As a result of the redundancy of the genetic code, adjacent pairs of amino acids can be encoded by as many as 36 different pairs of synonymous codons. A species-specific "codon pair bias" provides that some synonymous codon pairs are used more or less frequently than statistically predicted. We synthesized de novo large DNA molecules using hundreds of over-or underrepresented synonymous codon pairs to encode the poliovirus capsid protein. Underrepresented codon pairs caused decreased rates of protein translation, and polioviruses containing such amino acid-independent changes were attenuated in mice. Polioviruses thus customized were used to immunize mice and provided protective immunity after challenge. This "death by a thousand cuts" strategy could be generally applicable to attenuating many kinds of viruses.

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Virus attenuation by genome-scale changes in codon pair bias.

Poliomyelitis has appeared in epidemic form, become endemic on a global scale, and been reduced to near-elimination, all within the span of documented medical history. Epidemics of the disease appeared in the late 19th century in many European countries and North America, following which polio became a global disease with annual epidemics. During the period of its epidemicity, 1900-1950, the age distribution of poliomyelitis cases increased gradually. Beginning in 1955, the creation of poliovirus vaccines led to a stepwise reduction in poliomyelitis, culminating in the unpredicted elimination of wild polioviruses in the United States by 1972. Global expansion of polio immunization resulted in a reduction of paralytic disease from an estimated annual prevaccine level of at least 600,000 cases to fewer than 1,000 cases in 2000. Indigenous wild type 2 poliovirus was eradicated in 1999, but unbroken localized circulation of poliovirus types 1 and 3 continues in 4 countries in Asia and Africa. Current challenges to the final eradication of paralytic poliomyelitis include the continued transmission of wild polioviruses in endemic reservoirs, reinfection of polio-free areas, outbreaks due to circulating vaccine-derived polioviruses, and persistent excretion of vaccine-derived poliovirus by a few vaccinees with B-cell immunodeficiencies. Beyond the current efforts to eradicate the last remaining wild polioviruses, global eradication efforts must safely navigate through an unprecedented series of endgame challenges to assure the permanent cessation of all human poliovirus infections.

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From Emergence to Eradication: The Epidemiology of Poliomyelitis Deconstructed

Received 22 December 2009/Accepted 25 March 2010 Metagenomics and a panmicrobial microarray were used to examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides. Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles, while simian retrovirus (SRV) was present as genetically defective DNA. Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids. Deep sequencing and microarrays can therefore detect attenuated virus sequence changes, minority variants, and adventitious viruses and help maintain the current safety record of live-attenuated viral vaccines.

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Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus.

This article summarizes the status of environmental surveillance (ES) used by the Global Polio Eradication Initiative, provides the rationale for ES, gives examples of ES methods and findings, and summarizes how these data are used to achieve poliovirus eradication ES complements clinical acute flaccid paralysis (AFP) surveillance for possible polio cases ES detects poliovirus circulation in environmental sewage and is used to monitor transmission in communities If detected, the genetic sequences of polioviruses isolated from ES are compared with those of isolates from clinical cases to evaluate the relationships among viruses To evaluate poliovirus transmission, ES programs must be developed in a manner that is sensitive, with sufficiently frequent sampling, appropriate isolation methods, and specifically targeted sampling sites in locations at highest risk for poliovirus transmission After poliovirus ceased to be detected in human cases, ES documented the absence of endemic WPV transmission and detected imported WPV ES provides valuable information, particularly in high-density populations where AFP surveillance is of poor quality, persistent virus circulation is suspected, or frequent virus reintroduction is perceived Given the benefits of ES, GPEI plans to continue and expand ES as part of its strategic plan and as a supplement to AFP surveillance

Environmental Surveillance for Polioviruses in the Global Polio Eradication Initiative

The genus Enterovirus (EV) of the family Picornaviridae includes poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These diverse viruses cause a variety of diseases, including non-specific febrile illness, hand-foot-and-mouth disease, neonatal sepsis-like disease, encephalitis, paralysis and respiratory diseases. In recent years, several non-polio enteroviruses (NPEVs) have emerged as serious public health concerns. These include EV-A71, which has caused epidemics of hand-foot-and-mouth disease in Southeast Asia, and EV-D68, which recently caused a large outbreak of severe lower respiratory tract disease in North America. Infections with these viruses are associated with severe neurological complications. For decades, most research has focused on poliovirus, but in recent years, our knowledge of NPEVs has increased considerably. In this Review, we summarize recent insights from enterovirus research with a special emphasis on NPEVs. We discuss virion structures, host-receptor interactions, viral uncoating and the recent discovery of a universal enterovirus host factor that is involved in viral genome release. Moreover, we briefly explain the mechanisms of viral genome replication, virion assembly and virion release, and describe potential targets for antiviral therapy. We reflect on how these recent discoveries may help the development of antiviral therapies and vaccines.

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The life cycle of non-polio enteroviruses and how to target it.

As the global eradication of wild poliovirus nears, the World Health Organization (WHO) is addressing challenges unprecedented in public health. The live, attenuated oral poliovirus vaccine (OPV), used for more than four decades to interrupt poliovirus transmission, and the vaccine of choice for developing countries, is genetically unstable. Reversion of the small number of substitutions conferring the attenuated phenotype frequently occurs during OPV replication in humans and is the underlying cause of the rare cases of vaccine-associated paralytic poliomyelitis (VAPP) in OPV recipients and their close contacts. Whereas VAPP has long been recognized, two other adverse events have been identified more recently: (a) long-term excretion of highly evolved vaccine-derived polioviruses (VDPVs) in persons with primary immunodeficiencies, and (b) polio outbreaks associated with circulating VDPVs in areas with low rates of OPV coverage. Developing a posteradication strategy to minimize the risks of VDPV emergence and spread has become an urgent WHO priority.

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Walter R. Dowdle

Papers

Vaccine-derived polioviruses and the endgame strategy for global polio eradication.