Polio and Its Epidemiology

Shulman, Lester M.

doi:10.1007/978-1-4419-0851-3_839

Lester M. Shulman^2,3

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A Brief Definition of Polio and Its Importance

The word “polio ” has been used to describe both a disease and the disease agent. Among current methods to measure the importance of or interest in a topic is to run a general web search for the term and to search the scientific literature in PubMed. A Google web search of the word “polio” in Aug 2010 yielded 31,100,000 hits, while a search in PubMed yielded 22,000 articles and 826 review articles. This review will concentrate on those aspects of the epidemiology of polio as it relates to disease eradication and the sustainability of this effort. The terms “polio” and “poliomyelitis ” will be used when describing the disease and “poliovirus ” and related terms such as “polio vaccine ” will be used to describe the agent that causes the disease.

In order to understand the epidemiology of polio, it is important to understand the adversary. Toward this goal, this chapter starts with a detailed physical characterization of polioviruses and the pathological effects caused by poliovirus infections that are most relevant to understanding the epidemiology of polio. This is followed by a description of the global efforts to eradicate poliomyelitis and the viral agent causing the disease, and concludes with a discussion of the future directions needed to achieve and sustain eradication and prevent reemergence. Smallpox was the first human disease to be eradicated and we are currently in the endgame of eradication of polio as the second. Polio eradication is currently the largest public health program in the world and has involved both health professionals and more than ten million volunteers in all countries since the inception of the Global Poliomyelitis Eradication Initiative by the World Health Assembly in 1988 [1].

Introduction

The road toward polio eradication has been long [2] and by no means smooth. Important milestones along the march toward recognition and understanding the disease, identification of its causative agent, and toward prevention and eradication will be briefly discussed in the introduction (see also Fig. 1). A poliovirus isolate is classified as vaccine, vaccine-derived (VDPV), or wild-type poliovirus based on the percent nucleotide sequence homology between its capsid protein VP1 and that of the corresponding OPV vaccine serotype. An isolate with VP1 homology of 99–100% is classified as vaccine virus, 85–99% as VDPV, and > 85% as wild-type poliovirus [3]. This rule of thumb for classifying polioviruses as VDPVs has recently been modified for serotype 2 to include isolates with ≥6 nucleotide changes (i.e., <1%) and the upper limit of 15% for VP1 divergence has been eliminated (Summary of the 16th Informal Consultation on the Global Polio Laboratory Network, Geneva, Switzerland, 2010).

**Polio and Its Epidemiology. Figure 1**

The earliest record attributed to polio comes from an Egyptian Stele from 1400 BCE that depicts an Egyptian high priest or official with a walking stick and withered leg that bears a striking resemblance to a recent picture of a man with poliomyelitis (Fig. 2). Polio infections from this time to the nineteenth century were endemic and usually occurred in young children where most infections were probably asymptomatic. While early descriptions of “acquired clubfoot” by Hippocrates and Galen were consistent with polio, the first modern medical characterization of polio includes descriptions of “Debility of the Lower Extremities” by Underwood in 1789, polio by Monteggia in 1813, “infantile paralysis” by Heine in 1840, and involvement of motor neurons in infantile paralysis by Duchenne in 1855. Involvement of motor neurons was confirmed by biopsy of the brain and spinal cord of a polio victim by Cornil in 1863 and by a detailed description of physiological changes in the anterior horn of the spinal cord by Charcot and Joffroy in 1870.

**Polio and Its Epidemiology. Figure 2**

A new epidemiological aspect of polio emerged in the nineteenth century, namely, the appearance of outbreaks that increasingly affected adults as well as children [4]. Paradoxically this shift from an endemic to an outbreak pattern of disease transmission may have been facilitated by a “hygiene barrier” derived from improved community sanitation that may have resulted in a shift from fecal–oral to oral–oral transmission, an increase in naïve individuals especially among older cohorts, and primary exposure of increasingly older cohorts where disease manifestation were more severe. These epidemics became more frequent by the mid-twentieth century and involved growing numbers of people. Wickman described the acute infectious nature of polio in his analysis of a 1905 polio outbreak in New York, confirming Medin’s realization in 1889 that paralytic cases were only a small part of epidemics and that even persons with mild illness could infect others. Further complications were the observation by Burnet and MacNamara in 1931 [5] that different strains of poliovirus caused disease, but infection with some strains did not protect against subsequent infection with other strains and the observation in the 1950s that poliomyelitis could be triggered by physical injury during a poliovirus infection and that there was an increased risk of paralysis in limbs that received a mechanical stress or after tonsillectomies [6]. Two important new concepts were the establishment of a national center for treatment of poliomyelitis victims at Warm Springs, Georgia, and the use of professional fund-raisers by President Roosevelt supported by others in the late 1920s. The nonpartisan National Foundation for Infantile Paralysis and the March of Dimes established in 1937 institutionalized this fundraising effort. The iron lung, developed by Drinker in 1929, and the concept of supportive rehabilitation involving the use of hot moist packs to relieve muscle spasm and physiotherapy to maintain strength of unaffected muscle fibers promoted by Kenny in the 1940s were important advances for treatment of poliomyelitis.

The study of the pathological organism that caused poliomyelitis was enabled by the discovery of a bacteria-free “filterable” etiological agent, the poliovirus, which could pass disease from one primate to another by Landsteiner and Popper in 1909. Burnet and MacNamara realized in 1931 that there was more than one type of poliovirus since exposure to some isolates did not protect against exposure to others. By 1951, the National Foundation for Infantile Paralysis concluded that there were only three serotypes of poliovirus . The study of poliovirus was aided by (a) the first passages of poliovirus in a non-primate rodent system by Armstrong in 1939, (b) passage in tissue cultures by Enders, Weller, and Robbins in 1949 [7], (c) development of plaque assays for quantification of polio by Dulbecco and Vogt in 1954 [8], (d) the use of microcarrier cell systems for vaccine production by van Wezel in 1967 [9], (e) development of monkey neurovirulence tests in 1979 [10], (f) the use of pathogen-free diploid MRC5 cells (human fetal cells derived from normal lung tissue) and permanent cell lines like Vero (a cell line prepared from the kidney of a normal adult African green monkey) for vaccine production in the early 1990s, (g) identification and cloning of the poliovirus receptor CD155 [11], (h) development of the transgenic PVr-mouse model which expresses the human poliovirus receptor as an alternative to monkeys for neurovirulence testing [12], (i) preparation of a murine cell line, L20B, expressing the human poliovirus receptor for selective growth of poliovirus [13], and (j) the development of the immunological and molecular tools (discussed in detail below) that provide the identity the serotype of the isolate, distinguish whether its origin was from a vaccine or wild strain, and provide phylogenetic information on the evolutionary relationship to other isolates.

Advances in culturing polioviruses outlined in the previous paragraph laid the foundation for developing the vaccines that have turned polio into a vaccine-preventable disease and a candidate for eradication (see below). Early experiments and clinical trials such as those in 1935–1936 with inactivated poliovirus by Brodie and Park [14] and attenuated live vaccine by Kolmer [15] were hampered by lack of awareness until 1951 that there were three serotypes. Afterward, effective inactivated vaccine was developed and tested by Salk and coworkers starting in 1953–1954 [16, 17], while Koproswski, Sabin, and Cox developed and tested attenuated oral vaccines in 1950 [18, 19], 1956–1957 [20], and 1958 [21], respectively. Between 1951 and 1962, 12.9 million children were vaccinated with Koprowski strains and 11 million with Sabin strains [19]. A number of important epidemiological observations were made during that time that continue to guide current vaccination strategies. For attenuated oral vaccines these included (a) the first demonstration by Koprowski of interference between poliovirus serotypes during coinfection [19], (b) a demonstration that maternal antibodies did not prevent an immune response in vaccinees under 6 months of age [19], (c) the observation by Koprowski and especially Bottiger that live vaccine spread to contacts [19], (d) a demonstration of persistence of antibodies at the same levels in vaccinated children for at least 3 years [19], (e) proof of concept by Koprowski that live polio vaccine could be effective in containing large outbreaks [19], and (f) documentation of high vaccine safety with both the Koprowski and Sabin OPV strains [4, 19, 22]. Safety issues relating to both the live and inactivated viral strains will be mentioned in discussions starting on pages 8150 and 8160. After extensive evaluation in hundreds of monkeys at Baylor College of Medicine, and the Division of Biological Standards at the NIH, the Sabin strains were chosen for licensure primarily on the basis of lower neurotropism, but also based on genetic stability on passage in humans and a lower ability to spread to contacts (reviewed in Sutter et al. [4] and Furesz [22]). Efforts to eradicate polio and to prevent reemergence are presented in detail in the following section. Initial paradigms attributed to the different properties of the individual vaccines have not always held true in all circumstances [23].

The Epidemiology of Polio

Epidemiological studies to discover the means of preventing a disease usually begin with the recognition of a new pattern of similar symptoms among those affected and the establishment of a case definition. Discovering means for preventing the disease may start before the disease agent is discovered and characterized, but is certainly accelerated once this characterization becomes available together with the means of quantifying intervention strategies. It is much less common to start with an agent and then search for a disease as in the case of human anelloviruses [24]. Human anelloviruses are small circular DNA viruses considered to be orphan viruses. They were initially discovered in a patient with hepatitis, but subsequent research indicated no causal link to hepatitis and it has been very difficult to associate them with any other specific disease. However, this section of the review will start with a description of those physical aspects of poliovirus that have the most impact on epidemiology of the disease. This is because there is already a clear case definition for polio and poliomyelitis, polioviruses have been recognized as the causative agents of these diseases, numerous methods for characterizing poliovirus and preventing poliovirus infections have been developed and tested, and the disease is approaching elimination or eradication.

Structural and Functional Organization of the Poliovirus Genome

Polioviruses belong to the Picornaviridae virus family. The Picornaviridae genome consist of a single strand of positive-sense RNA approximately 7,500 nucleotides located within a protein capsid made up of 60 capsomeres that forms a virion 27–30 nm in diameter. The genome is organized from its 5′ end to its 3′end into a number of functional regions (Fig. 3) that include a 5′ untranslated region (5’UTR) that regulate translation and replication [25], a long open reading frame that encodes a single large polypeptide that is cleaved after translation into four structural capsid proteins and a number of nonstructural proteins including an RNA polymerase, and a short 3′ untranslated region that is attached to a poly-A tail in both viral mRNA and genomic RNA in the virion [25] (see reviews by Wimmer et al. [26], Racaniello [25], and Sutter et al. [4]). The positive-sense single strand of genomic RNA in the virion, serves directly as an mRNA template for translation to viral proteins once the virion penetrates its host cell membrane. Later it serves as a template for synthesis of a complimentary negative sense strand. The current understanding of the physical and genetic aspects of polio was greatly facilitated by the development of and the current commercial availability of methods for easily extracting viral nucleic acids from poliovirus and poliovirus-infected cells and analyzing and manipulating these sequences. Some of these studies led to the unanticipated conclusion that poliovirus capsid proteins and the sequences that encode them define polioviruses, whereas all other elements in the poliovirus genome may be substituted by genomic recombination with equivalent sequences from closely related isolates of enterovirus species C in vivo and even more distantly related rhinoviruses in the laboratory as long as functionality is maintained (reviewed by Kew et al. [3]). Finally, advances in molecular biology also enabled poliovirus to be the first virus to be synthesized from nucleotides in a test tube [27, 28].

**Polio and Its Epidemiology. Figure 3**

The 5’UTR was first subdivided into a highly conserved region (nucleotides 1–650) and a hypervariable region (nucleotides 651–750) based on an analysis of 33 wild-type 3 polioviruses [29]. A series of stem-loop structures with a high degree of secondary structure were proposed to be present within the conserved region by Pilipenko et al. [30] and Skinner et al. [31]. A single nucleotide substitution in a loop structure in stem-loop V of the 5’UTR significantly influenced the neurovirulence of poliovirus isolates from all three serotypes and affected the maximum temperature at which viral isolate replicate efficiently (see reviews by Kew et al. [3] and Sutter et al. [4] and discussions on poliovirus evolution starting on page 8137). The hypervariable region appears to be much less structured, reflecting the high degree of variation and the U nucleotide richness [29].

An Internal Ribosome Entry Site [32, 33], IRES, enables uncapped RNA from Picornaviridae to be translated in eukaryotic cells by host ribosomes [25]. One of the first steps in initiation of viral translation is the binding [34] of cellular RNA binding proteins PCB₁ and PCB₂ to stem-loop IV of the IRES. This enables the 40S ribosomal unit to bind to the IRES and continue the process of translation as if the RNA was a capped eukaryotic mRNA. Functional IRES elements can be interchanged among Picornaviridae [3]. Nucleotide differences in the conserved 5’UTR among different isolates were unevenly distributed [29] with changes tending to conserve the stem structures. In contrast, the hypervariable region did not seem to have a highly conserved secondary structure and nucleotide differences appeared to be more or less evenly spread throughout [29]. While the length of the hypervariable region was generally conserved suggesting an unknown function [29], small deletions were tolerated [35].

Picornaviridae have a genome of approximately 7,200–7,400 nt with a single open reading frame (ORF). While this ORF encodes four capsid proteins and at least seven viral proteins (Fig. 3), these proteins are only produced after the initial translation product, a single polypeptide, is enzymatically cleaved into smaller and smaller polyproteins during and after translation (posttranslational processing). The polypeptide is cleaved in an ordered series of steps (Fig. 3b), by viral encoded protease activity within the nascent polypeptide (self-cleavage) and in trans from viral proteases released after cleavage. Interestingly some of the intermediate cleavage products have unique activities by themselves that contribute to the replication cycle of the virus, but which differ from those of the final cleavage products (reviewed by Racaniello [36] and Krausslich et al. [37]). Properties of the polioviral capsid proteins define the epidemiology of polioviruses. The most important aspects of the structure of the four capsid proteins, their assembly into capsomeres and organization within adjacent capsomeres that relate to the epidemiology of polio, will be discussed above on page 8131. The 900–906 nucleotide sequence of the VP1 of polioviruses has become the minimum standard for determining the evolutionary relationship among polioviruses and the rate at which they evolve [4, 38, 39].

Many of the nonstructural proteins and intermediate cleavage products are multifunctional and act at a number of steps in RNA synthesis (reviewed in [4, 25]). Most of the nonstructural proteins will only be mentioned in passing since equivalent nonstructural proteins from other related picornaviruses may replace all of the nonstructural viral proteins as long as functional sites including cleavage recognition sites are maintained (reviewed in [3]). The resultant chimeric recombinants behave as polioviruses. One nonstructural protein, the RNA polymerase, will be discussed in some detail (see page 8135) because of its profound effect on polio epidemiology regardless of its source.

The secondary structure in the 3’UTR that may play a role in translation and replication of picornaviral genomic RNA has been reviewed [25]. A nucleotide difference between Sabin serotype 1 and its wild parent influences the temperature at which serotype 1 can replicate [40]. A poly-A tail is present on both genomic and mRNA that stimulates the cap-independent, internal ribosome entry site (IRES)-driven translation of poliovirus RNA in a mammalian cell-free system by tenfold [41].

Both genomic and minus strand RNA are linked to the small viral encoded protein, VPg, (Fig. 3a) through pUpU bound to tyrosine, the third amino acid from NH terminal end of VPg, by a phosphodiester bond [42]. VPg is also present in infected cells in an unmodified form and bound to pUpU through the same 0⁴-phosphotyrosine bond found in the covalently linked forms [42, 43]. The uridylylation of VPg takes place on the opposite side of the polymerase that binds RNA. A host encoded unlinking enzyme that cleaves the 0⁴-phosphotyrosine bond between VPg and RNA has been described [44] although its role in replication has not been established. The poliovirus encoded VPg can be replaced by VPg from echoviruses [43].

A mature infectious poliovirus consists of a single sense strand of polyadenylated RNA covalently linked to a viral encoded protein, VPg, surrounded by an icosahedral protein coat, the capsid, made up of 60 capsomeres that each contain a single copy of each of the four viral capsid proteins. Adjacent capsomeres are organized around both fivefold and threefold axes of symmetry and the surface around these axes is organized into a series of regular protrusions and depressions (Fig. 4). The capsid structure is metastable [45] rather than rigid and internal parts of capsid may even be transiently expressed on the surface ([46], review in [25]) exposing additional epitopes such as PALTAVE inVP1 [47].

**Polio and Its Epidemiology. Figure 4**

Capsid proteins are the first viral encoded proteins to appear on the nascent poliovirus polyprotein and are cleaved from the nascent polyprotein into an intermediate polyprotein, P1, by 2A^pro while the full-length polyprotein is still being synthesized. P1 is processed into final cleavage products VP1 and VP3 and an intermediate cleavage product VP0. VP0 is only cleaved into VP2 and VP4 during the final stages of maturation of the virion. The protein chains of VP1, VP2, and VP3 are arranged in wedge-like structures with extruding loops that interact to form the major (NAgIa, NAgIIa, and NAgIIIa) and minor (NAgIb, NAgIIb, and NAgIIIb) neutralizing antigenic epitopes [48]. Amino acid differences within neutralizing antigenic sites divide polioviruses into three serotypes with limited cross-reactivity [49]. The amino acids of the neutralizing antigenic sites have been mapped onto the three-dimensional structures of the viral capsid of type 2 poliovirus as colored spheres, Fig. 4. Those that are unique to the neutralizing antigenic epitopes are colored yellow. Some amino acid residues in and adjacent to these neutralizing antigenic sites (red spheres in Fig. 4) are also involved in receptor binding and this may have restricted the number of serotypes [50] and influenced evolution in these epitopes in the absence of immunoselection especially during the emergence of vaccine-derived polioviruses (VDPVs) (see page 8159).

The three-dimensional view of the structure of the capsomeres at the fivefold axis of symmetry reveals an elevated central plateau with a hole in the middle surrounded by a depression called the canyon [51, 52]. The fivefold axis of symmetry for type 2 poliovirus is shown in Fig. 4. A number of conserved amino acids and amino acids within and adjacent to the serotype-specific neutralizing epitopes are located on the surface of the canyon walls and have been implicated in interaction with the poliovirus receptor [26, 50].

The human encoded, poliovirus receptor , CD155, belongs to the immunoglobulin super gene family and has one variable and two constant immunoglobulin-like domains (residues 28–337) [11]. This human encoded gene has alternative splice sites that result in two membrane-bound and two secreted isoforms [53]. The variable domain 1 penetrates the canyon and binds to amino acid residues from all three external capsid proteins and the principal binding sites are at the bottom of the canyon above the hydrophobic pocket (blue spheres in Fig. 4) and on the outer side of the canyon rim [50, 54]. The residues of type 1 poliovirus involving receptor virion binding include residues 102–108, 166–169, 213–214, 222–236, 293–297, 301–302 in VP1, residues 140–144, 170–172 in VP2, and 58–62, 93, and 182–186 in VP3. The equivalent residues for serotype 2 poliovirus have been mapped onto the three-dimensional capsid structure as red (shared with neutralizing antigenic epitopes) and magenta spheres for those associated only with the receptor binding sites (Fig. 4). Cryo-electron microscope studies have shown the binding of the poliovirus to the virion to be a two-step process [54]. The initial binding of the receptor to amino acid residues along the canyon wall results in little or no change in virion structure. However, this binding rapidly sets into motion conformational changes leading to the 135 S or A particle state that initiates uncoating and the start of the infections cycle [45, 54].

The human poliovirus receptor has been cloned and used to establish a murine cell line, L20B, where expression of the poliovirus receptor allows infection and growth of polio from clinical and other samples but not most other human non-polio enteroviruses [13, 55, 56]. Transgenic mice, PVR Tg-21 mice that express the human poliovirus receptor, not only support poliovirus infection and present with neurological symptoms, but allow determination of the relative neurovirulence of the isolates [12, 57–59].

A hydrophobic pocket (blue spheres in Fig. 4) located below the canyon floor is normally occupied by pocket factors such as sphingosine-like molecules including palmitic and myristic acids and hydrophobic compounds, that stabilize the capsid, enable receptor docking and whose removal is a necessary prerequisite for uncoating [25, 45, 54, 60].

Small molecules such as pleconaryl and isoflavenes can bind in this hydrophobic pocket and exert antiviral effects by affecting the binding of the receptor or enhancing the stability of the virion and preventing uncoating [25]. Because of the metastable nature of the capsid, mutations distant from the receptor and drug binding sites can compensate mutations in the respective binding sites [45, 61].

Molecular analysis studies of isolates shed during persistent infections of immunodeficient patients [62, 63] and from phylogenetically related aVDPVs from environmental samples help pinpoint amino acid substitutions in capsid proteins that determine antigenicity, receptor recognition, attenuation of neurovirulence, and properties of the hydrophobic pocket. Other changes, some of which are at interfaces between the threefold or fivefold interfaces of capsomeres may also affect these properties indirectly.

In order for single stranded, positive-sense genomic RNA to be incorporated into progeny of the infecting virus, a complimentary negative RNA strand must first be synthesized using the original single stranded positive-sense RNA genome as template, and this complimentary negative strand must then be used as template for synthesis of new positive-strand RNAs. While some positive-sense copies are incorporated into progeny virions as genomic RNA, other newly synthesized positive-strand RNAs serve as templates to repeat and amplify RNA replication and/or for translation to produce more viral proteins. Eukaryotic cells that serve as the host for poliovirus replication lack a polymerase that can synthesize complimentary RNA from an RNA template. Therefore the virus must encode its own polymerase. Since the single stranded positive-sense RNA genome of the infecting virion is also an mRNA that is immediately translated, the virion does not have to incorporate the polymerase into the virion itself to start replication. The translation product of the 3D^pol gene (Fig. 3) is the required RNA-primed RNA polymerase. Both the intermediate cleavage products that contain the 3D^pro and the final cleavage product are multifunctional and the crude replication complex also contains other viral proteins and protein cleavage intermediates such as 2BC, 2C, and 3AB as well as host proteins (reviewed in [4, 25]). The binding site for RNA template and primer are on one face of 3D^pol and a binding site for the uridylylation of VPg, a prerequisite for covalent linking of VPg to viral RNA, is on the opposite face [25].

One important contrast between genomic DNA replication in eukaryotic host cells and genomic RNA replication in Picornaviridae relates to the fidelity of replication. Specifically, there is an elaborate proofreading mechanism combined with pathways for correcting misincorporations during replication of Eukaryotic DNA that is lacking in the RNA-primed RNA polymerase complex for viral replication [64, 65]. This leads to such a high evolutionary rate for polioviruses that it borders on error catastrophe [66, 67] (discussed further below).

Poliovirus Infections in Cells, Individuals, and Populations

This section will deal with the epidemiological aspects of poliovirus infections at three levels, infections in single cells, infections in a single individual, and infections in populations of individuals. The normal infectious cycle of a poliovirus starts with recognition and attachment to poliovirus-specific cell receptors on susceptible cells of human or closely related primate origin. It continues with penetration and uncoating, translation of viral RNA, posttranslational processing of viral polyproteins, replication of viral genomes, assembly and maturation of progeny viruses culminating in the release of infectious polioviruses. During this process, the virus employs and modifies host cell functions to optimize viral yield. The observation that viral RNA and cDNA is infectious when transfected into permissive host cells has allowed recovery of virus from extracted genomic RNA, cloned cDNA or RNA translated from cloned DNA [68–70], genomic RNA immobilized on FTA paper (WHO 16th Informal Consultation Of The Global Polio Laboratory Network, September 2010, Geneva, Switzerland), and from polioviral RNA synthesized in a test tube from individual nucleotides [27, 71]. The infectious cycle has been reviewed extensively. The reader is referred to the following reviews for further reading [4, 25, 72].

Poliovirus Infections at the Level of the Host Cell

All polioviruses recognize a single host cell receptor [50], CD155, also known as the poliovirus receptor (PVR). Identification and cloning of the poliovirus receptor CD155 [11] allowed the creation of cell lines [13] and animal models [58, 59] for the study of polioviral infections in non-primate hosts. The interaction of virion and receptor is complex [25]. The capsid structure is dynamic allowing the transient presence of internal portions and epitopes of capsid proteins on the outer surface of the virion [25, 46] including the N-terminus of VP1 even before uncoating. The shape of the receptor and its position relative to the host cell membrane and the canyon on the virion into which it fits bring the fivefold axis of capsomeres in close proximity to the cell membrane [54].

Conformational changes, induced shortly after the virion–receptor interaction, are required to initiate the uncoating process (reviewed in Racaniello [25]). The capsid begins to disassociate during a transition to the A particle. The A particle contains the viral RNA but has lost its VP4 capsid proteins. The N-terminal of the VP1 externalizes and may insert into the plasma membrane. Viral RNA is believed to enter the cell at or near the fivefold axis through a continuous channel formed in part by VP1 that continues through the cell membrane [54]. VP4 plays a part in formation of the pore. The pore for poliovirus entry is probably not formed within endosomes [25]. Small molecules that sit in the hydrophobic pocket may influence these conformational changes without affecting receptor binding [61, 73–75].

The only viral proteins in the virion are the four capsid proteins and the VPg covalently linked to the genomic RNA. The internal ribosomal entry site (IRES) on uncoated polioviral RNA enables translation of the viral polyprotein on host cell ribosomes (reviewed in [3, 25]). VPg appears to be cleaved from this RNA and subsequently synthesized viral RNA that will be used as mRNA [44]. Nuclear trafficking of cellular proteins is downregulated shortly after infection resulting in accumulation of host nuclear proteins in the cytoplasm that could function alone or in combination with viral encoded proteins in viral RNA translation, synthesis, and packaging [73]. Downregulation may be due in part to specific degradation of two host transporters, Nup 153 and p62. A full-length polyprotein is not observed in spite of being encoded by the single long open reading frame since posttranslational cleavage of the polyprotein is initiated as soon as the portion encoding the 2A^pro has been translated. Many of the nonstructural proteins and intermediate cleavage products are multifunctional and act directly or indirectly at a number of steps in the RNA synthesis pathway (reviewed in [4, 25]). One example is the aforementioned viral encoded protease, 2A^pro, that also shuts off host protein synthesis by cleaving eIF4G. eIF4G is required for translation of capped eukaryotic mRNAs, while the C-terminal of the cleavage product enhances IRES activity [25]. 2A^pro is also important for negative strand but not positive-strand RNA synthesis [76]. Another example is the intermediate cleavage product, 3CD^pro, that also participates in the posttranslational processing of the polyprotein.

The last protein of the polyprotein to be translated is the 3D polymerase. RNA-primed RNA synthesis is initiated once the 3D has been released from the polyprotein reviewed in [25]. VPg-pUpU or VPg itself could act as a precursor for RNA synthesis by hybridizing to template RNA [44, 77]. The binding site for template and primer are on one side and that for VPg is on the other side. A replicate intermediate is formed and consists of a positive-sense RNA with 6–8 nascent negative strand RNAs. The negative sense strand serves in turn as template for synthesis of a 30-fold excess of new sense strand RNAs. Full-length dsRNAs can be isolated from infected cells. Altogether the genomic RNA is amplified up to 50,000-fold. VPg is bound to both genomic RNA and negative sense RNA.

Poliovirus and other picornaviruses employ a quasispecies reproductive strategy [64, 78] where the lack of proofreading rapidly results in a mixture of progeny with modified genomes containing randomly positioned single nucleotide substitutions. Genomic recombination is a second method of evolution where a single event results in substitutions of many nucleotides from a different poliovirus or closely related non-polio enterovirus for the equivalent sequence in the original poliovirus. The majority of single nucleotide substitutions are deleterious or neutral; however some may confer a reproductive advantage for progeny for growth in the current or future host and/or for host-to-host transmission. Evolutionary changes become “fixed” by selective outgrowth of individual members of the quasispecies that pass through bottlenecks within and between hosts [79]. Two evolutionary pathways, the very high number of progeny (>10,000 per infected cell) and outgrowth by chance selection and/or a selective advantage, result in one of the highest observed rates of molecular evolution [39].

RNA is synthesized from four nucleotides, two pyrimidines (uracil and cytosine) and two purines (adenine and guanine). The most common route for polioviral evolution is by nucleotide misincorporation (single nucleotide substitution) in the absence of both proofreading and post-incorporation excision–repair pathways. The nucleotide position that is substituted is probably random but may be influenced to some extent by secondary structure and the adjacent nucleotides. Quasispeciation arises from the fact that the remaining progeny retain the original nucleotide at the position of each unique substitution in an individual progeny virus, while within the cloud of progeny each isolate may have a unique substitution at a different position in the genome.

Among the isolates that make up the quasispecies, substitutions should be found at each position in the genome at an equal frequency, at least in theory. However, substitutions are much more frequently observed in some positions than in others. Two related factors contribute significantly to the nonuniform distribution (see page 8140) of observed substitutions along the genome. The first is that almost all observations have been made with RNA extracted from the quasispecies that arose during replication of a viable virus directly in the primary host or after amplification of one or more isolates from the quasispecies in vivo in a second host or ex vivo in tissue culture. The second is that substitutions in some positions produce nonviable or less fit offspring that are eliminated during this amplification process.

Sequence-specific variability, based on the individual nucleotide base and its nearest neighbors [67], and inherent characteristics of the polymerase are other factors that contribute to the nonuniform distribution. If misincorporations were unbiased, transversions (the substitution of a pyrimidine by a purine or vice versa) would be expected to occur at twice the rate of transition (the substitution of a purine with a purine or a pyrimidine with a pyrimidine). However empiric observations have revealed a polymerase-based bias of approximately ten to one in favor of transitions [39]. To currently include sequence data from the genomes of nonviable progeny requires either amplification of individual genomes by a process that does not require an active poliovirus infection but that includes high fidelity with proofreading and excision-repair (reverse transcribing the genomic RNA and cloning the cDNA of all viable and nonviable poliovirus progeny into plasmids that can be amplified in bacterial strains with high fidelity, proofreading, and error correction) or by direct sequencing of individual gnomes without amplification (chip/array sequencing technology) [66, 80]. Neither approach is currently very easy to apply since both would require individually processing large numbers of genomes equivalents, although Crotty et al. were able to calculate a rate of 2.1 × 10⁻² substitutions per site by direct measurement of mutations in the VP1 of 55 cloned genomes after a single cycle of in vitro virus growth. Massive parallel next-generation sequencing may offer the best approach for analyzing viable and nonviable members of a quasispecies [289].

Wild poliovirus genomes frequently recombine (recombination) with polioviruses and closely related non-polio enterovirus genomes [81, 82]. This recombination can only occur during concurrent infection of a single cell by both parental isolates. Intratypic recombination may occur even within capsid proteins [83–85].

The noncapsid regions of polioviruses are most similar in sequence to other members of the enterovirus C genotype that includes Coxsackie A virus (CAV) serotypes 1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24, and these sequences are readily shuffled among polioviruses and the other members of this group [86, 87]. In fact polioviruses show evidence of having evolved from C-cluster Coxsackie A viruses and may reemerge from them after eradication [87]. Interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of circulating vaccine-derived polioviruses, cVDPVs [88].

Recombination is not site specific, does not require extensive homology between genomes at the crossover site, and most likely occurs by an exchange of templates by the synthesis of complimentary RNA by the RNA-primed RNA polymerase rather than by breakage rejoining [89]. Intratypic (same serotype) and intertypic (different serotype) recombination in in vitro occurred at 1.3 × 10⁻³ and 7.6 × 10⁻⁶, respectively [89], while recombinations between polio and NPEVs occurred at a frequency of 10⁻⁶ [87]. Administration of trivalent oral vaccine anywhere and in areas where wild polioviruses and genotype C viruses co-circulate provides the conditions for concurrent infections and polio vaccine-polio vaccine, polio vaccine-wild polio, and polio vaccine-NPEV, as well as endemic wild polio-NPEV recombinations. For examples of such recombinations see molecular analyses of isolates from the cVDPV outbreaks in Haiti and Dominican Republic [90] and Indonesia [91] and in individual cases [92].

Molecular epidemiology is the study of disease and factors controlling the presence or absence of a disease or pathogen using molecular data (DNA, RNA, or protein sequences). The next portion of this section will concentrate on those aspects of molecular epidemiology that impact on the epidemiology of polio.

Polioviruses and other enteroviruses are among the organisms with the highest rate of misincorporation ([67, 78], and reviewed [39]). Misincorporation comes at a high cost, namely, only approximately 10% of the >10,000 progeny from a single infected cell are viable [67]. This high frequency of misincorporation helps to explain the high ratio of physical to infectious particles [93]. Studies with ribavirin [66, 94], an antiviral drug acting as a nucleoside analogue, have shown that the misincorporation rate of polioviruses is close to the catastrophe error rate, that is, the transition point where a modest increase in misincorporation results in a drastic decrease in viability. In these experiments a 9.7-fold increase in mutagenesis resulted in a 99.3% loss in viral genome infectivity after a single round of replication, while a less than twofold increase in the natural mutational frequency resulted in a 50% loss of viability.

Fitness is based on the overall performance during viral replication [67], a complex process, involving recognition of and binding to the host cell, uncoating/entry, initiation of protein synthesis before RNA replication takes place, regulation of replication and translation once RNA replication is initiated, culminating with assembly, maturation and externalization of mature virus and survivability until subsequent infection of the next host cell or organism. Changes can affect more than one of these processes. For example, an increase in the mutational rate in infections in the presence of the nucleoside analog ribavirin not only led to an increase in nonviable genomes, but also caused a reduction in the total number of viral genomes produced [66]. One of the advantages of the quasispecies nature of poliovirus offspring is that isolates with a selective advantage to new growth conditions may already exist in the population [66]. Studies on mixed infections in PVR Tg21-transgenic mice suggest that random selection may play a role in the selection of which genomic variants within a mixed infection in the gut infect the CNS since virus isolated from the CNS was not always the most neurovirulent [95]. Other experiments showed that increased fidelity of the polymerase reduced viral fitness in the PVR Tg21-transgenic mice [96] or in tissue culture [97]. An alternate explanation for selection was proposed by Andino and colleagues [98]. They provide evidence that the quasispecies is not just a collection of individual variants, but a group of interactive variants and that fitness and selection may occur at the level of the population rather than at the level of individual genomes. In their study, an increase in polymerase fidelity affected viral adaptation and pathogenesis in addition to genome variability. Data supporting the suggestion that minor components can alter the phenotype of quasispecies comes from retroviral infections [99] and studies with VSV [100].

A number of studies have shown that single nucleotide misincorporations by the polio RNA-primed RNA polymerase accumulate at a more or less constant rate that can be used as a “molecular clock ” to estimate evolutionary time between isolates and to determine whether sequence differences between two polioviruses isolated within a given time interval are consistent with a shared, direct evolutionary pathway between them [38, 39, 101, 102]. In general, the rate of accumulation and fixation of single nucleotide substitutions appears to be similar for all isolates regardless of kind (all three serotypes of wild, vaccine, or vaccine-derived polioviruses), type of polymerase (original intact polymerase or chimeric or complete recombinant from the same serotype, a different serotype or even a group C non-polio enterovirus), or type of infection (transient in immune competent individuals, persistent in immunodeficient patients, or even in the very elderly where waning immunity may play a role in selection) [4, 39, 79, 90, 101, 103–110]. Moreover, the rate of third codon position synonymous substations appeared to be fairly constant throughout the period of virus excretion in a persistently infected individual [107]. Using molecular observations from full-length genome sequences from viruses isolated during a 10 year long outbreak established from a single imported founder virus, Jorba et al. [39] calibrated five clocks based on five different classes of nucleotide substitutions. The constants for total substitutions (K_t), synonymous third position substitutions in coding regions (K_s), synonymous transitions (A_s), synonymous transversions (B_s), and non-synonymous substitutions (K_a) were 1.03 ± 0.10 × 10⁻², 1.00 ± 0.08 × 10⁻², 0.96 ± 0.09 _ 10⁻², 0.10 ± 0.03 _ 10⁻², and 0.03 ± 0.01 × 10⁻² substitutions/site/year, respectively. The rates were similar whether calculated using linear regression, a maximum likelihood/single-rate dated tip method, and Bayesian inference. The first two constants were mostly controlled by the third. As for saturation, third position synonymous transitions become evident by 10 years and complete saturate within 65 years while saturation of synonymous transversions was predicted to be minimal at 20 years and incomplete even at 100 years. This wide variation in calculated time constants depending on the type of substitutions together with differences in the estimated time until all possible changes become saturated, provides a flexibility that allows one or more clock to be applied to characterize the range from evolution in outbreaks between very closely related isolates with short intervals between isolations, to comparison between much more distantly related polioviruses or related enteroviruses. It is interesting to note that the molecular clocks are fairly constant given that intratypic and intrageneous recombination can result in complete or partial substitution of the polymerase whose intrinsic properties presumably govern the rate of misincorporation. Mutations may increase non-synonymous mutation rates [111] while others decrease them [96]. Multiple recombination events [83, 85] must be ruled out or taken into account when calculating time clocks based on the number of nucleotide differences.

Different factors that affect fitness and determine the viability of individual viral offspring result in differences in observed substitution rates and patterns in the different functional elements of the genome shown in Fig. 3. Namely, using the rate of substitutions in the VP1 capsid protein as reference, the rates of substitutions are approximately half in the conserved region of the 5’UTR, approximately threefold higher in the hypervariable region of the 5’UTR, and equivalent or somewhat lower in the remainder of the ORF [4, 39, 101, 109]. The data for nucleotide substitutions in the nonstructural P2 and especially the P3 regions of the ORF and the 3’UTR are less accurate and less informative due to frequent recombinations among polioviruses and between polioviruses and non-polio enteroviruses within these regions.

The genetic code introduces a bias in the position of observed substitutions. Substitutions in the third position of a codon are least likely to result in an amino acid change and these synonymous substitutions are by far the most abundantly observed in wild poliovirus , polio vaccine and VDPV infections [4, 79]. Non-synonymous substitutions that occur in the initial stages of vaccine infections restore replicative fitness and in many cases neurovirulence [3, 112] while those in persistent infections may influence receptor–virus interaction (see page 8133, 8159).

Three-dimensional requirements also bias the observed distribution of substitutions. Maintenance of stem of the stem-loop structure in the conserved region of the 5’UTR especially within the IRES appears to be one of the major constraints on viability. For example, complimentary paired double substitutions that maintained stem structure were frequently found in the loop V of evolutionarily related environmental isolates [109] whereas a single nucleotide substitution in a loop of Loop V is a dominant determinant of attenuation of neurovirulence and growth at elevated temperatures. Other examples of three-dimensional effects are mutations that occur at distances from functional sites that influence the viral response to antiviral drugs in the hydrophobic pocket [61] and mutations that occur at the interfaces between proteins and at the N-terminals of VP1 and VP4 that may affect structural stability and the receptor-induced transitions [45].

Due to the complex nature of polioviral replication and multi-functionality of viral enzymes and viral three-dimensional structures, selective pressures that operate on one structure or function may affect another seemingly unrelated property. One of these apparent paradoxes is the fact that some RNA viruses including poliovirus may diverge antigenically in the absence of immune selection [113]. One of the features that distinguishes polio vaccine evolution during persistent infections in total B-cell deficient immunodeficient patients from evolution during person-to-person transmission in immune competent but naïve individuals is that isolates from the former but not the latter have high numbers of amino acid substitutions in and around neutralizing antigenic sites [3, 4, 62, 114]. Antibody titers tend to wane in the elderly. The finding of amino acid substitutions at or near neutralizing antigenic sites during infection of the elderly with type 1 monovalent mOPV [103] may suggest that waning immunity may create a situation resembling the early stages in establishment of persistence in immunodeficient patients. Since some of the amino acids and structural organizations are shared by neutralizing antigenic sites and receptor binding sites, the high mutation rate in neutralizing antigenic sites is more likely the result from selective pressures governing receptor–virus interaction during establishment and maintenance of persistence than on non-humoral mediated immune selection or selection by variations in anti-polio antibodies in the IVIg regimens these immunodeficient patients receive to compensate for their B-cell deficiencies. This sharing of functions has also been suggested to be one of the reasons why there are only three serotypes of poliovirus [26, 50]. It is commonly accepted that the evolution of a fourth serotype would require receptor switching of a non-polio enterovirus to the use of the PVR, CD155. A somewhat paradoxical alternative for emergence of a fourth serotype may be through antigenic evolution during persistent infections in immunodeficient individuals as a result of selective pressures relating to receptor binding. Consistent with this is the observation that cohorts of immunized individuals who had high titers against vaccine strains had significantly reduced geometric mean titers against highly diverged neurovirulent vaccine-derived viruses that were isolated from environmental samples [109, 115] and individual titers against some of these isolates were <1:8 in 7% of the adults [109, 116].

Any discussion on molecular evolution of polioviruses and their effects on polio epidemiology would be incomplete without a discussion on vaccine-derived polioviruses, VDPVs (see page 8157 and the section on future directions). Evolution of live attenuated polio vaccine occurs by the same processes as in wild polioviruses, namely, by accumulation of single nucleotide substitutions and through genomic recombination. Evolution of VDPVs occurs during person-to-person circulation in cohorts of naïve or under-immunized individuals especially after interruption of vaccination, or during persistent infections of immunodeficient individuals [3, 80, 106, 114, 117, 118]. The letters “c” or “i” for viruses that evolved during person-to-person circulation or during persistent infections of immunodeficient individuals, respectively, are appended before “VDPV” when the evolutionary pathway is known. The prefix “a” is added instead when the pathway is ambiguous or unknown.

One of the goals of the Global Eradication Initiative (see page 8150) is to reach a stage where all wild poliovirus transmission is terminated and vaccination can be discontinued. “Emergence of VDPVs,” “failure to vaccinate,” and “vaccine failure” discussed below have been the three major reasons for the delay in achieving eradication of poliomyelitis . Providing that enough money and effort can be mobilized, current vaccines and vaccine strategies are probably sufficient to enable immediate solutions for “vaccine failure” and “failure to vaccinate.” Promising alternatives applying experience with adjuvants and better applicators have revived the possibility of using techniques explored in the 1950s such as fractional subdermal doses of inactivated virus, IPV [16, 119–121].

Vaccine-derived viruses consistently emerge as a consequence of the inherent genetic instability of poliovirus [122]. Moreover, many of the first sites that mutate restore replicative fitness, reverse attenuation of neurovirulence, and restrictions on growth at elevated temperatures. cVDPVs behave like wild polio [106, 112]. These cVDPVs clearly present a serious health threat [114]. The minimal amino acid changes in neutralizing antigenic sites that occur during person-to-person transmission of cVDPVs [3, 4, 123] allow rapid control through OPV immunization campaigns [106, 114, 124]. In contrast, iVDPV infections have not always been curable [63, 125], and the numbers and identities of anonymous persistent secretors are unknown [109]. The problem of reemergence of poliomyelitis through cVDPVs and especially iVDPVs requires a coordinated global program to discontinue the use of OPV with substitution of alternative vaccination strategies to prevent the appearance of large cohorts of unimmunized individuals during the period when OPV or cVDPVs may still circulate and iVDPV and aVDPV infections persist [116, 122, 126–128]. In fact, eradication should be redefined to include the elimination of both wild and vaccine-derived viruses [122].

OPV strains, like their wild counterparts, readily recombine the noncapsid encoding portions of their genomes with other polioviruses and related non-polio enteroviruses at very early stages in emergence. Evidence for this comes from analysis of poliovirus RNA from vaccine-associated paralytic poliomyelitis cases, VAPP, where, for example, >50% of polioviruses isolates had recombinant genomes [81, 129–132]. Supporting this is evidence from environmental surveillance where vaccine viruses with minimal divergence (0.5–1%) in their VP1 sequences had already recombined with polio and non-polio enteroviral genomes in regions encoding nonstructural proteins [133]. The ability to simultaneously reverse multiple mutations by recombination could foil efforts to develop improved oral vaccines. Introducing mutations that decrease the chance for reversal by single nucleotide replacement such as incorporation of polymerases with improved fidelity or a total redesign of the genome of each serotype based on rare codon usage [3] could be bypassed by recombination.

Finally, the general consensus is that selective pressure or a higher mutation rate due to local sequence or secondary structure leads to a higher frequency of mutations at certain “hot spots” [93]. However after reviewing the pattern of changes in substitution frequencies throughout the genome it may be more accurate to think of the real frequency of substitutions as that observed in the so-called hyper variable region of the 5’UTR which may be under minimal selective pressure, and consider all other regions as “cold or colder spots” with lower observed rates of substitution derived from negative selection driven by the requirement for viability.

Virion assembly, maturation, and release in picornaviral infections have been reviewed [25]. The ratio of viral particles to infection particles ranges between 30:1 and 1,000:1. Many of the viral particles are noninfectious due to lethal mutations in their genomic RNA and/or incomplete maturation.

Poliovirus Infections at the Level of the Individual Host

The incidence of poliovirus infections is significantly higher in summer and autumn in temperate zones, becoming less seasonal as the environment becomes more tropical (reviewed in [3]). Improved sanitation and vaccination have reduced natural endemic infections in the very young and together with incomplete vaccine coverage has led to an increasing number of infections in older individuals.

There are two major routes of host-to-host transmission. The most common and most efficient is fecal–oral, followed by oral–oral transmission as Dowdle et al. described for the fate of poliovirus in the environment and their review of the infectious dose for transmission in humans [134]. It has been postulated that there has been a shift from the former to the latter route, as the level of community hygiene improved [56]. The infective dose after ingestion of Sabin vaccine strains is approximately 100-fold higher than that for wild poliovirus, 1000 CCID₅₀ compared to 10 CCID₅₀, respectively [3, 134, 135]. Nerve damage in the lower spinal cord results in paralysis of the lower limbs (spinal poliomyelitis ), whereas damage in the upper spinal cord and medulla may result in bulbar poliomyelitis and paralysis of breathing [72]. The percent of infections ending in paralytic poliomyelitis is further reduced in highly immunized populations. This ratio of asymptomatic cases to paralytic cases has implications for surveillance strategies (see page 8154) based on investigation of all AFP cases. Between these two extremes falls the “minor disease” [72, 136], approximately 5% of infections with wild polio that result in abortive poliomyelitis with fever, fatigue headache, sore throat, and/or vomiting, and another 1–2% result in non-paralytic poliomyelitis with aseptic meningitis, pain, and muscle spasms. The incubation period is between 7 and 14 days but ranges between 2 and 35 days [72]. Virus can be recovered from the throat, blood, and feces by 3–5 days. It was initially thought that viremia was infrequent, but this was based on observations in patients with paralytic poliomyelitis who most likely already had high circulating titers of neutralizing antibodies [72, 137]. However when observations were made early after exposure, for example, in contacts of cases, a high frequency of viremia was demonstrated, implying that the viremia might play a vital role in the development of paralytic poliomyelitis [136, 138]. This was strengthened by concurrent experiments that demonstrated a protective effect against CNS lesions by antiserum in experimentally infected primates. The genetic basis for neurovirulence of poliovirus isolates is addressed below on page 8146.

Paralytic poliomyelitis, encephalitis, and aseptic meningitis occur after a biphasic infection where viremia in some systemic infections is followed by infection of the CNS [136, 138] (Fig. 5). Studies of virus in the CNS and stools in VAPP patients suggested that the virus that invades the CNS was randomly selected [95]. Acute flaccid paralysis (AFP) is a direct result of destructive replication in motor neurons followed by atrophy of de-enervated muscles. Skeletal muscles are affected when nerves in the anterior horn of the spinal cord are infected and bulbar paralysis occurs when cranial nerves are infected [4, 139]. The maximum effect on muscles occurs within a few days after the start of symptoms. Muscle recovery can occur when infection only results in temporary loss of nerve function. Residual paralysis may last from months to the life of the infected individual [72].

**Polio and Its Epidemiology. Figure 5**

Poliovirus infections are not the only cause of AFP. Non-polio AFP occurs with an incidence of 1 per 100,000 children (see page 8153 for the implication this has for surveillance). Guidelines that help epidemiological investigators distinguish AFP caused by polio from AFP caused by other causes are reviewed in Sutter et al. [4]. Final diagnosis requires laboratory confirmation of a poliovirus infection.

Poliovirus infections start as a local infection of cells in the tonsil, intestinal M cells, Peyer’s patch of the ileum, and the mesenteric lymph nodes [3, 72]. This replication in the gut results in the excretion of poliovirus during defecation by all individuals with asymptomatic as well as symptomatic infections and is the basis for fecal–oral transmission . It also provides the rational for supplementary environmental sewage surveillance (see page 8154) for poliovirus infections. A review of publications between 1935 and 1995 on excretion of polioviruses by Alexander and associates [140] indicated that in most infections of naïve children, wild polioviruses were excreted for 3–4 weeks with a mean rate of 45% at 28 days, and 25% of the cases were still excreting during the sixth week. In contrast, fewer than 20% excreted vaccine strains after 5 weeks. Excretion of polio ranged from a few days to several months [141]. The highest probability of detecting poliovirus positive stool samples was reported to be at 14 days after the onset of paralysis [140] and is the basis of stool sample collection for diagnosis of polio AFP surveillance (see page 8153). The disappearance of poliovirus from sewage samples and from stool samples of immunized children within 6–8 weeks after an immunization campaign [142] or after transition to exclusive immunization with IPV [143] provides additional confirmation for the short duration of excretion. Persistent poliovirus infections are the exception and will be discussed in more detail below. Interestingly, more than one evolutionarily linked lineage of the same serotype may co-circulate in the gut of such persistently infected individuals [79, 104, 107, 109].

Excretion and the duration of excretion are dependent on host factors and on vaccination history of the infected individual. Immunization history may start with passive immunization from maternal antibodies. However, maternal antibodies have an estimated half-life of approximately 1 month [144]. Based on a comparison between titers in cord blood and at 6 weeks, the half-lives for maternal neutralizing antibodies against type 1, 2, and 3 polio were 30.1 days, 29.2 days, 34.6 days, respectively [119]. Immunization history obviously also includes polio vaccinations and natural exposure to endemically circulating wild poliovirus and waning immunity in aging cohorts.

Skeletal muscle injury, including injury caused from intramuscular injections, increases the likelihood of poliomyelitis in children infected with wild or vaccine poliovirus. Mouse model studies have suggested that in this provocative poliomyelitis, the muscle injury facilitates viral entry to nerve axons and subsequent damage to the motor neurons in the spinal cord [145].

Some individuals who had poliomyelitis develop new muscle pains, hypoventilation, new or increased weakness or fatigue and paralysis decades later after a period of relative stability. This reappearance of polio-related symptoms is referred to as postpolio syndrome . There is a large body of literature relating to postpolio syndrome that will be left up to the reader to pursue. Suggested starting points include the websites of the Post-Polio Health International (www.post-polio.org), the Mayo Clinic (www.MayoClinic.com), a 1992 paper on the “Epidemilogy of the post-polio syndrome” by Ramlow et al. [146], and a 2010 review on the pathophysiology and management of postpolio syndrome by Gonzalez et al. [147]. There is still a debate whether persistent poliovirus or mutated poliovirus contribute to the development of postpolio syndrome [147]. The risk factors include the extent of permanent residual impairment after recovery from the poliovirus infection, an increased recovery after AFP possibly related to the extra stress on compensatory neural pathways and overuse of weakened muscles, the age of onset of the initial illness, and physical activity performed to the point of exhaustion.

Natural infections with poliovirus stimulate both humoral and cell-mediated immunity (see [149, 150] and reviews [4, 148]). Neutralizing antibodies appear in exposed individuals around the time that paralytic symptoms become evident in the few individuals who develop symptomatic infections [72]. Neutralizing IgG and IgM antibodies are also induced in response to immunization with inactivated polio vaccine. The neutralizing antibodies induced after exposure to live or inactivated poliovirus prevent disease by blocking virus spread to motor neurons of the central nervous system [3]. Once seroconversion occurs after vaccination, individuals are protected from disease for life, although circulating antibody titers may wane late in life and may drop below protective levels against one or more serotype in some individuals.

The epitopes on vaccine-derived and wild poliovirus strains that induce neutralizing antibodies may differ from those on vaccine strains. Neutralizing antibody titers ≥1:8 against each of the three Sabin OPV serotypes are considered protective; however higher titers may be needed to compensate for the relatively lower antigenicity of wild and vaccine-derived strains [151]. For example, the highest serum neutralizing antibody titers were recorded from individuals immunized exclusively with OPV or IPV when the live challenge virus was the same as that used in vaccination, slightly lower for the respective heterologous strain, and significantly lower for wild and vaccine-derived strains. Serum from some individuals who had titers of >1:50 against Sabin vaccine strains had titers of <1:8 against some wild or vaccine-derived of at least one serotype, suggesting that titers of 1:64, 1:32, and 1:16 against Sabin serotypes 1, 2, and 3, respectively, might be more appropriate to ensure minimal protective coverage [151].

Primary infection in the intestinal tract by wild poliovirus or live attenuated polio vaccine induces secretory IgA antibodies in addition to IgM and IgG antibodies. One of the rationales for the use of live attenuated polio vaccine was that while disease would be prevented by humoral antibody production stimulated by either OPV or IPV, the extent of infection or reinfection and shedding would also be reduced by induction of secretory IgA antibodies by active infection of intestinal cells with live vaccine in mimicry of the natural route of infection [148, 152–155]. IgA induced in the gut plays an important role in terminating primary infection in the intestinal tract and the tonsils [3, 152] affecting both fecal–oral and oral–oral transmission. In practice IPV also induce some intestinal immunity although less than OPV and the duration of excretion in individuals immunized with IPV appears to be longer [23, 56, 156–158].

There is some indication that the duration and possibly memory of intestinal protection is relatively short and the time for clearance of virus relatively longer than the 3–6 or 7–14 days incubation period of the minor and paralytic diseases [152, 156, 159]. Complete blockage of replication in the intestines may occur in only 25–40% of fully immunized children [158, 159]. The rapid decline in intestinal immunity means that polio can establish transient infections even in persons with adequate humoral immunity and circulate silently in that community. Lower efficiency of oral vaccines under certain conditions further complicates efforts to break chains of poliovirus transmission . Passive immunization with maternal antibodies, which has a short half-life, also affects oral vaccine efficacy (see page 8143).

It is not clear what role cell-mediated immunity may play in the control of polioviral infections. Cell-mediated immune responses were observed early after wild poliovirus infections by the macrophage migration inhibition (MIF) technique but were not observed a later time [160], whereas intradermal administration of subfractional doses caused induration and erythema of 3 mm diameter or above, in 14 of 18 vaccinees that indicated a cell-mediated immune response [161]. In addition, at least in a mouse model, all three serotypes stimulated cross-reactive and serotype-specific T helper cell responses detected by both in vitro proliferation and interleukin (IL)-2/IL-4 production [162].

How can poliovirus infections be prevented? The main tools in the global eradication of poliomyelitis have been the introduction of universal vaccination (vaccine) and improvements in hygiene. The primary goal of routine immunization is to protect the individual [163]. The secondary goal is to immunize a high enough proportion of the population so that the entire population will become protected. As eradication approaches completion, it is becoming more and more apparent that additional approaches will need to be employed in parallel with and perhaps instead of vaccination to extinguish the last pockets of endemic person-to-person transmission and persistent infections. All of these approaches will be necessary to prevent and control reemergence of polio after eradication. For more information, the readers are referred to excellent reviews on inactivated poliovirus vaccine by Plotkin and Vidor [164] and live oral poliovirus vaccines by Sutter et al. [4]. Sources for early history can be found in A History of Poliomyelitis by Paul [165] and Polio Vaccine: The First 50 Years and Beyond edited by E. Griffiths et al. [166].

The road to the development of effective vaccines against poliovirus was long and paralleled the growing understanding and ability to manipulate viral infections in the laboratory. Most of the important early milestones were listed in the last paragraph of the introduction and in Fig. 1. Mass vaccination trials and studies involving millions of vaccinees played an early and important part in acceptance of universal polio vaccination as a means for fighting poliomyelitis [19]. It must be stressed that problems and other difficulties during this progression stimulated numerous basic and epidemiological research studies that have resulted in improvements culminating in the current safe high-potency oral and inactivated vaccines that have reduced the number of annual paralytic poliomyelitis cases from >350,000 per year in 1988 to approximately 1,500 in the last few years. Difficulties in reducing this further are discussed below on page 8166. Criteria for quality control for production of polio vaccines introduced by the WHO in 1962 have been updated in relation to newly acquired knowledge about the epidemiology of poliovirus and polio vaccine. One of the major risks associated with the use of live vaccine is that progenies of the vaccine readily accumulate mutations some of which may reverse attenuation. The highest risk for vaccine-associated paralytic paralysis, VAPP, comes from Sabin 3, the vaccine serotype that also has the highest variability across production lots [3]. However the risk of OPV-associated polio is less than 0.3 per million doses [22] with the risk highest in naive children receiving their first dose [81]. The risk (see page 8156) of not using oral vaccine for global eradication compared to its use at the current stage in the Global Poliovirus Eradication Initiative remains overwhelmingly in favor of its use [1].

Three incidents nearly derailed early efforts to develop and employ effective vaccines. The most glaring of these, primarily from the point of views of negative publicity for use of polio vaccines, was the “Cutter Incident” in 1955 where wild poliovirus was inadequately inactivated probably because of failure to remove clumps that may have sequestered and protected infective vaccine virus, and a nonlinear tailing-off of inactivation at low titers [22, 167, 168]. Altogether more than 400,000 children were inoculated with an inadequately inactivated vaccine batch produced by the cutter vaccine production facility which resulted in 94 cases of poliomyelitis among primary vaccinees, 126 cases in family contacts, and another 40 cases among community contacts and 10 deaths. The publicity caused great concern throughout the world until the cause was discovered and corrective measures applied. The second, apparent failure of early vaccines to protect against subsequent infection and paralytic disease due to an initial lack of awareness in the 1950s that there were three non-cross-reacting serotypes of polio has already been mentioned. The third problem, the contamination of live polio vaccine with SV40 virus, a simian virus, continues to raise concerns about long-term effects from human zoonotic infection with this virus that was shown to cause cancer in mice [22, 169–171]. The SV40 was inadvertently introduced through the use of SV40-infected simian cell cultures in some early vaccine production batches. So far there is little evidence for any contribution to the incidence of tumors in the humans who received SV40.

The many vaccination formulation and vaccination schedules that have been employed during the effort to eradicate polio and the rational for their use have been reviewed in depth [4, 164]. Changes in schedules and formulations mean that in any one region different cohorts in the total population will have received different vaccine formulations and immunization schedules. This complicates determining duration of protection and interpretation of events. The evolution of vaccination policy in Israel [172–174], a graph showing the history of poliomyelitis in Israel (Fig. 6), and the two disagreeing discussions that were published within the same report on the underlying causes that enabled the last outbreak in Israel in 1988 [175] are a good example of this difficulty.

**Polio and Its Epidemiology. Figure 6**

Isolation of poliovirus with attenuated neurovirulence was a prerequisite for the development of oral polio vaccines (OPV; see reviews [3, 4, 166]). Vaccine candidates were either derivatives of neurovirulent or even highly neurovirulent (e.g., Sabin 3) isolates selected for attenuation after passage in primates, primate cell cultures, and/or non-primate cell cultures or starting from isolates with low neurovirulence (e.g., Sabin 2). Neurovirulence refers to the ability of an isolate to cause an infection adversely affecting functions of the CNS, keeping in mind that for any neurovirulent isolate, only 5% of infections cause transitory adverse CNS effects and less than 1% cause permanent paralytic poliomyelitis. The total number of nucleotide differences between vaccine strains and their respective parental strains was found to be 57 nucleotides and 21 amino acids for serotype 1 [176–178], 2 nucleotide differences and 1 amino acid difference for serotype 2 [179, 180], and 10 nucleotide differences and 3 amino acid differences for serotype 3 [181, 182]. Sequence analysis coupled with genetic manipulation has allowed investigators to pinpoint which of these nucleotide differences between vaccine candidates and vaccine strains account for the loss of neurovirulence (Fig. 3) [93, 183]. “Quantitative determination of the contributions of each substitution is complicated by several factors: (a) The role of minor determinants of attenuation is difficult to measure, (b) some substitutions have pleiotropic effects on phenotype, (c) some Sabin strain phenotypes require a combination of substitutions, (d) second-site mutations can suppress the attenuated phenotype in various ways, and (e) the outcome of experimental neurovirulence tests may vary with the choice of experimental animals (monkeys versus transgenic mice) or the route of injection (intraspinal versus intracerebral)” [3]. The propensity of vaccine to evolve and revert to neurovirulent phenotype is discussed throughout the current review.

The safety and effectiveness of live attenuated polio vaccine strains in preventing poliomyelitis was very clearly demonstrated in large clinical studies involving millions of children in the 1950s [19]. A number of factors including vaccination schedules, the presence of maternal antibodies, hygiene, and nutritional status of the individual influence the efficiency of induction of seroconversion by OPV strains. Early studies showed that viral interference between strains in the trivalent vaccine and from concurrent infections with non-polio enteroviruses also influences vaccination outcome [19]. Multiple doses of OPV are recommended to ensure seroconversion rather than to boost waning immunity [184]. The number of OPV doses that is needed to reach 90–95% seroconversion rates in naïve children is not the same for all populations. For example, three doses will seroconvert 90–95% of naïve children in developing countries, whereas in certain regions within developing countries such as India, the same three doses will only seroconvert a maximum of 60% of vaccinees [184]. Supplemental immunization activities (SIAs) employed sometimes more than once a year are needed to ensure adequate primary vaccination coverage and to fight endemic circulation of wild poliovirus or reintroductions of wild poliovirus. In SIAs, all children in national or subnational regions are immunized in national immunization day (NID) and/or subnational immunization day (SNID) campaigns , respectively, with a dose of OPV irrespective of vaccine history. The costs of the additional doses needed to raise seroconversion rates to above 90%, significantly raise the cost for effective immunization with OPV and require the coordination and use of many paid and voluntary staff. In fact, in the end it may actually be easier to immunize three times with IPV (even at current costs) than with the additional number of doses of OPV especially when access to populations is difficult and environmental conditions challenge maintenance of viability of the live vaccine. This counters both the lower cost and difficulty of administration rationales for using OPV instead of IPV. Mass immunization campaigns have rapidly boosted herd immunity [3].

The take of OPV is negatively influenced by the presence of maternal antibodies. Nonetheless, when infants are fed OPV at birth, 30–60% excrete virus, 20–40% of infants seroconvert, and the subsequent take of OPV is better when a birth dose is given (reviewed in [184, 185]).

Vaccination formulation must also take into account differences in the efficacy of induction of intestinal immunity by the different vaccine serotypes [155]. For example, type two was 100% effective with two doses, whereas types 1 and 3 needed more than three doses. Since the elimination of wild type 2 in 1999 [186], and the significant decrease in the number of endemic regions where wild type 1 and 3 co-circulate, SIAs have increasingly turned to the use of monovalent and divalent OPV. Monovalent OPV vaccines improve seroconversion rates compared with tOPV [187]. However routine immunization still requires the use of tOPV to prevent the accumulation of large cohorts of individuals who are naïve to type 2 poliovirus and who could serve as a reservoir for transmission of neurovirulent type 2 VDPV as has occurred in Nigeria [106]. New guidelines for the use of mOPV1, mOPV2, and dOVP1+3 have been recently issued [188].

The use of inactivated poliovirus is an alternative approach to vaccination against polio (reviewed in [164]). Salk developed an inactivated polio vaccine , IPV, using neurovirulent strains of the three serotypes of poliovirus. IPV was successfully tested by a placebo-controlled trial in over 400,000 children and in unblinded observations on another 1,000,000 children before certification for use in the mid-1950s [16, 17, 189]. A relatively higher difficulty in production, greater production costs, higher difficulty in administration, and the initial belief that only live vaccine would efficiently evoke intestinal immunity led to the choice of OPV for most routine national vaccination programs [122]. Countries are currently switching to vaccination with IPV in combination with OPV or more often in place of OPV because of its relative safety record (no VAPP cases), improvements in manufacture that have increased effectiveness and reduced the cost difference between a dose of OPV and IPV, and the paradoxical success of OPV in reducing poliomyelitis as an epidemic disease in most countries [164]. Additional motivation has come from the increasing awareness that fully neurovirulent vaccine-derived polioviruses behave like wild polioviruses [133, 190, 191] that must be eliminated and prevented from emerging in order to attain final success for poliomyelitis eradication.

Early studies on genetic and antigenic variation such as a study of Sauket strains, the type 3 used in production of IPV [192] were instrumental in the establishment of rigorous standards for seed stocks for vaccine production. The original IPV formulation has since been improved. This enhanced IPV, eIPV, has a higher immunogenicity and protective efficacy than IPV [157]. A number of factors contributed to this improvement. These included new production protocols, new tissue culture techniques including a microcarrier-based technology, and a more optimal balanced formulation of the three serotypes. It can be administered alone or can be combined with other vaccines such as DTP. The immunogenicity of eIPV was at least as good as that of OPV and there was good long-term immunity [157]. Subdermal administration of fractional doses of IPV was one of the approaches tried in the early 1950s [16]. Subsequently seroconversion rates from fractional doses were shown to be adequate but somewhat lower than for full dose intramuscular injections fractional doses [119, 193]. In another trial, similar seroconversion rates were observed but there were lower median titers in those receiving fractional doses [120]. Fractional doses effectively boost titers in previously immunized individuals [194]; however there is no long-term information on the rate of waning immunity in individuals treated with these fractional doses. Large non-inferiority studies testing subdermal administration of fractional doses of IPV using needle-free devices such as recently by Mohammed et al. [120] and Resik et al. [119] offer one quite promising practical solution for realizing cessation of use of live OPV with affordable alternative vaccines as recommended by the Advisory Committee on Polio Eradication in 2004 [195].

Antiviral drugs offer a promising complimentary or alternative approach to the use of vaccine to control poliovirus infections especially for persistent infections in immunodeficient individuals, during the final stages of eradication, and for post-eradication reemergence [122]. Presumably theses drugs may also work to control severe infections by non-polio enteroviruses or have been chosen because they have been shown to do so. Drugs with unique virus-specific targets such as capsid proteins, the hydrophobic pocket, the RNA-primed RNA polymerase, protease inhibitors, protein 3A inhibitors, nucleoside analogs, proteinase 2c inhibitors, and compounds with unknown mechanisms of action have been reviewed [196]. There is still a long way to go to find truly effective universal anti-polio or anti-enteroviral drugs, thus only a few examples will be provided.

Pocket factor drugs such as WIN 51711 [74], isoflavenes [61], pleconaryl [197], and capsid inhibitor V-037 [198] prevent viral entry by interfering with receptor binding or by preventing conformational changes needed for viral capsid uncoating. One of the difficulties in developing pocket factor drugs comes from the quasispecies nature of enteroviral infections, where mutants may rapidly emerge [61, 63] or there may be viral isolates already present in the quasispecies that have mutations in the capsid that may either render the isolate resistant or even dependent on the drug for growth. Furthermore these resistance mutations may not even have to be at the drug binding site (see, e.g., [61]).

Ribavirin is a drug that normally interferes with mRNA capping. While enterovirus mRNA is uncapped, the polio polymerase can incorporate ribavirin into both negative and positive-strand progeny RNA molecules increasing mutagenesis above the catastrophe limit causing a decrease in the reproductive capacity of the viruses [66, 94] (discussed above on page 8138).

Passive immunization has also been tested as a means of preventing polio . Administration of immunoglobulin shortly after exposure to polio may reduce the incidence or severity of paralytic disease although its general use is not practical due to the short time during which it is effective [199]. Intravenous preparations of immunoglobulin prepared from human populations exposed to enteroviral infections have however helped to decrease chronic meningoencephalitis infections by these enteroviruses in agammaglobulinemic patients [200]. Regular intravenous treatment may help prevent poliomyelitis in immunodeficient individuals but may not prevent virus replication and shedding [62]. Passive immunization with immunoglobulin or human milk rich in anti-polio IgA together with another antiviral pleconaryl may have helped to resolve at least one persistent poliovirus infection [125]. However, efforts to cure another persistent poliovirus infection with human milk and ribavirin, or other antiviral treatments, did not prove successful [63] and this individual has continued to excrete highly diverged vaccine-derived poliovirus for more than 20 years [62]. Anecdotally, shedding of intestinal mucosa associated with a superinfection with Shigella sonnei may have helped to cure another persistent excretor [62].

Poliovirus Infections in Populations

Poliovirus infections in populations have been the subject of many reviews over the years. The older reviews are still of interest not only because of the information they review but because they also provide a picture of policies and knowledge available at the time. The following paragraphs will concentrate on those aspects of poliovirus infections in populations that impact the most on the endgame strategy of poliomyelitis eradication. The discussion will start with a brief overview of the changing nature of the epidemiology of poliovirus infections. This will be followed by a description of the Global Polio Eradication Initiative and will end with a discussion of the three main problems that have led to a delay in its realization, namely, “failure to vaccinate,” “vaccine failure,” and “vaccine-derived polioviruses.” When reading this section which will highlight some of the current problems and their solutions, the reader must keep in mind the overwhelming success of the Global Polio Eradication Initiation to date: a major reduction in the number of endemic countries where polio is still transmitted from 126 to 4; a decrease in the number of annual cases by >99% that prevented life-long paralysis in more than five million children between 1988 and 2005; eradication of one wild poliovirus serotype in 1999; and elimination of the majority of wild lineages throughout the world.

The nature of poliovirus infections in populations has gone through a number of phases. Before the appearance of outbreaks of poliomyelitis starting in the nineteenth century, poliovirus circulated endemically. Infections occurred in the very young, and conferred lifelong immunity against reinfections with the same serotype. The epidemics became more frequent, grew in size, and infections included older children and adults who were not naturally immunized. Vaccination has drastically reduced the number of people who have been exposed to natural infection with wild polioviral strains. There also appears to be a shift in person-to-person transmission routes. Oral–oral transmission has increased in importance while fecal–oral transmission decreased as a result of improved hygiene [56]. Control of poliomyelitis requires breaking all chains of wild poliovirus transmission by immunizing all children with three doses of polio vaccine or at least enough children so that herd immunity protects the remaining population. The percent of the population that needs to be immunized for establishment of herd immunity against wild poliovirus is >85% for developed countries and >90–95% in tropical developing countries. In 2010, there were still three groups of countries: four “endemic countries ,” Afghanistan, India, Nigeria, and Pakistan where the transmission of wild viruses has not yet been completely halted, “polio-free countries ” where vaccination has broken all endemic chains of wild poliovirus circulation and there have been no cases other than VAPP within the last 3 or more years, and “importation countries ” that were formerly polio-free, but where there are poliomyelitis cases caused by wild poliovirus imported from one of the “endemic countries” and where there may be local transmission of the imported wild poliovirus. Between 2002 and 2006, there were 26 importation counties, 7 with viruses imported from India and 19 with viruses imported from Nigeria [4]. There were 21 importation countries in 2009 and 13 in 2010. These included ongoing outbreaks in Tajikistan and the Russian Federation and apparently expanding outbreaks in Angola and the DRC. The former represents the first cases from wild poliovirus in the European region since regional eradication was declared in 2006 and the latter could potentially spread to polio-free countries in Africa and other regions. The list of importation countries is updated on a weekly and monthly basis and can be accessed at the web page of the Global Polio Eradication Initiative, www.polioeradication.org. Transmission routes are dependent on population movements. One or more outbreak founders may be introduced by infected persons coming from an endemic or importation country or by returning travelers from such countries. One event with very high risk for spreading poliovirus from one country to another is the Hajj in Saudi Arabia. To counter this threat, it is now mandatory for foreign pilgrims coming on Hajj and Umrah to be vaccinated for communicable diseases including polio, especially pilgrims with young children arriving from countries with polio cases. The children must have received a dose of OPV 6 weeks before their arrival and another upon arrival.

Smallpox was eliminated as a circulating human pathogen in 1977 after an 11-year extensive vaccination and surveillance program [201]. Only two sources of smallpox virus have been reserved for research purposes, one in the United States and one in Russia. Final eradication will be achieved when these last two remaining, contained sources of smallpox virus are finally destroyed. Proposing a similar approach in 1988, the World Health Assembly set a goal of eradicating poliomyelitis by the year 2000 (resolution WHA41.28). A group of experts at the global, regional, and country level set the criteria and conduct the process of certification of eradication [202]. These experts must be independent from the vaccine administration and polio laboratories. Global polio eradication, first targeted for completion by 2000, was limited to the eradication of all wild polioviruses with the caveat that “the occurrence of clinical cases of poliomyelitis caused by other enteroviruses, including attenuated polio vaccine viruses, does not invalidate the achievement of wild poliovirus eradication” (Report of the first meeting of the Global Commission for the Certification of the Eradication of Poliomyelitis. Geneva: World Health Organization; 1995. WHO document WHO/EPI/GEN/95.6). OPV vaccination would cease within a few years after eradication of poliomyelitis from wild polio and industrialized nations would save not only the large sums of money needed for the maintenance and rehabilitation of individuals with paralytic poliomyelitis but the costs of vaccine and vaccine administration as well [203–206].

In order to easily eradicate an infectious agent, (a) the agent should replicate in a single host with no intermediate vector, alternative reservoir species, or carrier state, (b) vaccines and/or anti-infectious agents must be available to break chains of transmission whether directly between susceptible organisms or after exposure to the infectious agent in the environment, (c) if transmission involves environmental exposure, there must be a finite and relatively short survival time for the infectious agents in the environment, (d) all or the majority of infections should be clinically apparent with unique symptoms, and (e) there must be an easy and cost-effective surveillance system for detection of the infectious agent, identifying infected individuals, and for determining the efficacy of treatments in individuals and populations [3, 207]. Deviations from some of these requirements for eradication have made eradication of polio more difficult from the start [56]. In particular, most (>95%) poliovirus infections are clinically asymptomatic while symptoms associated with the few infections that result in poliomyelitis are not unique to poliovirus infections and although effective vaccines were available, there is no easy and cost-effective method to determine the effectiveness of vaccination in vaccinated individuals. This makes surveillance and the identification and isolation of infected individuals much more difficult. When the GPEI resolution was passed in 1988 and even as late as 1996, it was stated [208] that there was no indication of chronic excretors; however, persistent infections (see page 8161) do exist and have emerged as one of the difficulties in achieving eradication. Differences between smallpox and polio have made it relatively more difficult to achieve polio eradication . For example, live polio vaccine is made from three temperature-sensitive serotypes of poliovirus because there were three non-cross-reacting poliovirus serotypes, whereas the vaccine for smallpox was a single more stable unrelated bovine virus. This has complicated vaccine formulation and administration. In addition since the vaccine contains live poliovirus there are also a number of more severe safety issues concerning pre- and post-eradication vaccine production compared with the smallpox vaccine.

Specific eradication strategies for polio included (a) high routine immunization with at least three doses of vaccine for all children and an additional birth dose in countries where polio has remained endemic (b) SIAs, either national or subnational immunization campaigns targeting children under 5 years of age (c) surveillance, primarily investigation of all cases of AFP with an increase in the number of supplementary surveillance programs such as sewage surveillance and enterovirus surveillance as the endgame of eradication approaches, and (d) house-to-house mopping-up immunization campaigns to block final chains of wild polio transmission [1, 163, 203]. The WHO requires genomic sequencing of all isolates of potential interest to the Polio Eradication Initiative. An isolate is of interest when results from either standard immunological and/or molecular tests conducted by accredited laboratories using standard methods (see next section) indicate that the isolate has behaved differently than standard Sabin strains of the corresponding serotype.

A Global Polio Laboratory Network, GPLN , was established to monitor poliovirus infections throughout the world using standardized methods, cell lines, reagents, and reporting methods [209–213]. These standard methods (WHO/EPI.CDS/POLIO/90.1) have been reviewed and revised and improved as knowledge about the epidemiology of polio expands and as new analytical methods become available (Fig. 7). This includes even the flow charts or “algorithms” for culturing viruses, identifying polioviruses, and characterizing the serotype (Typic Differentiation) and determining wild, vaccine, or vaccine-derived virus within specific time frames (Intratypic Differentiation). The current fourth version of the Polio Laboratory Manual (WHO/IVB/04.10) was adopted in 2004. The three levels of laboratories, National and Sub-national Laboratories, Regional Reference Laboratories, and Global Specialized Laboratories, are certified each year through on-site visits, after accurate testing and timely reporting of a minimum number of relevant assays, and by results from proficiency tests. The requirements for certification, quality assurance, and safety and the responsibilities of the three types of laboratory are spelled out in the Polio Laboratory Manual (WHO/IVB/04.10). By the end of 2009, the GPLN consisted of 146 laboratories of which 139 were fully accredited by the WHO, and another 5 in the process of accreditation (WHO 16th Informal Consultation Of The Global Polio Laboratory Network, September 2010, Geneva, Switzerland).

**Polio and Its Epidemiology. Figure 7**

The Polio Laboratories are coordinated on a regional basis by Regional Laboratory Coordinators who report to the Global WHO Polio Coordinator in Geneva. Identification tasks such as intratypic differentiation and sequence analysis originally assigned to the more specialized labs are now being certified for use in National and Regional Reference Labs as expertise increases and methods – especially molecular methods are simplified. This trend has been accelerated by the increasing difficulty and costs of shipping material that may contain live infectious wild polioviruses between the different levels of laboratories and the need for decreasing the time between isolation and final notification of characterization of the poliovirus isolates. Rapid identification is especially critical for eradication efforts in endemic regions and for identifying introductions to polio-free regions from these endemic regions.

The laboratories work in close coordination with epidemiologists and medical staff in the investigation of all cases of AFP and/or supplementary enterovirus surveillance and with municipal employees and epidemiologists where supplemental environmental surveillance is utilized to screen for poliovirus presence and circulation. In late 2010, a commercially available method for preparing noninfective viral RNA suitable for molecular analysis based on automatic nucleic acid extraction, immobilization, and storage on Flinders Technology Associates (FTA) filter papers was being evaluated to increase safety and drastically reduce costs of shipping material between laboratories (Summary and Recommendations of the 16th Informal Consultation Of The Global Polio Laboratory Network, 2010, Geneva). Using this technology, viable virus could be reconstituted from the RNA after it is transfected into eukaryotic cells.

Intratypic differentiation (determination of the serotype of an isolate) was based on results from one immunological ELISA test [214] and one molecular-based test, either probe hybridization [215], diagnostic RT-PCR [216], RT-PCR and RFLP [217], RealTime-RT-PCR [218], or micro-array-based systems [80]. At the 16th Informal Consultation Of The Global Polio Laboratory Network, 2010, Geneva, the recommendation for ITD testing from ITD-accredited laboratories was modified to one of the following three options: (a) two RealTime RT-PCR procedures, one for ITD and one for detecting vaccine-derived poliovirus and sending all non-Sabin-like viruses or Sabin-like viruses with non-Sabin-like VP1 to higher level labs for full-length sequencing and molecular analysis of VP1, (b) one validated ITD method (ITD, or molecular) and shipment of all viruses to higher level labs for full-length sequencing and molecular analysis of VP1 or (c) on-site full-length VP1 sequencing of all isolates or referral of all virus isolates to higher level labs for full-length sequencing and molecular analysis of VP1. Results are confirmed by sequence analysis of the entire VP1 capsid gene. Molecular analysis of the sequences of the genomic RNA encoding the VP1 capsid gene is in fact the definitive method to determine whether an isolate is a vaccine strain, a VDPV, or wild isolate. Sequence analysis of the VP1, all four capsid genes (e.g., P1), and even the entire genome, infers evolutionary relatedness to other isolates in endemic or external reservoirs. The methodology and results from such analyses that help trace the origin of viruses founding outbreaks have been clearly presented in reviews by Kew et al. [101] and Sutter et al. [4]. Sequence data is kept in databases maintained by the specialized laboratories of the Polio Laboratory Network, such as the CDC in Atlanta, GA, Pasteur Institute in Paris, and the HTL in Finland. Phylogenetic comparisons of sequences from new isolates, routinely provided by the CDC, indicate evolutionary relationships to previously isolated polioviruses from the same region and trace importations to or from external reservoirs. Important epidemiological information can be obtained from this phylogenetic analysis. For example, a significant gap between a new sequence and all other known sequences indicates a gap in surveillance while an importation implies that there are cases or silent circulation of related viruses in the region containing the reservoir that it is most closely related to. Knowledge obtained about time clocks [39] for nucleotide substitutions (see page 8139) allow investigators to infer whether nucleotide differences between two isolates are consistent with local transmission or represent separate introductions (e.g., see Manor et al. [102]).

The currently recommended standard method for poliomyelitis surveillance is based on the isolation and molecular and serological analysis of all viruses from all cases of acute flaccid paralysis , AFP, to rule-in or rule-out polioviral etiology [219]. The definition of an outbreak varies depending on whether endogenous poliovirus transmission has remained unbroken or the area has been found to be polio-free. In the latter, given the goal of eradication, a single AFP case can be considered to be an outbreak. The previous section describes what is needed for timely high quality testing of all poliovirus isolates from cases and from other sources such as environmental surveillance. Much time, effort, and money has been spent on maintaining and improving lab quality assurance and performance of laboratories in the Global Laboratory Network. However two factors outside of the control of the laboratories strongly influence the final result. The first is sample collection and the second is the conditions under which the sample is stored and shipped to the first processing lab. The most appropriate sample with the highest probability of detecting poliovirus is a 5-g stool sample. For AFP cases, two stool samples (not rectal swabs) should be collected 1–2 days apart within 14 days of onset of paralysis. This is based on a review of studies measuring the timing of viral excretion (discussed on page 8143) in infected individuals [140] and the fact that detectable viral excretion is sometimes intermittent. Standardized tissue culture conditions using limited passages of poliovirus sensitive L20B, HEp2C, and RD cells provided by Specialized Laboratories of the Global Polio Laboratory Network are used according to standard operating procedures to isolate polioviruses from clinical samples [219]. An amended algorithm for isolating polioviruses designed to reduce the workload and the time between receipt of sample and identification of viruses of interest has been successfully evaluated in a number of National Poliovirus Laboratories (WHO 16th Informal Consultation Of The Global Polio Laboratory Network September 2010, Geneva, Switzerland). Standard typing and intratypic differentiation assays are based on results from serological assays and molecular assays as described above with final characterization based on the full-length sequence of VP1. Additional regions such as the 5’UTR, the entire P1 region encoding all four capsid proteins, the 3D polymerase, or the entire genome may be sequenced for higher resolution and to determine whether and to what extent genomic recombination has occurred.

Molecular data from any polioviral isolates recovered from the stool samples provides information about the serotype of the isolate or isolates, and differentiates between VAPP, persistent VDPV, and circulating VDPV or wild polioviruses . The different time clocks for single nucleotide substitutions [39] and unique recombination patterns are important tools for these analyses. Timely AFP surveillance also provides the necessary critical information about the temporal and geographic distribution of the isolates for efficient and economical infection and outbreak response. The rational for AFP surveillance is based on the observation that AFP from all non-polioviral causes occurs with an incidence of 1 per 100,000 in children up to the age of 15. When all AFP cases are investigated and the AFP incidence is within the range for non-polio causes, the absence of poliovirus in the stool samples from any AFP case is considered to indicate absence of circulating poliovirus in the region under surveillance. A surveillance area is considered to be wild poliovirus-free when adequate AFP surveillance levels for greater than 3 years indicate absence of wild poliovirus, and entire WHO-designated regions are considered to be free from endogenous wild polioviruses when all countries within that region are wild poliovirus-free.

Wild poliovirus positive AFP cases in previously polio-free areas or WHO regions can occur. Molecular analysis then reveals the most likely external reservoir from which the virus was transmitted [101]. Two recent examples of country-to-country transmission (see page 8156) which have seriously impacted the eradication initiative are the spread of wild polioviruses to >21 polio-free countries [220] as consequences of the temporary cessation of vaccination in Nigeria starting in 2003 and the spread of wild polio into the European region in 2010 [221] enabled by low vaccine coverage in Tajikistan. Sequence analysis of poliovirus isolates from cases in Mumbai confirmed cessation of local chains of transmission and the reintroduction of viral lineages still circulating in the north of India [222].

Most countries employ AFP surveillance. However not all are able to reach the required incidence of AFP investigations. Some of these countries supplement AFP surveillance with enteroviral surveillance and/or environmental surveillance (Fig. 7). Enteroviral surveillance is the systematic identification of the enterovirus genotypes in all clinical infections in general or more specifically from all cases with associated meningitis and encephalitis , symptoms that may appear more frequently than AFP in patients with poliovirus infections (approximately 5% of poliovirus infections compared to 0.5–1% for AFP). In some countries enteroviral surveillance and/or environmental surveillance are used exclusively.

A number of different sampling techniques have been used to obtain environmental samples including grab sampling during peak sewage usage, trapping with silicates or gauze, and automatic composite sampling of sewage aliquots at given time intervals over a 24-h period (Guidelines for environmental surveillance of poliovirus circulation, WHO/V&B/03.03 [223]). All sample storage and shipment must be at low temperatures (4–8°C) to maintain viability since currently certified tests require an amplification step in tissue culture. The FTA technology trial referred to above may eliminate the need for maintaining low temperatures.

The usefulness of L20B cells to isolate polio isolates in the presence of high titers of non-polio enteroviruses [222] has already been mentioned. It is still important to characterize the viruses that grow since L20B cells can also support growth of some other human and bovine enteroviruses, as well as less well-characterized viruses [56]. Additional steps involving molecular screening and growth at elevated temperatures has enabled investigators to more easily identify and characterize wild and vaccine-derived polio in the presence of high titers of vaccine viruses [102, 215, 216, 218, 224, 225]. Selective growth of non-vaccine poliovirus at elevated temperatures [226] is based on a relative small decrease of titer for these viruses compared to a much higher reduction in yield for vaccine strains at elevated temperatures. The main molecular determinant responsible for this difference is a single nucleotide change in loop V of the 5’UTR which can revert or be modified by other changes, hence some polioviruses of interest may escape detection and some minimally diverged vaccine virus may be included among the selected isolates. Confidence that polioviruses isolated by environmental surveillance reflect circulating viruses comes from the high sequence homology between environmental samples and isolates from cases [38, 143, 227]. One of the major contributions of environmental surveillance reviewed by Hovi et al. [143] is that it can be used to establish the presence and/or circulation of wild or vaccine-derived polioviruses before the appearance of AFP cases [102, 222, 227–229]. Environmental surveillance has also revealed the presence of presumptive primary vaccinees excreting OPV in Switzerland where vaccination is by exclusive use of IPV [56].

Different methods for analyzing environmental samples are also currently employed in different laboratories [223] since unlike AFP surveillance [230] there is as of yet no single standard method. The probability of detecting poliovirus in environmental samples [229] depends on the duration and amount of poliovirus excreted by one or more infected individuals (see page 8143), the effect of physical and mechanical factors on the dilution and survival of poliovirus in the sewage system (reviewed by Dowdle [134]), and the frequency of collection and laboratory processing of the environmental samples [223]. A model based on these factors [229] predicted that environmental surveillance could outperform AFP surveillance for small outbreaks as well as detect circulation before the appearance of cases. The location of the sampling site relative to the excretor and the number of excretors (Fig. 7) also determines the probability of detection [143]. In general polioviruses can be quantitatively recovered from the environment [231]. Decreasing this distance between the excretor and the sample collection site is more effective in increasing the probability of detection and less labor intensive than increasing the sampling frequency [109]. Environmental surveillance is resource and labor intensive and may require large capacity high-speed centrifuges that are not commonly present in most National Poliovirus Laboratories [143]. It requires judicious choice of potential target populations, a competent laboratory, a plan for routine surveillance and reporting, and the cooperation of municipal authorities. The WHO has recommended principles for selecting sites, sampling strategies, and interpretation of results [223]. Lengthy periods of poliovirus-free monitoring are needed to confirm that poliovirus transmission has stopped since even the most comprehensive surveillance covers only subgroups of the entire population of potential excretors [232].

Many poliovirus positive environmental samples contain one or at best a few polioviruses of interest indicating that the surveillance is operating at the lower limits of detection. Thus while negative findings cannot rule-out the presence of polioviruses at levels below detection, they gain significance when they are part of a long sequence of negative results from frequent routine surveillance at the given site. A positive finding of a wild poliovirus or a VDPV can trigger a response ranging from public announcements to remind individuals scheduled for routine vaccinations to be vaccinated in time in areas with high vaccine coverage to scheduling NIDS or SNIDS in regions where immunization coverage is below that required for establishing herd immunity. Sequence analysis can differentiate between multiple importations and local circulation when more than one poliovirus is isolated within a short interval of time [102].

Detection of “orphan polioviruses ” or virus that are not closely linked to previously sequenced isolates indicates gaps or suboptimal surveillance. The length of the gap is roughly proportional to 1% single nucleotide divergence per year [39]. Orphan viruses [91, 105, 191] were responsible for most cVDPV outbreaks. This is in contrast to the situation in Nigeria where intensive AFP surveillance triggered by the circulating wild polioviruses also revealed the initial stages in circulation of multiple lineages of predominantly type 2 cVDPVs [106]. The presence of type 2 cVDPVs, iVDPVs, and aVDPVs is of concern since despite elimination of transmission of wild type 2 in 1999, neurovirulent serotype 2 poliovirus is still among us [124, 128].

A number of countries that switched from OPV or combinations of OPV and IPV to exclusive use of IPV conducted environmental surveillance for OPV after the transition (reviewed in [143]). The OPV rapidly disappeared from the environmental samples but imported OPV-like isolates have been isolated from time to time presumably imported from OPV-using countries [233].

One of the important milestones toward achieving eradication is the containment of all potential sources of the pathogen. In 1999, a process for containing all laboratory stocks of wild poliovirus was initiated by the World Health Assembly entitled Global action plan for the laboratory containment of wild polioviruses or GAP 1 (WHO/V&B/99.32). A revised plan, GAP II, included two phases: (1) the identification in all facilities of all known poliovirus stocks and any material that could potentially contain live wild poliovirus, for example, any stool specimens that were collected at times when poliovirus was endemic, and (2) the containment of these stocks by destroying them, rendering them noninfectious, or transferring them to a minimal number of laboratories certified by the WHO as having appropriate BSL3/polio biosafety facilities and justification to work with wild viruses. A draft of the next version, GAP III, extends containment of wild polio to now also include containment of OPV/Sabin strains, and concentrates on minimizing risks associated from facilities that work with polioviruses and vaccine production and storage facilities after eradication of wild poliovirus transmission and cessation of OPV vaccination. Pathways of exposure from these facilities and assessment of the risks from a literature review have been calculated [134]. After risk analysis, a goal was set to reduce the number of such facilities globally to <20 essential facilities that meet required safeguards.

The original target date for polio eradication was not met. By 2001, the WHO Global Commission for the Certification of the Eradication of Poliomyelitis extended eradication to include elimination of circulating VDPVs (Certification of the Global Eradication of Poliomyelitis Report of the sixth meeting of the Global Commission for the Certification of the Eradication of Poliomyelitis. Washington D.C., 28–29 March 2001 WHO/V&B/01.15). The current target date for interruption of all wild poliovirus has been moved to 2013 (Global Polio Eradication Initiative – Programme of Work 2009 and financial resource requirements 2009–2013. WHO/POLIO/09.02). This section will conclude with a discussion of the three major problems that have accounted for the delay in completing the GPEI, “failure to vaccinate,” “vaccine failure,” and the emergence of “vaccine-derived viruses.”

Among the reports available online at the WHO website for polio eradication , www.polioeradication.org, is a report on the annual percentage of children in each country who received a minimum of three doses of polio vaccine annually since 1980. This report provides a complete picture of current polio immunization status. However the variability in coverage between countries and the annual fluctuation within countries illustrates the problem of failure to vaccinate and is also an indication of problems in sustaining the high coverage necessary for successful eradication.

Wild and vaccine-derived poliovirus can penetrate and circulate within areas where vaccination coverage is low or where vaccination has been discontinued [106, 117, 234]. When this occurs this is an example of “failure to vaccinate.” The temporary cessation of vaccination in Nigeria in 2003 [235, 236] is usually presented as the classic example for the consequences of a failure to vaccinate. The situation was complicated by suboptimal coverage within Nigeria when vaccination resumed within 12 months and the suboptimal coverage in other countries that had person-to-person contacts with infected Nigerians. Thirteen countries with 52% coverage have had multiple introductions of wild poliovirus from Nigeria, while another eight countries with higher 83% coverage did not have repeated outbreaks [1]. Use of type 1 mOPV was successful in reducing the number of cases from wild type 1 [190, 234] but the decreased use of tOPV lead to a significant increase in cases due to wild type 3. Subsequent use of mOPV1, mOPV3, and bOPV has effectively reduced the number of cases due to wild types 1 and 3 [237]. Unfortunately suboptimal immunization with any vaccine that contained type 2, presented fertile conditions for the emergence of multiple lineages of type 2 CVDPVs some of which have continued to circulate well into 2010 [106] (see discussion on page 8157, 8161).

A 2010 outbreak that started from a wild type 1 virus imported into Tajikistan from India spread into the Russian Federation. This was the first outbreak due to wild poliovirus in the WHO European region since it was declared polio-free in 2002 [221]. Again failure to vaccinate with coverage sufficient to maintain herd immunity was the main factor that facilitated the outbreak. As of June 2010, wild poliovirus cases from this outbreak accounted for more than 70% of all wild polio cases reported in 2010. Four NIDS with mOPV1 were conducted since the start of the outbreak.

When poliomyelitis occurs in vaccinated individuals it is categorized as “vaccine failure .” Current reports on India (20th Meeting of the India Expert Advisory Group for Polio Eradication Delhi, India, 24–25 June 2009 www.polioeraication.org) indicate that most of India is poliomyelitis-free with the exception of the north where both wild type 1 and type 3 still circulate. Type 1 and 3 mOPV have helped to reduce the number of circulating lineages and to constrict the areas within which the wild polioviruses are still circulating and causing cases. However, lack of cases in the south does not mean absence of wild poliovirus as environmental surveillance has revealed silent wild polio in Mumbai. The reservoir is not only a problem for India. Populations with suboptimal coverage in other countries are also at risk, as shown by the 2010 outbreak in Tajikistan and the Russian Federation that was traced back to northern India [221].

Vaccine failure in children in India refers to the finding that antibody response or seroconversion in children required more than the recommended three doses of tOPV [238, 239]. In Uttar Pradesh and Bihar in north India, local conditions exist where even administration of five doses of OPV does not induce the expected seroconversion rates. Approximately 15 doses were required to reach population immunity [240]. The fact that the age when the disease is acquired had not shifted upward was taken as an additional indication of vaccine failure [1]. Various trials of efficacy of mOPVs and bOPV and fractional IPV are underway to evaluate their short-term effectiveness in halting endemic transmission and their long-term performance in maintaining protective titers. In India mucosal immunity in response to vaccination with OPV varied depending on location, serotype, and vaccine formulation [241].

The high number of additional doses needed to achieve herd immunity in some regions such as in northern India combined with the additional cost of OPV in annual and sub-annual vaccination campaigns must be taken into account when comparing the cost effectiveness of OPV and IPV in inducing effective herd immunity.

A less serious type of vaccine failure is based on observations that do not completely confirm the paradigm that OPV prevents replication during subsequent exposures to poliovirus. Israeli children who had concluded a primary immunization schedule consisting of three doses of OPV and three doses of IPV by 18 months of age had seroconverted for all three serotypes with geometric mean titers >1,000 [159]. One month after the last vaccination they were challenged with an additional dose of OPV. Up to 60% of children excreted at least one OPV serotype between 1 and 3 weeks, the upper range of similar studies reviewed in that report. There was no evidence of transmission to siblings or mothers of these children, most likely because of good hygiene [159]. These rates were comparable to other similar studies [159]. Hygiene and high coverage probably also contributed to the fact that there was also no evidence for OPV circulation in IPV-vaccinated populations in the United States living adjacent to OPV-vaccinated populations in Mexico [242].

A number of comprehensive reviews on vaccine-derived polioviruses have been published [3, 62, 79, 105, 114, 243–246]. As described above (page 15), vaccine-derived polioviruses evolve either during person-to-person transmission (cVDPVs) or during relatively rare [247] persistent infections in immunodeficient patients (iVDPVs) (see reviews [3, 4, 105, 114]). To date (2010) there have been 12 cVDPV outbreaks [106]. Using the definition of outbreak in the context of eradication (i.e., even a single case), the number of outbreak may be even higher. For example, in 2009, 21 cases due to cVDPVs were found in four countries in addition to 153 in Nigeria and a case in Guinea traced back to Nigeria [248], and the cases in Nigeria represent emergence of multiple independent lineages [106].

Most outbreaks caused by cVDPVs have been caused by a single lineage that spread rapidly through a susceptible cohort within the general population. The outbreaks were only discovered after silent circulation of the VDPVs for more than 1 year or more indicating gaps in surveillance. By the time such outbreaks became evident, the genomes of the isolates had usually recombined with those of the progeny of other vaccine serotypes or closely related non-polio enteroviruses. When OPV is introduced or reintroduced into a population with cohorts of naïve individuals as in Nigeria, adequate surveillance revealed that in early stages of reemergence more than one independent lineage may emerge [106]. There is also a potential for recombination of cVDPVs with wild-type viruses in areas where both co-circulate as shown from retrospective molecular analysis of isolates during endemic circulation of wild polioviruses [82]. Luckily from the point of view of eradication, cVDPV outbreaks resemble outbreaks of poliomyelitis from wild polioviruses introduced into polio-free areas and their chains of transmission can be broken by similar vaccination responses [106, 234].

In certain circumstances poliovirus can establish persistent infection in immunodeficient individuals. The types of immune deficiencies of known chronic excretors have been reviewed [3, 4, 62, 105]. The genomes of the Sabin strains are unstable [114] and reversion of nucleotide changes that attenuated neurovirulence appear even among the progeny virus excreted by primary OPV vaccinees. These reversions are believed to improve the replicative fitness of the isolates [123] and are responsible for the rarity of vaccine-associated paralytic poliomyelitis cases (VAPP; 1 per 500,000–1,000,000 vaccinations of naïve infants, and 7,000 times higher for some immunodeficient individuals) and cVDPV outbreaks [3, 105]. Thus it is not surprising that reversion of attenuation also occurs at an early stage in chronic excretors [62]. During 4 months of observation of long-term excretion in a healthy child [62] type 1 virus diverged by 1.1% and evolved toward full reversion to wild-type phenotypic properties similar to the Mahoney parent of the Sabin 1 strain. It is less obvious why these isolates so quickly predominate in the quasispecies of persistently infected immunodeficient individuals. The process by which persistence is established and maintained may present selection through bottlenecks within a single individual that is similar to the bottleneck by which only a single progeny or a subpopulation of the quasispecies is passed onto the next host in person-to-person transmission among immune competent hosts. Selection by passage through bottlenecks was also suggested to explain evolution of wild poliovirus during long-term expression [249]. Examination of the genomes of iVDPV isolates differentiates them from cVDPVs in that significantly fewer heterotypic recombinations occur [4, 110] and intrageneous recombination appears to be largely absent [4, 105]. Interestingly, more than one highly divergent lineage may be recovered from a single stool sample from persistently infected individuals [3, 110]. This suggests that persistence and evolution occur in separate sites although intratypic recombination indicates that some mixed infections in a single cell must occur. Only a single serotype was detected in most (30 of 33) long-term excretors identified between 1962 and 2006 [4] and this pattern has continued to date. Amino acid changes in capsid proteins may allow polo to establish persistence in cells of the CNS [250].

Molecular analysis of phylogenetically related highly diverged (>10%) aVDPVs isolated from sewage in Finland, Slovakia, and Israel reveals a pattern of amino acid substitutions in or near neutralizing antigenic epitopes and lack of intrageneous recombination that resembles the pattern of changes in iVDPVs and is qualitatively different from evolutionary changes in cVDPVs [109, 251]. This pattern and the extended periods of time over which phylogenetically related polioviruses have been isolated from the same sewage systems and surveillance sites within those systems strongly suggests that replication of the related viruses has taken place in one immunodeficient host, or a very limited number of individuals in contact with such a host. Routine monthly sewage surveillance of catchment areas representing 35–40% of the population in Israel intermittently and repeatedly revealed the presence of highly diverged type 2 VDPVs 2 between 1998 and 2010. Phylogenetic analysis indicated that the isolates came from two different and unrelated persistent infections. Isolates form one foci have been isolated intermittently for 12 years and the second for 4 years. In addition there was a single, respectively, and a single isolation of a highly diverged type 1 VDPV. The situation in Finland is particularly interesting and unusual, since evidence suggests that the infected individual is simultaneously and persistently infected with three highly diverged VDPV serotypes [251].

Most mutations in iVDPVs and aVDPVs are synonymous and are observed in third position codons. These synonymous substitutions occur at similar rates to those for poliovirus during person-to-person transmission [39, 101, 107, 109]. Non-synonymous amino acid substitutions affect antigenicity, neurovirulence, receptor binding motifs, hydrophobic pockets, and drug sensitivity.

The prevalence of aVDPV excretors is unknown, but additional countries with excretors of aVDPVs are being reported as environmental surveillance is introduced into more and more regions [143, 246, 252–256]. Hovi et al. [143] have proposed expanding the suggestion that the GPLN include regular monitoring for cVDPVs [257] through increasing the number of laboratories that employ routine environmental surveillance. It is important to determine the exact nature of the immune status of these types of persistent excretors since it may be different than that for identified persistently infected individuals. Unfortunately the individuals infected with these aVDPS remain unidentified, and will most likely remain so for a long time [143]. The most frustrating attempt to locate such an excretor occurred in Slovakia where moving sampling sites progressively upstream successively restricted the excretor to a population of 500 individuals before detection ceased [143].

There is no consensus on the extent that persistent VDPVs may affect the realization of eradication [133, 163]. Determining the number of unidentified persistent infections is becoming more urgent as eradication of wild polio approaches (WHO 16th Informal Consultation Of The Global Polio Laboratory Network. 22–23 September 2010, Geneva, Switzerland, WHO/HQ. Summary of Discussions and Recommendations). Some researchers believe that VDPVs may pose an insurmountable problem [114, 127] while others feel that the problem is less severe [3, 4, 126]. Most of the identified persistent excretors had primary B-cell-related immunodeficiencies [4, 105]. While molecular epidemiological analysis has indicated that highly diverged neurovirulent anonymous VDPVs isolated from sewage in Finland, Slovakia, and Israel [109, 233, 258] resemble the molecular epidemiology of poliovirus isolates excreted over time by identified excretors of iVDPVs, the exact nature of the immune status that has presumably enabled infection to persist in these aVDPV excretors remains unknown. The time course of excretions, the rate of nucleotide substitutions in virus isolated from identified persistent excretors, and genomic recombination patterns have been consistent with the establishment of persistence and evolution of the virus in these individuals rather than transmission of an iVDPV. Highly diverged iVDPVs (as opposed to cVDPVs and less diverged iVDPVs) have not been found during routine AFP surveillance of cases of immune competent individuals [3]. One clear indication that iVDPV-like aVDPVs are transmissible comes from a study of silent transmission in infected children in an under-vaccinated community in Minnesota [259] where 8 of 23 infants had evidence of type 1 poliovirus of VDPV infection. While this absence of documented transmissibility of very highly diverged VDPVs is encouraging, it may only be circumstantial, since most of the highly diverged neurovirulent aVDPVs have been found in the environment of countries with high vaccine coverage and good hygiene barriers that have also prevented circulation and appearance of wild poliovirus cases even after neurovirulent wild polio was introduced from external reservoirs [102].

The amino acid substitutions in neutralizing antigenic epitopes/receptor binding residues in iVDPVs and iVDPV-like aVDPVs may have helped specialize these virus isolates for microenvironments within the gut during persistent infections. These same changes might affect/reduce transmission via the oral–oral route in communities where there is high vaccine coverage and good hygiene. If true, this might significantly reduce the threat to eradication, despite the highly neurovirulent nature of these isolates in animal model systems and the decreased geometric mean neutralizing antibody titers against some of these excreted iVDPV and aVDPV isolates in the general public in the highly immunized communities where these isolates are found [109, 233]. It must also be taken into account that identified and unknown excretors are free to travel to communities with poor vaccine coverage and substandard hygiene where the fecal–oral transmission route is more important and continuous person-to-person transmission easier to maintain.

Future Directions: The Endgame Stage of Eradication and Sustainability of Postpolio Eradication

This section will start with an overview of current accomplishments to provide a suitable background for the discussion of future directions and sustainability. One of the best online sources for keeping up to date on eradication can be found at www.polioeradication.org.

There has been a >99% overall reduction in the number of cases since adoption of the Global Poliovirus Eradication Initiative in 1988 [248]. The Region of the Americas (AMR) was certified to be free from all three serotypes of indigenous wild polioviruses in 1994 [260, 261] and the last case anywhere in the world from wild type 2 polio occurred in India in 1999 [186, 262]. Subsequently the Western Pacific Region (WPR) in 2000 [263] and the European Region (EUR) in 2002 [264] have also been certified to be free from indigenous wild polioviruses. These successes have been due to the dynamic nature of the eradication program where vaccination strategies have been adapted in response to specific problems and to changing conditions emerging as the endgame approached [1]. Four countries remain where indigenous poliovirus has continued, Afghanistan and Pakistan in the WHO Eastern Mediterranean Region (EMR), Nigeria in the WHO African Region (AFR), and India in the WHO South-East Asia Region (SEAR).

The accumulated costs for the vaccination program have exceeded 4.5 billion US dollars. National governments (list by alphabetical order: Australia, Austria, Belgium, Canada, Denmark, Finland, Germany, Ireland, Italy, Japan, Luxembourg, the Netherlands, Norway, the United Kingdom, and the United States) have provided a significant portion of the necessary funding. NGOs (WHO, UNICEF, Rotary International, the Bill and Melinda Gates Foundation, and the International Red Cross and Red Crescent societies), the World Bank, and corporate partners (Aventis Pasteur, De Beers) have also made significant contributions toward purchase of vaccines and for applied and basic polio research. In addition to paid professional staff, more than ten million volunteers have assisted in the global vaccination program. Their knowledge of local practices and beliefs has provided a significant asset to the GPEI [1].

One of the goals of eradication was to reach a stage where vaccination could be discontinued, as was the case for smallpox vaccinations after eradication of smallpox [265]. The estimated annual savings would be enormous and could be used to fund other global health initiatives. Similarly the organizational capabilities experience expertise and facilities of member Laboratories in the Global Polio Laboratory Network would also be employed to solve other health-related problems. The three main problems that have delayed eradication originally scheduled for 2000 have been discussed. Among these problems, chronic excretion of vaccine-derived viruses probably remains the most serious obstacle since the number of excretors remains unknown and there are no universally recognized methods of curing chronic excretion in those chronic excretors who have been identified.

Between January 2009 and June 2010, the Global Polio Laboratory Network analyzed 258,000 fecal specimens from 130,000 AFP cases for the presence of poliovirus, and between January 2009 and September 2010 it detected introductions of wild poliovirus into 23 previously “polio-free countries,” countries where indigenous polio transmission had been interrupted. Nineteen were in the African region and included seven countries (Burkina Faso, Benin, Chad, Côte d’Ivoire, Mauritania, Niger, and Togo) where the outbreak isolates were related to previous importations into those countries as was the case for Sudan in the Eastern Mediterranean Region. One of the more serious setbacks for eradication was the introduction of wild poliovirus into Tajikistan from Uttar Pradesh in India marking the first outbreak in the European region since it was certified poliovirus-free in 2002 [221]. The large outbreak (>450 confirmed cases) ensued spread into the Russian Federation and resulted in an immediate tenfold increase in the amount of samples that needed to be processed by the Polio Laboratories in the region. In all of these importation countries and/or regions, large-scale coordinated SIAs were conducted. The spread of wild poliovirus to poliovirus-free countries from Nigeria and India via Tajikistan illustrate the need to maintain high population immunity until all transmission of wild virus has ceased. Similarly, the emergence of multiple lineages of neurovirulent VDPVs in Nigeria and the increasing frequency of isolation of aVDPVs as more countries adopt environmental surveillance reinforce the need to discontinue use of OPV globally in a coordinated effort or staged manner. See Ehrenfeld et al. [266] for a review and discussion of key issues that have affected and will affect the GPEI, including: safety for volunteers in areas of strife, the low efficiency of OPV to induce herd immunity in certain settings, the requirement for maintaining high coverage even after eradication, the inherent mutability of OPV, problems for establishing the safety and efficacy and costs of new vaccines, new vaccine formulations, and scaling up alternatives to OPV.

The saying “May you live in interesting times,” often attributed to an ancient Chinese proverb or curse, appears appropriate for describing the current status in the quest to eradicate wild polioviruses. Eradication, which is tantalizingly close, will require substantial changes in vaccination policy and practice [117]. It must also include appropriate emergency response measures to control reemergence. “The ideal vaccine choice for the stockpile should be effective in any outbreak scenario, protect all vaccinees with one dose, spread to and protect the unvaccinated population, and have no detrimental effect” [267]. Long-term effects should be considered. While mOPV might be the most effective in rapidly controlling an outbreak and spreading and protecting unvaccinated individuals [267], plans that preferably do not require use of OPV adjacent to areas with high concentrations of unvaccinated individuals would be better in the long run [117, 268]. The reader is referred to the website of poliovirus eradication (www.polioeradication.org) for the latest information on past, current, and future policies.

Three problems have delayed the realization of eradication as has been discussed. Currently available vaccines can overcome “failure to vaccinate,” provided that enough doses of vaccines are made available, that there is the political will to use them, and that natural or man-made disasters do not prevent reaching the children for vaccination. Preliminary results from newly approved monovalent and bivalent oral polio vaccines and clinical studies using fractional doses of IPV indicate that there may already be a solution for “vaccine failure ” which is exemplified by the poor seroconversion rates for OPV in northern India [240]. The third major problem, “vaccine-derived polioviruses” is more complex, since VDPVs can evolve by person-to-person transmission (cVDPVs) or during persistent infections (iVDPVs). The spread of cVDPVs can be interrupted using the same methods as used to stop transmission of wild poliovirus (paradoxically including use of OPV in vaccination campaigns), since cVDPVs behave like wild virus [106, 234]. Moreover, while it is easy to say that current GPEI plans to coordinate global cessation of the use of OPV will prevent VAPP [206] and emergence of new cVDPVs, at this juncture the actual process is quite complicated and associated with a number of risks. The main problem that will need to be solved is the shedding of highly neurovirulent VDPVs into the environment for prolonged periods by identified and unidentified, persistently infected individuals. There is currently no universal solution to this problem [63, 125]. As long as shedding persists (perhaps as long as some of these individuals remain alive), containment as envisioned in GAP III will be incomplete and high vaccination coverage will need to be maintained. A related but more difficult problem that will need to be solved is (a) to determine the prevalence of unidentified, presumably persistently infected individuals who are responsible for shedding the highly diverged aVDPVs that have been isolated from environmental surveillance, and (b) to identify the presumably persistently infected individuals to determine the physiological conditions that enabled persistence and to try and clear the persistent infection with current or future antiviral treatments. As the endpoint of eradication of wild poliovirus is approached, the number of cases of poliomyelitis will decrease while the number of silent infections may increase as a result of high vaccine coverage. Under these conditions supplemental surveillance programs such as enterovirus surveillance and environmental surveillance will become an even more important tool for providing geographical information for designing NIDS, SNIDS, and final mopping-up campaigns for eradication and for monitoring for post-eradication reemergence.

“Although OPV has been the mainstay of the eradication program, its continued use is ironically incompatible with the eradication of paralytic disease (since) vaccine-derived viruses consistently emerge as a consequence of the inherent genetic instability of poliovirus [122].” “Eradication of vaccine” suggested in 1997 [204, 205] has become recommended policy on condition that provisions of GAP III for safety in vaccine production and polio laboratories are met [264, 269]. A model describing the impact of cVDPVs on eradication indicated that the probability of eradicating polio with continuous use of OPV was not very likely [270]. Alternative vaccination should be continued during and especially after the transition to maintain high coverage and to avoid the buildup of large susceptible populations during the time when there is the highest risk for reemergence of OPV strains [117, 134, 270–272]. Low population immunity remains the main known risk factor for the emergence and spread of cVDPVs [234, 243]. Since most of the cVDPVs in outbreaks circulated silently for months or years (VP1 divergence >2%) before detection in AFP cases, it is imperative that surveillance be improved and expanded to high-risk regions to detect silent circulation of VDPVs as early as possible.

Endgame vaccination strategies have been reviewed [3, 122, 163, 266, 268] and include (1) indefinite use of OPV, (2) cessation of all polio immunization (3) transition to use of IPV, by synchronous coordinated cessation of all use of OPV with (a) limited use of IPV or (b) replacement of all OPV with IPV, (4) country-by-country cessation of OPV use with options (3a) or (3b), (5) sequential removal of Sabin strains from OPV, as eradication proceeds, (6) development of new vaccines, and (7) indefinite use of IPV or new vaccines. The synchronous cessation of OPV has several problems particularly if inexpensive alternatives are not in place when it occurs, since this vacuum may result in large populations of naïve individuals, in whom, polio could reemerge, after periods of silent circulation, with high force and rapid spread. Such a scenario also does not address the potential risks of unidentified chronic excretors. A gradual shift to IPV may avoid some of the programmatic disadvantages that coordinating a synchronous shift would have on vaccination programs and vaccination production facilities. It also potentially provides a longer window for industry to increase production, integrate information from current fractional vaccine dosage and alternative routes of administration trials, and overcome problems of biocontainment and antigenicity associated with optional use of killed OPV as a substitute for the wild strains used in IPV production.

The WHO and UNICEF regularly consult informally with vaccine manufacturers to discuss the implications and practicality of vaccine policy decisions (summaries are available from the Internet using variations of a search for “WHO/UNICEF Informal Consultation with IPV and OPV Manufacturers”). For example, the 3rd WHO/UNICEF Informal Consultation with IPV and OPV Manufacturers (2003) included a discussion of post-eradication needs and biocontainment requirements and the 5th (2006) included updated information on progress of the GPEI and OPV cessation strategies.

The financial requirements for the transition period are complicated and have been set forth by the WHO (WHO Global Polio Eradication Initiative – Programme of Work 2009 and financial resource requirements 2009–2013 WHO/POLIO/09.02). The bottom line is that alternatives to OPV must be affordable [234]. Three recent reports deal in depth with the economics and practicality of universal replacement of OPV with IPV: (a) Global Post-eradication IPV Supply and Demand Assessment: Integrated Findings, March 2009, and (b) The supply landscape and economics of IPV-containing combination vaccines: Key findings, May 2010, both commissioned by the Bill & Melinda Gates Foundation and prepared by Oliver Wyman, Inc., and (c) Improving the affordability of inactivated poliovirus vaccines (IPV) for use in low- and middle-income countries – An economic analysis of strategies to reduce the cost of routine IPV immunization, April 20, 2010, prepared for PATH by Hickling, Jones, and Nundy. The second report [273] presents a thorough review of the current options and risks for new vaccines and vaccine formulations for achieving and maintaining eradication. New generations of inactivated polio vaccines may need to be developed for post-eradication use [266, 274] and they may have to be used indefinitely.

A number of decisions must be made now, some based on incomplete knowledge, because of the long lead time needed between planning facilities and final production of regulatory agency-approved products. For example, while fractional doses significantly reduce costs, they are less effective than full doses and there is little data on kinetics of waning, while questions still exist concerning sufficient antigenicity of Sabin IPV. Additional complications involve testing and regulatory approval of new products or formulations (see discussion on regulations and standardization of IPV and IPV combination vaccines in Baca-Estrada and Griffiths [275] and the views of vaccine producers [276, 277]). The good news is that when “new” polio vaccine, type 1 mOPV, was needed, it was produced by two companies and licensed in three countries in a relatively short time, 6 months [135, 278, 279]. (Quotation marks were used around the word new since in actuality millions of monovalent doses of each serotype had been used before introduction of tOPV [280] when old licenses had been left to expire). Licensing was also aided by the fact that monovalent batches were produced and safety tested before being combined to produce tOPV and only qualified tOPV producers were approached to provide mOPVs [279]. Ironically if mOPVs are more effective than respective serotypes in tOPV because of increased titers and longer replication times, the increased number of nucleotide substitutions may increase the potential for cVDPV outbreaks by the serotype used [103] or conversely from the remaining serotypes (or serotype if bOPV is used). Supporting this is the emergence of significantly higher numbers of type 1 viruses with increased antigenic divergence from Sabin 1 after a birth dose of mOPV1 and a second exposure to Sabin 1 [111]. Most (71%) were isolated from stools from infants who did not seroconvert after the birth dose [111]. Rapid licensing of bOPV on January 10, 2010, followed release of efficacy results on June 2009 (issue 6 PolioPipeline, summer 2010). The bad news is that combination vaccines containing IPV cannot be frozen raising questions about long-term stability and appropriate reference standards [275].

There have been a number of attempts to rationally redesign the sequence of vaccine seed strains to make them more stable and safer to use in vaccine production facilities in the post-eradication period [3]. One drawback is that there is no empirical data on how these new viruses will behave in the field especially in relation to genome stability and the ability to recombine with heterotypic or intragenic enteroviruses. Changing codons to equivalent but rarely used synonymous codons based on studies of codon use bias or increasing the frequency of CpG and UpA dinucleotides are methods to change the substitution rate [97, 281, 282]. Others modifications have led to polioviruses that can grow in nonhuman cell lines for production but have very low ability to infect human cells.

Widespread vaccination will continue at least during the 3-year period between the last case due to wild poliovirus and certification that wild poliovirus transmission has been interrupted globally. However, global vaccination should be continued for much longer since by one model [283], after 3 years there would only be a 95% certainty that all silent circulation had in fact ceased and the probability after 5 years ranged between 0.1% and 1%, while a more recent model has predicted a very high probability of reemergence within 10 years after eradication by VDPVs or accidental release of virus from vaccine production facilities, a polio laboratory, or bioterror [272]. Consequently vaccination will need to continue for at least 10 years after eradication. A special issue of the journal Risk Analysis (Volume 26, Issue 6, 2006) has been devoted to risks associated with polioviruses before, during, and after eradication of wild poliovirus. Finally vaccination with IPV may be continued indefinitely at least in countries where aVDPVs continue to be isolated from the environment with attendant risk from a polio vaccine production facility operating in a polio-free era (see discussion above on GAP III). To reiterate, current contingency plans for use of OPV in response to reemergence need to be revised based on the data on circulation of live vaccine strains after temporary and/or partial cessation of vaccination [117].

The final and one of the most important problems that must be successfully dealt with is to answer the question: “How can current achievements and eradication be sustained once the endgame has been concluded?” Ideally a major public health undertaking such as eradication requires a cost-benefit analysis, sufficient funds at the beginning, the means for achievement at hand, and the political and social will to carry the process through to the end. Delays and problems with fund-raising, especially when they occur during the endgame, may derail the entire effort [1]. Some problems with sustainability are associated with management and not scientific problems [1, 284]. However, new unanticipated scientific problems may appear which further delay polio eradication. After all, awareness of the potential problems from cVDPVs in communities with low vaccine coverage and chronic excretors of VDPV primarily appeared during the endgame of eradication when the global burden of cases had decreased by >99% and only after appropriate analytic tools to easily document and confirm VDPV had became widely available [3]. An example is the revelation of the 10-year circulation of cVDPVs in Egypt starting in 1983 [285] by retrospective phylogenetic examination of VP1 sequences.

The means to prevent disease and contain the spread of virus transmission when it emerges are already in hand. Safer and more cost-effective measures are in the pipeline that include schedule reduction and fractional doses, adjuvant use, optimizing of processing, Sabin or modified Sabin IPV, and noninfectious IPV [119, 273]. Sustainability for achieving eradication will depend on vaccine policy decisions made today, on the length of time it takes to eliminate all wild poliovirus transmissions, on political will and advocacy, on the motivation of volunteers and the level of local community involvement, program-related fatigue, and on the absence of complications [286] from bio-error, bioterror, or mother nature [291]. However the major determining factor will probably be the availability of financial resources [135]. Limited resources mean competition between routine immunization and eradication efforts during endgame. A predicted 1.3 billion USD funding gap in June 2010 is already forcing a reprioritization of planned activities (Global Polio Eradication Initiative Monthly Situation Report June 2010 www.polioeracdication.org.) “Even if there are no competing health needs, it is unlikely that immunization could be maintained indefinitely against a non-existent disease at a level that is sufficient to prevent vaccine-derived viruses evolving to cause epidemics”[163]. Programmatic setbacks such as those associated with failure to vaccinate (Nigeria and Tajikistan), vaccine failure (northern India), the frequency of repeated vaccination campaigns, or post-eradication reemergence must not be allowed to derail the current momentum and lead to program-related fatigue [1]. Detailed planning must be made for any post-eradication outbreaks (see Jenkins and Modlin [267] and Tebbins et al. [268]) and provisions to implement them including stockpiling must be in place. Finally it is well worth reading “The pathogenesis of poliomyelitis: what we don’t know” by Nathanson [287] and “Gaps in scientific knowledge for the post-eradication world” by Minor [288].

Note

Polio is the second human pathogen for which there is an ongoing global program for eradication that has reached the endgame. The first, smallpox, successfully completed the endgame and is now in the stage of post-eradication sustainability. Bioterror is the main threat to sustainability of smallpox eradication. This chapter will describe some of the difficulties with completing the endgame of polio eradication and then in sustaining postpolio eradication. More than the usual number of items are included in the Glossary to make it easier for the reader to follow the progress of eradication as it unfolds in the large number of official documents that deal with a global eradication program and which contain the usual copious number of professional acronyms.

Abbreviations

ACPE:: The Advisory Committee on Polio Eradication
AFP:: Acute flaccid paralysis.
AFP surveillance:: Characterization of enteroviruses in stool samples from all AFP cases especially in individuals under 15 years of age to rule-in or rule-out etiology by polioviruses.
aVDPV:: A vaccine-derived poliovirus isolate whose evolutionary path is unknown or ambiguous.
bOPV:: Bivalent oral polio vaccine (usually containing serotypes 1 and 3).
BSL:: Biosafety standard level.
Capsid:: The protein shell that surrounds a virus particle.
Capsomere:: One of the individual morphological units that make up the viral capsid.
CDC:: US Centers for Disease Control and Prevention
CD155 or PVr:: The human encoded cell receptor for poliovirus, a member of the immunoglobulin superfamily.
Codon:: A sequence of three adjacent nucleotides on a strand of DNA or RNA that specifies which specific amino acid will be incorporated into a protein.
Codon bias:: Unequal usage of synonymous codons (different codons that specify the same amino acid)
CPE:: Cytopathic effect.
cVDPV:: A circulating vaccine-derived poliovirus, that is, a poliovirus that has evolved from vaccine during person-to-person transmission.
eIPV:: Enhanced inactivated polio vaccine.
Emergence:: The appearance of a pathogen in a previously pathogen-free area.
Endemic:: The constant presence of a disease to a greater or lesser extent in a particular locality.
Enteroviruses:: Any of >80 different species of polioviruses, coxsackie viruses, echoviruses, and enteroviruses belonging to the genus Enterovirus in the family Picornaviridae.
Environmental surveillance (as related to polioviruses):: Investigation of sewage and recreational water for the presence of poliovirus as an indication of the presence of poliovirus-infected individuals in a community.
EPI:: Expanded Program on Immunization.
Epidemic:: A rapid spread of disease into a disease-free area or spread of a disease to more than the usual number of persons affected in a region with disease.
Epitopes:: The component of an antigen that is recognized by and binds to an antibody.
Eradication:: The complete elimination of all incidence of disease and/or the presence of the agent that causes the disease.
Evolution:: Change in the genetic composition of a population or the genome of a given organism during successive generations.
GAP:: Global action plan for laboratory containment.
GAP I:: GAP phase I – plan for identifying all known and potential sources of poliovirus especially wild polioviruses within each country.
GAP II:: GAP phase II – plan for laboratory containment of wild polioviruses.
GAP III:: GAP phase III – plan to minimize post-eradication poliovirus facility-associated risks.
GAVI:: Global alliance for vaccines and immunization
Genetic recombination (of polioviruses):: A situation where one portion of the genome of a poliovirus is replaced through a covalent linkage with the equivalent segment from another poliovirus or non-polio enterovirus.
Genotype:: He genetic makeup of an organism as distinguished from its physical characteristics.
GMT:: Geometric mean titer, usually calculated according to Karber.
GOARN:: Global Outbreak Alert and Response Network.
GPEI or GEI:: The Global Poliomyelitis Eradication Initiative of the WHO, adopted in 1988.
GPLN:: Global Polio Laboratory Network.
Hydrophobic pocket:: A hydrophobic space located under the binding site for the host encoded viral receptor that is located on the bottom of the canyon surrounding the fivefold axis of symmetry of the enteroviral capsid.
Hydrophobic pocket factors:: Small hydrophobic molecules that occupy the hydrophobic pocket and that may regulate the host receptor viral capsid interaction.
Immunodeficient:: Lacking one of the components of the immune system.
Immunogenicity:: The relative ability of a molecule to elicit an immune response.
i.d.:: Intradermal or under the skin.
i.m.:: Intramuscular.
i.n.:: Intranasal.
i.p.:: Intraperitoneal.
i.t.:: Intrathecal.
Infection:: Establishment and growth of an infectious agent in the body.
IPV:: Inactivated poliovirus vaccine.
IRES:: Internal ribosome entry site.
ITD:: Intratypic differentiation (determination if a virus isolate is vaccine, vaccine-derived, or wild).
iVDPV:: A vaccine-derived poliovirus that has diverged from its respective oral poliovirus serotype during persistent infection of an immunodeficient host.
Lineages:: A group of organisms that are closely related genetically.
MAPREC:: Mutant analysis by PCR and restriction fragment enzyme cleavage to measure reversion of attenuation sites in vaccine strains.
MNVT:: Monkey neurovirulence test, an in vivo neurovirulence test in monkeys.
mOPV:: Monovalent OPV.
Neurovirulence:: The ability of the poliovirus to infect and damage nerve cells causing disease of the nervous system.
Neurovirulence attenuation sites:: Specific nucleotide positions along the poliovirus genome where the specific nucleotide present at that site will influence whether or not an individual polioviral isolate will be neurovirulent.
Neutralizing antigenic sites:: Epitopes of the poliovirus that induce neutralizing antibodies.
NID:: National immunization day.
NSL:: Non-Sabin-like (wild) virus of vaccine-derived poliovirus (based on results of certain ITD tests).
Major disease:: Poliomyelitis, AFP, or cases of infection with polio that involves invasion and permanent damage to the nervous system.
Microarray:: A technology used to study many genes at once using thousands of different short molecular sequences at known position on solid support to hybridize to complementary nucleic acid sequences from different sources.
Minor disease:: Nonspecific illness caused by poliovirus that may include upper respiratory tract symptoms (sore throat and fever), gastroenteritis (nausea vomiting, abdominal pain, constipation or diarrhea), and influenza-like illness.
NCCs:: National Certification Committees.
NGOs:: Nongovernmental organizations.
NIDs:: National immunization days.
NPEV:: Non-polio enteroviruses.
Nonstructural genes:: Viral genes encoding proteins that are not incorporated into the structure of the capsid.
Oligonucleotide:: A short sequence of nucleotides frequently synthetic.
OPV:: Live attenuated oral poliovirus vaccine.
Outbreaks:: (Under eradication conditions) even the presence of a single case of paralytic poliomyelitis.
Persistent poliovirus infection:: An infection associated with an immunodeficient host where virus is not cleared but continues to replicate for an indefinite period of time.
Phylogenetic tree:: A diagram with branches showing the inferred evolutionary relationships among various biological entities.
Picornaviridae :: A viral family made up of the small (18–30 nm) ether-sensitive single stranded, positive-sense RNA viruses that lack an envelope.
Poliomyelitis:: The infectious disease caused by poliovirus involving inflammation of motor neurons of the spinal cord and brainstem that leads to acute paralysis followed by atrophy of the muscles enervated by the infected motor neurons.
Poliovirus:: One of three serotypes of picornaviruses that can cause acute flaccid paralysis and whose cell receptor is CD155.
Polypeptide:: A small molecule constructed from linked amino acids.
Postpolio syndrome:: Slow progressive muscle pain and weakness that reappears 30 or 40 years after paralysis caused by a poliovirus infection affecting muscles previously affected by polio as well as muscles that may not have been affected.
Posttranslational processing:: Any modification of a protein after it has been translated.
Provoked poliomyelitis:: Poliomyelitis resulting from physical trauma during infection with poliovirus.
Proofreading:: An enzymatic process that checks whether a newly incorporated nucleotide in a nascent chain is the correct compliment of its corresponding nucleotide in the template.
PVR:: The poliovirus receptor, CD155.
PVR Tg21 transgenic mouse:: A mouse that has been genetically modified to express the human poliovirus receptor.
Quasispecies:: A term used to describe a cluster, cloud, or swarm of viruses with minor differences in nucleotide sequence that arise during replication as a consequence of polymerase incorporation errors.
Rearrangement (in relation to polioviruses):: A structural alteration in the genomic sequence occurring during coinfection with two or more viruses resulting in a new genome in which parts are from different parental polio or non-polio enteroviruses
Reemergence:: Emergence after an absence.
RCC:: Regional Certification Committees.
RCT:: Reproductive capacity temperature, the temperature at which viruses can replicate.
RNA-dependent RNA polymerase:: A viral encoded polymerase that synthesizes a complimentary RNA strand from an RNA template.
RRL:: Regional Reference Laboratory of the Global Polio Laboratory Network.
SAGE:: Strategic Advisory Group of Experts on Immunization.
Serotype:: A group of closely related virus expressing a common set of antigens.
Seroconversion:: Appearance of antibodies following exposure to antigen in seronegative person, or ≥4-fold increase in titer of previously immune person.
Seroconversion index:: The mean seroconversion rate against all three poliovirus serotypes.
SL:: Sabin-like poliovirus (based on result from some ITD tests).
SIA:: Supplemental immunization activities (such as NIDs, SNIDS, and mop-ups).
Silent circulation:: Person-to-person transmission of virus in a community in the absence of cases of AFP.
Silent infection:: Asymptomatic infection.
Silent presence:: The presence of a virus in the absence of clinical cases and the absence of person-to-person transmission.
SNIDS:: Sub-national Immunization Days.
Stakeholders:: All governmental and nongovernmental agencies involved in the GPEI.
Structural genes (in relation to polio):: Genes encoding viral capsid proteins.
Synonymous nucleotide substitutions:: Changes in the nucleotide sequence that do not result in a change in encoded amino acid.
TAG:: Technical Advisory Group.
TD:: Typic differentiation (determination of the serotype of a poliovirus isolate).
TOPV:: Trivalent oral polio vaccine containing all three serotypes of attenuated poliovirus.
Transition:: The substitution of a purine nucleotide with the other purine, or a pyrimidine nucleotide with the other pyrimidine.
Transversions:: The substitution of a pyrimidine nucleotide by a purine nucleotide or vice versa.
UNICEF:: United Nations Children’s Fund.
Vaccine strains:: Poliovirus strains approved by the WHO for production of live and inactivated polio vaccines.
VAPP:: Vaccine-associated paralytic poliomyelitis.
VDPV:: A vaccine-derived poliovirus that has diverged through evolution from its respective live poliovirus vaccine strain serotype by more than 1% (serotypes 1 and 3) or more than 0.6% (serotype 2) of its respective VP1 capsid protein.
Viremia:: The presence of virus in the bloodstream during an infection.
VPg:: Viral protein genome linked – 22 amino acid protein covalently linked to genome and complimentary negative strand.
VP1:: Viral capsid protein 1.
VP2:: Viral capsid protein 2.
VP3:: Viral capsid protein 3.
VP4:: Viral capsid protein 4.
WHA:: World Health Assembly.
WHO:: World Health Organization.
WPV or wild poliovirus:: Any poliovirus that is not derived from attenuated oral polio vaccine strains
3’UTR:: The untranslated region of the polioviral genome that is located 3′ of the open reading frame that encodes the viral polyprotein.
3D^pol :: Viral encoded RNA-dependent RNA polymerase.
5’UTR:: A highly structured, untranslated area of the polioviral genome located 5′ to the open reading frame that encodes the viral polyprotein. The 5’UTR is covalently linked on its 5′ base to viral protein VPg.

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Central Virology Laboratory, Laboratory of Environmental Virology at Sheba Medical Center, Public Health Services, Israel Ministry of Health, 52621, Ramat Gan, Israel
Dr. Lester M. Shulman (Head, Laboratory of Environmental Virology, Senior Lecturer)
Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel
Dr. Lester M. Shulman (Head, Laboratory of Environmental Virology, Senior Lecturer)

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Robert A. Meyers

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Shulman, L.M. (2012). Polio and Its Epidemiology . In: Meyers, R.A. (eds) Encyclopedia of Sustainability Science and Technology. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-0851-3_839

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