Circulation of a type 1 recombinant vaccine-derived poliovirus strain in a limited area in Romania.

After intensive immunisation campaigns with the oral polio vaccine (OPV) as part of the Global Polio Eradication Initiative, poliomyelitis due to wild viruses has disappeared from most parts of the world, including Europe. Here, we report the characterization of a serotype 1 vaccine-derived poliovirus (VDPV) isolated from one acute flaccid paralysis (AFP) case with tetraplegia and eight healthy contacts belonging to the same small socio-cultural group having a low vaccine coverage living in a small town in Romania. The genomes of the isolated strains appeared to be tripartite type 1/type 2/type 1 vaccine intertypic recombinant genomes derived from a common ancestor strain. The presence of 1.2% nucleotide substitutions in the VP1 capsid protein coding region of most of the strains indicated a circulation time of about 14 months. These VDPVs were thermoresistant and, in transgenic mice expressing the human poliovirus receptor, appeared to have lost the attenuated phenotype. These results suggest that small populations with low vaccine coverage living in globally well-vaccinated countries can be the origin of VDPV emergence and circulation. These results reaffirm the importance of active surveillance for acute flaccid paralysis and poliovirus in both polio-free and polio-endemic countries.

Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

Genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees.

The trivalent oral poliomyelitis vaccine (OPV) contains three different poliovirus serotypes. It use therefore creates particularly favorable conditions for mixed infection of gut cells, and indeed intertypic vaccine-derived recombinants (VdRec) have been frequently found in patients with vaccine-associated paralytic poliomyelitis. Nevertheless, there have not been extensive searches for VdRec in healthy vaccinees following immunization with OPV. To determine the incidence of VdRec and their excretion kinetics in primary vaccinees, and to establish the general genomic features of the corresponding recombinant genomes, we characterized poliovirus isolates excreted by vaccinees following primary immunization with OPV. Isolates were collected from 67 children 2 to 60 days following vaccination. Recombinant strains were identified by multiple restriction fragment length polymorphism assays. The localization of junction sites in recombinant genomes was also determined. VdRec excreted by vaccinees were first detected 2 to 4 days after vaccination. The highest rate of recombinants was on day 14. The frequency of VdRec depends strongly on the serotype of the analyzed isolates (2, 53, and 79% of recombinant strains in the last-excreted type 1, 2, and 3 isolates, respectively). Particular associations of genomic segments were preferred in the recombinant genomes, and recombination junctions were found in the genomic region encoding the nonstructural proteins. Recombination junctions generally clustered in particular subgenomic regions that were dependent on the serotype of the isolate and/or on the associations of genomic segments in recombinants. Thus, VdRec are frequently excreted by vaccinees, and the poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes.

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

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

[Intense campaign for vaccination with the oral polio vaccine: what are the repercussions on the enterovirus world?]. / Campagnes intensives de vaccination avec le vaccin polio oral: quelles répercussions sur le monde des entérovirus? Groupe d'Etude Entérovirus.

To eradicate poliomyelitis and poliovirus, intensive vaccination campaigns with oral polio-vaccine (OPV) have been organised. Eradication campaigns may well be successful because the antiviral immunity and the local intestinal immunity due to OPV in particular avoids and/or limits poliovirus circulation. These campaigns give interesting opportunities for studying the impact of viral vaccines on the viral world in terms of ecological and genetic virology. The pre-eradication phase we are now entering brings with it two kinds of problems. First, the major disadvantage of OPV is the genetic and phenotypic variability of the vaccine strains. This variability leads to the spread of potentially pathogenic strains, which can be implicated in vaccine-associated paralytic poliomyelitis (VAPP). Genetic changes are characterised by point mutations and by genetic exchanges among OPV strains, between OPV and wild strains and perhaps between poliovirus and non-polio enteroviruses (ENPV). The fact that a few OPV mutant strains have been shown to multiply and/or to circulate for long periods suggests that OPV could sustain a reservoir of pathogenic poliovirus strains. Second, there are ecological considerations. The disappearance of wild poliovirus through OPV vaccination could be due not only to antiviral local immunity but also to competition between OPV strains and wild strains for infecting the digest tract. Moreover, a competition between OPV and other enteroviruses may take place in a common ecological niche. To our knowledge, the possible impact of intensive OPV vaccination campaigns on the ENPV populations has never been considered. Because the goal of poliovirus eradication may be reached in the near future, there is worry as to the possible evolution of ENPV towards highly epidemic and pathogenic strains. This is leading those laboratories involved in poliomyelitis surveillance not only to search for remaining wild poliovirus strains but also to study the possible long-term circulation of OPV strains and to develop efficient ENPV surveillance.

Natural genetic exchanges between vaccine and wild poliovirus strains in humans.

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.

Molecular epidemiology of poliovirus infection in Tunisia.

This report is an overview of poliomyelitis surveillance in Tunisia from 1991 to 1996. In all, 2088 stool specimens, collected from 152 acute flaccid paralysis (AFP) cases and from 1747 of their healthy contacts were investigated. Virus isolation was done systematically in RD and HEp-2C cell lines and isolated viruses were typed by sero-neutralisation as polioviruses or non-polio enteroviruses. Poliovirus isolates were analysed systematically for their wild or vaccine-related origin by two methods--one based on antigenic differences and one on genetic differences between strains. All type 2 polioviruses were vaccine-related and most wild viruses belonged to polio serotype 3. Wild polio type 3 viruses were detected in 1991 and 1992 in six cases of paralytic polio. A silent circulation of wild polio 1 and wild polio 3 was detected in 1994. No wild virus was detected in Tunisia from 1995 onwards. Wild polioviruses were sequenced and compared with Tunisian wild strains isolated during the 1980s, as well as other genotypes from the international database. These investigations revealed a single Tunisian polio 3 genotype that has been circulating from 1985 to 1994 and two different polio 1 genotypes. These results reflect effective control strategies within the country and contribute to the improvement of the polio eradication programme effectiveness at national and global levels.

The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses.

The development of media free of serum and animal or human proteins is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al., 1994), by replacing the animal derived products by plant extracts. The new serum and animal protein free medium (MDSS2N) can be efficiently used for biomass production of various cell lines. These cells grow equally well or better in this new serum-free medium than in the old formulation (MDSS2):* BHK-21/BRS cells, adapted to MDSS2N, showed an overall specific growth rate of 0.0197 h-1 (mu_max = 0.0510+/-0.0058 h-1), whereas those cultivated in MDSS2 grew with an average specific growth rate of 0.0179 h-1 (mu_max = 0.0305+/-0.0177 h-1).* Vero cells grew with an average specific growth rate of 0.0159 h-1 and 0.0153 h-1 in MDSS2 and MDSS2N, respectively. Very similar growth rates were obtained in microcarrier cultures in stirred tank reactors: the specific growth rates were 0.0161 h-1 and 0.0166 h-1 for MDSS2 and MDSS2N cultures, respectively.* For MDCK cells, when cultured on microcarriers in bioreactors, a higher average specific growth rate was observed in MDSS2N than in MDSS2; values of 0.0248 h-1 and 0.0168 h-1, respectively, were obtained.The capacity of MDSS2N to support the production of different viruses was equally evaluated and it could be established that for certain viruses there are no or insignificant differences between MDSS2N and MDSS2 (influenza and polio virus), whereas, the production of rabies virus is somewhat reduced in MDSS2N when compared to MDSS2. The use of MDSS2N for cell culture and the production of various viruses is discussed.

Antigenic and molecular characterization of wild type 1 poliovirus causing outbreaks of poliomyelitis in Albania and neighboring countries in 1996.

Mass vaccination has led poliomyelitis to become a rare disease in a large part of the world, including Western Europe. However, in the past 20 years wild polioviruses imported from countries where polio is endemic have been responsible for outbreaks in otherwise polio-free European countries. We report on the characterization of poliovirus isolates from a large outbreak of poliomyelitis that occurred in Albania in 1996 and that also spread to the neighboring countries of Yugoslavia and Greece. The epidemics involved 145 subjects, mostly young adults, and caused persisting paralysis in 87 individuals and 16 deaths. The agent responsible for the outbreak was isolated from 74 patients and was identified as wild type 1 poliovirus by both immunological and molecular methods. Sequence analysis of the genome demonstrated the involvement of a single virus strain throughout the epidemics, and genotyping analysis showed 95% homology of the strain with a wild type 1 poliovirus strain isolated in Pakistan in 1995. Neutralization assays with both human sera and monoclonal antibodies were performed to analyze the antigenic structure of the epidemic strain, suggesting its peculiar antigenic characteristics. The presented data underline the current risks of outbreaks due to imported wild poliovirus and emphasize the need to improve vaccination efforts and also the need to implement surveillance in countries free of indigenous wild poliovirus.

Activation of the envelope proteins by a metalloproteinase enables attachment and entry of the hepatitis B virus into T-lymphocyte.

Previously, we identified an HBV binding factor (HBV-BF), a 50-kDa serum glycoprotein which interacts with HBV envelope proteins and which is also located in the membrane of normal human hepatocyte (A. Budkowska et al. (1993) J. Virol. 67, 4316). Here we show that HBV-BF is a neutral metalloproteinase which shares substrate specificity and properties with a newly described family of membrane type matrix metalloproteinases. HBV-BF treatment of the HBV resulted in the cleavage of the N-terminal part of the middle HBV envelope protein at the pre-S2(136-141) amino acid sequence VRGLYF/L (containing a single arginine cleavage site). HBV-BF affected the reactivity of the large HBV protein with pre-S1-specific MAbs, probably inducing the conformational change of the pre-S1 domain. The HBV-BF-digested virus remained morphologically intact with unchanged S antigenic determinants. The structural modifications of the viral envelope proteins induced by HBV-BF enabled cell membrane attachment and viral entry into the T-lymphocyte. Both processes were blocked by the metalloproteinase inhibitor 1,10 phenanthroline. Thus, the host-dependent proteolytic activation of the envelope proteins seems to be essential for the HBV entry into the cell. HBV-BF under a membrane bound or a secreted form could be (one of) the molecule(s) responsible for the HBV proteolytic activation.

Potency of wild-type or sabin trivalent inactivated poliovirus vaccine, by enzyme-linked immunosorbent assay using monoclonal antibiotics specific for each antigenic site.

Potency testing of inactivated poliovirus vaccine (IPV) is hampered by the absence of a standardized in vitro test, as well as the lack of a generally accepted quantitative animal test. In vitro tests must be able to measure selectively the content of the "D" antigen in the vaccine which includes virus neutralizing antibodies. We tested 12 poliovirus type 1, 12 type 2 and six type 3, D antigen-specific monoclonal mouse antibodies (mAb) for use in the enzyme-linked immunosorbent assay (ELISA). We characterized the site-specific reactivities of three mAbs, one for each poliovirus type. The reactivity of the complete mAb panel encompassed the important antigenic sites on the virus surface of each of the poliovirus serotypes. Some of the mAbs were cross-reactive between wild-type and Sabin strain IPV. At least one mAb of each poliovirus type that was D antigen-specific and reacted with both wild-type and Sabin IPV was directed against an antigenic site thought to be immunogenic in humans. These reagents may be useful for improved standardization of the ELISA for IPV.

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.

Random selection: a model for poliovirus infection of the central nervous system.

Mixed infections occur in the natural environment, and also result from the use of mixed live vaccines. Some recipients of the trivalent oral poliovirus vaccine develop vaccine-associated paralytic poliomyelitis (VAPP). Numerous serotypes and recombinant genotypes of vaccine-derived polioviruses may be found in stool samples from such cases. To investigate the relationship between the multiplication of various genotypes at the primary replication site in the gut and the infection outcome in the central nervous system (CNS), the viruses excreted on consecutive days by two patients with VAPP were compared with the viruses isolated from the CNS. The genotypes from stools were numerous and varied with time in both cases, suggesting a multiplication of the viruses in multiple foci in the gut. Where the CNS isolated virus clearly corresponded to one of the many viruses detected in stool, this virus was unexpectedly less neurovirulent than others isolated from stool. To assess the mechanism by which viruses with different degrees of neurovirulence are selected in the CNS, transgenic mice sensitive to poliovirus infection were inoculated extraneurally with mixtures of two phenotypically different viruses at different neuropathogenic doses. The virus(es) inducing neurological disease was then isolated from the CNS. At less than 100% input neuropathogenic dose of both inoculated viruses, individual mice were affected stochastically by the virus variants from the mixture. Extrapolated to humans, this selection pattern might explain the occurrence of CNS infections with less neurotropic viruses derived from an extraneural pool containing also highly neurotropic viruses.

Influence of host related factors on the antibody response to trivalent oral polio vaccine in Tunisian infants.

The low efficiency of trivalent oral polio vaccine (TOPV) in inducing protective antibody titres to polio3 is a problem of great importance in many regions of the world. A prospective study was conducted in 121 Tunisian infants aged 3 months during routine immunization with TOPV under carefully controlled conditions. Seroconversion rates to polio1, polio2 and polio3, one month after the third dose, were 94.7, 100 and 89.5%, respectively. The kinetics of the antibody response showed delayed and more difficult responses to polio3 compared to polio2 and polio1. The following host related factors, previously suggested to interfere with the immune response, were assessed: maternal antibodies; breast-feeding; concurrent enteric infections; and other illnesses. The main factor associated with the lack of seroconversion was concurrent infection with non-polio enteroviruses (NPE) which was found in 50% of non-responders to polio1 and/or to polio3 during the vaccination protocol whereas no NPE was isolated in vaccine responders. The other studied factors seemed not to interfere in the infants according to the locally adopted vaccination schedule and to the specific socio-economic conditions.

Elucidating mechanisms of thermostabilization of poliovirus by D2O and MgCl2.

To understand a significant reduction in the loss of poliovirus infectivity by D2O and a combination of D2O and MgCl2 at 37-45 degrees C, this paper attempts to elucidate the mechanisms underlying the thermostabilization of poliovirus. Three serotypes of Sabin oral poliovirus vaccine strains were investigated. Temperature-dependent fluorescence emission intensity studies showed that the effects of D2O and MgCl2 on the stability and conformation of poliovirus are correlated with those of the infectivity of poliovirus. Fluorescence steady-state polarization revealed that the conformation of poliovirus capsid is sensitive to D2O medium and MgCl2 salt, and that the rigidity of poliovirus conformation is increased in their presence. The exposure of poliovirus tryptophan residues to water is modified by D2O and MgCl2, as evidenced by changes in fluorescence emission intensity excited at 295 nm. The involvement of hydrogen bonding in the D2O effect was demonstrated by the greatly increased value of relative fluorescence intensity. Conformational alteration was also shown by changes in the positive band (193-230 nm) of circular dichroism spectra. D2O and MgCl2 were also found to reduce the interaction of virus with water as examined by differential scanning microcalorimetry, leading to a decline in the extent of water penetration into the poliovirus capsid. All these observations were found to be more profound in a combination of D2O with MgCl2 than D2O or MgCl2 alone. By inducing a conformation favorable to maintaining the poliovirus assembly and by reducing virus-water interaction to decrease water penetration into the poliovirus capsid, D2O, MgCl2, or D2O-MgCl2 is able to exert its thermostabilization effect. Thus, to maintain the virus assembly and conformation of the virus and to reduce the swelling of the virus capsid are key factors in increasing the thermostability of poliovirus. These two factors are mutually complementary. The latter can provide a favorable environment for the formers and the formers, in turn, lead to the latter.

Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors.

The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h, respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of (106.75) and 10(6.67) TCID50 per 50 µl; signifying a specific productivity of 0.89 and 1.07 TCID50/c.Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×10(6)c/ml. After infection with virus (multiplicity of infection (MOI) 0.1-0.3) titers of about 6.3×10(8) TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and 2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981, 47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×10(9) TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine, and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to the modification of the medium composition.

The replacement of water with deuterium oxide significantly improves the thermal stability of the oral poliovirus vaccine.

The poliomyelitis eradication programme relies largely on the massive administration of the oral poliovirus vaccine (OPV). The major difficulty in assuring good vaccine coverage, especially in hot climates, is the thermostability of the vaccine. Several attempts have been made to stabilize the OPV with limited benefits. In this report, we describe a heavy water based stabilization procedure, which has been shown to increase the thermostability of the vaccine, notably at temperatures which are commonly encountered during usual transportation in conditions of cold chain failure. Safety considerations regarding the human use of heavy water containing bioproducts are discussed.

Comparative study of five methods for intratypic differentiation of polioviruses.

A coded panel of 90 poliovirus isolates, 30 of each of the three known serotypes, was used to evaluate five methods for the intratypic differentiation of polioviruses: (i) an enzyme-linked immunosorbent assay with polyclonal cross-absorbed antisera (PAb-E), (ii) a neutralization assay with type-specific monoclonal antibodies (MAb-N), (iii) a restriction fragment length polymorphism (RFLP) assay, (iv) a Sabin vaccine strain-specific PCR assay, and (v) a Sabin vaccine strain-specific cRNA probe hybridization (ProHyb) assay. Sequence analysis was used for the definitive characterization of the strains. The panel was distributed to five laboratories; each laboratory analyzed the strains by at least two methods. Each method was used by three or four laboratories. The total performance scores (percentage correct results per number of tests) of the five methods were 96.7% for PAb-E, 93.9% for MAb-N, 91.9% for RFLP assay, 93.3% for Sabin vaccine strain-specific PCR, and 97.4% for Sabin vaccine strain-specific ProHyb. Consistent results were obtained by each laboratory for 88 of 90 isolates (97.8%) examined by PAb-E, 81 of 90 isolates (90.0%) examined by MAb-N, 78 of 90 isolates (86.7%) examined by RFLP assay, 81 of 90 isolates (90.0%) examined by PCR, and 89 of 90 isolates (98.9%) examined by ProHyb assay. Six strains were classified differently by different methods. It is recommended that at least two methods be used for the intratypic differentiation of poliovirus isolates, and each method should be based on a different principle (i.e., antigenic properties and nucleotide sequence composition). If two assays yield discrepant results, further characterization, preferably by partial sequence determination, will be required for correct identification.

Tripartite genome organization of a natural type 2 vaccine/nonvaccine recombinant poliovirus.

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.

Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus.

The temperature-sensitive and attenuated phenotypes of the Sabin type 1 vaccine strain of poliovirus result from numerous point mutations which occurred in the virulent Mahoney virus parent. One of these mutations is located in a 3D polymerase (3Dpol) codon (U-6203-->C, Tyr-73-->His) and is involved in attenuation in common mice (M. Tardy-Panit, B. Blondel, A. Martin, F. Tekaia, F. Horaud, and F. Delpeyroux, J. Virol. 67:4630-4638, 1993). This mutation also appears to contribute to temperature sensitivity, in association with at least 1 other of the 10 mutations of the 3'-terminal part of the genome including the 3Dpol coding and 3' noncoding regions. To map the other mutation(s), we constructed poliovirus mutants by mutagenesis and recombination of Mahoney and Sabin 1 cDNAs. Characterization of these poliovirus mutants showed that a second mutation in a 3Dpol codon (C-7071-->U, Thr-362-->Ile) contributes to temperature sensitivity. A mutation in the 3' noncoding region of the genome (A-7441-->G), alone or linked to another mutation (U-7410-->C), also appeared to be involved in this phenotype. The temperature-sensitive effect associated with the 3'-terminal part of the Sabin 1 genome results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the His-73 and Ile-362 codons of 3Dpol and nucleotide G-7441. Sequence analysis of strains isolated from patients with vaccine-associated paralytic poliomyelitis showed that these genetic determinants are selected against in vivo, although the Ile-362 codon appeared to be more stable than either the His-73 codon or G-7441. These genetic determinants may contribute to the safety of Sabin 1 in vaccines.