The global polio eradication initiative was launched in 1988. Substantive progress has been made toward this goal, but further work is still required (11,12,21,29). Successful eradication of the cause of an infectious disease, exemplified by that of Variola virus, has at least four fundamental requirements: (i) absence of an animal reservoir; (ii) effective vaccines and, in the case of liveattenuated viruses, genetically stable vaccines; (iii) absence of long-term virus carriers; and (iv) sensitive tools to detect the presence of the causal agent. In the case of poliovirus eradication, these requirements have not been completely met. The basic strategy of the polio eradication initiative involves achieving high levels of routine immunization, mass vaccination, supplementary mop-up immunization activity, and poliovirus surveillance based on virological investigation of acute flaccid paralysis (AFP) cases (51, 56). There are two effective vaccines: inactivated polio vaccine (IPV), originally developed by Jonas Salk and colleagues, and the live-attenuated oral polio vaccine (OPV) of Albert Sabin (39). Both vaccines provide effective protection from poliomyelitis. However, IPV induces less mucosal immunity in the gut than OPV, a prerequisite for reducing intestinal reinfection, virus shedding, and transmission to susceptible contacts (33,37,41,44,45). OPV, which better protects from infection, suffers from other inadequacies that complicate the ongoing initiative (11,12,21,29). One inadequacy is the emergence of virulent vaccine-derived polioviruses (VDPVs) upon long-term replication in immunodeficient persons (iVDPV) or following sustained circulation in populations with immunity gaps (cVDPV). Ambiguous VDPVs, such as environmental isolates, represent a category of virulent polioviruses that cannot easily be assigned to iVDPV or cVDPV (29). Additional challenges for the initiative are poorly understood deficiencies in vaccine efficacy (24) and the low clinical attack rate following infection with virulent polioviruses (Յ1:100 paralytic case/infections). This calls for specific and sensitive virus-tracking tools. AFP surveillance is presently the gold standard for meeting those requirements (56).The three serotypes of poliovirus are members of the family Picornaviridae (20,40,55,61). The viruses possess a singlestranded RNA genome of approximately 7,400 nucleotides (nt). Upon infection, the genomic RNA is translated as a polyprotein, which is then processed by proteases into functional proteins. The structural proteins are located within the N-terminal P1 region and the nonstructural proteins within the succeeding P2 and P3 regions of the polyprotein. Similar to some other RNA viruses, the virus-encoded RNA-dependent RNA polymerase is error prone and lacks proofreading activity. This results in a high mutation frequency for these viruses. The molecular evolution of polioviruses is characterized by selective propagation of virus variants that are generated through mutations and heterotypic or intragenus recombination...