RESUMEN
Detection of enteroviruses and rhinoviruses has traditionally been based on laborious and time-consuming virus isolation. Recently, rapid and sensitive assays for detecting enterovirus and rhinovirus genomic sequences by reverse transcription-polymerase chain reaction (RT-PCR) have been introduced. An RT-PCR assay is described that amplifies both enteroviral and rhinoviral sequences, followed by liquid-phase hybridization carried out in a microtiter plate format. In the hybridization assay, amplicons are identified by enterovirus- or rhinovirus-specific probes carrying lanthanide chelate labels, which can be detected simultaneously by time-resolved fluorometry. The sensitivity and specificity of the RT-PCR-hybridization method were evaluated with a representative collection of enteroviruses and rhinoviruses and tested further its applicability to the clinical setting with cerebrospinal fluid samples and nasopharyngeal aspirates. The RT-PCR assay amplified all enteroviruses and rhinoviruses tested, and all but one amplicon gave a positive result in the subsequent hybridization assay. The RT-PCR-hybridization method was more sensitive than virus isolation for the detection of enteroviruses and rhinoviruses in the clinical samples. High sensitivity, rapidity, and easy performance make the assay suitable for the routine diagnosis of enterovirus and rhinovirus infections.
Asunto(s)
Infecciones por Enterovirus/diagnóstico , Enterovirus/aislamiento & purificación , Fluorometría/métodos , Lantano/metabolismo , Infecciones por Picornaviridae/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Rhinovirus/aislamiento & purificación , Animales , Líquido del Lavado Bronquioalveolar/virología , Línea Celular , Enterovirus/genética , Infecciones por Enterovirus/líquido cefalorraquídeo , Infecciones por Enterovirus/virología , Células HeLa , Humanos , Infecciones por Picornaviridae/líquido cefalorraquídeo , Infecciones por Picornaviridae/virología , ARN Viral/análisis , Rhinovirus/genética , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Coxsackievirus A9 (CAV9), a human pathogen causing symptoms ranging from common colds to fatal infections of the central nervous system, is an icosahedral single-stranded RNA virus that belongs to the genus Enterovirus of the family Picornaviridae. One of the four capsid proteins, VP1, includes the arginine-glycine-aspartate (RGD) motif within its C-terminal extension. This region binds to integrin alpha v beta 3, the only receptor for CAV9 to be conclusively identified to date. RESULTS: The crystal structure of CAV9 in complex with the antiviral compound WIN 51711 has been solved to 2.9 A resolution. The structures of the four capsid proteins, VP1 to VP4, resemble those of other picornaviruses. The antiviral compound is bound in the VP1 hydrophobic pocket, and it is possible that the pocket entrance contains a second WIN 51711 molecule. Continuous electron density for the VP1 N terminus provides a complete picture of the structure close to the fivefold axis. The VP1 C-terminal portion is on the outer surface of the virus and becomes disordered five-residues N-terminal to the RGD motif. CONCLUSIONS: The RGD motif is exposed and flexible in common with other known integrin ligands. Although CAV9 resembles coxsackie B viruses (CBVs), several substitutions in the areas implicated in CBV receptor attachment suggest it may recognise a different receptor. The structure along the fivefold axis provides new information on the uncoating mechanism of enteroviruses. CAV9 might bind a larger natural pocket factor than other picornaviruses, an observation of particular relevance to the design of new antiviral compounds.
Asunto(s)
Cápside/química , Enterovirus/fisiología , Enterovirus/ultraestructura , Secuencia de Aminoácidos , Cápside/metabolismo , Cristalización , Cristalografía por Rayos X/métodos , Enterovirus/aislamiento & purificación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos , Estructura Secundaria de Proteína , Receptores de Vitronectina/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.
Asunto(s)
Infecciones por Coxsackievirus/etiología , Enterovirus Humano B/patogenicidad , Enterovirus/patogenicidad , Animales , Animales Recién Nacidos , Infecciones por Coxsackievirus/microbiología , Modelos Animales de Enfermedad , Enterovirus/clasificación , Enterovirus/genética , Enterovirus Humano B/clasificación , Enterovirus Humano B/genética , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , ARN Viral/genética , ARN Viral/metabolismo , Serotipificación , Especificidad de la EspecieRESUMEN
Although echovirus 22 is presently classified as a member of the enterovirus group in the family of picornaviruses, it has been reported to have exceptional biological properties when compared with other representatives of the group. We have determined the complete nucleotide sequence of the echovirus 22 (Harris strain) genome, which appears to be significantly different from all the other studied picornaviruses. However, the organization of the genome [7339 nucleotides, excluding the poly(A) tract] is similar to that of previously sequenced picornaviruses. This genome includes a 5' untranslated region, relatively well-conserved when compared with aphtho- and cardioviruses, followed by an open reading frame coding for a 2180-amino acid-long polyprotein. The amino termini of capsid polypeptides VP1 and VP3 were determined by direct sequencing, and the other proteolytic cleavage sites in the polyprotein were predicted by comparison with other picornavirus proteins. The amino acid identities of echovirus 22 polypeptides with the corresponding proteins of other picornaviruses are in the 14-35% range, similar to those percentages seen when representatives of the five picornavirus groups (entero-, rhino-, cardio-, aphtho-, and hepatoviruses) are compared. Our results suggest that echovirus 22 belongs to an independent group of picornaviruses.
Asunto(s)
Enterovirus Humano B/genética , Genes Virales , Picornaviridae/clasificación , ARN Viral/genética , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Enterovirus Humano B/clasificación , Datos de Secuencia Molecular , Picornaviridae/genética , Proteínas Virales/genéticaRESUMEN
We have used enzymic amplification of specific nucleic acid sequences followed by hybridization, for the rapid detection and typing of human picornaviruses after cell culture isolation. The test is based on the synthesis of cDNA, the polymerase chain reaction and the use of oligonucleotide probes. The primers were selected from the 5' non-coding region of the genome representing highly conserved regions. Sequences specific to enteroviruses and rhinoviruses were used as probes. The assay was able to identify all the picornavirus reference strains analysed and it was also possible to discriminate between enteroviruses and rhinoviruses by the hybridization procedure. When 29 picornavirus clinical isolates were analysed, all except one were detected by gel electrophoresis and a specific hybridization signal was obtained with all except three strains using the oligonucleotide probes.
Asunto(s)
Amplificación de Genes , Picornaviridae/genética , Reacción en Cadena de la Polimerasa , Secuencia de Bases , ADN Viral/análisis , Enterovirus/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Picornaviridae/clasificación , Picornaviridae/aislamiento & purificación , Rhinovirus/genéticaRESUMEN
Forty-eight different enterovirus serotypes were analysed by a nucleic-acid hybridization test using probes derived from the 3' end of coxsackievirus A21 (CA21) and B3 (CB3), poliovirus 3 (P3) and enterovirus 70 (E70). More than 90% of the serotypes could be detected with this collection of reagents. The CB3 probe reacted with all the coxsackie B viruses, with all three poliovirus serotypes, and with almost all of the 30 ECHO virus types tested. In addition some of the coxsackie A viruses and the BrDr 73 strain of enterovirus 71 gave a positive signal with this probe. The P3 probe detected all the poliovirus strains and also some coxsackievirus A isolates but no coxsackie B or ECHO viruses. A similar hybridization pattern as with the P3 probe was obtained when the CA 21 probe was used. The E70 probe appeared to be strain-specific. The results indicate that nucleic-acid hybridization is a useful method for rapid detection and subgrouping of enteroviruses during virus isolation, and that the test could be further developed for typing of the strains.