PubMed Health⌕ Search

Biomedical subjects

R Crainic

Publications and source records attributed to R Crainic.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Environmental Microbiology↗
PubMed1995

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.

Animals↗
PubMed1994

Point mutations involved in the attenuation/neurovirulence alternation in type 1 and 2 oral polio vaccine strains detected by site-specific polymerase chain reaction.

One of the problems raised by the use of the attenuated oral poliovirus vaccine (OPV) Sabin strains is the genetic instability of the attenuated phenotype upon multiplication in vivo. Nucleotide sites critical for attenuation were identified for each of the three poliovirus serotypes. One important position lies in the 5' non-coding region of the genome of each of the three OPV strains, at nucleotide 480 in type 1, 481 in type 2 and 472 in type 3. Point mutations at these positions were usually selected upon multiplication in vivo as substitutions of the vaccine-type residue. The reversion was found to correlate with an increased degree of neurovirulence. To screen easily for this mutation in a great number of strains, we developed a site-specific polymerase chain reaction method based on the property of the Taq polymerase to elongate only primers with a perfect homology at the 3' extremity. We screened for this mutation in five type 1 and nine type 2 polio vaccine-derived strains isolated from vaccine-associated paralytic poliomyelitis (VAPP) cases and in 16 such strains isolated from healthy vaccinees. All 14 strains isolated from VAPP presented the reversion. Of the eight pairs of type 1 isolates from healthy vaccinees, four presented the reversion 3 days after vaccine administration and all but one at 7 days postvaccination. These results support the involvement of the 5' non-coding specific nucleotide sites in the reversion to neurovirulence of attenuated polio vaccine strains upon multiplication in the human gut.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

High diversity of poliovirus strains isolated from the central nervous system from patients with vaccine-associated paralytic poliomyelitis.

To establish the etiology of vaccine-associated paralytic poliomyelitis (VAPP), isolates from the central nervous system (CNS) from eight patients with VAPP were compared with stool isolates from the same patients. The vaccine (Sabin) origin was checked for all of the available isolates. Unique and similar strains were recovered from paired stool and CNS samples for five of the eight VAPP cases and the three wild-type cases included in the study. In the remaining three VAPP cases, the stool samples and, in one case, the CNS samples contained mixtures of strains. In two of these cases an equivalent of the CNS isolate was found among the strains separated by plaque purification from stool mixtures, and in one case different strains were isolated from CNS and stool. This shows that the stool isolate in VAPP might not be always representative of the etiologic agent of the neurological disease. A wide variety of poliovirus vaccine genomic structures appeared to be implicated in the etiology of VAPP. Of nine CNS vaccine-derived strains, four were nonrecombinant and five were recombinant (vaccine/vaccine or even vaccine/nonvaccine). The neuropathogenic potential of the isolates was evaluated in transgenic mice sensitive to poliovirus. All of the CNS-isolated strains lost the attenuated phenotype of the Sabin strains. However, for half of them, the neurovirulence was lower than expected, suggesting that the degree of neurovirulence for transgenic mice is not necessarily correlated with the neuropathogenicity in humans.

Animals↗
PubMed1993

Evolution and polymorphism of poliovirus genomes.

The three poliovirus serotypes are very stable. Breakthrough of the serotype barrier has never been observed in the natural evolution of poliovirus. This serotype stability contrasts with the high level of genomic and phenotypic variability that occurs within the bounds of serotype. The efficient control of poliomyelitis by immunization is based upon type-specific immunity and serotype stability. The development of attenuated strains by Albert Sabin was possible because of the high variability of poliovirus genomes. The three Sabin strains, one for each serotype, were selected as variants of non-attenuated wild polioviruses, and each represents a unique poliovirus genotype. A consequence of poliovirus variability is the polymorphic character of its genome. This polymorphism makes possible the identification of poliovirus genotypes upon which studies on poliovirus evolution, virologic surveillance, and poliomyelitis diagnostics are based. The antigenic and genomic peculiarities of the Sabin strains are used to distinguish them from wild polioviruses among field isolates. The mechanisms of poliovirus variation and their significance to the evolution of both wild and vaccine poliovirus strains are the subjects of this article. The natural evolution of polioviruses is discussed in the context of the global initiative to eradicate poliomyelitis, which relies on the worldwide use of Sabin's vaccine.

Animals↗
PubMed1993

Polioviruses with natural recombinant genomes isolated from vaccine-associated paralytic poliomyelitis.

To determine how oral poliovirus vaccine (Sabin) strains evolve during replication in humans and to confirm the etiology of vaccine-associated paralytic poliomyelitis (VAPP), we examined 70 vaccine-derived strains isolated from VAPP cases. Two distant sequences of the poliovirus genome were targeted for a double restriction fragment length polymorphism assay (RFLP) of reverse-transcribed genomic segments amplified by PCR, an extension of the method that we described previously (Balanant et al., 1991). One (RFLP-1) was a 480-long nucleotide sequence coding for the N-terminal part of the VP1 capsid polypeptide, situated in the 5' third of the viral genome (nucleotides 2401-2880). The other (RFLP-3D1) was a 291-long nucleotide sequence coding for a part of the viral polymerase, situated near the 3' end of the genome (nucleotides 6086-6376). Strain-specific restriction profiles could be generated for different field isolates by using three restriction enzymes in each case: HaeIII, DdeI, and HpaII for RFLP-1 and HaeIII, DdeI and RsaI for RFLP-3D1. With few exceptions, the vaccine-specific RFLP profiles were found to be conserved in both regions during replication of these viruses in humans. Thus, RFLP could be used as a marker so as to identify the origin of viral isolates at both ends of their genome. Whether viral isolates were vaccine-derived was determined by using strain-specific monoclonal antibodies and RFLP-1. Among the 70 isolates, 21 of the 43 type 2 strains and 15 of the 22 type 3 strains had a recombinant genome. None of the 5 type 1 Sabin-derived isolates was found to be recombinant. Both intertypic vaccine/vaccine and vaccine/non-vaccine recombinants were detected. Partial nucleotide sequencing confirmed the RFLP results in all cases that were investigated. In one case, it was possible to predict the recombination junction site from the restriction profiles. This site was more precisely localized by sequencing. The C6203 > U nucleotide substitution, which is suspected to contribute to the reversion toward neurovirulence of the attenuated Sabin 1 strain, was detected in almost all the recombinant genomes containing Sabin 1-specific sequences at the 3' extremity. This mutation was detected by identification of the modified RsaI profile in the RFLP-3D1. The results presented in this paper suggest that recombination, alone or together with mutation, might be one of the mechanisms of the reversion toward neurovirulence of attenuated vaccine strains and of the natural evolution of poliovirus.

Base Sequence↗
PubMed1993

Molecular epidemiology of type 1 polioviruses isolated in Israel and defined by restriction fragment length polymorphism assay.

The genomic variability of 27 type 1 polioviruses (PV-1) isolated in Israel during 1980-1991 was examined by restriction fragment length polymorphism (RFLP) analysis of a reverse-transcribed genomic fragment amplified by polymerase chain reaction. By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles were generated for the PV-1/Mahoney and PV-1/Sabin strains and 27 wild-type isolates. The profile observed for PV-1 isolated during an outbreak in 1988 was also observed for PV-1 isolated from different places in Israel in 1982 and 1983, 1987, and 1991. This profile, characterized by the lack of the DdeI site, was different from the DdeI profile of PV-1 isolated in 1984 or in 1986 from sporadic cases of poliomyelitis. The diversity of circulating PV-1 in Israel was also confirmed by nucleotide sequence analysis. The epidemiologic information provided by the RFLP and sequence data establishes a clear epidemiologic link between epidemic and sporadic virus strains and demonstrates the power of this molecular approach to epidemiology.

Base Sequence↗
PubMed1993

Genomic modifications in naturally occurring neurovirulent revertants of Sabin 1 polioviruses.

Five type 1 Sabin-like poliovirus strains were isolated from paralytic cases of poliomyelitis. Their Sabin origin was confirmed by antigenic analysis with monoclonal antibodies and restriction fragment length polymorphism (RFLP) assay. Several nucleotide positions known to play a role in the reversion towards neurovirulence were examined: viz 480, 2741, 6203, 7441. Analysis was performed by enzymatic restriction and partial sequencing of PCR-amplified genomic segments. All the strains bore the reversion G > A at residue 480; in four of the five viruses, only this reversion was found. The fifth strain presented four additional reversions and several new mutations. These results indicate that, during multiplication in the human gut, Sabin 1 poliovirus carrying one or several mutations related to reversion towards neurovirulence can be selected.

Humans↗
PubMed1991

Characterization of persistent poliovirus mutants selected in human neuroblastoma cells.

Six Sabin-derived persistent poliovirus mutants were selected in human neuroblastoma IMR-32 cells. The mutants had a titer 30 to 10(5) times lower in nonneural HEp-2c cells than in IMR-32 cells. When the growth cycles of persistent viruses in the two cell lines were compared, the most striking feature was a delay of 2 to 4 hr in virus release from HEp-2c cells. In Hep-2c cells, type 1 mutants could spontaneously establish a persistent infection in the absence of any exogenous viral inhibitor. Mutations at a rate of 1 every 210 nucleotides had accumulated in the genome of the type 1 mutants selected in neuroblastoma cells, modifying cell specificity and conferring the ability to persist in some nonneural cells. These results indicate that mutants of poliovirus with highly modified biological properties can be selected in vitro in cells of neural origin.

Blotting, Northern↗
PubMed1991

The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay.

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.

Base Sequence↗
PubMed1991

Antibodies may act synergistically or additively with interferon to inhibit poliovirus.

We investigated the protective effects of combinations of interferon-alpha (IFN-alpha) and neutralizing monoclonal antibodies (mAbs) and serum antibodies against poliovirus type 1 (PV-1) in vitro. Our results indicate that the antiviral effects of IFN-alpha and most neutralizing mAbs to PV-1 act synergistically to inhibit PV-1. However, the antiviral effects of IFN-alpha and one type specific mAb to PV-1 were additive. Further, the protective effects observed with combinations of IFN-alpha and rabbit, monkey or human serum containing effects observed with combinations strains Mahoney (Mah) and Sabin (Sab) were similar to those observed with combinations of IFN-alpha and mixtures of mAbs with synergistic and additive activities. Our studies suggest that the antiviral activity of neutralizing antibody acts with the antiviral activity of IFN to inhibit virus infection synergistically or additively and that the different antibody activities are associated with the mechanism of neutralization.

Animals↗
PubMed1991

Analysis of neutralization-escape mutants selected from a mouse virulent type 1/type 2 chimeric poliovirus: identification of a type 1 poliovirus with antigenic site 1 deleted.

A chimeric type 1/type 2 poliovirus (v510), in which the antigenic site 1 (Ag1) of poliovirus type 1 (PV-1) Mahoney was replaced by the corresponding site of poliovirus type 2 (PV-2) Lansing, is known to be neurovirulent for mice and neutralized by both type 1 and type 2 monoclonal antibodies. Neutralization-escape mutants to monoclonal antibodies specifically recognizing the PV-2 sequence were obtained from v510. The nucleotide sequence and the mouse neurovirulence of mutants were determined. Amino acid substitutions obtained inside the replaced sequence, at positions 95 and 99, and outside this site, at positions 93 or 104, rendered the virus attenuated for mice. One of the escape mutants harboured a deletion of the entire substituted nonapeptide sequence in v510. This particular virus, which is a PV-1 Mahoney lacking the natural Ag1 loop, does not react with PV-2-specific monoclonal antibodies, has a ts phenotype, is heat-labile and is devoid of neurovirulence for mice.

Amino Acid Sequence↗
PubMed1990

Poliovirus permissivity and specific receptor expression on human endothelial cells.

To test the role of the endothelial cells (EC) in the poliomyelitis pathogenesis, their sensitivity to poliovirus infection was determined at different times after isolation from the human umbilical vein. While 80% of EC were permissive for poliovirus after 4 days of in vitro primary cultures, only 6% of freshly isolated EC were susceptible to poliovirus infection. A progressive development of this susceptibility was observed during the first 3 days of culture. In an attempt to explain the mechanism of the appearance of cell permissivity for poliovirus, the expression of the poliovirus receptor on EC was studied by cytofluorometric analysis using an anti-receptor monoclonal antibody. The number of poliovirus receptor molecules in the EC population was found to increase with time. This paralleled the increase of the poliovirus-binding capacity of EC cultures. In contrast, the efficiency of viral internalization did not appear to be dependent on the age of culture. These results indicate that the development of EC permissivity for poliovirus in vitro is mainly dependent on the expression of poliovirus receptor on the cell surface.

Cells, Cultured↗
PubMed1990

Structural factors modulate the activity of antigenic poliovirus sequences expressed on hybrid hepatitis B surface antigen particles.

We have studied the functional expression of antigenic poliovirus fragments carried by various hybrid hepatitis B surface antigen (HBsAg) particles. Several constructions were made by using two different insertion sites in the HBsAg molecule (amino acid positions 50 and 113) and two different sequences, one derived from poliovirus type 1 (PV-1) and the other from PV-2. The inserted fragments each encompassed residues 93 to 103 of the capsid protein VP1, a segment which includes the linear part of the neutralization antigenic site 1 of the poliovirus. The antigenicity and immunogenicity of the hybrid particles were evaluated and compared in terms of poliovirus neutralization. A high level of antigenic and immunogenic activity of the PV-1 fragment was obtained by insertion at position 113 but not at position 50 of HBsAg. However, a cooperative effect was observed when two PV-1 fragments were inserted at both positions of the same HBsAg molecule. Antibodies elicited by the PV-2 fragment inserted at amino acid position 113 did not bind or neutralize the corresponding poliovirus strain. They did, however, bind a chimeric poliovirus in which the homologous antigenic fragment of PV-1 had been replaced by that of PV-2. The only virions that were neutralized by these antibodies were certain mutants carrying amino acid substitutions within the PV-2 fragment. These results show that position, constraints from the carrier protein, and nature of the inserted sequences are critically important in favoring or limiting the expression of antigenic fragments as viral neutralization immunogens.

Amino Acid Sequence↗
PubMed1989

Molecular pathogenesis of type 2 poliovirus in mice.

Lansing strain of poliovirus type 2 (PV-2) produces a fatal paralytic disease in mice after intracerebral inoculation. To identify virus-containing nerve cells in poliovirus-infected mice, we developed a technique of in situ hybridization for viral RNA with a poliovirus-specific riboprobe. Large numbers of genomes were found in motoneurons and their processes, as well as in ghosts of neurons with or without inflammatory cells. This indicates that the death of motoneurons is due to a direct effect of viral replication. The presence of viral RNA in neuronal processes suggests an axonal dissemination of poliovirus within the central nervous system (CNS). Detection of viral genomes in small cells located in anterior and in posterior horns shows that motoneurons are not the only cells susceptible to poliovirus in the CNS. To see if PV-2/Lansing still exits in nature, PV-2 isolated from the CNS and from the gut of two human cases of paralytic poliomyelitis were characterized by determining epitope maps and phenotypic markers and by sequencing regions of the genome coding for antigenic sites and the 5' non-coding region. Comparison of virus isolated from the intestine and from the CNS of the same subject revealed a high degree of homology, suggesting that virus isolated from the gut is representative of virus in the neuronal lesions. The isolate from a case had more homology to PV-2/Lansing, whereas the other isolate showed more homology to PV-2/Sabin. These results show that, despite intensive vaccination with oral polio vaccine for 25 years, wild PV-2/Lansing-like viruses, pathogenic for mice, still circulate in nature ad induce human paralytic cases.

Animals↗
PubMed1989

Expression of a poliovirus type 1 neutralization epitope on a diphtheria toxin fusion protein.

The diphtheria toxin (DT) secreted by Corynebacterium diphtheriae is used after formolization as an efficient vaccine against diphtheria. In an attempt to evaluate its capacity to present heterologous peptide sequence in a recognized form, we created in-phase insertion in the gene encoding the non-toxic mutant protein CRM228 of DT. The sequence chosen for insertion was the synthetic DNA fragment encoding a poliovirus neutralization epitope. Tripartite fusion proteins comprising the mutant DT, the poliovirus peptide and beta-galactosidase were obtained in E. coli and purified by affinity chromatography. These fusion proteins reacted both with antibodies directed against the DT and a poliovirus specific monoclonal antibody. Moreover, these hybrid toxins induced protective antibodies against the lethal effect of DT and neutralizing antibodies against poliovirus. We conclude that the modification of highly immunogenic DT may provide a means for the presentation of foreign peptide sequences to the immune system.

Animals↗
PubMed1989

Persistent poliovirus infection of human neuroblastoma cells.

Two human neuroblastoma cell lines were persistently infected with poliovirus strains of all three serotypes. In persistently infected IMR-32 cells, which were studied in greatest detail, viral antigens were present in most cells, and over a 9-month period virions were found in the medium at high titers. Persistently infected cells were resistant to superinfection by Sabin 1, 2, and 3 poliovirus but sensitive to coxsackievirus B3. The viruses recovered from persistently infected cells were studied for conservation of epitopes, host cell specificity, and temperature resistance phenotype. The antigenic site 1 carried by the major capsid protein VP1 was modified on the persistent viruses of all three serotypes. This was confirmed for one virus by sequencing the corresponding genomic region in which two mutations were detected. The titers of persistent viruses were 1-3 log10 units higher on IMR-32 cells than on nonneuronal HEp-2 cells, while parental viruses had similar titers on both lines. When thermosensitive viruses were used to initiate the infection, the persistent viruses were found to be thermoresistant at 39 degrees C. Together the results indicate that the persistent infection correlated with the selection of highly mutated viral strains. Poliovirus-infected neuroblastoma cell lines thus constitute an in vitro model of chronic viral infections, which are increasingly implicated in human neural diseases.

Cell Line↗