A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

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.

Prevalence of rhinoviruses in young children of an unselected birth cohort from the Netherlands.

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.

Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis.

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.

Genetic and antigenic structural characterization for resistance of echovirus 11 to pleconaril in an immunocompromised patient.

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.

Clinical relevance of positive human parechovirus type 1 and 3 PCR in stool samples.

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.

Evaluation of two (semi-)nested VP1 based-PCRs for typing enteroviruses directly from cerebral spinal fluid samples.

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.

Comprehensive full-length sequence analyses of human parechoviruses: diversity and recombination.

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.

Widespread recombination within human parechoviruses: analysis of temporal dynamics and constraints.

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.

Human parechovirus infections in Dutch children and the association between serotype and disease severity.

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.