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PubMed1983

Antibody to a synthetic nonapeptide corresponding to the NH2 terminus of poliovirus genome-linked protein VPg reacts with native VPg and inhibits in vitro replication of poliovirus RNA.

A synthetic nonapeptide corresponding to the N-terminal sequence of poliovirus genome-linked protein (VPg) was linked to bovine serum albumin and used to raise antibodies in rabbits. The antipeptide antibodies specifically precipitated the nonapeptide, native VPg, and VPg-linked poliovirion RNA. The antipeptide antibodies inhibited host factor-stimulated, poliovirus replicase-catalyzed in vitro synthesis of full-length (35S) RNA in response to virion RNA. Oligouridylic acid-stimulated RNA synthesis was not affected by the antipeptide antibodies. Preincubation of the antibodies with excess nonapeptide reversed the antipeptide antibody-mediated inhibition of host factor-stimulated RNA synthesis by the poliovirus replicase. A role for VPg in the in vitro replication of poliovirus RNA genome is discussed.

Antibodies, Viral↗
PubMed1993

Enzymatic activity of poliovirus RNA polymerases with mutations at the tyrosine residue of the conserved YGDD motif: isolation and characterization of polioviruses containing RNA polymerases with FGDD and MGDD sequences.

The poliovirus RNA-dependent RNA polymerase (3Dpol) shares a region of homology with all RNA polymerases, centered around the amino acid motif YGDD, which has been postulated to be involved in the catalytic activity of the enzyme. Using oligonucleotide site-directed mutagenesis, we substituted the tyrosine at this motif of the poliovirus RNA-dependent RNA polymerase with cysteine, histidine, isoleucine, methionine, phenylalanine, or serine. The enzymes were expressed in Escherichia coli, and in vitro enzyme activity was tested. The phenylalanine and methionine substitutions resulted in enzymes with activity equal to that of the wild-type enzyme. The cysteine substitution resulted in an enzyme with approximately 50% of the wild-type activity, while the serine substitution resulted in an enzyme with approximately 10% of the wild-type activity; the isoleucine and histidine substitutions resulted in background levels of enzyme activity. To assess the effects of the mutants in viral replication, the mutant polymerase genes were subcloned into the infectious cDNA clone of poliovirus. Transfection of poliovirus cDNA containing the phenylalanine mutation in 3Dpol gave rise to virus in all of the transfection trials, while cDNA containing the methionine mutation resulted in virus in only 3 of 40 transfections. Transfection of cDNAs containing the other substitutions at the tyrosine residue did not result in infectious virus. The recovered viruses demonstrated kinetics of replication similar to those of the wild-type virus, as measured by [3H]uridine incorporation at either 37 or 39 degrees C. RNA sequence analysis of the 3Dpol gene of both viruses demonstrated that the tyrosine-to-phenylalanine or tyrosine-to-methionine mutation was still present. No other differences in the 3Dpol gene between the wild-type and phenylalanine-containing virus were found. The virus containing the methionine mutation also contained two other nucleotide changes from the wild-type 3Dpol sequence; one resulted in a glutamic acid-to-aspartic acid change at amino acid 108 of the polymerase, and the other resulted in a C-to-T base change at nucleotide 6724, which did not result in an amino acid change. To confirm that the second amino acid mutation found in the 3Dpol gene of the methionine-substituted virus allowed for replication ability, a mutation corresponding to the glutamic acid-to-aspartic acid change was made in the polymerase containing the methionine substitution, and this double-mutant polymerase was expressed in E. coli. The double-mutant enzyme was as active as the wild-type enzyme under in vitro assay conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.

Animals↗
PubMed1997

Inhibition of host cell transcription by poliovirus: cleavage of transcription factor CREB by poliovirus-encoded protease 3Cpro.

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. Previous studies from our laboratory showed that activated transcription from a cyclic AMP-responsive element (CRE)-containing promoter was severely inhibited in extracts prepared from poliovirus-infected HeLa cells compared to those from mock-infected cells. Here we demonstrate that the CRE-binding protein, CREB, is specifically cleaved by the poliovirus-encoded protease 3Cpro both in vitro and in virus-infected cells. The proteolytic cleavage of CREB leads to a significant loss of its DNA binding as well as transcriptional activity. Additionally, we demonstrate that the phosphorylated, transcriptionally active form of CREB is cleaved by the viral protease in vitro. The results presented here suggest that a direct cleavage of CREB by the viral protease 3Cpro leads to inhibition of CREB-activated transcription in poliovirus-infected HeLa cells.

Amino Acid Sequence↗
PubMed1994

Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding.

Poliovirus proteinase 3CDpro by itself is not an RNA-binding protein. Two cellular proteins have been purified from HeLa cells (p50 and p36) which interact with purified 3CDpro but only p36-3CDpro bind to the 5'-terminal 110 nucleotides of polioviral RNA genome, an RNA segment whose secondary structure resembles a cloverleaf. The identity of these factors was determined by microsequencing tryptic digests of the purified proteins. Host protein p50 is the eukaryotic elongation factor EF-1 alpha, and p36 an N-terminal fragment thereof. p36, referred to as host factor, did not appear to interact with purified 3Cpro or 3Dpol. Significantly, the formation of a 3CDpro-cloverleaf complex was also observed in the presence of purified poliovirus polypeptide 3AB, the precursor of VPg. 3AB by itself does not stably bind to the cloverleaf. Competition experiments have demonstrated that the RNA-protein interactions are specific for the full-length cloverleaf. UV cross-linking studies were employed to examine the protein components of the cloverleaf ribonucleoproteins. RNA footprinting was used to determine the site on the cloverleaf where the viral and cellular factors bind. Finally, we have discovered that 3AB-3CDpro also interacts with the 3'-terminal sequence of poliovirus RNA. In contrast to the 5'-terminal cloverleaf, the 3'-terminal RNA can bind 3AB in the absence of other proteins. A model for initiation of poliovirus RNA synthesis is presented.

3C Viral Proteases↗
PubMed2003

Serological response and poliovirus excretion following different combined oral and inactivated poliovirus vaccines immunization schedules.

A controlled study was conducted in Karachi, Pakistan to compare humoral and mucosal immune responses against polioviruses in infants who received oral poliovirus vaccine (OPV) at birth and at 6, 10, and 14 weeks according to the Expanded Program on Immunization (EPI) with infants who received either three doses of inactivated poliovirus vaccine (IPV) at 6, 10, and 14 weeks together with OPV or one additional dose of IPV at 14 weeks together, with the last dose of OPV. A total of 1429 infants were enrolled; 24-week serum specimens were available for 898 infants (63%). They all received a challenge dose of OPV type 3 at 24 weeks of age. The addition of three doses of IPV to three doses of OPV induced a significantly higher percentage of seropositive children at 24 weeks of age for polio 1 (97% versus 89%, P<0.001) and polio 3 (98% versus 92%) compared to the EPI schedule. However, the one supplemental dose of IPV at 14 weeks did not increase the serological response at 24 weeks. Intestinal immunity against the challenge dose was similar in the three groups. Combined schedules of OPV and IPV in the form of diphtheria-pertussis-tetanus-IPV vaccine (DPT-IPV) may be useful to accelerate eradication of polio in developing countries.

Antibodies, Viral↗
PubMed1997

Humoral and mucosal immunity in infants induced by three sequential inactivated poliovirus vaccine-live attenuated oral poliovirus vaccine immunization schedules. Baltimore Area Polio Vaccine Study Group.

The relative immunity induced by sequential administration of inactivated poliovirus vaccine (IPV) produced in human diploid cells and live attenuated oral poliovirus vaccine (OPV) was evaluated by randomization of 510 infants to receive IPV and OPV sequentially according to one of three experimental schedules, IPV only, or OPV only. The antibody response to two IPV doses was lower than expected. However, for each of the IPV-OPV sequential schedules, the first OPV dose significantly enhanced seroconversion rates and geometric mean microneutralization antibody titers. Three months after the final dose, 96%-99%, 99%-100%, and 81%-100% of infants had antibodies to poliovirus types 1, 2, and 3, respectively, and subjects with two or more prior OPV doses were significantly less likely than those with none or one prior OPV dose to excrete virus in feces after an OPV challenge. Sequential IPV-OPV immunization is now recommended for routine use in the United States. The optimal schedule consists of two IPV doses followed by two OPV doses.

Antibodies, Viral↗
PubMed1996

A poliovirus-susceptible transgenic mouse model as a possible replacement for the monkey neurovirulence test of oral poliovirus vaccine.

Two poliovirus-susceptible transgenic mouse (Tg PVR) strains, Tg1 and Tg21, were compared with the monkey test for their sensitivity to neurovirulence of live oral poliovirus vaccine (OPV). Intracerebral (i.c.) and intraspinal (i.s.) routes of inoculation were investigated to determine the most suitable combination of mouse strain and route. Evaluation of the mouse tests was performed using several indicators; clinical score and failure time were selected as the most efficient. Tg1 and Tg21 mice inoculated i.s. with type 2, and Tg21 mice inoculated i.s. with type 3 OPV were determined to be the most appropriate systems, whereas they are shown not to be suitable for type 1 OPV. The sensitivity of each of the two mouse models was at least equal to that of the monkey test, suggesting that these mouse systems might be considered as a potential replacement for the monkey test of OPV. However, more data are needed to establish regulatory criteria of acceptability for vaccine lots tested in Tg PVR mice. This is the first study conducted with Tg PVR mice with all three types of poliovirus vaccine preparations.

Animals↗
PubMed1998

Genetic stability of Sabin 1 strain of poliovirus: implications for quality control of oral poliovirus vaccine.

The Sabin vaccine strains of poliovirus, like all RNA viruses, exist as a quasispecies of genomic sequences whose composition can be altered during virus propagation. Since changes in vaccine virus during manufacture can enhance the neurovirulent potential of the vaccine, each monovalent lot of oral poliovirus vaccine (OPV) undergoes several tests to ensure consistency of manufacture, including the monkey neurovirulence test (MNVT). Recently, we proposed a new molecular approach for direct quantification of vaccine variants with neurovirulent potential as an alternative way to monitor consistency of OPV production. Analysis of the Sabin 1 genome allowed us to identify a limited number of specific loci that exhibit significant change during viral propagation in vitro and in vivo. Here we explore the possible roles of these changes and show that 7427-U-->C and 7441-G-->A alterations in the 3'-UTR of the Sabin 1 virus do not increase monkey neurovirulence. These, as well as our previous results, suggest that only mutations in the 5'-UTR play a significant role in the limited increase in Sabin 1 monkey neurovirulence observed after extended propagation of the virus beyond the passage level used in vaccine production. Our studies with high-passage batches of the Sabin 1 strain confirmed the stability of this strain, which retains acceptable levels of monkey neurovirulence even after serial passages at elevated temperature. Compared to the MNVT, molecular analysis of the genetic composition of Sabin 1 poliovirus provides a more sensitive analytical approach to monitor consistency of vaccine production.

Animals↗
PubMedDate not supplied

Vaccination with inactivated poliovirus vaccine and oral poliovirus vaccine in Denmark.

In Denmark a polio vaccination program including both inactivated poliovirus vaccine (IPV) and oral poliovirus vaccine ( OPV ) has been in use since 1968. Three injections of IPV are given when the children are five, six, and 15 months of age. Subsequently, three vaccinations with trivalent OPV are administered at the age of three, four, and five years. The acceptance rate is high-93%-98%-and greater than 95% of the population has antibodies to poliovirus. The geometric mean titer of serum antibodies is much greater than 10 IU for all three types. The epidemiology of poliomyelitis and the background for the development of the present vaccination schedule are reviewed.

Antibodies, Viral↗
PubMed1991

Efficacy and safety of oral poliovirus vaccine and inactivated poliovirus vaccine.

Inactivated poliovirus vaccine (IPV) is the vaccine of choice for protection against paralytic poliomyelitis provided that it is used within the context of a program to increase and sustain the level of uptake to as close to 100% as possible. This means targeting the disadvantaged in society as well as those who have their own pediatrician. The reasons are that enhanced-potency killed polio vaccine is safe, whereas oral poliovirus vaccine (OPV) is associated with a low, but definite, risk of paralysis, especially after the first dose. The immunity, as measured by antibody concentrations, is at least as good as and, in some circumstances, such as in the tropics or for booster doses, better than that provided by OPV. IPV reduces the replication of living poliovirus and produces herd immunity, as exemplified by experience in Sweden and Holland. The immunity, whether induced by OPV or IPV, involves memory cells and is long lasting, as seen by the rapid secondary response to a booster dose. IPV also can be mixed with other vaccine components to provide immunity against an increasing range of childhood infections.

Humans↗
PubMed2002

Novel design architecture for genetic stability of recombinant poliovirus: the manipulation of G/C contents and their distribution patterns increases the genetic stability of inserts in a poliovirus-based RPS-Vax vector system.

Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus env gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.

Amino Acid Sequence↗
PubMed2002

Laboratory surveillance for wild poliovirus and vaccine-derived poliovirus, 2000-2001.

After the 1988 World Health Assembly resolution to eradicate poliomyelitis, the Global Laboratory Network for Poliomyelitis Eradication (the laboratory network) was established by the World Health Organization (WHO). The laboratory network is one component of the international surveillance system for detecting polio through notification of cases of acute flaccid paralysis (AFP). As progress is made in eradicating polio worldwide, the laboratory network provides critical virologic evidence about where poliovirus is circulating as a guide to supplementary vaccination activities aimed at interrupting transmission. This report summarizes the laboratory data for documenting the decline in wild poliovirus transmission and the occurrence of vaccine-derived polioviruses and highlights the expanding role of the laboratory network in global polio eradication.

Clinical Laboratory Techniques↗
PubMed2006

Assessment of efficacy of a live oral poliovirus vaccine for virulent Sabin-like poliovirus 1 strains in Japan.

Virulent Sabin-like poliovirus (VSLP) was isolated from river and sewage waters between October 1993 and September 1995 in Toyama Prefecture, Japan (Yoshida et al., Lancet 356, 1461-1463, 2000). In this study, to assess the possibility of an epidemic of poliomyelitis caused by a VSLP in Japan under the current vaccination policy of administration of live attenuated oral poliovirus vaccine (OPV), we determined titers of serum neutralizing antibodies to poliovirus 1 (PV-1) strains Sabin (vaccine strain), Mahoney (wild-type strain) and G4-12 (VSLP) in various groups of residents of Toyama Prefecture, Japan. The seropositivity and geometric mean neutralizing antibody titers against these strains in the individuals who obtained two doses of OPV were 99.1%, 94.5% and 95.5%, respectively, and 564, 186 and 194, respectively. Although the antibody titers to G4-12 were lower compared with those to Sabin, these results indicate that the OPV vaccination policy in Japan has been effective in preventing poliomyelitis caused by VSLPs. These results also suggest that (i) an epidemic of poliomyelitis caused by a VSLP has not occurred in Japan due to herd immunity, and (ii) the possibility of reemergence of VSLPs will be prevented if sufficient herd immunity is acquired immediately after completion of the OPV vaccination in accordance with the poliomyelitis eradication program.

Adolescent↗
PubMed1997

Childhood immunizations: position on the enhanced inactivated poliovirus vaccine and live attenuated oral poliovirus vaccine dilemma.

Recent review of the polio vaccines (live attenuated oral poliovirus vaccine [OPV] and enhanced inactivated poliovirus vaccine [eIPV]) for children has generated much debate between infectious disease experts and public health officials. Poliomyelitis was a common medical condition in the 1940s and 1950s, and the success of OPV in eradicating poliomyelitis from the United States and even the Western hemisphere cannot be disputed. However, the adverse condition of vaccine-associated paralytic poliomyelitis (VAPP) has been reported in eight to nine cases per year as a result of exclusively using OPV in the United States. The dilemma has been how to continue the elimination of wild-type poliovirus paralytic poliomyelitis in the United States and worldwide while minimizing the occurrence of VAPP. Clinical trials have supported that eIPV and OPV provide similar protection for humoral immunity. However, OPV provides superior gastrointestinal immunity, which is a public health benefit for vulnerable populations. Recommendations among experts have concluded that the sequential eIPV/OPV is the preferred schedule, with eIPV only or OPV only as alternative equally acceptable schedules. Therefore, factors such as cost, compliance, and access to health care must be considered by parents and providers when selecting a polio vaccine regimen, especially among underserved populations.

Age Factors↗
PubMed1983

Antibody to host factor precipitates poliovirus RNA polymerase from poliovirus-infected HeLa cells.

An antibody to a host cell protein (host factor) which inhibited the host factor-dependent transcription of poliovirion RNA was used to characterize the immunoprecipitated proteins from both uninfected and poliovirus-infected HeLa cells. The antibody specifically precipitated a major protein of approximately 67,000 Da and a 40,000-Da minor protein from uninfected HeLa cells. The immune serum also specifically precipitated poliovirus RNA polymerase (P63, NCVP4) from poliovirus-infected HeLa cells indicating an association of the viral RNA polymerase with the host factor in vivo. A physical interaction between purified host factor and P63 could also be demonstrated in vitro. The antibody was further used to determine the distribution of host factor among different subcellular fractions of HeLa cells. Most of the cellular host factor was present in the cytoplasmic fraction (70%). Approximately 30% of the total host factor was found to be associated with the ribosomes.

Antibodies↗
PubMed1987

Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants.

A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.

Animals↗
PubMed2000

Three-dimensional structure of poliovirus receptor bound to poliovirus.

Poliovirus initiates infection by binding to its cellular receptor (Pvr). We have studied this interaction by using cryoelectron microscopy to determine the structure, at 21-A resolution, of poliovirus complexed with a soluble form of its receptor (sPvr). This density map aided construction of a homology-based model of sPvr and, in conjunction with the known crystal structure of the virus, allowed delineation of the binding site. The virion does not change significantly in structure on binding sPvr in short incubations at 4 degrees C. We infer that the binding configuration visualized represents the initial interaction that is followed by structural changes in the virion as infection proceeds. sPvr is segmented into three well-defined Ig-like domains. The two domains closest to the virion (domains 1 and 2) are aligned and rigidly connected, whereas domain 3 diverges at an angle of approximately 60 degrees. Two nodules of density on domain 2 are identified as glycosylation sites. Domain 1 penetrates the "canyon" that surrounds the 5-fold protrusion on the capsid surface, and its binding site involves all three major capsid proteins. The inferred pattern of virus-sPvr interactions accounts for most mutations that affect the binding of Pvr to poliovirus.

Amino Acid Sequence↗