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.

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 ϳ10-fold range, and virus yields decreased over an ϳ65-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 ϳ3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased ϳ18-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 (ϳ50 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.The use of synonymous codons at unequal frequencies, the codon usage bias, is characteristic of all biological systems (26,27). The strength and direction of codon usage bias are related to the genomic GϩC content and the relative abundance of different isoaccepting tRNAs (reviewed in references 1, 16, and 53). Codon usage can affect the efficiency of gene expression. In bacteria (Escherichia coli) (26, 75), yeast (Saccharomyces cerevisiae) (5, 27), plants (Arabidopsis thaliana) (12), nematodes (Caenorhabditis elegans) (16), and insects (Drosophila melanogaster) (50), the most highly expressed genes use codons matched to the most abundant tRNAs (2). In contrast, in humans and other vertebrates, codon usage bias is much more strongly correlated with the GϩC content of the isochore where the gene is located (51, 71) than with the breadth or level of gene expression (16) or the number of corresponding tRNA genes (28, 30). Despite the weak correlation between codon usage and the levels of gene expression in mammalian cells (16,71), imbalances between codon usage and tRNA abundance can sharply reduce the levels of gene expression.Optimization of codon composition is frequently required for the efficient expression of genes in heterologous host systems (3,31,67,76). For example, the expression of human immunodeficiency virus type 1 ...

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5 untranslated region (5 UTR) and noncapsid-3 UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.

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 ϳ9% 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°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.Diversification of genomic sequences is constrained in all biological systems. At the level of primary sequences, the range of variability in coding regions is restricted by the codon usage bias (CUB), whereby a subset of synonymous codons are preferentially used in translation (24,53,69). The intensity of the CUB and the specific set of preferred codons vary widely across biological systems (39). Intertwined with the CUB is the suppression of the dinucleotides CpG and TpA (or UpA in RNA viruses) in the genomes of higher eukaryotes (4,7,26,61) and many of their RNA viruses and small DNA viruses (28,49). Variation in the primary sequences of RNA virus genomes is further constrained by requirements to maintain essential secondary and higher-order structures (42, 54, 68).We previously described the modulation of the replicative fitness of the Sabin type 2 oral poliovirus vaccine (OPV) strain (Sabin 2) by systematically changing the CUB in the capsid region, replacing the naturally occurring preferred codons with an unpreferred synonymous codon (isocodon) for each of nine amino acids (8). We called our approach "codon deoptimization" to contrast with the process of codon optimization, which is frequently used to maximize expression of foreign proteins in he...

We have calibrated five different molecular clocks for circulating poliovirus based upon the rates of fixation of total substitutions (K t ), synonymous substitutions (K s ), synonymous transitions (A s ), synonymous transversions (B s ), and nonsynonymous substitutions (K a ) into the P1/capsid region (2,643 nucleotides). Rates were determined over a 10-year period by analysis of sequences of 31 wild poliovirus type 1 isolates representing a well-defined phylogeny derived from a common imported ancestor. Similar rates were obtained by linear regression, the maximum likelihood/single-rate dated-tip method, and Bayesian inference. . Nonsynonymous substitutions at all P1/capsid sites, including the neutralizing antigenic sites, appeared to be constrained by purifying selection. Simulation of the evolution of third-codon positions suggested that saturation of synonymous transitions would be evident at 10 years and complete at ϳ65 years of independent transmission. Saturation of synonymous transversions was predicted to be minimal at 20 years and incomplete at 100 years. The rapid evolution of the K t , K s , and A s clocks can be used to estimate the dates of divergence of closely related viruses, whereas the slower B s and K a clocks may be used to explore deeper evolutionary relationships within and across poliovirus genotypes.Poliovirus is one of the most rapidly evolving viruses known (17,31,38,52,58). Estimates of the rates of total nucleotide substitution into poliovirus capsid regions average ϳ10 Ϫ2 substitutions per site per year (31, 39, 52-54, 89, 90). The rates appear to be similar across the three poliovirus serotypes and for both circulating polioviruses and polioviruses associated with chronic infections. This very rapid rate of genomic evolution has facilitated high-resolution molecular epidemiologic studies, permitting the unambiguous identification of the sources of imported viruses (10, 41) and the resolution of separate lineages during outbreaks (39,43,74,75), during endemic transmission (23,52,90), during prolonged poliovirus replication in immunodeficient patients (9,31,53,89), and from environmental surveillance (23). However, the rapid accumulation of nucleotide substitutions, most of which are synonymous transitions, obscures deeper evolutionary relationships (46,70).Underlying the rapid pace of poliovirus genomic evolution are the high rates of base misincorporation (in the range of 10 Ϫ5 to 10 Ϫ3 per base per replication) by the poliovirus RNAdependent RNA polymerase (2,15,19,81,82,84). These high mutation rates are attributable to the absence of 3Ј35Ј exonuclease proofreading mechanisms for the viral RNA polymerases (19), although other mechanisms may also be involved (17, 18). The strong transition bias of the poliovirus polymerase (2, 46) is reflected in the pattern of fixation nucleotide substitutions into poliovirus genomes.In this report, we have calibrated five different molecular clocks based upon the rates of fixation of total substitutions (K t ), synonymous substitutions (K s ),...

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We determined the complete genomic sequences of nine type 1 immunodeficient vaccine-derived poliovirus (iVDPV) isolates obtained over a 337-day period from a poliomyelitis patient from Taiwan with common variable immunodeficiency. The iVDPV isolates differed from the Sabin type 1 oral poliovirus vaccine (OPV) strain at 1.84% to 3.15% of total open reading frame positions and had diverged into at least five distinct lineages. Phylogenetic analysis suggested that the chronic infection was initiated by the fifth and last OPV dose, given 567 days before onset of paralysis, and that divergence of major lineages began very early in the chronic infection. Key determinants of attenuation in Sabin 1 had reverted in the iVDPV isolates, and representative isolates of each lineage showed increased neurovirulence for PVR-Tg21 transgenic mice. None of the isolates had retained the temperature-sensitive phenotype of Sabin 1. All isolates were antigenic variants of Sabin 1, having multiple amino acid substitutions within or near neutralizing antigenic sites 1, 2, and 3a. Antigenic divergence of the iVDPV variants from Sabin 1 followed two major independent evolutionary pathways. The emergence of distinct coreplicating lineages suggests that iVDPVs can replicate for many months at separate sites in the gastrointestinal tract. Some isolates had mosaic genome structures indicative of recombination across and within lineages. iVDPV excretion apparently ceased after 30 to 35 months of chronic infection. The appearance of a chronic VDPV excretor in a tropical, developing country has important implications for the strategy to stop OPV immunization after eradication of wild polioviruses.The central strategy of the World Health Organization Global Polio Eradication Initiative is widespread use of oral poliovirus vaccine (OPV) at high rates of coverage. This strategy has reduced the global incidence of polio by over 99% since the start of the Initiative in 1988 and restricted wild poliovirus circulation to countries in western and central Africa and southern Asia (87). However, use of OPV is associated with some rare adverse events, including the appearance of cases of vaccine-associated paralytic poliomyelitis among OPV recipients and contacts (76), and the occurrence of polio outbreaks associated with circulating vaccine-derived poliovirus (cVDPV) (36). While cVDPV outbreaks can be prevented by maintenance of high rates of OPV coverage, the occurrence of vaccine-associated paralytic poliomyelitis is associated with the inherent genetic instability of the live, attenuated OPV strains (56).In immunocompetent individuals, the risk of vaccine-associated paralytic poliomyelitis is very low, estimated in the United States at 1 case per 2.4 million OPV doses distributed (75,76).