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Inactivated poliomyelitis vaccine made from Sabin strains (sIPV) has been encouraged to introduce in the "Global Polio Eradication & Endgame Strategic Plan" and increasingly used worldwide. Attenuated Sabin strains used in manufacture of oral poliovirus vaccine (OPV) and sIPV may regain full or partial neurovirulence during growth in vaccine recipients and the vaccine manufacturing processes. Ensuring the molecular consistency of sIPV batches and that no mutation accumulates beyond the level present in past batches are important for quality control of vaccine manufacture process. Direct deep-sequencing allows the construction of a library of virus RNA and the detection of genetic mutations throughout the viral genome. In the present study, direct deep-sequencing was conducted to detect molecular mutations in virus passages, multiple sIPV monovalent lots, and virus monovalent lots from different polio type III strains. The results indicated that direct deep-sequencing can be used to identify and quantify small amounts of mutant viruses in vaccine preparations, trace the source of a specific virus seed, and monitor the batch-to-batch consistency of vaccines, suggesting that this technique could be suitable for the quality control and consistency monitoring of sIPV production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The poliovirus eradication initiative has spawned global immunization infrastructure and dramatically decreased the prevalence of the disease, yet the original virus eradication goal has not been met. The suboptimal properties of the existing vaccines are among the major reasons why the program has repeatedly missed eradication deadlines. Oral live poliovirus vaccine (OPV), while affordable and effective, occasionally causes the disease in the primary recipients, and the attenuated viruses rapidly regain virulence and can cause poliomyelitis outbreaks. Inactivated poliovirus vaccine (IPV) is safe but expensive and does not induce the mucosal immunity necessary to interrupt virus transmission. While the need for a better vaccine is widely recognized, current efforts are focused largely on improvements to the OPV or IPV, which are still beset by the fundamental drawbacks of the original products. Here we demonstrate a different design of an antipoliovirus vaccine based on in situ production of virus-like particles (VLPs). The poliovirus capsid protein precursor, together with a protease required for its processing, are expressed from a Newcastle disease virus (NDV) vector, a negative-strand RNA virus with mucosal tropism. In this system, poliovirus VLPs are produced in the cells of vaccine recipients and are presented to their immune systems in the context of active replication of NDV, which serves as a natural adjuvant. Intranasal administration of the vectored vaccine to guinea pigs induced strong neutralizing systemic and mucosal antibody responses. Thus, the vectored poliovirus vaccine combines the affordability and efficiency of a live vaccine with absolute safety, since no full-length poliovirus genome is present at any stage of the vaccine life cycle. IMPORTANCE A new, safe, and effective vaccine against poliovirus is urgently needed not only to complete the eradication of the virus but also to be used in the future to prevent possible virus reemergence in a postpolio world. Currently, new formulations of the oral vaccine, as well as improvements to the inactivated vaccine, are being explored. In this study, we designed a viral vector with mucosal tropism that expresses poliovirus capsid proteins. Thus, poliovirus VLPs are produced in vivo , in the cells of a vaccine recipient, and are presented to the immune system in the context of vector virus replication, stimulating the development of systemic and mucosal immune responses. Such an approach allows the development of an affordable and safe vaccine that does not rely on the full-length poliovirus genome at any stage., (Copyright © 2018 American Society for Microbiology.)

Background: Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods., Methods: We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process., Results: All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants., Conclusion: Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Until 2008 in Romania poliomyelitis has been controlled by predominantly using trivalent oral poliovirus vaccine (TOPV). The alternative vaccination schedule (formalin inactivated poliovirus vaccine IPV/OPV) has been implemented starting September 2008 and at the begining of 2009 was decided only vaccination with IPV. Between 1995-2006 the risk of the vaccine-associated paralytic poliomyelitis (VAPP) decreased with an average of less than 2 VAPP cases/year and no VAPP case between 2007 - September 2009. Begining with 2007 the number of the poliovirus strains isolated was less. All 9 poliovirus strains (PV) isolated between 2007-2009 and investigated by RT-PCR-RFLP in VP1-2A and VP3-VP1 coding regions showed Sabin-like profiles, and only one strain poliovirus type 3 showed Sabin 2-like profile by RFLP in 3D coding ARN polymerase region. The study about the seroprevalence of antibodies against poliovirus types in serum samples from the acute flaccid paralysis (AFP), facial paralysis (FP) cases showed that the seroprevalence of antibodies against types 1 and 2 Sabin strains was higher (>90%) than for type 3 Sabin strains (average 85%). It was confirmed the necessity of maintaining a proper vaccine coverage in population, after the switch in the vaccination strategy in Romania until all threats of poliovirus are eliminated globally.

Live attenuated vaccines remain the safest, most cost-effective intervention against viral infections. Because live vaccine strains are generated empirically and the basis for attenuation is usually ill defined, many important viruses lack an efficient live vaccine. Here, we present a general strategy for the rational design of safe and effective live vaccines that harnesses the microRNA-based gene-silencing machinery to control viral replication. Using poliovirus as a model, we demonstrate that insertion of small miRNA homology sequences into a viral genome can restrict its tissue tropism, thereby preventing pathogenicity and yielding an attenuated viral strain. Poliovirus strains engineered to become targets of neuronal-specific miRNAs lost their ability to replicate in the central nervous system, leading to significant attenuation of neurovirulence in infected animals. Importantly, these viruses retained the ability to replicate in nonneuronal tissues. As a result, these engineered miRNA-regulated viruses elicited strong protective immunity in mice without producing disease.

An assay for the evaluation of protective properties of inactivated poliovirus vaccines (IPVs) in transgenic (Tg) mice susceptible to poliovirus has been developed and optimized for type 2 IPV. This method was used to compare the immunogenicity and protective properties of experimental IPV produced from the attenuated Sabin strain (sIPV) with those of conventional IPV (cIPV) produced from the wild-type (wt) poliovirus MEF-1 strain. Modified enzyme-linked immunosorbent assays (ELISAs) were used to measure immune response in serum and saliva samples from test mice. Tg mice were vaccinated and were challenged either with wt poliovirus or virulent poliovirus derived from the vaccine strain. Compared with cIPV, sIPV induced lower levels of antibodies and did not completely protect mice against challenge with wt virus but did protect mice against challenge with the virulent vaccine-derived strain. This may be due to an 18% nucleotide difference between the MEF-1 and Sabin 2 strains, resulting in 72 amino acid substitutions and leading to antigenic dissimilarity. Immunological properties of both strains, revealed by cross-neutralization tests and ELISAs, confirmed that MEF-1 possesses broader immunogenicity than does Sabin 2. This animal model may be used for the assessment of new IPVs and of combination vaccines containing an IPV component., (Copyright 2004 Infectious Diseases Society of America)

A book published in 1999 hypothesized that the scientists who worked with the CHAT type 1 attenuated poliomyelitis strain, tested in the former Belgian Congo in the late 1950s, had covertly prepared the vaccine in chimpanzee kidney cells contaminated with a simian immunodeficiency virus, which evolved into human immunodeficiency virus type 1 group M. This article summarizes the results of the investigation conducted by the author to determine the legitimacy of the accusation. Testimony by eyewitnesses, historical documents of the time, epidemiological analysis, and analysis of ancillary phylogenetic, virological, and polymerase chain reaction data all indicate that this hypothesis is false.

SV40 was first identified as a contaminant of poliovaccines used from 1955 until 1963. Recently, SV40 has been detected in several human tumors. The virus detected in human tumors often contained only one 72-bp enhancer in the regulatory region, in contrast to the SV40 originally isolated from poliovaccines, which contained two 72-bp enhancers. The origin of viruses with one 72-bp enhancer in humans was unknown, because it was thought that these viruses were not present in poliovaccines. It was also thought that all poliovaccine vials produced from 1955 until 1963 had been discarded, thus the possibility that one 72-bp virions contaminated those vials could not be tested. We unexpectedly obtained what appear to be the last available vials of poliovaccine produced in 1955. In these vials, we detected and sequenced SV40 containing only one 72-bp enhancer in the regulatory region. The tissue culture cytopathic test currently used in the United States to screen oral poliovaccines was designed to detect rapidly proliferating SV40 virions containing two 72-bp enhancers. We found that this test is not sensitive enough to detect low amounts of the slow-replicating SV40 virions containing one 72-bp enhancer. This virus was easily detected in the same cells by immunostaining and PCR. Twelve current vials of poliovaccines tested uniformly negative for SV40, suggesting that the precaution of preparing poliovaccines from kidneys obtained from monkeys bred in isolated colonies prevented SV40 contamination. Our data demonstrate that humans were exposed to SV40 viruses with both one 72-bp enhancer and two 72-bp enhancers SV40 through contaminated vaccines. Our data also suggest that instead of cytopathic tests, immunohistochemical and/or molecular studies should be used to screen poliovaccines for SV40 to completely eliminate the risk of occasional contamination.

We investigated the neurovirulence of a chimeric poliovirus consisting of the coding region of Lansing type 2 poliovirus and the 5'NCR of type 3 poliovirus. Specifically we carried out studies on the effects of stable base pairing, between nucleotides 472 and 537, on neurovirulence. Mice were injected intracranially with the attenuated chimeric virus MAS 27 plaque 1 having the following nucleotide base pair at 472-537, G-G. Mutants recovered from the CNS of inoculated mice were divided into three groups according to the nucleotide sequence of the 5'NCR; MAS 27C type viruses having a single base change (G-C) at the position 472, MAS 27G type mutants having a single base change (G-C) at the position 537, and MAS 27U type viruses having a single base change (G-U) at the position 537. The isolate MAS 27C had back-mutated to the wild type, and 100 000 fold more virulent than attenuated MAS 27G and MAS 27U. MAS 27C type mutants were predominant, suggesting that base C at position 472 is favoured to form a stable secondary structure with guanine at position 537. Attenuated MAS 27G, however, carries guanine and cytosine at nucleotides 472 and 537 respectively, and was a stable attenuated virus following passage in four serial generations of mice. Furthermore, attenuated MAS 27G poliovirus produced viral proteins less efficiently and had slower growth rates than the revertant MAS 27C. The stable attenuated base paired MAS 27G might provide the basis for a prototype for a live attenuated stable type 3 poliovaccine., (Copyright 1998 Academic Press.)

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

During the fall and winter of 1992-1993 an outbreak of wild poliovirus type 3-associated poliomyelitis involving 71 patients occurred in The Netherlands. Almost all of the individuals involved in the outbreak belonged to an orthodox religious denomination that prohibits vaccination. A surveillance was initiated to determine if there had been an importation of this same strain of wild poliovirus into a southern Alberta community with a similar religious affiliation. Viral culture of stool samples from consenting individuals in the community resulted in viral isolates which typed as poliovirus type 3. Sequencing of amplicons generated from both the 5' nontranslated region and the VP1/2A portion of the genomes from representative poliovirus isolates indicated a greater than 99% genetic similarity to the strain from The Netherlands. The results of this study show that the utilization of PCR-based diagnostics offers an important molecular tool for the concise and rapid surveillance of possible cases of wild poliovirus importation into communities with individuals at risk for infection.

We have developed a method for differentiating polioviruses from nonpolio enteroviruses using PCR. A pair of panpoliovirus PCR primers were designed to match intervals encoding amino acid sequences within VP1 that are strongly conserved among polioviruses. The initiating primer hybridizes with codons of a 7-amino-acid sequence that has been found only in polioviruses; the second primer matches codons of a domain thought to interact with the cell receptor. The panpoliovirus PCR primers contain mixed-base and deoxyinosine residues to compensate for the high degeneracy of the targeted codons. All RNAs from 48 vaccine-related and 110 wild poliovirus isolates of all three serotypes served as efficient templates for amplification of 79-bp product. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions. Sensitivities of poliovirus detection were as low as 100 fg (equivalent to approximately 25,000 genomic copies or 25 to 250 PFU) when the amplified products were visualized by ethidium bromide fluorescence. These degenerate PCR primers should aid in the detection of all polioviruses, including those wild poliovirus isolates for which genotype-specific reagents are unavailable.

Poliomyelitis has been largely controlled by the use of attenuated live vaccine strains. The molecular basis of attenuation is beginning to be understood, but the interactions between the virus and its human host remain mysterious.

Transgenic mice that express the human receptor for poliovirus and mutant analysis by PCR followed by restriction enzyme cleavage (MAPREC) are two new approaches to poliovirus vaccine neurovirulence testing. This review assesses the validity and current status of these tests. Both approaches require further rigorous validation and development. They are most likely to be used as corollary rather than replacement tests for the current neurovirulence tests.

Intertypic vaccine/vaccine recombinant polioviruses are frequently isolated from vaccine-associated paralytic poliomyelitis cases (VAPP). We identified a vaccine/nonvaccine poliovirus recombinant as the causative agent of a lethal VAPP. Partial RNA sequencing revealed a tripartite recombinant structure of the viral genome. This consisted of a central capsid core of vaccine origin flanked by two units of nonvaccine origin. The first nonvaccine genomic unit spanned the whole 5' noncoding region, and the second one almost the entire nonstructural protein-coding region and the 3' noncoding region. Amino acid and nucleotide sequence similarities in the 3' and 5' unidentified regions indicated that the viral donor(s) were poliovirus species, suggesting recombination between a vaccine-derived and a wild poliovirus. The nonvaccine donor(s) could not be identified among the investigated wild polioviruses cocirculating in the same geographical area. This is the first report of a natural recombination event occurring in the 5' genomic extremity of poliovirus. The neurovirulence for transgenic mice and the pathogenicity for humans of the recombinant suggested that the modular genomic organization of this virus might have conferred a selective advantage over its vaccine parent.

The sequence of the poliovirus genome encoding 3CD (a protease) was transferred to the yeast Saccharomyces cerevisiae on expression vectors with either a constitutive or an inducible promoter. Transformants could only be obtained with vectors carrying the inducible transcription unit. Extracts of induced cells were able to cleave cell-free synthesized P1, the precursor of the poliovirus capsid proteins, into VP0, VP3 and VP1. In yeast cells constitutively expressing P1, induction of 3CD expression resulted in only trace amounts of processed products. Processing could be improved considerably by simultaneous induction of both P1 and 3CD expression. Analysis of extracts of such induced cells revealed the presence of particles that resembled authentic subviral particles.

Procedures for differentiation between wild and vaccine-derived strains of poliovirus are required, particularly in countries where wild and vaccine-related strains coexist. For this differentiation, we tested the method of Inouye and Hondo (S. Inouye and R. Hondo, Arch. Virol. 129:311-316, 1993) for discrimination of closely related viruses by using stringent microplate hybridization of PCR products. We used a pair of primers with enterovirus common sequences (between these primers there is a variable region for capsid proteins) for PCR using templates from wild and vaccine-derived poliovirus strains which were isolated in tissue culture and serotyped by neutralization assay. We also used the same primers for preparation of probes, which were labelled by incorporation of biotin-dUTP in the PCR, with the three original Sabin vaccine virus strains used as templates. The amplified DNAs from the isolates were immobilized on microplate wells and were then hybridized with the labelled probes. We found that, under the usual hybridization conditions, the Sabin vaccine virus strain probes hybridized with both wild and vaccine-derived viruses, but under stringent conditions, they reacted only with vaccine-derived viruses of the same serotype, clearly differentiating these from wild-type viruses.

Fecal shedding of virulent revertant polioviruses was examined in isolates from infants previously immunized with > or = 1 dose of orally administered live attenuated polio vaccine (OPV) alone, enhanced-potency inactivated polio vaccine (EIPV) alone, or a combination of both. After administration of OPV alone, vaccine poliovirus serotypes were recovered in feces within 1 week and for as long as 31-60 days in 30%-80% of subjects after 1 or 2 doses and in 30%-50% after immunization with > or = 3 doses. No revertant poliovirus shedding was observed after OPV challenge in subjects immunized previously with > or = 3 doses of OPV. However, fecal shedding of revertant poliovirus after OPV challenge was observed in 50%-100% of subjects previously immunized with > or = 3 doses of the EIPV. These findings suggest that prior immunization with EIPV does not prevent fecal shedding of revertant polioviruses after subsequent reexposure to OPV.

The genomic variability of poliovirus was examined by analyzing the restriction fragment length polymorphism of a reverse-transcribed genomic fragment amplified by the polymerase chain reaction. The fragment was a 480-nucleotide sequence of the poliovirus genome coding for the N-terminal half of the capsid protein VP1, including antigenic site 1. The identification of a pair of generic primers flanking this fragment allowed its amplification in practically all the poliovirus strains tested so far (more than 150). By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles could be generated for the amplified genomic fragment of each of the six reference poliovirus strains tested: one representative wild poliovirus of each of the three serotypes (P1/Mahoney, P2/Lansing, and P3/Finland/23127/84) and the three Sabin vaccine strains. When 21 poliovirus field isolates previously identified as Sabin vaccine-related were tested, they showed restriction profiles identical to those of the originating homotypic Sabin virus, demonstrating the conservation of these profiles during virus replication in humans. These profiles could thus be used as markers for Sabin-derived genotypes. To compare the distribution of poliovirus genotypes in nature before and after the introduction of poliovirus vaccines, the restriction profiles of the amplified genomic fragment of a total of 72 strains of various geographic and temporal origins were determined. Strains isolated before the introduction of polio vaccines displayed a wide diversity of genotypes. In contrast, wild (Sabin unrelated) strains isolated after vaccine introduction, during a single epidemic in a particular geographic area, showed identical or very similar restriction profiles, indicating the circulation of predominant regional genotypes. Our results indicate that the assay we developed for the analysis of the restriction fragment length polymorphism of the poliovirus genome may be used to identify and characterize poliovirus genotypes circulating in nature.

In order to begin to elucidate the genomic basis of the attenuation of mouse-adapted, poliovirus type 2 strain W-2 (PV2/W-2), we have cloned and sequenced the virus and compared it with the virulent, mouse-adapted PV2/Lansing strain. In addition, we have performed computer-generated comparisons of PV2/W-2 to the non-mouse-adapted, attenuated PV2/Sabin strain to determine whether mutational patterns occur that result in attenuation. The PV2/W-2 genome is 7434 nucleotides in length, which is three bases shorter than PV2/Lansing. The 5' non-coding region of PV2/W-2 is 747 nucleotides in length (compared to 744 in PV2/Lansing) and shares 98.8% identity with PV2/Lansing and 82.3% identity with PV2/Sabin. Overall, the PV2/W-2 polyprotein (2205 amino acids) is two amino acids shorter than that of either PV2/Lansing or PV2/Sabin (2207 amino acids). These contiguous deletions fall within the P3-D region (polymerase). Within these 2205 amino acid residues only 26 differences were observed between PV2/W-2 and PV2/Lansing (98.8% identity), whereas 92 occurred between PV2/W-2 and PV2/Sabin (95.8% identity). The 3' non-coding region of PV2/W-2 is 72 nucleotides in length and shares 100% identity with PV2/Lansing and 98.6% identity with PV2/Sabin. Amino acid changes in the capsid protein region occurred in neutralization sites 1 and 3, areas previously shown to be important for pathogenicity. The cleavage site between non-structural proteins P2-C/P3-A consisted of a glutamine-serine pair, in contrast to other sequenced polioviruses which have a glutamine-glycine dipeptide.

Poliovirus type 1 cDNA was prepared from viral RNA encoding the VP1 capsid region of the virus by using a specific DNA primer and was cloned in Escherichia coli. DNA fragments corresponding to VP1 amino acid positions 129 to 302 (pPM5k3), 52 to 302 (pPMhae3), and 24 to 129 (pPMDxba) were incorporated into plasmid vectors designed to express Trp LE-poliovirus VP1 fusion proteins under the control of the inducible tryptophan promoter-operator system. Induction of bacterial cultures containing the plasmids resulted in the production of fusion proteins which accounted for 21% (pPMhae3), 68% (pPM5k3), and 27% (pPMDxba) of the total cell protein. The proteins were purified, and each reacted with polyclonal antibodies raised against intact virions as measured by an enzyme-linked immunosorbent assay. The sera from rabbits immunized with the bacterially produced fusion proteins pPMDxba and pPMhae3 contained poliovirus-neutralizing antibodies.