RESUMEN
VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by approximately 10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of approximately 3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started approximately 9.3 years earlier. This estimate is about the time (6. 9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication.
Asunto(s)
Síndromes de Inmunodeficiencia/complicaciones , Filogenia , Poliomielitis/inmunología , Vacuna Antipolio Oral , Poliovirus/clasificación , Poliovirus/fisiología , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Cápside/genética , Proteínas de la Cápside , Niño , Evolución Molecular , Heces/virología , Estudios de Seguimiento , Variación Genética , Humanos , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/virología , Masculino , Datos de Secuencia Molecular , Poliomielitis/complicaciones , Poliomielitis/virología , Poliovirus/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ensayo de Placa Viral , Replicación ViralRESUMEN
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.
Asunto(s)
Poliovirus/clasificación , Poliovirus/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Aminoácidos , Elementos sin Sentido (Genética) , Cápside/genética , Cápside/inmunología , Proteínas de la Cápside , Codón , Humanos , Datos de Secuencia Molecular , Poliomielitis/inmunología , Poliovirus/inmunología , Vacuna Antipolio de Virus Inactivados/genética , ARN Viral/análisis , ARN Viral/genética , Análisis de Secuencia de ARN , Serotipificación , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Poliovirus/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Aminoácidos , Secuencia de Bases , Cápside/genética , Proteínas de la Cápside , Codón/genética , Secuencia Conservada , Cartilla de ADN/genética , Enterovirus/clasificación , Enterovirus/genética , Enterovirus/aislamiento & purificación , Estudios de Evaluación como Asunto , Humanos , Inosina/análogos & derivados , Inosina/genética , Datos de Secuencia Molecular , Poliomielitis/prevención & control , Poliomielitis/virología , Poliovirus/clasificación , Poliovirus/aislamiento & purificación , Vacuna Antipolio de Virus Inactivados/genética , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Serotipificación , Especificidad de la Especie , Virología/métodos , Virología/estadística & datos numéricosRESUMEN
We developed RNA probes for the identification of poliovirus isolates by blot hybridization. Two sets of vaccine strain-specific probes were prepared. They complemented variable genomic domains within (i) the 5'-untranslated region and (ii) the amino-terminal codons of VP1. An enterovirus group probe (EV/5UT) matching highly conserved 5'-untranslated region sequences was used to estimate the quantities of poliovirus (or enterovirus) RNA in the samples. Poliovirus sequences amplified from Sabin strain virion RNA templates by PCR were inserted into the pUC18 plasmid vector. The antisense PCR primer for each probe set contained sequences encoding a T7 promoter. Hybrids were detected by a sensitive nonisotopic method. RNA probes were labeled by incorporation of digoxigenin-uridylate into the transcripts. The binding of probe to immobilized poliovirus RNAs was visualized by hydrolysis of the chemiluminescent substrate 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamant ane) catalyzed by alkaline phosphatase conjugated to anti-digoxigenin (Fab) fragments. The specificities of the probes were evaluated with a panel of poliovirus isolates that had previously been characterized by sequence analysis. The RNAs of vaccine-related isolates hybridized with the appropriate probe sets. Wild polioviruses representing a broad spectrum of contemporary genotypes were recognized by the inabilities of their genomes to form stable hybrids with the Sabin strain-specific probes.
Asunto(s)
Vacuna Antipolio Oral/aislamiento & purificación , Poliovirus/aislamiento & purificación , Sondas ARN , Secuencia de Bases , ADN Viral , Ensayo de Inmunoadsorción Enzimática/métodos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Poliovirus/genética , Vacuna Antipolio Oral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
All entero- and rhinovirus RNAs sequenced thus far possess A and U residues at positions corresponding to nucleotides 480 and 525, respectively, of poliovirus type 1. These two nucleotides have been proposed previously to form a base pair. The single exception to this rule appears to be the Sabin type 1 strain, which has a G480. Isolates of the Sabin 1 virus from healthy vaccinees were shown to have either a reversion to A480 or a second-site mutation U525----C, both restoring a potential for efficient base pairing. In vitro translation experiments demonstrated that poliovirus type 1 RNAs with either A480-U525 or G480-C525 are more efficient in promoting translation initiation as compared with the Sabin 1 RNA (G480-U525). The Sabin 2 strain has a U and an A at position 398 and 481, respectively, while its predecessor, strain P712, is shown to have C398 and G481. All the derivatives of the Sabin 2 isolated from vaccine-associated paralytic poliomyelitis cases shown reversion to G481, and most of them reverted also to C398. It is proposed that bases at positions 398 and 481 may be involved in a tertiary interaction. The in vitro template activity of the Sabin type 2 RNA (A481) is significantly lower than that of the isolate RNAs with G481, thus confirming the relation between attenuation and translation efficiency demonstrated previously for the type 1 and type 3 Sabin strains. The C----U change at position 398 exerted only a minor effect on the RNA template activity.
Asunto(s)
Vacuna Antipolio Oral/genética , Poliovirus/genética , ARN Viral/genética , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Genes Virales/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis , Nucleótidos/genética , Poliovirus/crecimiento & desarrollo , Poliovirus/aislamiento & purificación , Biosíntesis de Proteínas , Relación Estructura-ActividadRESUMEN
Determination of the patterns of genomic variation among RNA virus isolates is a powerful approach for establishing their epidemiologic interrelationships. The standard technique for such studies, ribonuclease T1 oligonucleotide fingerprinting, can detect similarities only among very closely related isolates. The rapid evolution of the poliovirus genome during transmission in humans requires the application of alternate methods to identify more distant relationships. To obtain a substantially broader view of the distribution of wild poliovirus type 1 genotypes in nature, we compared 150 bases of genomic sequence information (encoding parts of the capsid protein VP1 and the noncapsid protein 2A) from 62 isolates obtained from poliomyelitis patients in five continents. The partial sequence information allowed us to (1) identify numerous geographic foci of endemic circulation of wild type 1 polioviruses, (2) reveal previously unsuspected links between cases in distant communities, (3) monitor the displacement from the environment of preexisting polioviruses by viruses from other regions, and (4) recognize the recombinant (vaccine-wild; wild-wild) origins of some epidemic polioviruses.
Asunto(s)
Poliomielitis/epidemiología , Poliovirus/genética , ARN Viral/genética , Secuencia de Bases , Brotes de Enfermedades , Genes Virales , Geografía , Datos de Secuencia Molecular , Mapeo Nucleótido , Poliomielitis/microbiología , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/genéticaRESUMEN
Poliovirus isolates can be identified according to their genotypes with use of the technique of oligonucleotide fingerprinting. Fingerprint analysis is performed by cleaving the viral RNA genome with ribonuclease T1 and separating the fragments (oligonucleotides) in two dimensions. The larger, structurally unique oligonucleotides distribute into patterns ("fingerprints") highly characteristic of a specific overall RNA sequence. Isolates from the same epidemic have very similar fingerprints. Isolates from distinct epidemics have very different fingerprints, a consequence of the rapid evolution of polioviruses during replication in humans. Similarity in the fingerprints of case isolates provides independent evidence for epidemiologic linkage. Fingerprinting can readily distinguish vaccine-related isolates from wild strains. Contemporary vaccine-related isolates are very probably vaccine-derived because their fingerprints contain characteristic vaccine-strain oligonucleotide spots (types 1 and 3) and because their wild-type parents are unlikely to have survived largely unaltered in the natural environment. Some examples of applications of this technique within different epidemiologic settings are described.
Asunto(s)
Oligonucleótidos/análisis , Poliovirus/genética , Humanos , Poliomielitis/microbiología , Poliovirus/aislamiento & purificación , Vacuna Antipolio de Virus Inactivados/análisis , Replicación ViralRESUMEN
Enterovirus 70 isolates obtained in Asia and the Americas between 1980 and 1981 from cases of acute hemorrhagic conjunctivitis were found to be very closely related by RNase T1 oligonucleotide fingerprinting. Two closely related isolates from the first acute hemorrhagic conjunctivitis epidemic (1969 to 1972) differed by many oligonucleotides from the 1980 to 1981 pandemic strains. The strong similarities of oligonucleotide patterns of isolates from the same epidemic but from distant regions of the world suggest that the genome of enterovirus 70 tends to be conserved during natural infection, a possible consequence of the transient nature of the disease.
Asunto(s)
Conjuntivitis/microbiología , Brotes de Enfermedades , Infecciones por Enterovirus/microbiología , Enterovirus/análisis , Hemorragia/microbiología , Oligonucleótidos/análisis , Enfermedad Aguda , Bangladesh , Enterovirus/genética , Enterovirus/aislamiento & purificación , Florida , Genes Virales , Honduras , Humanos , Japón , Marruecos , Oligonucleótidos/genética , Pakistán , Tailandia , VietnamRESUMEN
Poliovirus isolates of serotypes 2 and 3 from patients whose paralytic poliomyelitis cases were classified as oral vaccine-associated were analysed by oligonucleotide mapping of the virus genomes and by polyacrylamide gel electrophoresis of the virus proteins. Oligonucleotide maps of all isolates were similar to the maps of the corresponding oral vaccine strain. No two isolates gave identical maps. Most maps differed from that of the vaccine strain by at least one oligonucleotide spot. Maps of some isolates revealed numerous differences, indicating that multiple (greater than 100) genetic changes had occurred in the vaccine virus genomes during replication in one or two individuals. In contrast, maps of some neural tissue isolates showed minimal differences from the reference vaccine maps, raising the possibility that neurovirulence may be restored by a small number of genetic changes. For many isolates, changes were also detected in the mobilities of processing rates of the virus proteins.
Asunto(s)
Genes Virales , Poliomielitis/microbiología , Vacuna Antipolio Oral/efectos adversos , Poliovirus/crecimiento & desarrollo , Adulto , Preescolar , Humanos , Lactante , Oligorribonucleótidos/análisis , Poliomielitis/etiología , Poliovirus/genética , ARN Viral/análisis , Proteínas Virales/análisis , Replicación ViralRESUMEN
In 1979 there was a small outbreak of poliomyelitis among non-immunized Amish individuals in the United States. Epidemiological evidence linked these cases to those in Canada in 1978 which themselves were related to cases in the Netherlands in 1978. Poliovirus type 1 was found to be the cause both in the US and in the other countries. Strain characterization tests were carried out on virus isolates from all three outbreaks. All the type 1 strains were non-vaccine-like both with the absorbed strain specific antisera and with the modified Wecker test. However, the latter as well as the rct test used to characterize these strains are obviously not very discriminating marker tests. The oligonucleotide mapping procedure appears to be a much more definitive approach to recognizing relationships between polioviruses. The various marker tests provided laboratory data in support of the epidemiological evidence that the poliovirus type 1 spread from Holland to Canada in 1978 and from there to the US in 1979.
Asunto(s)
Poliomielitis/microbiología , Poliovirus/clasificación , Brotes de Enfermedades , Humanos , Oligopéptidos/análisis , Poliomielitis/epidemiología , Poliovirus/análisis , Poliovirus/genética , Estados UnidosAsunto(s)
Variación Genética , Poliovirus/genética , ARN Viral/genética , Proteínas Virales/genética , Adolescente , Adulto , Canadá , Niño , Preescolar , Brotes de Enfermedades , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Lactante , Kuwait , Masculino , Persona de Mediana Edad , Países Bajos , Oligorribonucleótidos/análisis , Poliomielitis/microbiología , Poliovirus/aislamiento & purificación , Ribonucleasa T1 , Estados Unidos , Replicación ViralRESUMEN
In 1973, a type 1 poliomyelitis epidemic in Kenya was curtailed at an early stage by two mass distributions of trivalent oral vaccine. It was considered useful to know the immunity status of the child population that had resulted from the vaccine distributions and that had presumably contributed to its control. We also wished to know to what extent wild and vaccine virus strains were in circulation after the mass vaccination campaign. Anal swabs and blood were collected from a sample of the children in four areas where the efficiency of vaccine distribution had varied, and the results of virus isolation attempts and antibody tests are reported. Three poliovirus strains were isolated. It was surprising that, in general, the herd immunity after two vaccination rounds did not substantially differ from that found in Kenya on other occasions. Possible reasons for these results are discussed.
Asunto(s)
Poliomielitis/prevención & control , Factores de Edad , Anticuerpos Antivirales/aislamiento & purificación , Preescolar , Humanos , Lactante , Kenia , Poliomielitis/epidemiología , Poliomielitis/inmunología , Poliomielitis/microbiología , Poliovirus/aislamiento & purificación , Vacuna Antipolio Oral/administración & dosificación , Vacuna Antipolio Oral/uso terapéutico , Factores de TiempoRESUMEN
The results of a long-term survey of poliomyelitis in Kenya are reported. Virus was isolated from patients and a limited number of sera were also tested for antibodies. The purpose of the investigation was to elucidate the epidemiological characteristics of the disease and to determine whether epidemics could be predicted. A 3-year rhythm was found in the circulation of type 1 poliovirus, making possible the prediction and hence, on two occasions, the curbing or prevention of epidemics.