RESUMEN
Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.
Asunto(s)
Infecciones por Enterovirus/diagnóstico , Enterovirus/clasificación , Parechovirus/clasificación , Infecciones por Picornaviridae/diagnóstico , ARN Viral/genética , Infecciones por Enterovirus/virología , Europa (Continente) , Dosificación de Gen , Humanos , Meningitis Viral/diagnóstico , Tipificación Molecular , Infecciones por Picornaviridae/virología , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Rhinovirus (RV) is a frequent pathogen in young children, eliciting symptoms ranging from common colds to wheezing illnesses and lower respiratory tract infections. The recently identified RV-C seems to be associated with asthma exacerbations and more severe disease, but results vary. We studied the prevalence and severity of infection with RV in an unselected birth cohort. Children with respiratory symptoms entered the symptomatic arm of the cohort and were compared with asymptomatic children. Severity of wheezing and other respiratory symptoms was registered. Respiratory viruses were evaluated using throat and nasopharyngeal swabs on first presentation and after recovery (wheezing children). RV genotyping was performed on RV-PCR positive samples. RV was the most prevalent respiratory virus and was found in 58/140 symptomatic children (41%), 24/96 (25%) control children and 19/74 (26%) wheezing symptomatic children after recovery (p <0.05) and did not differ between wheezing and non-wheezing symptomatic children-respectively, 42% (38/90) and 40% (20/50). RV-A was the most commonly detected species (40/68, 59%), followed by RV-C (22/68, 32%) and RV-B (6/68, 9%). RV-B was more frequently detected in asymptomatic children (5/6, p <0.05). There was no significant difference in the frequency of RV species between wheezing and non-wheezing symptomatic children. Children with RV mono-infection had more severe symptoms, but no association between RV species and severity of disease was seen. In an unselected birth cohort from the Netherlands with mild respiratory disease RV was the most prevalent respiratory virus. RV(-C) infection was not associated with more severe disease or wheezing.
Asunto(s)
Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/virología , Rhinovirus , Infecciones Bacterianas , Estudios de Casos y Controles , Preescolar , Estudios de Cohortes , Coinfección , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Países Bajos/epidemiología , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/tratamiento farmacológico , Prevalencia , Rhinovirus/clasificación , Rhinovirus/genética , Estaciones del Año , Índice de Severidad de la EnfermedadRESUMEN
UNLABELLED: The family Picornaviridae is a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies. IMPORTANCE: HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPeV1-specific neutralization and cross-neutralized HPeV2. One MAb also cross-neutralized other HPeVs. Surprisingly, this MAb also neutralized CV-A9. These MAbs provide a unique tool for further research and for the diagnosis (antigen detection) and possible treatment of HPeV infections.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Parechovirus/inmunología , Infecciones por Picornaviridae/inmunología , Linfocitos B/virología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Reacciones Cruzadas , Humanos , Países Bajos/epidemiología , Parechovirus/clasificación , Parechovirus/genética , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/terapia , PrevalenciaRESUMEN
Pleconaril is a capsid inhibitor used previously to treat enterovirus infections. A pleconaril-resistant echovirus 11 (E11) strain was identified before pleconaril treatment was given in an immunocompromised patient. The patient was also treated with intravenous Ig (IVIg) for a long period but remained unresponsive. The pleconaril-resistant strains could not be neutralized in vitro, confirming IVIg treatment failure. To identify the basis of pleconaril resistance, genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M and I188L) in the pleconaril-resistant viruses were found in the pocket region of VP1. Modelling suggested that V119M could confer resistance, most probably due to the protruding sulfate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterized by a different substitution (I183M), resistance was suggested to also result from a similar mechanism, i.e. due to a protruding sulfate side chain of methionine. Our results showed that resistant strains that arise in vivo display different markers from those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIg treatment failure, we predict that one of these factors could be immune related. Thus, both IVIg and capsid inhibitors target the viral capsid and can induce mutations that can be cross-reactive, enabling escape from both IVIg and the drug. This could limit treatment options and should be investigated further.
Asunto(s)
Antígenos Virales/metabolismo , Antivirales/farmacología , Farmacorresistencia Viral , Enterovirus Humano B/genética , Enterovirus Humano B/inmunología , Oxadiazoles/farmacología , Antígenos Virales/genética , Antivirales/uso terapéutico , Infecciones por Echovirus/virología , Regulación Viral de la Expresión Génica/fisiología , Humanos , Inmunoglobulinas Intravenosas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxadiazoles/uso terapéutico , OxazolesRESUMEN
Enteroviruses (EV) and human parechoviruses (HPeV) are endemic worldwide. These infections are a constant cause of hospitalisation and severe disease, predominantly in young children and infants. Coordinated monitoring and surveillance are crucial to control these infections. We have monitored EV and HPeV epidemiology in Amsterdam from 2007 to 2011 with real-time RT-PCR and direct genotyping, facilitating highly sensitive surveillance. Moreover, we conducted a literature survey of existing surveillance data for comparison. Only 14 studies were identified. While HPeV1 was most frequently detected in Amsterdam, EV-B viruses dominated nationally and internationally. Furthermore, the top 10 strains detected differed yearly and per study. However, detection and typing methods were too varied to allow direct comparison and comprehension of the worldwide distribution and circulation patterns of the different genotypes. This limited a direct response to anticipate peaks. Uniform European monitoring programmes are essential to aid prediction of outbreaks and disease management.
Asunto(s)
Líquido Cefalorraquídeo/virología , Infecciones por Enterovirus/diagnóstico , Enterovirus/genética , Heces/virología , Parechovirus/genética , Infecciones por Picornaviridae/diagnóstico , Enterovirus/aislamiento & purificación , Infecciones por Enterovirus/virología , Genotipo , Humanos , Países Bajos , Parechovirus/aislamiento & purificación , Infecciones por Picornaviridae/virología , Vigilancia de la Población , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Centros de Atención TerciariaRESUMEN
Enterovirus D68 (EV-D68) continued to circulate in a seasonal pattern in the Netherlands, after the outbreak in 2010. Outpatient EV-D68 cases, mainly in the under 20 and 5059 years age groups, presented with relatively mild respiratory disease. Hospital-based enterovirus surveillance identified more severe cases, mainly in children under 10 years of age. Dutch partial VP1 genomic region sequences from 2012 through 2014 were distributed over three sublineages similar to EV-D68 from the outbreak in the US in 2014.
Asunto(s)
Enterovirus Humano D/clasificación , Enterovirus Humano D/aislamiento & purificación , Infecciones por Enterovirus/virología , Vigilancia de Guardia , Adolescente , Adulto , Distribución por Edad , Anciano , Niño , Preescolar , Brotes de Enfermedades , Enterovirus Humano D/genética , Infecciones por Enterovirus/epidemiología , Hospitalización/estadística & datos numéricos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Filogenia , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Estudios Retrospectivos , Estaciones del Año , Análisis de Secuencia de ADN , Distribución por Sexo , Adulto JovenRESUMEN
Human parechoviruses (HPeV) cause symptoms ranging from severe neonatal infections to mild gastrointestinal and respiratory disease. Use of PCR and genotyping has markedly improved the detection rate of HPeV but has simultaneously raised questions about the clinical relevance of positive tests. This retrospective study correlates positive HPeV1 or HPeV3 PCR tests in stools from children with their symptoms to determine clinical relevance. Children with HPeV1- or HPeV3-positive stool samples, as detected by real time RT-PCR and direct genotyping, between 2004 and 2008 were selected. Clinical data were retrospectively collected from the patient's files and results were compared. One hundred and thirty-eight children with positive HPeV1 (n = 112) or HPeV3 (n = 26) stool samples were identified. Significantly more HPeV3-infected children were neonates or infants younger than 6 months of age. Meningitis or sepsis-like illnesses were diagnosed most frequently and were found in significantly younger children. Almost half of HPeV1-infected children had an underlying disease. Mild gastrointestinal disease was seen most frequently in these children. There was no clear correlation between viral load (Ct value) and severity of symptoms. In conclusion, HPeV3 detected by PCR in stool samples is associated with clinically relevant disease. For HPeV1, a positive stool sample is mainly associated with symptoms in children with underlying disease.
Asunto(s)
Heces/virología , Parechovirus/clasificación , Parechovirus/aislamiento & purificación , Infecciones por Picornaviridae/virología , Femenino , Enfermedades Gastrointestinales/epidemiología , Enfermedades Gastrointestinales/virología , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Meningitis/epidemiología , Meningitis/virología , Parechovirus/genética , Infecciones por Picornaviridae/clasificación , Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Sepsis/epidemiología , Sepsis/virología , Carga ViralRESUMEN
Human enteroviruses (EVs) are the leading cause of CNS-associated disease in childhood. Identification of the EV types that patients are infected with is essential for monitoring outbreaks, the emergence of new types or variants, epidemiological surveillance and contributes to patient management. Rapid and sensitive molecular detection methods are frequently used to detect EVs/HPeVs directly from CSF. This requires that sensitive EV typing methods from CSF material need to be developed. In the present study two nested PCR-based typing assays were evaluated. The performance of the EV-A and -B specific nested PCR protocol and the Codehop-based PCR protocol were analyzed with several TCID(50)-titrated EV-A to D strains and 22 EV positive CSF samples. The EV-A and -B protocol was found to be more sensitive than the Codehop protocol. The Codehop protocol showed a high degree of aspecific amplification products when run on a gel, and required additional gel purification. The detection limit of the two protocols varied between the types, ranging from 0.1TCID(50)/mL sample to 10(6)TCID(50)/mL sample. From the 22 EV positive CSF samples, 15 (68%) samples were typed using either protocol. All samples were characterized as members of species B (E30 (9), CAV9 (2), E6 (1), E11 (1), E21 (1), E25 (1)). Three samples (E30 (2) and E25 (1)) could only be typed using the EV-B protocol. In this study, selected EV strains could be typed using both assays at low virus concentrations, typically found in CSF. However, the EV-A and -B protocol was more sensitive than the Codehop protocol for primary typing of CSF samples.
Asunto(s)
Proteínas de la Cápside/análisis , Enterovirus Humano A/clasificación , Enterovirus Humano B/clasificación , Infecciones por Enterovirus/líquido cefalorraquídeo , Reacción en Cadena de la Polimerasa/métodos , Regiones no Traducidas 5' , Proteínas de la Cápside/genética , Electroforesis en Gel de Agar , Enterovirus Humano A/genética , Enterovirus Humano A/aislamiento & purificación , Enterovirus Humano B/genética , Enterovirus Humano B/aislamiento & purificación , Infecciones por Enterovirus/virología , Técnicas de Genotipaje/métodos , Humanos , Filogenia , ARN Viral/análisis , ARN Viral/genética , Sensibilidad y Especificidad , Carga ViralRESUMEN
Human parechoviruses (HPeVs) are highly prevalent RNA viruses classified in the family Picornaviridae. Several antigenically distinct types circulate in human populations worldwide, whilst recombination additionally contributes to the genetic heterogeneity of the virus. To investigate factors influencing the likelihood of recombination and to compare its dynamics among types, 154 variants collected from four widely geographically separated referral centres (UK, The Netherlands, Thailand and Brazil) were typed by VP3/VP1 amplification/sequencing with recombination groups assigned by analysis of 3Dpol sequences. HPeV1B and HPeV3 were the most frequently detected types in each referral region, but with marked geographical differences in the frequencies of different recombinant forms (RFs) of types 1B, 5 and 6. HPeV1B showed more frequent recombination than HPeV3, in terms both of evolutionary divergence and of temporal/geographical indicators of population separation. HPeV1 variants showing between 10 and 20% divergence in VP3/VP1 almost invariably fell into different recombination groups, compared with only one-third of similarly divergent HPeV3 variants. Substitution rates calculated by beast in the VP3/VP1 region of HPeV1 and HPeV3 allowed half-lives of the RFs of 4 and 20 years, respectively, to be calculated, estimates fitting closely with their observed lifespans based on population sampling. The variability in recombination dynamics between HPeV1B and HPeV3 offers an intriguing link with their markedly different seasonal patterns of transmission, age distributions of infection and clinical outcomes. Future investigation of the epidemiological and biological opportunities and constraints on intertypic recombination will provide more information about its influence on the longer term evolution and pathogenicity of parechoviruses.
Asunto(s)
Parechovirus/genética , Infecciones por Picornaviridae/virología , ARN Viral/genética , Recombinación Genética , Brasil , Análisis por Conglomerados , Evolución Molecular , Genotipo , Humanos , Datos de Secuencia Molecular , Países Bajos , Filogenia , Polimorfismo Genético , Análisis de Secuencia de ADN , Homología de Secuencia , Tailandia , Reino UnidoRESUMEN
Human parechoviruses (HPeVs) are highly prevalent pathogens among very young children. Although originally classified into two serologically distinct types, HPeV1 and -2, recent analyses of variants collected worldwide have revealed the existence of 12 further types classified genetically by sequence comparisons of complete genome sequences or the capsid (VP1) gene. To investigate the nature of HPeV evolution, its population dynamics and recombination breakpoints, this study generated 18 full-length genomic sequences of the most commonly circulating genotypes, HPeV1 and -3, collected over a time span of 14 years from The Netherlands. By inclusion of previously published full-length sequences, 35 sequences were analysed in total. Analysis of contemporary strains of HPeV1 and those most similar to the prototype strain (Harris) showed that HPeV1 variants fall into two genetically distinct clusters that are much more divergent from each other than those observed within other HPeV types. Future classification criteria for HPeVs may require modification to accommodate the occurrence of variants with intermediate degrees of diversity within types. Recombination was frequently observed among HPeV1, -4, -5 and -6, but was much more restricted among HPeV3 strains. Favoured sites for recombination were found to flank the capsid region, and further sites were found within the non-structural region, P2. In contrast to other HPeV types, the majority of the HPeV3 sequences remained monophyletic across the genome, a possible reflection of its lower diversity and potentially more recent emergence than other HPeV types, or biological and/or epidemiological constraints that limit opportunities for co-infections with potential recombination partners.
Asunto(s)
Variación Genética , Genoma Viral , Parechovirus/clasificación , Parechovirus/genética , ARN Viral/genética , Análisis de Secuencia , Análisis por Conglomerados , Genotipo , Humanos , Datos de Secuencia Molecular , Países Bajos , Parechovirus/aislamiento & purificación , Filogenia , Infecciones por Picornaviridae/virología , Recombinación GenéticaRESUMEN
Human parechoviruses (HPeVs), members of the family Picornaviridae, are classified into six types. To investigate the dynamics and likelihood of recombination among HPeVs, we compared phylogenies of two distant regions (VP1 and 3Dpol) of 37 HPeV isolates (types 1 and 3-5) and prototype sequences (types 1-6). Evidence for frequent recombination between HPeV1, 4, 5 and 6 was found. The likelihood of recombination was correlated with the degree of VP1 divergence and differences in isolation dates, both indicative of evolutionary times of divergence. These temporal dynamics were found to be most similar to those of human enterovirus species B variants. In contrast, HPeV3 remained phylogenetically distinct from other types throughout the genome. As HPeV3 is equally divergent in nucleotide sequence from the other HPeV types, its genetic isolation may reflect different biology and changed cellular tropisms, arising from the deletion of the RGD motif, and likely use of a non-integrin receptor.
Asunto(s)
Parechovirus/genética , Infecciones por Picornaviridae/virología , California , Finlandia , Humanos , Datos de Secuencia Molecular , Países Bajos , Filogenia , Recombinación Genética , Factores de Tiempo , Proteínas no Estructurales Virales/genética , Proteínas Estructurales Virales/genéticaRESUMEN
BACKGROUND: Human parechoviruses (HPeVs) are members of the family Picornaviridae and are classified into 3 known serotypes: HPeV1, HPeV2, and the recently identified HPeV3. HPeV1 and HPeV2 infections are most commonly associated with mild respiratory or gastrointestinal symptoms and occasionally with severe disease conditions, such as flaccid paralysis and encephalitis. HPeV3 infection has been associated with transient paralysis and neonatal infection and has until now only been reported in Japan and Canada. METHODS: Culture isolates considered to be enterovirus on the basis of cell culture but that were found to be enterovirus negative by 5' untranslated region reverse-transcriptase polymerase chain reaction (5'UTR RT-PCR) during the period December 2000 through January 2005 were selected. Isolates were tested by HPeV 5'UTR RT-PCR and were genotyped by sequencing the VP1 region. Phylogenetic analysis was performed, and the association with clinical symptoms was established. RESULTS: Thirty-seven (12%) of the 303 isolates that tested positive for enterovirus by cell culture were in fact HPeV. The majority of the HPeV-positive isolates (n = 27) could be identified as HPeV1. The remaining 10 isolates, which were grown from samples obtained in 2001, 2002, and 2004, could be typed as the recently identified HPeV3. HPeV was exclusively detected in children aged < 3 years. Children infected with HPeV3 were significantly younger than children infected with HPeV1, and sepsis-like illness and central nervous system involvement were more frequently reported in children infected with HPeV3. CONCLUSIONS: We report HPeV infections in young children during the period of 2000-2005 and show an association between HPeV3 infection and sepsis-like illness and central nervous system involvement in neonates.