdrawal of type 2-containing OPV (OPV2) in April 2016, cVDPV type 2 (cVDPV2) outbreaks have increased in number and geographic extent (4). During January 2018-March 2020, 21 countries reported 547 cVDPV2 cases. Complicating increased poliovirus transmission during 2020, the coronavirus disease 2019 (COVID-19) pandemic and mitigation efforts have resulted in suspension of immunization activities and disruptions to poliovirus surveillance. When the COVID-19 emergency subsides, enhanced support will be needed to resume polio eradication field activities.

Certification of global eradication of indigenous wild poliovirus type 2 occurred in 2015 and of type 3 in 2019. Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988 and broad use of live, attenuated oral poliovirus vaccine (OPV), the number of wild poliovirus cases has declined >99.99% (1). Genetically divergent vaccine-derived poliovirus* (VDPV) strains can emerge during vaccine use and spread in underimmunized populations, becoming circulating VDPV (cVDPV) strains, and resulting in outbreaks of paralytic poliomyelitis.† In April 2016, all oral polio vaccination switched from trivalent OPV (tOPV; containing vaccine virus types 1, 2, and 3) to bivalent OPV (bOPV; containing types 1 and 3) (2). Monovalent type 2 OPV (mOPV2) is used in response campaigns to control type 2 cVDPV (cVDPV2) outbreaks. This report presents data on cVDPV outbreaks detected during January 2018-June 2019 (as of September 30, 2019). Compared with January 2017-June 2018 (3), the number of reported cVDPV outbreaks more than tripled, from nine to 29; 25 (86%) of the outbreaks were caused by cVDPV2. The increase in the number of outbreaks in 2019 resulted from VDPV2 both inside and outside of mOPV2 response areas. GPEI is planning future use of a novel type 2 OPV, stabilized to decrease the likelihood of reversion to neurovirulence. However, all countries must maintain high population immunity to decrease the risk for cVDPV emergence. Cessation of all OPV use after certification of polio eradication will eliminate the risk for VDPV emergence.

Since the 1988 launch of global poliomyelitis eradication efforts, four of the six World Health Organization (WHO) regions have been certified polio-free. Nigeria is one of only three countries, along with Afghanistan and Pakistan, where transmission of wild poliovirus (WPV) has never been interrupted. During 2003-2013, northern Nigeria served as a reservoir for WPV reintroduction into 26 previously polio-free countries. In 2012, the Nigerian government launched a national polio eradication emergency plan to intensify efforts to interrupt WPV transmission. This report describes polio eradication activities and progress in Nigeria during January 2014-July 2015 and updates previous reports. No WPV cases have been reported to date in 2015, compared with a total of six cases reported during 2014. Onset of paralysis in the latest reported WPV type 1 (WPV1) case was July 24, 2014. Only one case of circulating vaccine-derived poliovirus type 2 (cVDPV2) has been reported to date in 2015, compared with 20 cVDPV2 cases during the same period in 2014. Pending final laboratory testing of 218 remaining specimens of 16,617 specimens collected since January 2015, Nigeria could be removed from the WHO list of polio-endemic countries in September 2015. Major remaining challenges to the national polio eradication program include sustaining political support and program funding in the absence of active WPV transmission, maintaining high levels of population immunity in hard-to-reach areas, and accessing children in security-compromised areas of the northeastern states.

Background Use of oral live-attenuated polio vaccines (OPV), and injected inactivated polio vaccines (IPV) has almost achieved global eradication of wild polio viruses. To address the goals of achieving and maintaining global eradication and minimising the risk of outbreaks of vaccine-derived polioviruses, we tested novel monovalent oral type-2 poliovirus (OPV2) vaccine candidates that are genetically more stable than existing OPVs, with a lower risk of reversion to neurovirulence. Our study represents the first in-human testing of these two novel OPV2 candidates. We aimed to evaluate the safety and immunogenicity of these vaccines, the presence and extent of faecal shedding, and the neurovirulence of shed virus. Methods In this double-blind, single-centre phase 1 trial, we isolated participants in a purpose-built containment facility at the University of Antwerp Hospital (Antwerp, Belgium), to minimise the risk of environmental release of the novel OPV2 candidates. Participants, who were recruited by local advertising, were adults (aged 18-50 years) in good health who had previously been vaccinated with IPV, and who would not have any contact with immunosuppressed or unvaccinated people for the duration of faecal shedding at the end of the study. The first participant randomly chose an envelope containing the name of a vaccine candidate, and this determined their allocation; the next 14 participants to be enrolled in the study were sequentially allocated to this group and received the same vaccine. The subsequent 15 participants enrolled after this group were allocated to receive the other vaccine. Participants and the study staff were masked to vaccine groups until the end of the study period. Participants each received a single dose of one vaccine candidate (candidate 1, S2/cre5/S15domV/rec1/hifi3; or candidate 2, S2/S15domV/CpG40), and they were monitored for adverse events, immune responses, and faecal shedding of the vaccine virus for 28 days. Shed virus isolates were tested for the genetic stability of attenuation. The primary outcomes were the incidence and type of serious and severe adverse events, the proportion of participants showing viral shedding in their stools, the time to cessation of viral shedding, the cell culture infective dose of shed virus in virus-positive stools, and a combined index of the prevalence, duration, and quantity of viral shedding in all participants. This study is registered with EudraCT, number 2017-000908-21 and ClinicalTrials. gov, number NCT03430349. Findings Between May 22 and Aug 22, 2017, 48 volunteers were screened, of whom 15 (31%) volunteers were excluded for reasons relating to the inclusion or exclusion criteria, three (6%) volunteers were not treated because of restrictions to the number of participants in each group, and 30 (63%) volunteers were sequentially allocated to groups (15 participants per group). Both novel OPV2 candidates were immunogenic and increased the median blood titre of serum neutralising antibodies; all participants were seroprotected after vaccination. Both candidates had acceptable tolerability, and no serious adverse events occurred during the study. However, severe events were reported in six (40%) participants receiving candidate 1 (eight events) and nine (60%) participants receiving candidate 2 (12 events); most of these events were increased blood creatinine phosphokinase but were not accompanied by clinical signs or symptoms. Vaccine virus was detected in the stools of 15 (100%) participants receiving vaccine candidate 1 and 13 (87%) participants receiving vaccine candidate 2. Vaccine poliovirus shedding stopped at a median of 23 days (IQR 15-36) after candidate 1 administration and 12 days (1-23) after candidate 2 administration. Total shedding, described by the estimated median shedding index (50% cell culture infective dose/g), was observed to be greater with candidate 1 than candidate 2 across all participants (2.8 [95% CI 1.8-3.5] vs 1.0 [0.7-1.6]). Reversion to neurovirulence, assessed as paralysis of transgenic mice, was low in isolates from those vaccinated with both candidates, and sequencing of shed virus indicated that there was no loss of attenuation in domain V of the 5 '-untranslated region, the primary site of reversion in Sabin OPV. Interpretation We found that the novel OPV2 candidates were safe and immunogenic in IPV-immunised adults, and our data support the further development of these vaccines to potentially be used for maintaining global eradication of neurovirulent type-2 polioviruses. Copyright (c) 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.

Widespread use of live attenuated (Sabin) oral poliovirus vaccine (OPV) has resulted in marked progress toward global poliomyelitis eradication (1). However, in underimmunized populations, extensive person-to-person transmission of Sabin poliovirus can result in genetic reversion to neurovirulence and paralytic vaccine-derived poliovirus (VDPV) disease (1). This report updates (as of February 26, 2019) previous reports on circulating VDPV type 2 (cVDPV2) outbreaks during 2017-2018 in the Democratic Republic of the Congo (DRC) and in Somalia, which experienced a concurrent cVDPV type 3 (cVDPV3) outbreak* (2,3). In DRC, 42 cases have been reported in four cVDPV2 outbreaks; paralysis onset in the most recent case was October 7, 2018 (2). Challenges to interrupting transmission have included delays in outbreak-response supplementary immunization activities (SIAs) and difficulty reaching children in all areas. In Somalia, cVDPV2 and cVDPV3 were detected in sewage before the detection of paralytic cases (3). Twelve type 2 and type 3 cVDPV cases have been confirmed; the most recent paralysis onset dates were September 2 (cVDPV2) and September 7, 2018 (cVDPV3). The primary challenge to interrupting transmission is the residence of >300,000 children in areas that are inaccessible for vaccination activities. For both countries, longer periods of surveillance are needed before interruption of cVDPV transmission can be inferred.

Polioviruses are positive-sense, single-stranded RNA picornaviruses and the principal cause of poliomyelitis. Global poliovirus surveillance has relied on poliovirus isolation in cells, which may take a minimum of 10 days, involves maintaining two cell lines, and propagates virus in high titers. With eradication underway, a major objective of the Global Polio Eradication Initiative (GPEI) is to develop culture-independent detection of polioviruses as an alternative method to complement the current virus isolation technique. A culture-independent method on poliovirus-positive stool suspensions was assessed with commercially available recombinant soluble poliovirus receptor (PVR) coupled to Histidine (His) tags. Viral RNA was screened by quantitative real-time reverse transcription PCR using the poliovirus intratypic differentiation kit. Poliovirus recovery was optimized with PVR-His–tagged protein and buffers supplemented with polyethylene glycol. To validate the poliovirus-PVR–His tag purification assay, 182 poliovirus-positive stools of programmatic importance were parallel tested against the GPLN-accepted virus isolation method. The PVR-His tag enrichment method detected poliovirus in 164 of 171 poliovirus-positive stools, whereas the virus isolation method misidentified 38 stools as poliovirus-negative (McNemar χ2 p

Delays in achieving the global eradication of wild poliovirus transmission continue to postpone subsequent cessation of all oral poliovirus vaccine (OPV) use. Countries must stop OPV use to end all cases of poliomyelitis, including vaccine-associated paralytic polio (VAPP) and cases caused by vaccine-derived polioviruses (VDPVs). The Global Polio Eradication Initiative (GPEI) coordinated global cessation of all type 2 OPV (OPV2) use in routine immunization in 2016 but did not successfully end the transmission of type 2 VDPVs (VDPV2s), and consequently continues to use type 2 OPV (OPV2) for outbreak response activities. Using an updated global poliovirus transmission and OPV evolution model, we characterize outbreak response options for 2019-2029 related to responding to VDPV2 outbreaks with a genetically stabilized novel OPV (nOPV2) strain or with the currently licensed monovalent OPV2 (mOPV2). Given uncertainties about the properties of nOPV2, we model different assumptions that appear consistent with the evidence on nOPV2 to date. Using nOPV2 to respond to detected cases may reduce the expected VDPV and VAPP cases and the risk of needing to restart OPV2 use in routine immunization compared to mOPV2 use for outbreak response. The actual properties, availability, and use of nOPV2 will determine its effects on type 2 poliovirus transmission in populations. Even with optimal nOPV2 performance, countries and the GPEI would still likely need to restart OPV2 use in routine immunization in OPV-using countries if operational improvements in outbreak response to stop the transmission of cVDPV2s are not implemented effectively.

Circulating vaccine-derived polioviruses (cVDPVs) can emerge in areas with low poliovirus immunity and cause outbreaks* of paralytic polio (1-5). Among the three types of wild poliovirus, type 2 was declared eradicated in 2015 (1,2). The use of trivalent oral poliovirus vaccine (tOPV; types 1, 2, and 3 Sabin strains) ceased in April 2016 via a 1-month-long, global synchronized switch to bivalent OPV (bOPV; types 1 and 3 Sabin strains) in immunization activities (1-4). Monovalent type 2 OPV (mOPV2; type 2 Sabin strain) is available for cVDPV type 2 (cVDPV2) outbreak response immunization (1-5). The number and geographic breadth of post-switch cVDPV2 outbreaks have exceeded forecasts that trended toward zero outbreaks 4 years after the switch and assumed rapid and effective control of any that occurred (4). New cVDPV2 outbreaks have been seeded by mOPV2 use, by both suboptimal mOPV2 coverage within response zones and recently mOPV2-vaccinated children or contacts traveling outside of response zones, where children born after the global switch are fully susceptible to poliovirus type 2 transmission (2-4). In addition, new emergences can develop by inadvertent exposure to Sabin OPV2-containing vaccine (i.e., residual response mOPV2 or tOPV) (4). This report updates the January 2018-June 2019 report with information on global cVDPV outbreaks during July 2019-February 2020 (as of March 25, 2020)† (2). Among 33 cVDPV outbreaks reported during July 2019-February 2020, 31 (94%) were cVDPV2; 18 (58%) of these followed new emergences. In mid-2020, the Global Polio Eradication Initiative (GPEI) plans to introduce a genetically stabilized, novel OPV type 2 (nOPV2) that has a lower risk for generating VDPV2 than does Sabin mOPV2; if nOPV2 is successful in limiting new VDPV2 emergences, GPEI foresees the replacement of Sabin mOPV2 with nOPV2 for cVDPV2 outbreak responses during 2021 (2,4,6).

Objective Primary immunodeficiency (PID) patients are prone to developing viral infections and should not be vaccinated with live vaccines. In such patients, prolonged excretion and viral divergence may occur and they may subsequently act as reservoirs in the community introducing mutated virus and jeopardizing polio eradication. One hundred and thirty PID cases were included for poliovirus detection in stool with assessment of divergence of detected polioviruses from oral polio vaccine (OPV) virus. Clinical presentations of PID patients with detectable poliovirus in stool specimens are described. Results Six PID patients (4.5%) had detectable vaccine-derived poliovirus (VDPV) excretion in stool specimens; of these, five patients had severe combined immunodeficiency (two with acute flaccid paralysis, one with meningoencephalitis and two without neurological manifestations), and one patient had X-linked agammaglobulinemia (paralysis developed shortly after diagnosis of immunodeficiency). All six case-patients received trivalent OPV. Five case-patients had type 2 immunodeficiency-related vaccine-derived polioviruses (iVDPV2) excretion; one had concomitant excretion of Sabin like type 3 virus and one was identified as iVDPV1 excretor. Surveillance for poliovirus excretion among PID patients is critical as these patients represent a potential source to reseed polioviruses into populations.

Since the 1988 inception of the Global Polio Eradication Initiative (GPEI), progress toward interruption of wild poliovirus (WPV) transmission has occurred mostly through extensive use of oral poliovirus vaccine (OPV) in mass vaccination campaigns and through routine immunization services (1,2). However, because OPV contains live, attenuated virus, it carries the rare risk for reversion to neurovirulence. In areas with very low OPV coverage, prolonged transmission of vaccine-associated viruses can lead to the emergence of vaccine-derived polioviruses (VDPVs), which can cause outbreaks of paralytic poliomyelitis. Although WPV type 2 has not been detected since 1999, and was declared eradicated in 2015,* most VDPV outbreaks have been attributable to VDPV serotype 2 (VDPV2) (3,4). After the synchronized global switch from trivalent OPV (tOPV) (containing vaccine virus types 1, 2, and 3) to bivalent OPV (bOPV) (types 1 and 3) in April 2016 (5), GPEI regards any VDPV2 emergence as a public health emergency (6,7). During May-June 2017, VDPV2 was isolated from stool specimens from two children with acute flaccid paralysis (AFP) in Deir-ez-Zor governorate, Syria. The first isolate differed from Sabin vaccine virus by 22 nucleotides in the VP1 coding region (903 nucleotides). Genetic sequence analysis linked the two cases, confirming an outbreak of circulating VDPV2 (cVDPV2). Poliovirus surveillance activities were intensified, and three rounds of vaccination campaigns, aimed at children aged

In 1988, when an estimated 350,000 cases of poliomyelitis occurred in 125 countries, the World Health Assembly resolved to eradicate polio globally. Transmission of wild poliovirus (WPV) continues uninterrupted in only three countries (Afghanistan, Nigeria, and Pakistan) (1), and among the three serotypes, WPV type 1 (WPV1) remains the only confirmed circulating type. This report describes global progress toward polio eradication during January 2016-March 2018, and updates previous reports (2). In 2017, 22 WPV1 cases were reported, a 41% decrease from the 37 WPV1 cases reported in 2016. As of April 24, 2018, eight WPV1 cases have been reported (seven in Afghanistan and one in Pakistan), compared with five cases during the same period in 2017. In Pakistan, continuing WPV1 transmission has been confirmed in multiple areas in 2018 by isolation from wastewater samples. In Nigeria, ongoing endemic WPV1 transmission was confirmed in 2016 (3); although WPV was not detected in 2017 or in 2018 to date, limitations in access for vaccination and surveillance in insurgent-held areas in northeastern Nigeria might permit continued undetected poliovirus transmission. Substantial progress toward polio eradication has continued in recent years; however, interruption of WPV transmission will require overcoming remaining challenges to reaching and vaccinating every missed child. Until poliovirus eradication is achieved, all countries must remain vigilant by maintaining high population immunity and sensitive poliovirus surveillance.

The last confirmed wild poliovirus (WPV) case in Democratic Republic of the Congo (DRC) had paralysis onset in December 2011 (1). DRC has had cases of vaccine-derived polioviruses (VDPVs) documented since 2004 (Table 1) (1-6). After an outbreak of 30 circulating VDPV type 2 (cVDPV2) cases during 2011-2012, only five VDPV2 cases were reported during 2013-2016 (Table 1) (1-6). VDPVs can emerge from oral poliovirus vaccine (OPV types 1, 2, or 3; Sabin) polioviruses that have genetically mutated resulting in reversion to neurovirulence. This process occurs during extensive person-to-person transmission in populations with low immunity or after extended replication in the intestines of immune-deficient persons following vaccination (1-6). During 2017 (as of March 8, 2018), 25 VDPV cases were reported in three provinces in DRC: in Tanganyika province, an emergence with one VDPV2 case (pending final classification) in Kabalo health zone and an emergence with one ambiguous VDPV type 1 (aVDPV1) case in Ankoro health zone; in Maniema province, an emergence with two cVDPV2 cases; and in Haut Lomami province, an emergence with 20 cVDPV2 cases that originated in Haut Lomami province and later spread to Tanganyika province (hereafter referred to as the Haut Lomami outbreak area) and an emergence with one aVDPV type 2 (aVDPV2) case in Lwamba health zone (Table 1) (Figure) (6). Outbreak response supplementary immunization activities (SIAs) were conducted during June-December 2017 (Table 2) (6). Because of limitations in surveillance and suboptimal SIA quality and geographic scope, cVDPV2 circulation is likely continuing in 2018, requiring additional SIAs. DRC health officials and Global Polio Eradication Initiative (GPEI) partners are increasing human and financial resources to improve all aspects of outbreak response.

Nearly three decades after the World Health Assembly launched the Global Polio Eradication Initiative in 1988, four of the six World Health Organization (WHO) regions have been certified polio-free (1). Nigeria is one of three countries, including Pakistan and Afghanistan, where wild poliovirus (WPV) transmission has never been interrupted. In September 2015, after >1 year without any reported WPV cases, Nigeria was removed from WHO's list of countries with endemic WPV transmission (2); however, during August and September 2016, four type 1 WPV (WPV1) cases were reported from Borno State, a state in northeastern Nigeria experiencing a violent insurgency (3). The Nigerian government, in collaboration with partners, launched a large-scale coordinated response to the outbreak (3). This report describes progress in polio eradication activities in Nigeria during January-December 2017 and updates previous reports (3-5). No WPV cases have been reported in Nigeria since September 2016; the latest case had onset of paralysis on August 21, 2016 (3). However, polio surveillance has not been feasible in insurgent-controlled areas of Borno State. Implementation of new strategies has helped mitigate the challenges of reaching and vaccinating children living in security-compromised areas, and other strategies are planned. Despite these initiatives, however, approximately 130,000-210,000 (28%-45%) of the estimated 469,000 eligible children living in inaccessible areas in 2016 have not been vaccinated. Sustained efforts to optimize surveillance and improve immunization coverage, especially among children in inaccessible areas, are needed.

In 1988, the World Health Assembly launched the Global Polio Eradication Initiative (GPEI) (1). Among the three wild poliovirus (WPV) serotypes, only type 1 (WPV1) has been detected since 2012. Since 2014, detection of WPV1 has been limited to three countries, with 37 cases in 2016 and 11 cases in 2017 as of September 27. The99.99% decline worldwide in polio cases since the launch of the GPEI is attributable to the extensive use of the live, attenuated oral poliovirus vaccine (OPV) in mass vaccination campaigns and comprehensive national routine immunization programs. Despite its well-established safety record, OPV use can be associated with rare emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (2). VDPVs can also emerge among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) can emerge very rarely among immunologically normal vaccine recipients and their contacts in areas with inadequate OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (3). During January 2016-June 2017, new cVDPV outbreaks were identified, including two in the Democratic Republic of the Congo (DRC) (eight cases), and another in Syria (35 cases), whereas the circulation of cVDPV type 2 (cVDPV2) in Nigeria resulted in cVDPV2 detection linked to a previous emergence. The last confirmed case from the 2015-2016 cVDPV type 1 (cVDPV1) outbreak in Laos occurred in January 2016. Fourteen newly identified persons in 10 countries were found to excrete iVDPVs, and three previously reported patients in the United Kingdom and Iran (3) were still excreting type 2 iVDPV (iVDPV2) during the reporting period. Ambiguous VDPVs (aVDPVs), isolates that cannot be classified definitively, were found among immunocompetent persons and environmental samples in 10 countries. Cessation of all OPV use after certification of polio eradication will eliminate the risk for new VDPV infections.

Vaccine-Derived Polioviruses (VDPVs) may emerge during person-to-person transmission (circulating VDPV [cVDPV]) or during prolonged replication in individuals with primary immunodeficiency (iVDPV). To prevent the emergence of type 2 vaccine-derived polioviruses (VDPV2s) which represents >90% of the VDPV during the past two years, WHO implemented a worldwide synchronized switch from trivalent oral poliovirus vaccine (tOPV; types 1, 2, and 3) to bivalent OPV (bOPV; types 1 and 3) in April 2016. When planning the scope of a post-switch outbreak response, it is important to know if the recently identified VDPV is likely to be cVDPV or iVDPV, as the public health responses dramatically differ in the two events: outbreak requires a community immunization response while chronic infection requires careful patient monitoring and potential individual treatment. Here we demonstrate that genetic distinctions between cVDPV and iVDPV sequences can be calculated (to some extent) to inform global poliovirus outbreak response, particularly for VDPV2. We performed a survey of cVDPV and iVDPV nucleotide sequences of the VP1 capsid region (∼900 nucleotides) from GenBank. A non-redundant dataset contains 33 cVDPV2 isolates and 33 iVDPV2 isolates was built. We compared each sequence to its Sabin 2 parental strain to determine the number of genetic nucleotide (NT) and amino acid (AA) substitutions. cVDPV2 and iVDPV2 sequences show similar profiles for NT and AA frequencies, codon usage, and AA conservation. Simple counts of NT, AA, and codon substitutions in comparative analyses between cVDPV2 and iVDPV2 were insufficient for a clear distinction between the two VDPV2 categories. However, the combined measures of NT and AA substitutions (from VP1 and neutralizing antigenic site sequences) were more informative, with more non-synonymous (AA changes) in iVDPV2 than cVDPV2. This work highlights that the genetic variations between iVDPVs and cVDPVs may be reflect the differences of the viral micro-environments, interactions between virus and host, and the selective pressures during person-to-person transmission compared with chronic infections in immune-deficient patients. The dataset was small but the issue is certainly deserving of attentions and suggesting the needs for further data collections from poliovirus surveillance. This is the first attempt using computational techniques to quantify the genetic difference between cVDPVs and iVDPVs.

Poliovirus (PV) environmental surveillance was established in Haiti in three sites each in Port-au-Prince and Gonaives, where sewage and fecal-influenced environmental open water channel samples were collected monthly from March 2016 to February 2017. The primary objective was to monitor for the emergence of vaccine-derived polioviruses (VDPVs) and the importation and transmission of wild polioviruses (WPVs). A secondary objective was to compare two environmental sample processing methods, the gold standard two-phase separation method and a filter method (bag-mediated filtration system [BMFS]). In addition, non-polio enteroviruses (NPEVs) were characterized by next-generation sequencing using Illumina MiSeq to provide insight on surrogates for PVs. No WPVs or VDPVs were detected at any site with either concentration method. Sabin (vaccine) strain PV type 2 and Sabin strain PV type 1 were found in Port-au-Prince, in March and April samples, respectively. Non-polio enteroviruses were isolated in 75-100% and 0-58% of samples, by either processing method during the reporting period in Port-au-Prince and Gonaives, respectively. Further analysis of 24 paired Port-au-Prince samples confirmed the detection of a human NPEV and echovirus types E-3, E-6, E-7, E-11, E-19, E-20, and E-29. The comparison of the BMFS filtration method to the two-phase separation method found no significant difference in sensitivity between the two methods (mid-P-value = 0.55). The experience of one calendar year of sampling has informed the appropriateness of the initially chosen sampling sites, importance of an adequate PV surrogate, and robustness of two processing methods.

The bag-mediated filtration system (BMFS) was developed to facilitate poliovirus (PV) environmental surveillance, a supplement to acute flaccid paralysis surveillance in PV eradication efforts. From April to September 2015, environmental samples were collected from four sites in Nairobi, Kenya, and processed using two collection/concentration methodologies: BMFS (> 3 L filtered) and grab sample (1 L collected; 0.5 L concentrated) with two-phase separation. BMFS and two-phase samples were analyzed for PV by the standard World Health Organization poliovirus isolation algorithm followed by intratypic differentiation. BMFS samples were also analyzed by a cell culture independent real-time reverse transcription polymerase chain reaction (rRT-PCR) and an alternative cell culture method (integrated cell culture-rRT-PCR with PLC/PRF/5, L20B, and BGM cell lines). Sabin polioviruses were detected in a majority of samples using BMFS (37/42) and two-phase separation (32/42). There was statistically more frequent detection of Sabin-like PV type 3 in samples concentrated with BMFS (22/42) than by two-phase separation (14/42,p = 0.035), possibly due to greater effective volume assayed (870 mL vs. 150 mL). Despite this effective volume assayed, there was no statistical difference in Sabin-like PV type 1 and Sabin-like PV type 2 detection between these methods (9/42 vs. 8/42,p = 0.80 and 27/42 vs. 32/42,p = 0.18, respectively). This study demonstrated that BMFS can be used for PV environmental surveillance and established a feasible study design for future research.

Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. The Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms were evaluated using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 510 chip runs produced more reads at a lower cost per sample than the highest output Ion Torrent PGM 318 chip run, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 and Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 and Illumina MiSeq V2) there is less of a difference in the cost per sample between the two sequencing platforms ($5.47-$10.25 more per sample for an Ion Torrent 530 chip run when multiplexing 24 samples). These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

Since the Global Polio Eradication Initiative (GPEI) began in 1988, transmission of wild poliovirus (WPV) has been interrupted in all countries except Afghanistan, Nigeria, and Pakistan. WPV type 2 (WPV2) was declared eradicated in 2015; WPV type 3 has not been detected since 2012 (1). After the certification of the eradication of WPV2, a global switch from trivalent oral poliovirus vaccine (tOPV, containing vaccine virus types 1, 2, and 3) to bivalent oral poliovirus vaccine (bOPV, containing types 1 and 3) was completed in April 2016. Nigeria last reported WPV type 1 (WPV1) cases in 2016. This report describes global progress toward poliomyelitis eradication during January 1, 2017-March 31, 2019, and updates previous reports (1,2). Afghanistan and Pakistan reported their lowest annual number of WPV cases (22) in 2017; however, 33 WPV1 cases were reported in 2018. During January-March 2019 (as of May 3), 12 WPV1 cases had been reported worldwide, four more than the eight reported during the corresponding period in 2018. The occurrence of polio cases caused by circulating vaccine-derived poliovirus (cVDPV) is rare and occurs where oral poliovirus vaccine (OPV) coverage has been low and vaccine virus reverts to neurovirulence (3). Eight countries (Democratic Republic of the Congo [DRC], Indonesia, Mozambique, Niger, Nigeria, Papua New Guinea, Somalia, and Syria) reported 210 cVDPV cases during 2017-2019 (as of May 3). Reaching children during supplemental immunization activities (SIAs), accessing mobile populations at high risk, and variations in surveillance performance represent ongoing challenges. Innovative efforts to vaccinate every child and strengthen coordination efforts between Afghanistan and Pakistan will help achieve eradication. For cVDPV outbreak responses to promptly stop transmission, intensified programmatic improvements are needed to make the responses more effective and limit the risk for generating future outbreaks.

Since the World Health Assembly's 1988 resolution to eradicate poliomyelitis, one of the main tools of the World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) has been the live, attenuated oral poliovirus vaccine (OPV). OPV might require several doses to induce immunity but provides long-term protection against paralytic disease. Through effective use of OPV, GPEI has brought polio to the threshold of eradication. Wild poliovirus type 2 (WPV2) was eliminated in 1999, WPV3 has not been detected since November 2012, and WPV1 circulation appears to be restricted to parts of Pakistan and Afghanistan. However, continued use of OPV carries two key risks. The first, vaccine-associated paralytic poliomyelitis (VAPP) has been recognized since the early 1960s. VAPP is a very rare event that occurs sporadically when an administered dose of OPV reverts to neurovirulence and causes paralysis in the vaccine recipient or a nonimmune contact. VAPP can occur among immunologically normal vaccine recipients and their contacts as well as among persons who have primary immunodeficiencies (PIDs) manifested by defects in antibody production; it is not associated with outbreaks. The second, the emergence of genetically divergent, neurovirulent vaccine-derived polioviruses (VDPVs) was recognized more recently. Circulating VDPVs (cVDPVs) resemble WPVs and, in areas with low OPV coverage, can cause polio outbreaks. Immunodeficiency-associated VDPVs (iVDPVs) can replicate and be excreted for years in some persons with PIDs; GPEI maintains a registry of iVDPV cases. Ambiguous VDPVs (aVDPVs) are isolates that cannot be classified definitively. This report updates previous surveillance summaries and describes VDPVs detected worldwide during January 2014-March 2015. Those include new cVDPV outbreaks in Madagascar and South Sudan, and sharply reduced type 2 cVDPV (cVDPV2) circulation in Nigeria and Pakistan during the latter half of 2014. Eight newly identified persons in six countries were found to excrete iVDPVs, and a patient in the United Kingdom was still excreting iVDPV2 in 2014 after more than 28 years. Ambiguous VDPVs were found among immunocompetent persons and environmental samples in 16 countries. Because the large majority of VDPV case-isolates are type 2, WHO has developed a plan for coordinated worldwide withdrawal of trivalent (types 1, 2, and 3) OPV (tOPV) and replacement with bivalent (types 1 and 3) OPV (bOPV) in April 2016, preceded by introduction of at least 1 dose of injectable inactivated poliovirus vaccine (IPV) into routine immunization schedules worldwide to maintain immunity to type 2 viruses.

Background Determining patterns of nucleotide and amino acid substitution is the first step during sequence evolution analysis. However, it is not easy to visualize the different phylogenetic signatures imprinted in aligned nucleotide and amino acid sequences. Results Here we present PoSE (Pattern of Sequence Evolution), a reliable resource for unveiling the evolutionary history of sequence alignments and for graphically displaying their contents. Substitutions are displayed by category (transitions and transversions), codon position, and phenotypic effect (synonymous and nonsynonymous). Visualization is accomplished using MATLAB scripts wrapped around PAML (Phylogenetic Analysis by Maximum Likelihood), implemented in an easy-to-use graphical user interface. The application displays inferred substitutions estimated by baseml or codeml, two programs included in the PAML software package. PoSE organizes patterns of substitution in eleven plots, including estimated non-synonymous/synonymous ratios (dN/dS) along the sequence alignment. In addition, PoSE provides visualization and annotation of patterns of amino acid substitutions along groups of related sequences that can be graphically inspected in a phylogenetic tree window. Conclusions PoSE is a useful tool to help determine major patterns during sequence evolution of protein-coding sequences, hypervariable regions, or changes in dN/dS ratios. PoSE is publicly available at https://github.com/CDCgov/PoSE

Stabilizing paper matrix methods for retaining nucleic acid from inactivated clinical specimens offer a solution for molecular diagnostics when specimens may be stored or shipped at ambient temperature. We developed cellulose disks (UNEXP) saturated with a total nucleic acid extraction buffer (UNEX) modified from a previously developed lysis buffer for multiple enteric pathogens. Infectivity of hepatitis A virus, adenovirus and poliovirus was destroyed after 2–3 h incubation at room temperature on the UNEXP disks. Norovirus RNA could be detected in UNEXP-eluted nucleic acids by reverse transcription-quantitative PCR (RT-qPCR) from 54 stool samples after 2 weeks storage at room temperature on disks; a subset of seven samples were positive after 3 months storage. Genotyping was successful in 76% of 54 samples tested including six of seven samples stored on the UNEXP disks for up to one month. Comparison of UNEXP with the FTA elute card in a subset of 10 samples demonstrated similar detection and genotyping rates after two weeks of storage at room temperature. UNEXP disks could be useful for epidemiologic investigations of disease outbreaks in resource-limited areas by simplifying specimen transport to regional diagnostic laboratories or shipment to international centers without the need to ship samples on dry ice.

We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. Four different functional domains were analyzed: (i) neutralizing antigenic (NAg) sites, (ii) residues binding the poliovirus receptor (PVR), (iii) VP1 residues 1 to 32, and (iv) the capsid structural core. Amino acid replacements mapped to 37 of 43 positions across all 4 NAg sites; the most variable and polymorphic residues were in NAg sites 2 and 3b. The most divergent of the 120 NAg variants had no more than 5 replacements in all NAg sites and were still neutralized at titers similar to those of Sabin 2. PVR-binding residues were less variable (25 different variants; 0 to 2 replacements per isolate; 30/44 invariant positions), with the most variable residues also forming parts of NAg sites 2 and 3a. Residues 1 to 32 of VP1 were highly variable (133 different variants; 0 to 6 replacements per isolate; 5/32 invariant positions), with residues 1 to 18 predicted to form a well-conserved amphipathic helix. Replacement events were dated by mapping them onto the branches of time-scaled phylogenies. Rates of amino acid replacement varied widely across positions and followed no simple substitution model. Replacements in the structural core were the most conservative and were fixed at an overall rate ∼20-fold lower than the rates for the NAg sites and VP1 1 to 32 and ∼5-fold lower than the rate for the PVR-binding sites. Only VP1 143-Ile, a non-NAg site surface residue and known attenuation site, appeared to be under strong negative selection. IMPORTANCE The high rate of poliovirus evolution is offset by strong selection against amino acid replacement at most positions of the capsid. Consequently, poliovirus vaccines developed from strains isolated decades ago have been used worldwide to bring wild polioviruses almost to extinction. The apparent antigenic stability of poliovirus obscures a dynamic of continuous change within the neutralizing antigenic (NAg) sites. During 7 years of a large outbreak in Nigeria, the circulating type 2 vaccine-derived polioviruses generated 120 different NAg site variants via multiple independent pathways. Nonetheless, overall antigenic evolution was constrained, as no isolate had fixed more than 5 amino acid differences from the Sabin 2 NAg sites, and the most divergent isolates were efficiently neutralized by human immune sera. Evolution elsewhere in the capsid was also constrained. Amino acids binding the poliovirus receptor were strongly conserved, and extensive variation in the VP1 amino terminus still conserved a predicted amphipathic helix.