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 ),...

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.Four methods are currently in routine use in the WHO Global Polio Laboratory Network (GPLN) (3, 14, 15) for differentiation between vaccine-related and wild poliovirus isolates (intratypic differentiation): (i) the enzyme-linked immunosorbent assay, using highly specific cross-absorbed antisera (13); (ii) nucleic acid hybridization, using Sabin vaccine strainspecific RNA probes (4); (iii) reverse transcription-PCR (RT-PCR), using vaccine strain-specific primers (16); and (iv) RT-PCR followed by restriction fragment length polymorphism analysis (1, 13). To achieve the required specificities for binding to variable target sequences, our RT-PCR primers were designed to contain mixed-base or inosine residues at positions of codon degeneracy (8, 9). In the poliovirus diagnostic RT-PCR kits currently distributed throughout the GPLN, identifications are based upon the mobilities of amplicons in polyacrylamide gels (8,9,15,16). This approach, while achieving the high levels of diagnostic accuracy and reliability required for global poliovirus surveillance, is especially laborious for GPLN laboratories with large workloads.Development of real-time RT-PCR (rRT-PCR) has opened the way for more-rapid and -accurate diagnostic assays (2). We have adapted our previously described poliovirus diagnostic RT-PCR methods (7-9) to the real-time format with an emphasis on high template specificities rather than quantitative determination of template concentrations. These new assays were tested against both Sabin vaccine-related isolates and wild poliovirus isolates representing all currently circulating genotypes.The enterovirus group-specific (panEV) primers used were essentially as described previously (10, 17). They target highly conserved sequences in the 5Ј untranslated region, and the antisense polarity primer (PCR-1; 10 pmol per assay) and TaqMan probe (panEV probe; 5 pmol) each have only one mixed-base residue, while the sense polarity primer (PCR-2; 10 pmol) is nondegenerate.To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan probes. Designing the degenerate TaqMan probes (a 15-pmol probe for each assay) was especially challenging because of the need to use longer sequences to obtain good hybrid stabilities while simultaneously compensating for the high level of degeneracy of sequences between primer binding sites. Although hybrid stabilities can be estimated by physicochemical calculations (12), development of the optimal primer and probe sets was a highly empirical process because variation within the target sequences was not predictable. The degen...

We developed RNA probes for the identification of poliovirus isolates by blot hybridization. Two sets of vaccine strain-specific probes were prepared. They complemented variable genomic domains within (i) the 5-untranslated region and (ii) the amino-terminal codons of VP1. An enterovirus group probe (EV/5UT) matching highly conserved 5-untranslated region sequences was used to estimate the quantities of poliovirus (or enterovirus) RNA in the samples. Poliovirus sequences amplified from Sabin strain virion RNA templates by PCR were inserted into the pUC18 plasmid vector. The antisense PCR primer for each probe set contained sequences encoding a T7 promoter. Hybrids were detected by a sensitive nonisotopic method. RNA probes were labeled by incorporation of digoxigenin-uridylate into the transcripts. The binding of probe to immobilized poliovirus RNAs was visualized by hydrolysis of the chemiluminescent substrate 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2-adamantane) catalyzed by alkaline phosphatase conjugated to anti-digoxigenin (Fab) fragments. The specificities of the probes were evaluated with a panel of poliovirus isolates that had previously been characterized by sequence analysis. The RNAs of vaccine-related isolates hybridized with the appropriate probe sets. Wild polioviruses representing a broad spectrum of contemporary genotypes were recognized by the inabilities of their genomes to form stable hybrids with the Sabin strain-specific probes.

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Vaccine-derived polioviruses (VDPVs) are associated with polio outbreaks and prolonged infections in individuals with primary immunodeficiencies. VDPV-specific PCR assays for each of the three Sabin oral poliovirus vaccine (OPV) strains were developed, targeting sequences within the VP1 capsid region that are selected for during replication of OPV in the human intestine. Over 2400 Sabin-related isolates and identified 755 VDPVs were screened. Sensitivity of all assays was 100%, while specificity was 100% for serotypes 1 and 3, and 76% for serotype 2. The assays permit rapid, sensitive identification of OPV-related viruses and flag programmatically important isolates for further characterization by genomic sequencing.

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We have developed RNA probes for the direct identification of wild poliovirus isolates by blot hybridization. The probes are complementary to sequences of the first 30 to 32 codons of VP1, which evolve more extensively (approximately 1.5-fold) than the rest of VP1. To illustrate our general approach, we describe the design of probes specific to each of four major genotypes recently endemic (1981 to 1991) to the Americas: Andean type 1, Brazil type 1, Brazil type 3, and Central America-Mexico type 3. A wild isolate of each genotype was selected according to molecular and epidemiologic criteria to be representative of the principal lineages in circulation. Variable VP1 sequences of the representative isolates were amplified by the reverse transcriptase PCR and were inserted into a plasmid vector containing a T7 promoter. The in vitro transcripts, labeled with digoxigenin, served as probes. These formed stable hybrids only with RNAs of isolates of the corresponding genotypes. Hybrids were detected by a sensitive chemiluminescence assay, capable under normal diagnostic conditions of detecting specific wild poliovirus sequences in samples containing up to a 100-fold excess of Sabin vaccine strain-related sequences of the same serotype.

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