Identification of two determinants that attenuate vaccine-related type 2 poliovirus.

The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.     

Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.     

A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice.

The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.     

Nucleotide sequence of a neurovirulent variant of the type 2 oral poliovirus vaccine.

Infectious cDNAs of the Sabin type 2 poliovirus vaccine virus and a vaccine-derived neurovirulent type 2 strain (P2/117) have been cloned in Escherichia coli. Nucleotide sequence analysis revealed that P2/117 differs from the vaccine strain by just 23 point mutations. Three occur in the 5' noncoding region. The remainder result in a total of 5 coding changes located in VP1, VP4, 2B, and 3D. The likely role of these mutations in the evolution to neurovirulence is discussed.     

A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.     

Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.     

Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y.

A base change from C to U at position 472 of the 5' noncoding region of the poliovirus genome is known to be a major determinant of attenuation in the P3/Sabin vaccine strain. To determine the biochemical basis for the attenuated phenotype imparted by this mutation, a cell line in which replication of neurovirulent and attenuated viruses could be distinguished was identified. A pair of P3/Sabin-P2/Lansing viral recombinants that differ only at position 472 was used; the viruses replicated equally well in HeLa cells, but the virus with a U at base 472 was attenuated in mice. In the human neuroblastoma cell line SH-SY5Y, recombinants with a U at base 472 replicated to approximately 10-fold-lower titers than did neurovirulent viruses with a C at this position. Analysis of viral RNA and protein synthesis indicated that translation of the attenuated viral RNA was specifically reduced in SH-SY5Y cells.     

Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature.

Poliovirus type 1 neurovirulence is difficult to analyze because of the 56 mutations which differentiate the neurovirulent Mahoney strain from the attenuated Sabin strain. We have isolated four neurovirulent mutants which differ from the temperature-sensitive parental Sabin 1 strain by only a few mutations, using selection for temperature resistance: mutant S(1)37C1 was isolated at 37.5 degrees C, S(1)38C5 was isolated at 38.5 degrees C, and S(1)39C6 and S(1)39C10 were isolated at 39.5 degrees C. All four mutants had a positive reproductive capacity at supraoptimal temperature (Rct+ phenotype). Mutant S(1)37C1 induced paralysis in two of four cynomolgus monkeys, and the three other mutants induced paralysis in four of four monkeys. The lesion score increased from the S(1)37C1 mutant to the S(1)39 mutants. To map the mutations associated with thermoresistance and neurovirulence, we sequenced all regions in which the Sabin 1 genome differs from the Mahoney genome. The S(1)37C1 mutant had one mutation in the 5' noncoding region and another in the 3' noncoding region. Mutant S(1)38C5 had these mutations plus another mutation in the 3D polymerase gene. The S(1)39 mutants had three additional mutations in the capsid protein region. The mutations were located at positions at which the Sabin 1 and Mahoney genomes differ, except for the mutation in the 5' noncoding region. The noncoding-region mutations apparently confer a low degree of neurovirulence. The 3D polymerase mutation, which distinguishes S(1)38C5 and S(1)39 mutants from S(1)37C1, is probably responsible for the high neurovirulence of S(1)38C5 and S(1)39 mutants. The capsid region mutations may contribute to the neurovirulence of the S(1)39 mutants, which was the highest among the mutants.     

Natural type 3/type 2 intertypic vaccine-related poliovirus recombinants with the first crossover sites within the VP1 capsid coding region

Background

Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008.

Principal Findings

Complete genomic sequences revealed their vaccine-related genomic features and showed that their first crossover sites were randomly distributed in the 3′ end of the VP1 coding region. The length of donor Sabin 2 sequences ranged from 55 to 136 nucleotides, which is the longest donor sequence reported in the literature for this type of poliovirus recombination. The recombination resulted in the introduction of Sabin 2 neutralizing antigenic site 3a (NAg3a) into a Sabin 3 genomic background in the VP1 coding region, which may have been altered by some of the type 3-specific antigenic properties, but had not acquired any type 2-specific characterizations. NAg3a of the Sabin 3 strain seems atypical; other wild-type poliovirus isolates that have circulated in recent years have sequences of NAg3a more like the Sabin 2 strain.

Conclusions

10 natural type 3/type 2 intertypic VP1 capsid-recombinant polioviruses, in which the first crossover sites were found to be in the VP1 coding region, were isolated and characterized. In spite of the complete replacement of NAg3a by type 2-specific amino acids, the serotypes of the recombinants were not altered, and they were totally neutralized by polyclonal type 3 antisera but not at all by type 2 antisera. It is possible that recent type 3 wild poliovirus isolates may be a recombinant having NAg3a sequences derived from another strain during between 1967 and 1980, and the type 3/type 2 recombination events in the 3′ end of the VP1 coding region may result in a higher fitness.     

重新评价G-480位点变异对Ⅰ型脊髓灰质炎疫苗病毒神经毒力的影响

在对分离于中国贵州省的9株Ⅰ型循环的疫苗衍生脊髓灰质炎病毒（cVDPVs）进行全基因组核苷酸序列分析后,发现已知最重要的决定病毒神经毒力的位点G-480和U-525并没有发生回复野生型突变;另外一些已知的神经毒力决定位点,如A-2438、A-2795、C-6203和G-7441等均已经发生了回复野生型突变。根据核苷酸序列的不同,从9株Ⅰ型cVDPVs毒株中选取5株病毒感染转人脊髓灰质炎病毒受体基因的小鼠进行神经毒力实验,发现它们的神经毒力都有所升高,其中CHN8184株和CHN8229-1．1株的神经毒力已经十分接近P1／Mahoney株,CHN8229-1．1株、CHN8229-2株和CHN8229-3株神经毒力依次递减,但仍处于较高水平,在它们的全基因组中分别只有7个和2个核苷酸的差异,而毒力却相差很多,提示有新的未鉴别的神经毒力决定位点的存在。对这些毒株5′非编码区（5′NCR）的第Ⅴ结构域进行二级结构预测,发现它们的二级结构很稳定。在G-480位点没有发生回复突变的情况下,部分毒株的神经毒力已经非常接近P1／Mahoney的水平,提示先前的研究中关于G-480突变对Ⅰ型脊髓灰质炎病毒神经毒力的作用可能被估计过高,G-480位点不是唯一重要的神经毒力决定位点,可能多个核苷酸联合突变才能达到减毒的效果。要真正全面了解P1／Sabin株的减毒机制,还需要进行更加深入的研究。     

A single base deletion in the 5' noncoding region of Theiler's virus attenuates neurovirulence.

Viral chimeras have been constructed through in vitro manipulations of the infectious cDNA clones of two prototypes of Theiler's murine encephalomyelitis virus: (i) the virulent GDVII strain and (ii) the less virulent BeAn and VL strains. Previous studies have suggested that the phenotypic differences in virulence between the BeAn and GDVII strains map to both the 5' noncoding and the coat protein regions of these viral genomes. It is shown here that attenuation mapped to the 5' noncoding region is due, at least in part, to an inadvertent deletion resulting from a cloning artifact of one C nucleotide out of four between positions 876 and 879 in the BeAn sequences. The in vitro growth characteristics in BHK-21 cells, however, do not reflect the large differences in neurovirulence between chimeras that are identical except for the deleted C. Another chimera with a mutation at position 877 and a deletion at 976 is also attenuated. The wild-type sequences from the less virulent strains BeAn and VL between nucleotides 1 and 933, in an otherwise GDVII chimera, do not attenuate virulence. Sequences of the 500 nucleotides of the 5' noncoding region proximal to the translation initiation codon were obtained for nine additional Theiler's virus strains. The attenuating deletions are discussed in the context of these sequences and the proposed secondary structures for the 5' noncoding region.     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.     

Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells

The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.     

Genetic analysis of the attenuation phenotype of poliovirus type 1.

Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation.     

Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5' noncoding sequence in viral replication.

A number of deletion and insertion sequences were introduced into the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of type 1 poliovirus by using an infectious cDNA clone of the virus strain. The genomes of all three poliovirus serotypes contained highly homologous sequences (nucleotide positions 509 to 639) as well as highly variable sequences (positions 640 to 742) in the 5' noncoding region. The viability of mutant viruses was tested by transfecting mutant cDNA clones into African green monkey kidney cells and then estimating the plaque sizes displayed on the cells. The results suggested that the highly variable sequence next to the VP4 coding region did not play an important role, at least in the in vitro culture system used, that the loci of highly conserved nucleotide sequences were not always expected to be the genome regions essential for viral replication, that the sequence between positions 564 and 599 carried genetic information to maintain the efficiency of certain steps in viral replication, and that the sequence between positions 551 to 563 might play an essential role in viral replication. Four-base deletion or insertion mutations were introduced into relatively variable sequences in the genome region upstream of position 509. The results suggest that variable sequences do not always indicate that the corresponding genome regions are less important. Apparent revertants (large-plaque variants) were easily generated from one of the viable mutants with the small-plaque phenotype. The determination of nucleotide sequences of the revertant genomes revealed the second mutation site. The results suggested that the different loci at around positions 200 and 500 might specifically interact with each other. This interaction may result in the formation of a functional structure that influences the efficiency of certain steps in the viral replication.     

Determinants in the 5' noncoding region of poliovirus Sabin 1 RNA that influence the attenuation phenotype.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.     

The nucleotide sequence of poliovirus type 3 leon 12 a1b: comparison with poliovirus type 1.

The complete nucleotide sequence of the genome of the Sabin vaccine strain of poliovirus type 3 (P3/Leon 12 a1 b) has been determined from cDNA cloned in E. coli. The genome comprises a 5' non-coding region of 742 nucleotides, a large open reading frame of 6618 nucleotides (89% of the sequence) and a 3' non-coding region of 72 nucleotides. There is 77.4% base-sequence homology and 89.6% predicted amino-acid homology between types 1 and 3. Conservation of all glutamine-glycine and tyrosine-glycine cleavage sites suggests a mechanism of polyprotein processing similar to that established for poliovirus type 1.     

Nucleic acid sequence of the region of the genome encoding capsid protein VP1 of neurovirulent and attenuated type 3 polioviruses

Three closely related strains of poliovirus type 3 have been used to study the molecular basis of attenuation in the currently used Sabin vaccine of this serotype. Plaque-purified derivatives of these strains possess closely similar serological and biochemical properties yet differ markedly in neurovirulence for monkeys. Molecular cloning via an RNA . cDNA method has facilitated comparative nucleotide sequencing. Initial efforts have concentrated on the region of the genome encoding VP1. Only minor structural differences between neurovirulent and attenuated type 3 strains were detected, in contrast to the major differences observed between the vaccine strains of poliovirus type 1 and its virulent precursor P1/Mahoney. These observations suggest that the molecular basis of attenuation of type 3 Sabin vaccine virus does not involve the VP1 polypeptide and, therefore, that mutations conferring the attenuated phenotype probably lie elsewhere in the genome.     

Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2.

Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.     

Reversion to neurovirulence of the live-attenuated Sabin type 3 oral poliovirus vaccine

The complete nucleotide sequence has been determined of a strain of poliovirus type 3, P3/119, isolated from the central nervous system of a victim of fatal vaccine-associated poliomyelitis. Comparison of this sequence with those obtained previously for the Sabin type 3 vaccine, P3/Leon 12a1b and its neurovirulent progenitor, P3/Leon/37, reveals that these three strains are on a direct geneaological lineage and therefore that P3/119 is a bona fide revertant of the vaccine. P3/119 differs in sequence from its attenuated vaccine parent at just seven positions. Only one of these differences, a mutation from U to C at position 472 in the presumed noncoding region of the genome, is a back mutation to the wild type sequence. Of the six other differences, three give rise to coding changes in virus structural proteins, two are silent changes in the major open reading frame of the genome and one affects the 3'-terminus just prior to the poly A tract. These differences indicate that there are three possible types of molecular change which could, singly or collectively, result in attenuation and reversion to neurovirulence of the Sabin type 3 vaccine.