Estimation of the neurovirulence of poliovirus by non-radioisotope molecular analysis to quantify genomic changes

Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has been developed for poliovirus to determine quantitatively for the presence of genomic changes in particular nucleotide sequences correlate with the characteristic of neurovirulence for monkeys. Currently the MAPREC is scheduled to be used as a routine safety test for oral poliomyelitis vaccine (OPV). Radioisotopes (RI) are used in MAPREC for quantitative determinations, a circumstance likely to limit its use. We investigated the possibility of developing a modified MAPREC, which did not require the use of radioisotopes, and developed a procedure designated NON-RI MAPREC. Conventional MAPREC and NON-RI MAPREC were then used in a series of studies in which analyses were performed on Sabin type 1 and Sabin type 3 attenuated vaccine polioviruses prepared under various conditions. Under the experimental conditions used, the stability of the genome of type 1 virus was shown to be markedly greater than that of the type 3 virus, and the frequency of mutants was observed to vary in relation to both the virus strain and the virus inoculum used. The results of the studies relating to the two analytical procedures used indicated that the reproducibility of both methods was of a similarly high order, but that MAPREC had a somewhat broader range of sensitivity than NON-RI MAPREC. As the quantity of genomic changes in OPV relating to neurovirulent properties are within the range of detection by NON-RI MAPREC, this procedure can be used as a quality control test for OPV.     

Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine.

The poliovirus type 3 Sabin oral poliovirus vaccine strain P3/Leon/12a1b differs in nucleotide sequence from its neurovirulent progenitor P3/Leon/37 by just 10 point mutations. The contribution of each mutation to the attenuation phenotype of the vaccine strain was determined by the construction of a series of recombinant viruses from infectious cDNA clones. The neurovirulence testing of recombinant viruses indicated that the attenuation phenotype is determined by just two point mutations: a C to U in the noncoding region at position 472 and a C to U at nucleotide 2034 which results in a serine-to-phenylalanine amino acid substitution in the structural protein VP3.     

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.     

A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating.

The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.     

A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans.

A mutation in the genome of poliovirus type 3 that is known to reduce neurovirulence in humans similarly reduces neurovirulence in mice when incorporated into a mouse-adapted-human poliovirus recombinant. Viral recombinants with a uracil at nucleotide position 472 in the 5'-noncoding regions of their genomes are unable to replicate in the mouse brain. Viral recombinants with a cytosine at this position are neurovirulent in mice. Neurovirulence of poliovirus in mice may therefore prove to be a useful indicator of the genetic stability of new attenuating mutations created by site-directed mutagenesis.     

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.     

Neurovirulence of type 1 polioviruses isolated from sewage in Japan.

Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use.     

Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and in rare cases cause vaccine-associated paralytic poliomyelitis (VAPP). Reversion of vaccine strains toward a pathogenic phenotype is probably one of the main causes of VAPP, a disease most frequently associated with type 3 and type 2 strains and more rarely with the type 1 (Sabin 1) strain. To identify the determinants and mechanisms of safety versus pathogenicity of the Sabin 1 strain, we characterized the genetic and phenotypic changes in six Sabin 1-derived viruses isolated from immunocompetent patients with VAPP. The genomes of these strains carried either few or numerous mutations from the original Sabin 1 genome. As assessed in transgenic mice carrying the human poliovirus receptor (PVR-Tg mice), all but one strain had lost the attenuated phenotype. Four strains presented only a moderate neurovirulent phenotype, probably due at least in part to reversions to the wild-type genotype, which were detected in the 5' noncoding region of the genome. The reversions found in most strains at nucleotide position 480, are known to be associated with an increase in neurovirulence. The construction and characterization of Sabin 1 mutants implicated a reversion at position 189, found in one strain, in the phenotypic change. The presence of 71 mutations in one neurovirulent strain suggests that a vaccine-derived strain can survive for a long time in humans. Surprisingly, none of the strains analyzed were as neurovirulent to PVR-Tg mice as was the wild-type parent of Sabin 1 (Mahoney) or a previously identified neurovirulent Sabin 1 mutant selected at a high temperature in cultured cells. Thus, in the human gut, the Sabin 1 strain does not necessarily evolve toward the genetic characteristics and high neuropathogenicity of its wild-type parent.     

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.     

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.     

2010年山东省环境污水中脊髓灰质炎病毒的监测及其基因特征分析

本研究在山东省开展了脊髓灰质炎病毒(Poliovirus,PV)的外环境监测,从济南、临沂两地采集污水标本,浓缩处理后进行病毒分离,对分离到的PV采用中和试验进行血清定型,并对其VP1及3D区进行序列测定,分析其基因突变和重组情况。2010年,共采集污水标本32份,PV阳性10份,阳性率31.3%;分离到18株PV(PV1型3株,PV2型9株,PV3型6株),均为疫苗相关株,VP1完整编码区核苷酸变异数在0~4个之间,在3株PV2型病毒和4株PV3型病毒的基因组中发现重组;对VP1区影响神经毒力的减毒位点分析发现,PV1型病毒中有1株在nt 2 749发生突变(A→G),PV2型病毒中有1株在nt2 908发生A→G突变,3株在nt2 909发生U→C突变,6株PV3型病毒全部在nt2 493发生C→U突变。环境污水中可以分离到PV,其基因重组率和主要减毒位点的回复突变率较高,未发现脊灰野毒株和疫苗衍生株脊灰病毒(Vaccine-derived poliovirus,VDPV)。     

Attenuation stem-loop lesions in the 5' noncoding region of poliovirus RNA: neuronal cell-specific translation defects.

The nucleotide at position 480 in the 5' noncoding region of the viral RNA genome plays an important role in directing the attenuation phenotype of the Sabin vaccine strain of poliovirus type 1. In vitro translation studies have shown that the attenuated viral genomes of the Sabin strains direct levels of viral protein synthesis lower than those of their neurovirulent counterparts. We previously described the isolation of pseudorevertant polioviruses derived from transfections of HeLa cells with genome-length RNA harboring an eight-nucleotide lesion in a stem-loop structure (stem-loop V) that contains the attenuation determinant at position 480 (A. A. Haller and B. L. Semler, J. Virol. 66:5075-5086, 1992). This stem-loop structure is a major component of the poliovirus internal ribosome entry site required for initiation of viral protein synthesis. The eight-nucleotide lesion (X472) was lethal for virus growth and gave rise only to viruses which had partially reverted nucleotides within the original substituted sequences. In this study, we analyzed two of the poliovirus revertants (X472RI and X472R2) for cell-type-specific growth properties. The X472RI and X472R2 RNA templates directed protein synthesis to wild-type levels in in vitro translation reaction mixtures supplemented with crude cytoplasmic HeLa cell extracts. In contrast, the same X472 revertant RNAs displayed a decreased translation initiation efficiency when translated in a cell-free system supplemented with extracts from neuronal cells. This translation initiation defect of the X472R templates correlated with reduced yields of infectious virus particles in neuronal cells compared with those obtained from HeLa cells infected with the X472 poliovirus revertants. Our results underscore the important of RNA secondary structures within the poliovirus internal ribosome entry site in directing translation initiation and suggest that such structures interact with neuronal cell factors in a specific manner.     

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.     

Transgenic mice carrying the human poliovirus receptor: new animal models for study of poliovirus neurovirulence.

Recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains of poliovirus type 1 were subjected to neurovirulence tests using a transgenic (Tg) mouse line, ICR-PVRTg1, that carried the human poliovirus receptor gene. The Tg mice were inoculated intracerebrally with these recombinant viruses and observed for clinical signs, histopathological lesions, and viral antigens as parameters of neurovirulence of the viruses. These parameters observed in the Tg mice were different for different inoculated viruses. Dose-dependent incidences of paralysis and of death were observed in the Tg mice inoculated with any viruses used. This indicates that values of 50% lethal dose are useful to score a wide range of neurovirulence of poliovirus. The neurovirulence of individual viruses estimated by the Tg mouse model had a strong correlation with those estimated by monkey model. Consequently, the mouse tests identified the neurovirulence determinants on the genome of poliovirus that had been identified by monkey tests. In addition, the mouse tests revealed new neurovirulence determinants, that is, different nucleotides between the two strains at positions 189 and 21 and/or 935 in the 5'-proximal 1,122 nucleotides. The Tg mice used in this study may be suitable for replacing monkeys for investigating poliovirus neurovirulence.     

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.     

Molecular Genetic Analysis of Revertants from a Poliovirus Mutant That Is Specifically Adapted to the Mouse Spinal Cord

SA virus, a mutant of the Mahoney strain of type 1 poliovirus (PV1/Mahoney), replicates specifically in the spinal cords of mice and causes paralysis, although the PV1/Mahoney strain does not show any mouse neurovirulence (Q. Jia, S. Ohka, K. Iwasaki, K. Tohyama, and A. Nomoto, J. Virol. 73:6041-6047, 1999). The key mutation site for the mouse neurovirulence of SA was mapped to nucleotide (nt) 928 of the genome (A to G), resulting in the amino acid substitution of Met for Ile at residue 62 within the capsid protein VP4 (VP4062). A small-plaque phenotype of SA appears to be indicative of its mouse-neurovirulent phenotype. To identify additional amino acid residues involved in the host range determination of PV, a total of 14 large-plaque (LP) variants were isolated from a single point mutant, Mah/I4062M, that showed the SA phenotype. All the LP variants no longer showed any mouse neurovirulence when delivered via an intraspinal inoculation route. Of these, 11 isolates had a back mutation at nt 928 (G to A) that restored the nucleotide of the PV1/Mahoney type. The reversions of the remaining three isolates (LP8, LP9, and LP14) were mediated by a second site mutation. Molecular genetic analysis involving recombinants between Mah/I4062M and the LP variants revealed that the mere substitution of an amino acid residue at position 107 in VP1 (Val to Leu) (LP9), position 33 in VP2 (Val to Ile) (LP14), or position 231 in VP3 (Ile to Thr) (LP8) was sufficient to restore the PV1/Mahoney phenotype. These amino acid residues are located either on the surface or inside of the virus particle. Our results indicate that the mouse neurovirulence of PV is determined by the virion surface structure, which is formed by all four capsid proteins.     

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.     

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.     

Attenuating mutations in the poliovirus 5' untranslated region alter its interaction with polypyrimidine tract-binding protein.

Mutations in the 5' untranslated regions (5'-UTRs) of all three serotypes of the Sabin vaccine strains are known to be major determinants of the attenuation phenotype. To further understand the functional basis of the attenuation phenotype caused by mutations in the 5'-UTR, we studied their effects on viral replication, translation, and the interaction of the viral RNA with cell proteins. A mutation at base 472 (C472U), which attenuates neurovirulence in primates and mice, was previously found to reduce viral replication and translation in neuroblastoma cells but not in HeLa cells. This mutation reduced cross-linking of the poliovirus 5'-UTR to polypyrimidine tract-binding protein (pPTB) in neuroblastoma cells but not in HeLa cells. These defects were absent in a neurovirulent virus with C at nucleotide 472. When C472U and an additional mutation, G482A, were introduced into the 5'-UTR, the resulting virus was more attenuated, had a replication and translation defect in both HeLa cells and neuroblastoma cells, and cross-linked poorly to pPTB from both cell types. A neurovirulent revertant of this virus (carrying U472C, G482A, and C529U) no longer had a replication defect in HeLa and SH-SY5Y cell lines and cross-linked with pPTB to wild-type levels. The results suggest that the attenuating effects of the mutation C472U may result from an impaired interaction of the 5'-UTR with pPTB in neural cells, which reduces viral translation and replication. Introduction of a second mutation, G482A, into the 5'-UTR extends this defect to HeLa cells.     

Experience in Canada with the new revised monkey neurovirulence test for oral poliovirus vaccine

Nine years of experience in our laboratory, using more than 1500 cynomolgus monkeys in 138 tests, has shown that the new neurovirulence test (NVT) adopted by the World Health Organization (WHO) for live, oral monovalent vaccine of each poliovirus type, was a reproducible and sensitive assay likely to ensure the safety of this vaccine in humans. Our findings were the following: (1) when the test vaccine and the appropriate homotypic reference vaccine were tested in a single group of monkeys, the concurrent use of the reference vaccine considerably increased the reproducibility of the NVT; (2) in the assessment of the degree of attenuation of each lot of vaccine, the use of 12 monkeys for types 1 and 2 vaccines and 20 monkeys for type 3 vaccine (inoculated intraspinally each for reference and test vaccine) was satisfactory; (3) the virus dose used per monkey (10(5.6) to 10(6.6) pfu per monkey) was found not to be critical, i.e. the lower virus dose yielded mean lesion scores in the central nervous system of monkeys at least as high or higher than the tenfold higher virus dose; (4) the statistical analysis of our data showed that the old intrathalamic (IT) assay was considerably less sensitive than the new intraspinal (IS) assay, i.e., a test vaccine with a twofold increase in monkey neurovirulence would have a 41% chance of failing in the IT test (using 30 monkeys per vaccine), while this chance increased to 99% in the WHO IS assay (using 12 or 20 monkeys per vaccine). Since the introduction of the WHO NVT in Canada, the laboratory findings in monkeys were confirmed by vaccine experience in humans; the number of vaccine-associated paralytic poliomyelitis cases in the population showed a further decline.