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.

High-Throughput Next-Generation Sequencing of Polioviruses.

The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.

Vaccine-derived poliomyelitis 12 years after infection in Minnesota.

A 44-year-old woman with long-standing common variable immunodeficiency who was receiving intravenous immune globulin suddenly had paralysis of all four limbs and the respiratory muscles, resulting in death. Type 2 vaccine-derived poliovirus was isolated from stool. The viral capsid protein VP1 region had diverged from the vaccine strain at 12.3% of nucleotide positions, and the two attenuating substitutions had reverted to the wild-type sequence. Infection probably occurred 11.9 years earlier (95% confidence interval [CI], 10.9 to 13.2), when her child received the oral poliovirus vaccine. No secondary cases were identified among close contacts or 2038 screened health care workers. Patients with common variable immunodeficiency can be chronically infected with poliovirus, and poliomyelitis can develop despite treatment with intravenous immune globulin.

Multiple independent emergences of type 2 vaccine-derived polioviruses during a large outbreak in northern Nigeria.

Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5'-untranslated region [5'-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5'-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.

Outbreak of type 2 vaccine-derived poliovirus in Nigeria: emergence and widespread circulation in an underimmunized population.

Wild poliovirus has remained endemic in northern Nigeria because of low coverage achieved in the routine immunization program and in supplementary immunization activities (SIAs). An outbreak of infection involving 315 cases of type 2 circulating vaccine-derived poliovirus (cVDPV2; >1% divergent from Sabin 2) occurred during July 2005-June 2010, a period when 23 of 34 SIAs used monovalent or bivalent oral poliovirus vaccine (OPV) lacking Sabin 2. In addition, 21 "pre-VDPV2" (0.5%-1.0% divergent) cases occurred during this period. Both cVDPV and pre-VDPV cases were clinically indistinguishable from cases due to wild poliovirus. The monthly incidence of cases increased sharply in early 2009, as more children aged without trivalent OPV SIAs. Cumulative state incidence of pre-VDPV2/cVDPV2 was correlated with low childhood immunization against poliovirus type 2 assessed by various means. Strengthened routine immunization programs in countries with suboptimal coverage and balanced use of OPV formulations in SIAs are necessary to minimize risks of VDPV emergence and circulation.

Genetic inactivation of poliovirus infectivity by increasing the frequencies of CpG and UpA dinucleotides within and across synonymous capsid region codons.

Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only approximately 9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5 degrees C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.

Neutralization capacity of highly divergent type 2 vaccine-derived polioviruses from immunodeficient patients.

The use of the oral poliovirus vaccine (OPV) in developing countries has reduced the incidence of poliomyelitis by >99% since 1988 and is the primary tool for global polio eradication. Spontaneous reversions of the vaccine virus to a neurovirulent form can impede this effort. In persons with primary B-cell immunodeficiencies, exposure to OPV can result in chronic infection, mutation, and excretion of immunodeficiency-associated vaccine-derived polioviruses, (iVDPVs). These iVDPVs may have the potential for transmission in a susceptible population and cause paralysis. The extent to which sera from OPV recipients are able to neutralize iVDPVs with varying degrees of antigenic site substitutions is investigated here. We tested sera from a population immunized with a combination vaccine schedule (both OPV and inactivated polio vaccine) against a panel of iVDPVs and found that increases in amino acid substitution in the P1 capsid protein resulted in a decrease in the neutralizing capacity of the sera. This study underscores the importance of maintaining high vaccine coverage in areas of OPV use as well as active surveillance of those known to be immunocompromised.

Development of a new oral poliovirus vaccine for the eradication end game using codon deoptimization.

Enormous progress has been made in global efforts to eradicate poliovirus, using live-attenuated Sabin oral poliovirus vaccine (OPV). However, as the incidence of disease due to wild poliovirus has declined, vaccine-derived poliovirus (VDPV) has emerged in areas of low-vaccine coverage. Coordinated global cessation of routine, type 2 Sabin OPV (OPV2) use has not resulted in fewer VDPV outbreaks, and continued OPV use in outbreak-response campaigns has seeded new emergences in low-coverage areas. The limitations of existing vaccines and current eradication challenges warranted development of more genetically stable OPV strains, most urgently for OPV2. Here, we report using codon deoptimization to further attenuate Sabin OPV2 by changing preferred codons across the capsid to non-preferred, synonymous codons. Additional modifications to the 5' untranslated region stabilized known virulence determinants. Testing of this codon-deoptimized new OPV2 candidate (nOPV2-CD) in cell and animal models demonstrated that nOPV2-CD is highly attenuated, grows sufficiently for vaccine manufacture, is antigenically indistinguishable from Sabin OPV2, induces neutralizing antibodies as effectively as Sabin OPV2, and unlike Sabin OPV2 is genetically stable and maintains an attenuation phenotype. In-human clinical trials of nOPV2-CD are ongoing, with potential for nOPV strains to serve as critical vaccine tools for achieving and maintaining polio eradication.

Are Circulating Type 2 Vaccine-derived Polioviruses (VDPVs) Genetically Distinguishable from Immunodeficiency-associated VDPVs?

Public health response to vaccine-derived poliovirus (VDPV) that is transmitted from person to person (circulating VDPV [cVDPV]) differs significantly from response to virus that replicates in individuals with primary immunodeficiency (immunodeficiency-associated VDPV [iVDPV]). cVDPV outbreaks require a community immunization response, whereas iVDPV chronic infections require careful patient monitoring and appropriate individual treatment. To support poliovirus outbreak response, particularly for type 2 VDPV, we investigated the genetic distinctions between cVDPV2 and iVDPV2 sequences. We observed that simple genetic measurements of nucleotide and amino acid substitutions are sufficient for distinguishing highly divergent iVDPV2 from cVDPV2 sequences, but are insufficient to make a clear distinction between the two categories among less divergent sequences. We presented quantitative approaches using genetic information as a surveillance tool for early detection of VDPV outbreaks. This work suggests that genetic variations between cVDPV2 and iVDPV2 may reflect differences in viral micro-environments, host-virus interactions, and selective pressures during person-to-person transmission compared with chronic infections in immunodeficient patients.

Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region.

We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (N(C)) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G+C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an approximately 10-fold range, and virus yields decreased over an approximately 65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced approximately 3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased approximately 18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (approximately 50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.

Outbreak of poliomyelitis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus.

An outbreak of paralytic poliomyelitis occurred in the Dominican Republic (13 confirmed cases) and Haiti (8 confirmed cases, including 2 fatal cases) during 2000-2001. All but one of the patients were either unvaccinated or incompletely vaccinated children, and cases occurred in communities with very low (7 to 40%) rates of coverage with oral poliovirus vaccine (OPV). The outbreak was associated with the circulation of a derivative of the type 1 OPV strain, probably originating from a single OPV dose given in 1998-1999. The vaccine-derived poliovirus associated with the outbreak had biological properties indistinguishable from those of wild poliovirus.