Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.     

History of polio vaccination

Poliomyelitis is an acute paralytic disease caused by three poliovirus (PV) serotypes. Less than 1% of PV infections result in acute flaccid paralysis. The disease was controlled using the formalin-inactivated Salk polio vaccine (IPV) and the Sabin oral polio vaccine (OPV). Global poliomyelitis eradication was proposed in 1988 by the World Health Organization to its member states. The strategic plan established the activities required for polio eradication, certification for regions, OPV cessation phase and post-OPV phase. OPV is the vaccine of choice for the poliomyelitis eradication program because it induces both a systemic and mucosal immune response. The major risks of OPV vaccination are the appearance of Vaccine-Associated Paralytic Poliomyelitis cases (VAPP) and the emergence of Vaccine Derived Polioviruses strains. The supplementary immunization with monovalent strains of OPV type 1 or type 3 or with a new bivalent oral polio vaccine bOPV (containing type 1 and type 3 PV) has been introduced in those regions where the virus has been difficult to control. Most countries have switched the schedule of vaccination by using IPV instead of OPV because it poses no risk of vaccine-related disease. Until 2008, poliomyelitis was controlled in Romania, an Eastern European country, predominantly using OPV. The alternative vaccination schedule (IPV/OPV) was implemented starting in September 2008, while beginning in 2009, the vaccination was IPV only. The risk of VAPP will disappear worldwide with the cessation of use of OPV. The immunization for polio must be maintained for at least 5 to 10 years using IPV.     

Growth kinetic analysis of bi-recombinant poliovirus vaccine strains

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and in rare cases may cause vaccine-associated paralytic poliomyelitis (VAPP). Mutations at specific sites of the genome and recombination between Sabin strains may result in the loss of the attenuated phenotype of OPV (Oral Poliovirus Vaccine) strains and the acquisition of traits characteristic of wild polioviruses, such as increased neurovirulence and loss of temperature sensitivity. In this study, we determined the phenotypic traits such as temperature sensitivity and growth kinetics of eight OPV isolates (six bi-recombinant and two non-recombinant). The growth phenotype of each isolate as well as of Sabin vaccine strains in Hep2 cell line at two different temperatures (37 and 40°C) was evaluated using two different assays, RCT test (Reproductive Capacity at different Temperatures) and one-step growth curve analysis. Moreover, the nucleotide and amino acid positions in the genomes of the isolates that have been identified as being involved in the attenuated and thermo sensitive phenotype of Sabin vaccine strains were investigated. Mutations that result in loss of the attenuated and thermo sensitive phenotype of Sabin vaccine strains were identified in the genomes of all isolates. Both mutations and recombination events correlated well with the reverted phenotypic traits of OPV-derivatives. In the post-eradication era of wild polioviruses, the identification and the characterization (genomic and phenotypic) of vaccine-derived polioviruses become increasingly important in order to prevent cases or even outbreaks of paralytic poliomyelitis caused by neurovirulent strains.     

Coupled mutations in the 5'-untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications.

All entero- and rhinovirus RNAs sequenced thus far possess A and U residues at positions corresponding to nucleotides 480 and 525, respectively, of poliovirus type 1. These two nucleotides have been proposed previously to form a base pair. The single exception to this rule appears to be the Sabin type 1 strain, which has a G480. Isolates of the Sabin 1 virus from healthy vaccinees were shown to have either a reversion to A480 or a second-site mutation U525----C, both restoring a potential for efficient base pairing. In vitro translation experiments demonstrated that poliovirus type 1 RNAs with either A480-U525 or G480-C525 are more efficient in promoting translation initiation as compared with the Sabin 1 RNA (G480-U525). The Sabin 2 strain has a U and an A at position 398 and 481, respectively, while its predecessor, strain P712, is shown to have C398 and G481. All the derivatives of the Sabin 2 isolated from vaccine-associated paralytic poliomyelitis cases shown reversion to G481, and most of them reverted also to C398. It is proposed that bases at positions 398 and 481 may be involved in a tertiary interaction. The in vitro template activity of the Sabin type 2 RNA (A481) is significantly lower than that of the isolate RNAs with G481, thus confirming the relation between attenuation and translation efficiency demonstrated previously for the type 1 and type 3 Sabin strains. The C----U change at position 398 exerted only a minor effect on the RNA template activity.     

Frequent isolation of intertypic poliovirus recombinants with serotype 2 specificity from vaccine-associated polio cases.

Five representatives from a collection of 21 Sabin type 2-like poliovirus strains isolated from paralytic poliomyelitis cases in two regions of the USSR have been subjected to limited nucleotide sequencing. All proved to be intertypic recombinants having the genes encoding capsid proteins of Sabin 2 origin and a 3'-end portion of the genome derived from either type 1 (3 isolates) or type 3 (2 isolates) Sabin strains. The crossover points in all the 5 genomes have been mapped to different loci of the P3 region. At least 6 additional isolates from the same collection (and 2 isolates from healthy contacts), appeared to have a type 2/type 1 recombinant genome, as judged by oligonucleotide mapping. The biological significance of frequent occurrence of recombinants among field isolates of vaccine-related strains is discussed.     

Sabin Type 2 Polioviruses with Intertypic Vaccine/Vaccine Recombinant Genomes

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and, in rare cases, cause vaccine-associated paralytic poliomyelitis. In the present study, 15 vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis and from healthy vaccinees were examined. Four distant sequences of the poliovirus genome (5^′ NCR, VP3/VP1, VP1/2A, and 3DPol/3^′ NCR) were targeted, and the reverse-transcribed segments were amplified by polymerase chain reaction followed by restriction fragment length polymorphism analysis with four restriction enzymes. Among the 15 isolates (11 Sabin type 2, 3 Sabin type 1, and 1 Sabin type 3), four Sabin type 2 isolates (36%) were found to be intertypic vaccine/vaccine recombinant in the 3DPol/3^′ NCR region of the viral genome. The recombinant genotypes identified were S2/S2/S1 for two isolates and S2/S2/S2/S3 and S2/S2/S1/S2 for each of the other two isolates, respectively. Recombinant viruses with unmodified segments in the 5^′ NCR and the VP3/VP1 regions of the viral genome, a modified segment in the VP1/2A region only for one strain, and an often recombinant segment in the 3DPol/3^′ NCR parts of the genome were so identified. These findings provide strong evidence that recombination is a frequent phenomenon in type 2 poliovirus vaccine strains and suggest that recombination may be an important mechanism of the natural evolution of polioviruses of Sabin type 2 origin, perhaps even one of the mechanisms of reversion of attenuated vaccine strains toward neurovirulence. Electronic Publication     

Natural variation of poliovirus neutralization epitopes.

Poliovirus-neutralizing monoclonal antibodies were prepared against type 1, type 2, and type 3 wild laboratory (Mahoney, MEF1, and Saukett) and Sabin vaccine strains. Fifty-five poliovirus laboratory strains and field isolates were assayed by neutralization index test with a panel of homotypic monoclonal antibodies. A total of 27 monoclonal antibodies were used. Two categories of neutralization epitopes were found, i.e., cross-reacting (K), which is present on almost all strains of the same serotype, and strain-specific (V, variable), either wild (VW) or Sabin (VS). Several distinct neutralization epitopes were defined for each of the three poliovirus serotypes in almost every category. The study of antigenic variation of the Sabin type 1 vaccine virus during replication in human intestine showed that the VS neutralization epitope may be lost and even replaced by the VW epitopes of the parental Mahoney virus. A late isolate from a vaccine-fed infant recovered the complete neutralization epitope pattern of the Mahoney strain. Upon in vivo virus replication, a different kind of antigenic variation was also detected in which an epitope lost its function in virus neutralization but kept its antigenic conformation unaltered. Neutralization epitope analysis demonstrated that the presence of VS epitopes on a field isolate suggests the Sabin origin of the strain when the isolate displays the same epitope pattern as the original Sabin virus, or confirms it when the VS epitope(s) is mutually exclusive of VW epitopes. The lack of VS epitopes on a field isolate does not rule out its being of Sabin origin.     

Genomic Analysis of Recombinant Sabin Clinical Isolates

Recombination in Poliovirus vaccine strains is a very frequent phenomenon. In this report 23 polio/Sabin strains isolated from healthy vaccinees or from VAPP patients after OPV administration, were investigated in order to identify recombination sites from 2C to 3D regions of the poliovirus genome. RT-PCR, followed by Restriction Fragment Length Polymorphism (RFLP) screening analysis were applied in four distant genomic regions (5′ UTR, VP1, 2C and 3C–3D) in order to detect any putative recombinant. The detected recombinants were sequenced from 2C to the end of the genome (3′ UTR) and the exact recombination sites were determined with computational analysis. Five of the 23 isolated strains were recombinant in one genomic region, two of them in 2C, isolates EP16:S3/S2, EP23:S3/S1, two in 3D isolates EP6:S2/S1, EP12:S2/S1 and one in 3A isolate EP9:S2/Sl. Point mutations were found in strains EP3, EP6, EP9 and EP12. Recombination specific types and sites re-occurrence along with point mutations are discussed concerning the polioviruses evolution.     

Genotype-specific in vitro amplification of sequences of the wild type 3 polioviruses from Mexico and Guatemala.

The extensive nucleotide sequence heterogeneity among independent genotypes of wild polioviruses permits the systematic design of genotype-specific molecular reagents. We have prepared two sets of polymerase chain reaction (PCR) primer pairs specific for the genotype of wild poliovirus type 3 recently endemic to Mexico and Guatemala. Nucleotide sequences of a representative wild type 3 virus isolated in Mexico in 1989 differed from the corresponding Sabin 3 (Leon 12 a1b) sequences at 167 of 900 positions within the VP1 region. From the sequence data, wild virus-specific primer pairs were designed to complement regions of high mismatch (greater than 33%) with Sabin 3 templates. Primer binding sites were spaced along the genome so that the predicted amplification products (142 bp and 163 bp) could be easily resolved electrophoretically from the products generated with our Sabin strain-specific primers (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 53 bp). RNAs of all wild type 3 poliovirus isolates from Mexico and Guatemala obtained over a 13-year period (1977-1990) served as efficient templates for amplification of the 142-bp and 163-bp products. Genomic templates derived from vaccine-related polioviruses and most heterologous wild polioviruses were inactive under equivalent reaction conditions. Amplifications generating a 114-bp product with a broadly reacting primer pair, matching highly conserved sequences in the 5'-noncoding region, provided a positive control for the presence in samples of poliovirus (or enterovirus) RNAs. Selective amplification of wild Mexico-Guatemala type 3 poliovirus sequences was obtained with either primer set in reactions containing large stoichiometric excesses (up to 10(6)-fold) of vaccine-related RNAs. We have used wild genotype-specific PCR primer sets to facilitate identification of wild polioviruses present in both clinical and environmental samples.     

Aggregation of enterovirus small plaque variants and polioviruses under low ionic strength conditions.

Virion aggregation in low ionic conditions was observed with small plaque variants of Coxsackievirus type B3 and Echovirus types 4 and 11 by sedimentation and filtration methods. Inclusion of salts or DEAE-dextran into the media prevented or reversed virion aggregation. The effect of pH on aggregate formation in low ionic strength solutions was also investigated with various strains of poliovirus. Type I Sabin strain formed aggregates even at high pH, while Mahoney strains did so only below pH 6.5. Type 2 virus, Sabin and MEF1 strains, and type 3 virus, Sabin, Saukett and Suwa strains, showed an intermediate behaviour between the two type 1 strains, except MEF1-LB strain, a clone obtained from MEF1 strain under acidic overlay, which showed little tendency to aggregate. These results were compared with the degree of the d character of the strains. Besides the effect of inhibiting virion aggregation, the inclusion of DEAE-dextran into a sucrose gradient slowed the sedimentation of some of the viruses in low ionic strength solutions.     

Comparative sequence analysis of the 5'terminal noncoding regions of poliovirus vaccine strain Sabin 1, Sabin 2, and Sabin 3 genomes

Complementary DNAs (cDNAs) were synthesized with virion RNAS from immunologically distinct vaccine strains of poliovirus, that is, Sabin 1 (LSc, 2ab), Sabin 2 (p712, Ch, 2ab), and Sabin 3 (Leon 12a₁b). Restriction endonuclease HaeIII cleavage patterns of these cDNAs suggested striking difference in RNA sequences among these poliovirus serotypes. The virion RNA of Sabin 3 was shown to have a genome-linked protein (VPg) and physical properties of the VPg were identical to those of VPgs of the other poliovirus strains. Virion RNA from each serotype was labeled with ¹²⁵I-Bolton-Hunter reagent, after proteinase K treatment to remove all but a small peptide of the VPg which covalently linked to the 5′ terminus. The specifically 5′-end-labeled RNAs were analyzed by rapid RNA sequencing techniques. Sequences of the first 20 nucleotides were identical in Sabin 1, Sabin 2, and Sabin 3 strains, that is, VPg-pU-U-A-A-A-A-C-A-G-C-U-C-U-G-GG-G-U-U-G. Complete sequence homology was observed between virion RNAs of Sabin 1 and Sabin 2 strains up to the 43rd nucleotide from the 5′ end except for the 25th nucleotide by a “wandering spot” analysis of ¹²⁵I-labeled RNA mixture of these two strains. These results suggested that serotypes of poliovirus were derived by numerous mutations from a putative prototype poliovirus, and that the homologous sequence at 5′ termini, conserved through a long evolutional process, may have essential roles for replication and viral function of poliovirus.     

Combined immunodeficiency presenting with vaccine-associated paralytic poliomyelitis: a case report and narrative review of literature

Neurologic abnormalities compatible with vaccine-related poliovirus infection (VAPP) may be a first presentation of some primary immunodeficient patients. The risk of VAPP rises from 1 case per 750?000 in normal population to 1 per 7000 times higher, particularly for persons with agammaglobulinemia and hypogammaglobulinemia. However, there is no appropriate estimation for VAPP occurrence in patients with cellular immunity defects. Herein we report a case of combined immunodeficiency with paralytic complication due to oral polio vaccine and we present a literature review on this topic.     

Evolution of poliovirus type I during 5.5 years of prolonged enteral replication in an immunodeficient patient

Poliovirus type 1 was isolated from an immunodeficient patient 4 days after onset of paresis (IS1) and after 5.5 years of prolonged enteral virus replication (IS2). Antigenic characterization revealed that IS1 was Sabin 1-like, whereas IS2 reacted like poliovirus 1 Mahoney. Complete genomic sequencing demonstrated the phylogenetic relationship (94.9% identity) of IS1 and IS2, which differed from the most closely related Sabin 1 by 5.4 and 8.3%, respectively. Both isolates had revertant-like mutations at nucleotides 480 and 6203. Deduced amino acid sequences indicated significant changes between IS1 and IS2 at the neutralizing antigenic site 1. Prolonged enteral replication, evolution, and shedding of poliovirus by immunodeficient patients should be considered in the poliovirus eradication strategy.     

Correlation between recombination junctions and RNA secondary structure elements in poliovirus Sabin strains

In order to test the hypothesis that RNA structural elements promote the distribution of certain types of recombination junctions in each one of the 2C and 3D poliovirus genomic regions (Sabin 3/Sabin 2 or Sabin 1 in 2C and Sabin 2/Sabin 1 or Sabin 3 in 3D), we searched in 2C and 3D regions of reference Sabin strains for high probability RNA structural elements that could promote recombination. Recombination junctions that were identified in clinical strains of this study, as well as in clinical strains of previous studies, were superimposed on RNA secondary structure models of 2C and 3D genomic regions. Furthermore, we created an in vitro model, based on double infection of cell-culture with two poliovirus strains, for the production and identification of recombinant Sabin strains in 2C and 3D regions. Our intention was to compare the results that refer to the correlation of recombination junctions and RNA secondary structures in 2C and 3D regions of clinical strains, with the respective results of the in vitro model. Most of the recombination junctions of the clinical strains were correlated with RNA secondary structure elements, which were identical between recombining Sabin strains, and also presented high predictive value. In consensus were, the respective results originated from the in vitro model. We propose that the distribution of specific types of recombination junctions in certain regions of Sabin strains is not fortuitous and is correlated with RNA secondary structure elements identical to both recombination partners. Furthermore, results of this study highlight an important role for the stem region of the RNA structure elements in promoting recombination.     

Molecular and antigenic characterization of a highly evolved derivative of the type 2 oral poliovaccine strain isolated from sewage in Israel

An unusual, highly diverged derivative of the Sabin type 2 oral poliovaccine (OPV) strain was recovered from environmental samples during routine screening for wild polioviruses. Virus was cultivated in L20B cells and then passaged on BGM cells at 40 degrees C (RCT [reproductive capacity at supraoptimal temperature]-positive marker) to select against most OPV strains. All but 1 of 25 RCT-positive OPV-derived environmental isolates were antigenically and genetically (>99.5% VP1 sequence match) similar to the respective Sabin strains. However, isolate PV2/4568-1/ISR98 (referred to below as 4568-1) escaped neutralization with Sabin 2-specific monoclonal antibodies and cross-adsorbed sera, and had multiple nucleotide substitutions (220 of 2,646; 8.3%) in the P1 capsid region. Fourteen of the 44 associated amino acid substitutions in the capsid mapped to neutralizing antigenic sites. Neutralizing titers in the sera of 50 Israeli children 15 years old were significantly lower to 4568-1 (geometric mean titer [GMT], 47) than to Sabin 2 (GMT, 162) or to the prototype wild strain, PV2/MEF-1/EGY42 (GMT, 108). Two key attenuating sites had also reverted in 4568-1 (A(481) to G in the 5' untranslated region and the VP1 amino acid I(143) to T), and the isolate was highly neurovirulent for transgenic mice expressing the poliovirus receptor (PVR-Tg21 mice). The extensive genetic divergence of 4568-1 from the parental Sabin 2 strain suggested that the virus had replicated in one or more people for approximately 6 years. The presence in the environment of a highly evolved, neurovirulent OPV-derived poliovirus in the absence of polio cases has important implications for strategies for the cessation of immunization with OPV following global polio eradication.     

A seroprevalence study of poliovirus antibody against a collection of recombinant and non-recombinant poliovirus vaccine strains in the population of southern Greece

In this study, the serological status of the southern Greek population in the 1–10-year, 11–20-year, 21–30-year and 31–40-year age groups with regard to Sabin vaccine strains and a collection of 15 recombinant and four non-recombinant poliovirus vaccine strains was determined. For all three poliovirus types, the highest neutralization test (NT) titres were observed in the 1–10-year age group, indicating a good response to vaccination. In general, the serological status of the population of southern Greece with regard to poliovirus is better for types 1 and 2 than for type 3. The presence of the lowest NT titre in the 21–30-year age group against poliovirus type 3 suggests the need for a booster dose of monovalent Sabin3 vaccine to ensure personal and herd immunity.     

Recombinants of Mahoney and Sabin strain poliovirus type 1: analysis of in vitro phenotypic markers and evidence that resistance to guanidine maps in the nonstructural proteins

A neutralizing monoclonal antibody-resistant variant of the Sabin vaccine strain of poliovirus type 1 and a guanidine-resistant variant of the virulent parent Mahoney strain were derived. The two variants were used, in a coinfection, to generate recombinant virus containing both resistance markers. Recombinants appeared on the order of 1.0 PFU for every 10(4) total PFU. Two independently derived recombinant viruses were isolated. Each isolate contained the P1 (structural protein) gene region of the Sabin strain virus and the P3 (nonstructural replicase protein) gene region of the Mahoney strain virus. The recombinant virus phenotypes were compared with certain characteristic in vitro phenotypes of both parents. It was found that the slow growth in cell culture, temperature sensitivity, and virion surface charge characteristics of the Sabin virus mapped entirely to the structural protein gene region whereas the phenotype of the actinomycin D sensitivity of the Sabin virus mapped to the gene region specifying the nonstructural replication proteins. Sequence analysis of the recombinant RNA revealed that the crossover occurred 3' of nucleotide 3919. This result showed that the resistance of poliovirus mutants to growth in 2 mM guanidine hydrochloride maps in the 3'-terminal region of the viral genome specifying the nonstructural proteins.     

A Sabin 2-related poliovirus recombinant contains a homologous sequence of human enterovirus species C in the viral polymerase coding region

A type 2 vaccine-related poliovirus (strain CHN3024), differing from the Sabin 2 strain by 0.44% in the VP1 coding region was isolated from a patient with vaccine-associated paralytic poliomyelitis. Sequences downstream of nucleotide position 6735 (3D ^pol coding region) were derived from an unidentified sequence; no close match for a potential parent was found, but it could be classified into a non-polio human enteroviruses species C (HEV-C) phylogeny. The virus differed antigenically from the parental Sabin strain, having an amino acid substitution in the neutralizing antigenic site 1. The similarity between CHN3024 and Sabin 2 sequences suggests that the recombination was recent; this is supported by the estimation that the initiating OPV dose was given only 36–75 days before sampling. The patient’s clinical manifestations, intratypic differentiation examination, and whole-genome sequencing showed that this recombinant exhibited characteristics of neurovirulent vaccine-derived polioviruses (VDPV), which may, thus, pose a potential threat to a polio-free world.     

Comparative study of molecular and antigenic characterization for intratypic differentiation (ITD) of poliovirus strains

This study was designed to compare the sensitivity of a Sabin vaccine strain‐specific PCR assay and an enzyme‐linked immunosorbent assay with polyclonal cross‐absorbed antisera (PAb‐E) for intratypic differentiation (ITD) of polioviruses (PVs). These were used for the definitive characterization of the strains. Poliovirus strains isolated in L20B and RD cell lines were subjected to both PCR and ELISA. Both PCR and ELISA identified 3 (13.6%) out of 22 isolates, respectively as poliovirus Sabin 1. PCR identified 4 (18.2%) out of 22 isolates as poliovirus Sabin 2 and ELISA identified 2 (9.1%) out of 22 isolates as poliovirus Sabin 2. None of the two assay identified poliovirus Sabin 3. Both PCR and ELISA identified 12 (54.5%) out of 22 isolates, respectively as wild poliovirus (WPV) 1. None of the assays identified any of the isolates as WPV 2 and 3. Only PCR assay was able to identify the mixture of two poliovirus Sabin serotypes (a mixture of Sabin 1 and 2) and two mixtures of poliovirus Sabin 2 and 3. In this study, only ELISA was able to identified two invalid results. Invalid results observed in this study are of important practical implication to the emergence of vaccine‐derived poliovirus. This may have epidemic potential. Hence, the two ITD assays are of paramount importance for identification of PVs. It is therefore recommended in line with WHO guideline that at least two methods be used for the ITD of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic and genetic properties). J. Med. Virol. 84:1975–1979, 2012. © 2012 Wiley Periodicals, Inc.     

Reevaluation of nucleotide sequences of wild-type and attenuated polioviruses of type 3

Published sequences of wild-type and attenuated Sabin strains of type 3 poliovirus (Leon/37 and Leon 12a(1)b) were derived from cDNA clones. Recent direct sequencing of Sabin 3 RNA showed that it differed from the published sequence in at least two sites. Here results of direct sequencing of genomes of three independently re-derived sub-strains of attenuated Sabin 3 poliovirus used for oral poliovirus vaccine (OPV) production in addition to the most widely used Pfizer sub-strain are reported. The results showed that all four sub-strains of attenuated type 3 poliovirus contain unique patterns of mutations. Two stocks of the wild-type progenitor Leon/37 strain were also sequenced. Analysis of the two samples of Leon/37 virus showed that one of them is much closer to the Sabin 3 strain, and is an intermediate product of the attenuation process. In addition, we created genetically engineered constructs which contained some of the mutations suspected for their possible role in neurovirulence, and tested them in monkeys and in transgenic mice sensitive to poliovirus. The results suggested that none of them increased neurovirulence of the virus, but some may improve virus replication. Therefore the only mutation occurring in Sabin 3 under vaccine production conditions that appears to affect neurovirulence of the virus is the well known U-->C reversion at nucleotide 472.