Poliovirus Evolution Research Articles

Underperformed and Underreported Testing for Persistent Oropharyngeal Poliovirus Infections in Primary Immune Deficient Patients-Risk for Reemergence of Polioviruses.

Individuals with primary immune deficiencies (PIDs) may excrete poliovirus for extended periods and remain a major reservoir for polio after eradication. Poliovirus can spread by fecal-oral or oral-oral transmission. In middle- and high-income countries, oral-oral transmission may be more prevalent than fecal-oral transmission of polioviruses where PIDs patients survive longer. Our aim was to determine the prevalence of prolonged or persistent oropharyngeal poliovirus infections in PIDs. We performed a literature search for reports of prolonged (excreting poliovirus for ≥6 months and ≤5 years) or persistent (excreting poliovirus for >5 years) poliovirus infections in PIDs. There were 140 PID cases with prolonged or persistent poliovirus infections. All had poliovirus-positive stools. Testing of oropharyngeal mucosa was only reported for 6 cases, 4 of which were positive. Molecular analyses demonstrated independent evolution of poliovirus in the gut and oropharyngeal mucosa in 2 cases. Seven PIDs had multiple lineages of the same poliovirus serotype in stools without information about polioviruses in oropharyngeal mucosa. Testing for persistence of poliovirus in oropharyngeal mucosa of PID patients is rare, with virus recovered in 4 of 5 cases in whom stools were positive. Multiple lineages or serotypes in 7 additional PID cases may indicate separate foci of infection, some of which might be in oropharyngeal mucosa. We recommend screening throat swabs in addition to stools for poliovirus in PID patients. Containment protocols for reducing both oral-oral and fecal-oral transmission from PID patients must be formulated for hospitals and community settings.

Poliovirus Evolution toward Independence from the Phosphatidylinositol-4 Kinase III β/Oxysterol-Binding Protein Family I Pathway.

Phosphatidylinositol-4 kinase III β (PI4KB) and oxysterol-binding protein (OSBP) family I provide a conserved host pathway required for enterovirus replication. Here, we analyze the role and essentiality of this pathway in enterovirus replication. Phosphatidylinositol 4-phosphate (PI4P) production and cholesterol accumulation in the replication organelle (RO) are severely suppressed in cells infected with a poliovirus (PV) mutant isolated from a PI4KB-knockout cell line (RD[Δ PI4KB]). Major determinants of the mutant for infectivity in RD(Δ PI4KB) cells map to the A5270U(3A-R54W) and U3881C(2B-F17L) mutations. The 3A mutation is required for PI4KB-independent development of RO. The 2B mutation rather sensitizes PV to PI4KB/OSBP inhibitors by itself but confers substantially complete resistance to the inhibitors with the 3A mutation. The 2B mutation also confers hypersensitivity to interferon alpha treatment on PV. These suggest that the PI4KB/OSBP pathway is not necessarily essential for enterovirus replication in vitro. This work supports a two-step resistance model of enterovirus to PI4KB/OSBP inhibitors involving unique recessive epistasis of 3A and 2B and offers insights into a potential evolutionary pathway of enterovirus toward independence from the PI4KB/OSBP pathway.

Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

Dynamics of Evolution of Poliovirus Neutralizing Antigenic Sites and Other Capsid Functional Domains during a Large and Prolonged Outbreak.

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

Phylogenetic Analysis of Poliovirus Sequences.

Comparative genomic sequencing is a major surveillance tool in the Polio Laboratory Network. Due to the rapid evolution of polioviruses (~1 % per year), pathways of virus transmission can be reconstructed from the pathways of genomic evolution. Here, we describe three main phylogenetic methods; estimation of genetic distances, reconstruction of a maximum-likelihood (ML) tree, and estimation of substitution rates using Bayesian Markov chain Monte Carlo (MCMC). The data set used consists of complete capsid sequences from a survey of poliovirus sequences available in GenBank.

Poliovirus Evolution: The Strong, Silent Type

The high mutation rates of poliovirus and other RNA viruses promote viral diversity by rapid sampling of mutations. In this issue, Lauring et al. (2012) report that underlying synonymous codon variation in virus populations can have a considerable impact on adaptive potential with implications for viral fitness and virulence.

Recombination between Poliovirus and Coxsackie A Viruses of Species C: A Model of Viral Genetic Plasticity and Emergence

Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV), an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C), in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs.

Analysis of codon usage and nucleotide composition bias in polioviruses

BackgroundPoliovirus, the causative agent of poliomyelitis, is a human enterovirus and a member of the family of Picornaviridae and among the most rapidly evolving viruses known. Analysis of codon usage can reveal much about the molecular evolution of the viruses. However, little information about synonymous codon usage pattern of polioviruses genome has been acquired to date.MethodsThe relative synonymous codon usage (RSCU) values, effective number of codon (ENC) values, nucleotide contents and dinucleotides were investigated and a comparative analysis of codon usage pattern for open reading frames (ORFs) among 48 polioviruses isolates including 31 of genotype 1, 13 of genotype 2 and 4 of genotype 3.ResultsThe result shows that the overall extent of codon usage bias in poliovirus samples is low (mean ENC = 53.754 > 40). The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in those polioviruses. Depending on the RSCU data, it was found that there was a significant variation in bias of codon usage among three genotypes. Geographic factor also has some effect on the codon usage pattern (exists in the genotype-1 of polioviruses). No significant effect in gene length or vaccine derived polioviruses (DVPVs), wild viruses and live attenuated virus was observed on the variations of synonymous codon usage in the virus genes. The relative abundance of dinucleotide (CpG) in the ORFs of polioviruses are far below expected values especially in DVPVs and attenuated virus of polioviruses genotype 1.ConclusionThe information from this study may not only have theoretical value in understanding poliovirus evolution, especially for DVPVs genotype 1, but also have potential value for the development of poliovirus vaccines.

Mechanisms of genetic variation in polioviruses

Polioviruses, as with all RNA viruses, are in a constant process of evolution driven by different mechanisms. With multiple mechanisms for genetic variability, they are successful conformists, adapting to changes in their habitat. The evolution of polioviruses may occur with generation of point mutations followed by genetic drift and selection. The mutation rate of polioviruses based on several studies is approximately 3 × 10(-2) mutations/synonymous site/year in the gene encoding viral protein 1. Genetic variation in polioviruses may also be increased by sharing of genetic data of two different poliovirus lineages by means of homologous recombination. According to the current view, recombination is considered usually to occur by strand-switching, but a non-replicative model has also been described. In recombination, polioviruses may either gain a set of advantageous mutations selected and fixed in previous generations of the parental viruses or get rid of deleterious ones. The prerequisites and constraints of the evolution mechanisms will be discussed. Furthermore, consequences of poliovirus evolution will be reviewed in the light of observations made on currently circulating polioviruses. We will also describe how polioviruses strike back: as wild type polioviruses approach eradication, vaccine derived strains increase their occurrence and genetic variability.

Co-Circulation and Evolution of Polioviruses and Species C Enteroviruses in a District of Madagascar

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5′-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3′-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3Dpol coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity.

Antigenic evolution of vaccine-derived polioviruses: changes in individual epitopes and relative stability of the overall immunological properties.

The Sabin oral poliovirus vaccine (OPV) readily undergoes changes in antigenic sites upon replication in humans. Here, a set of antigenically altered descendants of the three OPV serotypes (76 isolates) was characterized to determine the driving forces behind these changes and their biological implications. The amino acid residues of OPV derivatives that lie within or close to the known antigenic sites exhibited a marked tendency to be replaced by residues characteristic of homotypic wild polioviruses, and these changes may occur very early in OPV evolution. The specific amino acid alterations nicely correlated with serotype-specific changes in the reactivity of certain individual antigenic sites, as revealed by the recently devised monoclonal antibody-based enzyme-linked immunosorbent assay. In comparison to the original vaccine, small changes, if any, in the neutralizing capacity of human or rabbit sera were observed in highly diverged vaccine polioviruses of three serotypes, in spite of strong alterations of certain epitopes. We propose that the common antigenic alterations in evolving OPV strains largely reflect attempts to eliminate fitness-decreasing mutations acquired either during the original selection of the vaccine or already present in the parental strains. Variability of individual epitopes does not appear to be primarily caused by, or lead to, a significant immune evasion, enhancing only slightly, if at all, the capacity of OPV derivatives to overcome immunity in human populations. This study reveals some important patterns of poliovirus evolution and has obvious implications for the rational design of live viral vaccines.

Characterization of CHAT and Cox type 1 live-attenuated poliovirus vaccine strains.

CHAT and Cox type 1 live-attenuated poliovirus strains were developed in the 1950s to be used as vaccines for humans. This paper describes their characterization with respect to virulence, sensitivity for growth at high temperatures, and complete nucleotide and amino acid sequences. The results are compared to those for their common parental wild virus, the Mahoney strain, and to those for two other poliovirus strains derived from Mahoney, the Sabin 1 vaccine strain and the mouse-adapted LS-a virus. Analysis of four isolates from cases of vaccine-associated paralytic poliomyelitis related to the CHAT vaccine revealed genetic and phenotypic properties of the CHAT strain following replication in the human gut. CHAT-VAPP strain 134 contained a genome highly evolved from that of CHAT (1.1% nucleotide differences), suggesting long-term circulation of a vaccine-derived strain in the human population. The molecular mechanisms of attenuation and evolution of poliovirus in humans are discussed in the context of the global polio eradication initiative.

Sabin type 2 polioviruses with intertypic vaccine/vaccine recombinant genomes.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and, in rare cases, cause vaccine-associated paralytic poliomyelitis. In the present study, 15 vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis and from healthy vaccinees were examined. Four distant sequences of the poliovirus genome (5' NCR, VP3/VP1, VP1/2A, and 3DPol/3' NCR) were targeted, and the reverse-transcribed segments were amplified by polymerase chain reaction followed by restriction fragment length polymorphism analysis with four restriction enzymes. Among the 15 isolates (11 Sabin type 2, 3 Sabin type 1, and 1 Sabin type 3), four Sabin type 2 isolates (36%) were found to be intertypic vaccine/vaccine recombinant in the 3DPol/3' NCR region of the viral genome. The recombinant genotypes identified were S2/S2/S1 for two isolates and S2/S2/S2/S3 and S2/S2/S1/S2 for each of the other two isolates, respectively. Recombinant viruses with unmodified segments in the 5' NCR and the VP3/VP1 regions of the viral genome, a modified segment in the VPI/2A region only for one strain, and an often recombinant segment in the 3DPol/3' NCR parts of the genome were so identified. These findings provide strong evidence that recombination is a frequent phenomenon in type 2 poliovirus vaccine strains and suggest that recombination may be an important mechanism of the natural evolution of polioviruses of Sabin type 2 origin, perhaps even one of the mechanisms of reversion of attenuated vaccine strains toward neurovirulence.

Evolution of circulating wild poliovirus and of vaccine-derived poliovirus in an immunodeficient patient: a unifying model.

We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis.

Evolution of Poliovirus Type I during 5.5 Years of Prolonged Enteral Replication in an Immunodeficient Patient

Poliovirus type 1 was isolated from an immunodeficient patient 4 days after onset of paresis (IS1) and after 5.5 years of prolonged enteral virus replication (IS2). Antigenic characterization revealed that IS1 was Sabin 1-like, whereas IS2 reacted like poliovirus 1 Mahoney. Complete genomic sequencing demonstrated the phylogenetic relationship (94.9% identity) of IS1 and IS2, which differed from the most closely related Sabin 1 by 5.4 and 8.3%, respectively. Both isolates had revertant-like mutations at nucleotides 480 and 6203. Deduced amino acid sequences indicated significant changes between IS1 and IS2 at the neutralizing antigenic site 1. Prolonged enteral replication, evolution, and shedding of poliovirus by immunodeficient patients should be considered in the poliovirus eradication strategy.

Evolution and Polymorphism of Poliovirus Genomes

Abstract. The three poliovirus serotypes are very stable. Breakthrough of the serotype barrier has never been observed in the natural evolution of poliovirus. This serotype stability contrasts with the high level of genomic and phenotypic variability that occurs within the bounds of serotype. The efficient control of poliomyelitis by immunization is based upon type-specific immunity and serotype stability. The development of attenuated strains by Albert Sabin was possible because of the high variability of poliovirus genomes. The three Sabin strains, one for each serotype, were selected as variants of non-attenuated wild polioviruses, and each represents a unique poliovirus genotype. A consequence of poliovirus variability is the polymorphic character of its genome. This polymorphism makes possible the identification of poliovirus genotypes upon which studies on poliovirus evolution, virologic surveillance, and poliomyelitis diagnostics are based. The antigenic and genomic peculiarities of the Sabin strains are used to distinguish them from wild polioviruses among field isolates. The mechanisms of poliovirus variation and their significance to the evolution of both wild and vaccine poliovirus strains are the subjects of this article. The natural evolution of polioviruses is discussed in the context of the global initiative to eradicate poliomyelitis, which relies on the worldwide use of Sabin's vaccine.

Polioviruses with Natural Recombinant Genomes Isolated from Vaccine-Associated Paralytic Poliomyelitis

To determine how oral poliovirus vaccine (Sabin) strains evolve during replication in humans and to confirm the etiology of vaccine-associated paralytic poliomyelitis (VAPP), we examined 70 vaccine-derived strains isolated from VAPP cases. Two distant sequences of the poliovirus genome were targeted for a double restriction fragment length polymorphism assay (RFLP) of reverse-transcribed genomic segments amplified by PCR, an extension of the method that we described previously (Balanant et al., 1991). One (RFLP-1) was a 480-long nucleotide sequence coding for the N-terminal part of the VP1 capsid polypeptide, situated in the 5′ third of the vital genome (nucleotides 2401-2880). The other (RFLP-3D1) was a 291-long nucleotide sequence coding for a part of the viral polymerase, situated near the 3′ end of the genome (nucleotides 6086-6376). Strain-specific restriction profiles could be generated for different field isolates by using three restriction enzymes in each case: HaeIII, DdeI, and HpaII for RFLP-1 and HaeIII, DdeI and RsaI for RFLP-3D1. With few exceptions, the vaccine-specific RFLP profiles were found to be conserved in both regions during replication of these viruses in humans. Thus, RFLP could be used as a marker so as to identify the origin of viral isolates at both ends of their genome. Whether viral isolates were vaccine-derived was determined by using strain-specific monoclonal antibodies and RFLP-1. Among the 70 isolates, 21 of the 43 type 2 strains and 15 of the 22 type 3 strains had a recombinant genome. None of the 5 type 1 Sabin-derived isolates was found to be recombinant. Both intertypic vaccine/vaccine and vaccine/non-vaccine recombinants were detected. Partial nucleotide sequencing confirmed the RFLP results in all cases that were investigated. In one case, it was possible to predict the recombination junction site from the restriction profiles. This site was more precisely localized by sequencing. The C 6203 > U nucleotide substitution, which is suspected to contribute to the reversion toward neurovirulence of the attenuated Sabin 1 strain, was detected in almost all the recombinant genomes containing Sabin 1-specific sequences at the 3′ extremity. This mutation was detected by identification of the modified RsaI profile in the RFLP-3D1. The results presented in this paper suggest that recombination, alone or together with mutation, might be one of the mechanisms of the reversion toward neurovirulence of attenuated vaccine strains and of the natural evolution of poliovirus.

Genetic diversity and rapid evolution of poliovirus in human hosts.

Rapid changes in phenotypic markers of polioviruses during replication in man were already reported in the 1950s but studies examining the mode and rate of genetic variation of the viruses only started in the 1980s. Recent studies have revealed that like other RNA viruses, polioviruses show typical features of a quasispecies with a multitude of variants present in a host at a given time and drifts in the composition of the mixture in relation to time. In this review we discuss the pattern of genetic variation of polioviruses and its relation to virus-host relationships.

Evolution of Poliovirus During an Outbreak: Sequential Type 3 Poliovirus Isolates from Several Persons Show Shifts of Neutralization Determinants

An outbreak of poliomyelitis in Finland resulted in the widespread circulation of wild-type 3 poliovirus strains that had antigenic properties distinct from the strains used to produce the attenuated and inactivated vaccines. Considerable variation was observed in the ability of broadly reacting monoclonal antibodies directed against type 3 poliovirus to neutralize the 54 strains examined. Sequential isolates from several persons showed an antigenic drift with these monoclonal antibodies and selected human sera. In addition, some faecal specimens were found to contain more than one antigenic variant. Primer extension sequencing of genomic RNAs of three plaque-purified antigenic variants isolated from one patient showed base substitutions in the region coding for the major antigenic site of poliovirus type 3. The resulting difference in the amino acid sequence in the virion protein VP1 could explain the differences observed in the neutralization of these strains by the monoclonal antibodies. Whether the observed changes in the antigenic characteristics of the sequential isolates represent true antigenic drift under immunological pressure or whether the emergence of the new variants is based on other modes of selection during replication is not known.

Molecular Epidemiology of Polioviruses

Poliovirus isolates can be identified according to their genotypes with use of the technique of oligonucleotide fingerprinting. Fingerprint analysis is performed by cleaving the viral RNA genome with ribonuclease T1 and separating the fragments (oligonucleotides) in two dimensions. The larger, structurally unique oligonucleotides distribute into patterns ("fingerprints") highly characteristic of a specific overall RNA sequence. Isolates from the same epidemic have very similar fingerprints. Isolates from distinct epidemics have very different fingerprints, a consequence of the rapid evolution of polioviruses during replication in humans. Similarity in the fingerprints of case isolates provides independent evidence for epidemiologic linkage. Fingerprinting can readily distinguish vaccine-related isolates from wild strains. Contemporary vaccine-related isolates are very probably vaccine-derived because their fingerprints contain characteristic vaccine-strain oligonucleotide spots (types 1 and 3) and because their wild-type parents are unlikely to have survived largely unaltered in the natural environment. Some examples of applications of this technique within different epidemiologic settings are described.