Response to Vaccine-Derived Polioviruses Detected through Environmental Surveillance, Guatemala, 2019.

Guatemala implemented wastewater-based poliovirus surveillance in 2018, and three genetically unrelated vaccine-derived polioviruses (VDPVs) were detected in 2019. The Ministry of Health (MoH) response included event investigation through institutional and community retrospective case searches for acute flaccid paralysis (AFP) during 2018-2020 and a bivalent oral polio/measles, mumps, and rubella vaccination campaign in September 2019. This response was reviewed by an international expert team in July 2021. During the campaign, 93% of children 6 months <7 years of age received a polio-containing vaccine dose. No AFP cases were detected in the community search; institutional retrospective searches found 37% of unreported AFP cases in 2018‒2020. No additional VDPV was isolated from wastewater. No evidence of circulating VDPV was found; the 3 isolated VDPVs were classified as ambiguous VDPVs by the international team of experts. These detections highlight risk for poliomyelitis reemergence in countries with low polio vaccine coverage.

Monomeric YoeB toxin retains RNase activity but adopts an obligate dimeric form for thermal stability.

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress.

The structure and function of Mycobacterium tuberculosis MazF-mt6 toxin provide insights into conserved features of MazF endonucleases.

Toxin-antitoxin systems are ubiquitous in prokaryotic and archaeal genomes and regulate growth in response to stress. Escherichia coli contains at least 36 putative toxin-antitoxin gene pairs, and some pathogens such as Mycobacterium tuberculosis have over 90 toxin-antitoxin operons. E. coli MazF cleaves free mRNA after encountering stress, and nine M. tuberculosis MazF family members cleave mRNA, tRNA, or rRNA. Moreover, M. tuberculosis MazF-mt6 cleaves 23S rRNA Helix 70 to inhibit protein synthesis. The overall tertiary folds of these MazFs are predicted to be similar, and therefore, it is unclear how they recognize structurally distinct RNAs. Here we report the 2.7-Å X-ray crystal structure of MazF-mt6. MazF-mt6 adopts a PemK-like fold but lacks an elongated ß1-ß2 linker, a region that typically acts as a gate to direct RNA or antitoxin binding. In the absence of an elongated ß1-ß2 linker, MazF-mt6 is unable to transition between open and closed states, suggesting that the regulation of RNA or antitoxin selection may be distinct from other canonical MazFs. Additionally, a shortened ß1-ß2 linker allows for the formation of a deep, solvent-accessible, active-site pocket, which may allow recognition of specific, structured RNAs like Helix 70. Structure-based mutagenesis and bacterial growth assays demonstrate that MazF-mt6 residues Asp-10, Arg-13, and Thr-36 are critical for RNase activity and likely catalyze the proton-relay mechanism for RNA cleavage. These results provide further critical insights into how MazF secondary structural elements adapt to recognize diverse RNA substrates.

Importance of the E. coli DinJ antitoxin carboxy terminus for toxin suppression and regulated proteolysis.

Toxin-antitoxin genes play important roles in the regulation of bacterial growth during stress. One response to stress is selective proteolysis of antitoxin proteins which releases their cognate toxin partners causing rapid inhibition of growth. The features of toxin-antitoxin complexes that are important to inhibit toxin activity as well as to release the active toxin remain elusive. Furthermore, it is unclear how antitoxins are selected for proteolysis by cellular proteases. Here, we test the minimal structural requirements of the Escherichia coli DinJ antitoxin to suppress its toxin partner, YafQ. We find that DinJ-YafQ complex formation is critically dependent on the last ten C-terminal residues of DinJ. However, deletion of these 10 DinJ residues has little effect on transcriptional autorepression suggesting that the YafQ toxin is not a critical component of the repression complex in contrast to other toxin-antitoxin systems. We further demonstrate that loop 5 preceding these ten C-terminal residues is important for Lon-mediated proteolysis. These results provide important insights into the critical interactions between toxin-antitoxin pairs necessary to inhibit toxin activity and the regulated proteolysis of antitoxins.

mRNA bound to the 30S subunit is a HigB toxin substrate.

Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs.

Mechanism of endonuclease cleavage by the HigB toxin.

Bacteria encode multiple type II toxin-antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition.

Defining the mRNA recognition signature of a bacterial toxin protein.

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosome-dependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide.

Molecular basis of ribosome recognition and mRNA hydrolysis by the E. coli YafQ toxin.

Bacterial type II toxin-antitoxin modules are protein-protein complexes whose functions are finely tuned by rapidly changing environmental conditions. E. coli toxin YafQ is suppressed under steady state growth conditions by virtue of its interaction with its cognate antitoxin, DinJ. During stress, DinJ is proteolytically degraded and free YafQ halts translation by degrading ribosome-bound mRNA to slow growth until the stress has passed. Although structures of the ribosome with toxins RelE and YoeB have been solved, it is unclear what residues among ribosome-dependent toxins are essential for mediating both recognition of the ribosome and the mRNA substrate given their low sequence identities. Here we show that YafQ coordinates binding to the 70S ribosome via three surface-exposed patches of basic residues that we propose directly interact with 16S rRNA. We demonstrate that YafQ residues H50, H63, D67 and H87 participate in acid-base catalysis during mRNA hydrolysis and further show that H50 and H63 functionally complement as general bases to initiate the phosphodiester cleavage reaction. Moreover YafQ residue F91 likely plays an important role in mRNA positioning. In summary, our findings demonstrate the plasticity of ribosome-dependent toxin active site residues and further our understanding of which toxin residues are important for function.

Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops.

Maintenance of the correct reading frame on the ribosome is essential for accurate protein synthesis. Here, we report structures of the 70S ribosome bound to frameshift suppressor tRNA(SufA6) and N1-methylguanosine at position 37 (m(1)G37) modification-deficient anticodon stem loop(Pro), both of which cause the ribosome to decode 4 rather than 3 nucleotides, resulting in a +1 reading frame. Our results reveal that decoding at +1 suppressible codons causes suppressor tRNA(SufA6) to undergo a rearrangement of its 5' stem that destabilizes U32, thereby disrupting the conserved U32-A38 base pair. Unexpectedly, the removal of the m(1)G37 modification of tRNA(Pro) also disrupts U32-A38 pairing in a structurally analogous manner. The lack of U32-A38 pairing provides a structural correlation between the transition from canonical translation and a +1 reading of the mRNA. Our structures clarify the molecular mechanism behind suppressor tRNA-induced +1 frameshifting and advance our understanding of the role played by the ribosome in maintaining the correct translational reading frame.

Structural insights into translational recoding by frameshift suppressor tRNASufJ.

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5' or 3' direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA(SufJ), a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA(SufJ) contains an insertion 5' to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL(SufJ) or tRNA(SufJ) does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL(SufJ) and ASL(Thr) bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34-37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL(SufJ) imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA(SufJ) during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.

Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state.

After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 Å distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge--namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-Tu-GTP-aminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.

Structure of the Proteus vulgaris HigB-(HigA)2-HigB toxin-antitoxin complex.

Bacterial toxin-antitoxin (TA) systems regulate key cellular processes to promote cell survival during periods of stress. During steady-state cell growth, antitoxins typically interact with their cognate toxins to inhibit activity presumably by preventing substrate recognition. We solved two x-ray crystal structures of the Proteus vulgaris tetrameric HigB-(HigA)2-HigB TA complex and found that, unlike most other TA systems, the antitoxin HigA makes minimal interactions with toxin HigB. HigB adopts a RelE family tertiary fold containing a highly conserved concave surface where we predict its active site is located. HigA does not cover the solvent-exposed HigB active site, suggesting that, in general, toxin inhibition is not solely mediated by active site hindrance by its antitoxin. Each HigA monomer contains a helix-turn-helix motif that binds to its own DNA operator to repress transcription during normal cellular growth. This is distinct from antitoxins belonging to other superfamilies that typically only form DNA-binding motifs upon dimerization. We further show that disruption of the HigB-(HigA)2-HigB tetramer to a HigBA heterodimer ablates operator binding. Taken together, our biochemical and structural studies elucidate the novel molecular details of the HigBA TA system.

Mechanisms of toxin inhibition and transcriptional repression by Escherichia coli DinJ-YafQ.

Bacteria encounter environmental stresses that regulate a gene expression program required for adaptation and survival. Here, we report the 1.8-Å crystal structure of the Escherichia coli toxin-antitoxin complex YafQ-(DinJ)2-YafQ, a key component of the stress response. The antitoxin DinJ dimer adopts a ribbon-helix-helix motif required for transcriptional autorepression, and toxin YafQ contains a microbial RNase fold whose proposed active site is concealed by DinJ binding. Contrary to previous reports, our studies indicate that equivalent levels of transcriptional repression occur by direct interaction of either YafQ-(DinJ)2-YafQ or a DinJ dimer at a single inverted repeat of its recognition sequence that overlaps with the -10 promoter region. Surprisingly, multiple YafQ-(DinJ)2-YafQ complexes binding to the operator region do not appear to amplify the extent of repression. Our results suggest an alternative model for transcriptional autorepression that may be novel to DinJ-YafQ.

Validation of improved automated nucleic acid extraction methods for direct detection of polioviruses for global polio eradication.

Polioviruses (PV), the main causative agent of acute flaccid paralysis (AFP), are positive-sense single-stranded RNA viruses of the family Picornaviridae. As we approach polio eradication, accurate and timely detection of poliovirus in stool from AFP cases becomes vital to success for the eradication efforts. Direct detection of PV from clinical diagnostic samples using nucleic acid (NA) extraction and real-time reverse transcriptase polymerase chain reaction (rRT-PCR) instead of the current standard method of virus isolation in culture, eliminates the long turn-around time to diagnosis and the need for high viral titer amplification in laboratories. An essential component of direct detection of PV from AFP surveillance samples is the efficient extraction of NA. Potential supply chain issues and lack of vendor presence in certain areas of the world necessitates the validation of multiple NA extraction methods. Using retrospective PV-positive surveillance samples (n=104), two extraction kits were compared to the previously validated Zymo Research Quick-RNA™ Viral Kit. The Roche High Pure Viral RNA Kit, a column-based manual extraction method, and the MagMaX™ Pathogen RNA/DNA kit used in the automated Kingfisher Flex system were both non-inferior to the Zymo kit, with similar rates of PV detection in pivotal rRT-PCR assays, such as pan-poliovirus (PanPV), poliovirus serotype 2 (PV2), and wild poliovirus serotype 1 (WPV1). These important assays allow the identification and differentiation of PV genotypes and serotypes and are fundamental to the GPLN program. Validation of two additional kits provides feasible alternatives to the current piloted method of NA extraction for poliovirus rRT-PCR assays.

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Correction: Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.

[This corrects the article DOI: 10.1371/journal.pone.0255795.].

Stress granules form in Brachionus manjavacas (Rotifera) in response to a variety of stressors.

Many eukaryotes share a common response to environmental stresses. The responses include reorganization of cellular organelles and proteins. Similar stress responses between divergent species suggest that these protective mechanisms may have evolved early and been retained from the earliest eukaryotic ancestors. Many eukaryotic cells have the capacity to sequester proteins and mRNAs into transient stress granules (SGs) that protect most cellular mRNAs (Anderson and Kedersha, 2008). Our observations extend the phylogenetic range of SGs from trypanosomatids, insects, yeast and mammalian cells, where they were first described, to a species of the lophotrochozoan animal phylum Rotifera. We focus on the distribution of three proteins known to be associated with both ribosomes and SG formation: eukaryotic initiation factors eIF3B, eIF4E and T-cell-restricted intracellular antigen 1. We found that these three proteins co-localize to SGs in rotifers in response to temperature stress, osmotic stress and nutrient deprivation as has been described in other eukaryotes. We have also found that the large ribosomal subunit fails to localize to the SGs in rotifers. Furthermore, the SGs in rotifers disperse once the environmental stress is removed as demonstrated in yeast and mammalian cells. These results are consistent with SG formation in trypanosomatids, insects, yeast and mammalian cells, further supporting the presence of this protective mechanism early in the evolution of eukaryotes.

CaFÉ: A Sensitive, Low-Cost Filtration Method for Detecting Polioviruses and Other Enteroviruses in Residual Waters.

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.

Genome Sequences of 16 Enterovirus Isolates from Environmental Sewage in Guatemala, 2019 to 2021.

Enteroviruses can cause human infectious disease. We report 16 near-complete genome sequences of enteroviruses that were isolated through environmental surveillance of wastewater in Guatemala.

Culture-Independent Detection of Poliovirus in Stool Samples by Direct RNA Extraction.

Laboratory surveillance for poliovirus (PV) relies on virus isolation by cell culture to identify PV in stool specimens from acute flaccid paralysis (AFP) cases. Although this method successfully identifies PV, it is time-consuming and necessitates the additional biorisk of growing live virus in an increasingly polio-free world. To reduce the risk of culturing PV, the Global Polio Laboratory Network (GPLN) must switch to culture-independent diagnostic methods with sensitivity at least equivalent to that of cell culture procedures. Five commercial nucleic acid extraction kits and one enrichment method were tested for PV extraction efficiency. RNA yield was measured using real-time reverse transcription (RT)-PCR. Based on greater RNA yield, compared with the other kits, the Quick-RNA viral kit was selected for further testing and was optimized using an RNA extraction procedure for stool suspensions. RNA extraction was retrospectively tested with 182 stool samples that had previously tested positive for PVs, in parallel with the standard GPLN virus isolation algorithm. After virus isolation or RNA extraction, real-time RT-PCR assays were performed. RNA extraction was significantly more sensitive than virus isolation (McNemar's test, P < 0.001). Thereafter, the RNA extraction method was tested in parallel for 202 prospective samples; RNA extraction and virus isolation were not significantly different from each other (McNemar's test, P = 0.13). Direct RNA extraction was noninferior to current cell culture methods for detecting PV in stool samples. Our results show that direct RNA extraction can make downstream manipulation safer and can reduce the risk of accidental posteradication viral release. The method is amenable to implementation in a wide variety of polio laboratories. IMPORTANCE Successfully identifying poliovirus from acute flaccid paralysis (AFP) cases is a vital role of the Global Polio Laboratory Network to achieve the goals of the Global Polio Eradication Initiative. Currently, laboratory surveillance relies on virus isolation by cell culture to test for PV present in stool samples. Although this method can identify polioviruses, laboratories must switch to culture-independent methods to reduce the risk associated with growing live viruses in a soon-to-be polio-free world. By implementing this streamlined method, in combination with real-time RT-PCR, laboratories can quickly screen for and type polioviruses of programmatic importance to support the final stages of global polio eradication.