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 ...

Two systems for measuring embryo development in vitro were evaluated. One was a 1-4 scale based on a subjective evaluation of embryo quality (EQ) from microscopic appearance. In addition, a formula for scoring embryo growth rate in vitro was developed. The embryo development rating (EDR) was based on the ratio between the time at which embryos were observed at a particular stage after insemination and the time at which they would be expected to reach that stage in a hypothetical "ideal" growth rate with a cell cycle length of 11.9 hr. Using this scoring system, "normally" growing embryos scored 100. This approach was aimed at partially normalizing the data and allowed all embryos to be analyzed similarly regardless of the time of observation. Analysis of 1539 embryo replacements resulting in 232 clinical pregnancies showed that both EDR and embryo-quality scores were of value in predicting success, with clinical pregnancy most likely to eventuate from a combination of moderate to good EQ scores (2-4) coupled with average or above-average growth rates (EDR scores from 90 to 129). Poor-quality and very slowly or very rapidly growing embryos were underrepresented in cycles that proceeded to pregnancy. These inferences were based on all embryos transferred (mean, 2.73 per transfer cycle), and they were substantiated by an analysis of 33 pregnancies resulting from replacement of a single embryo and from 18 pregnancies in which all embryos scored the same with both systems. EQ and EDR were significantly associated with each other and together provide a valuable guide in predicting pregnancy, in selecting embryos for freezing, and in monitoring day-to-day performance in the in vitro fertilization (IVF) program.