Gerrit Koen

Detection of human enterovirus and human parechovirus (HPeV) genotypes from clinical stool samples: polymerase chain reaction and direct molecular typing, culture characteristics, and serotyping

Molecular (polymerase chain reaction [PCR]) methods are increasingly used to detect and type human enteroviruses (HEVs) and parechoviruses (HPeV). Here, we assessed their value in comparison to virus culture and serotyping for detection and typing of HEV and HPeV in stool samples from hospitalized patients. By use of real-time PCR, 221/1174 patients (18.8%) were found positive for HEV/HPeV. By cell culture, a virus could be isolated from 107 of the HEV/HPeV PCR-positive samples. Culture efficiency was correlated to the Ct value, (geno)type, and cell lines used. Of the HEV/HPeV PCR-positive samples, 47% could be genotyped by VP1 genotyping and 25% by serotyping. In conclusion, PCR detection of HEV/HPeV from stool is more sensitive than virus culture, particularly for coxsackieviruses A and HPeVs. However, the genotyping method used here could identify only 47% of the HEV/HPeV strains. Further optimization and validation of direct genotyping are needed, and clinical relevance of HEV/HPeV detection in stool needs to be determined.

Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes

ABSTRACT The “gold standard” for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed.

Specific cell tropism and neutralization of human parechovirus types 1 and 3: implications for pathogenesis and therapy development

Human parechoviruses (HPeVs) are picornaviruses frequently infecting humans. While HPeV1 is associated with mild disease, HPeV3 is the cause of neonatal sepsis and meningitis. To test whether in vitro replication kinetics of HPeV1 and HPeV3 could be related to pathogenicity, HPeV1 and HPeV3 strains isolated from patients were cultured on cell lines of gastrointestinal, respiratory and neural origin, and replication kinetics were measured by real-time PCR. No relationship was found between clinical symptoms and in vitro replication of the HPeV1 strains. In contrast, the HPeV3 strains showed faster replication in neural cells and there was a relationship between higher in vitro replication kinetics and neuropathogenicity in the patient. Furthermore, HPeV1 could be neutralized efficiently by its specific antibody and by intravenous immunoglobulins (IVIG), while most HPeV3 strains could not be neutralized. In IVIG, very low neutralizing antibody (nAb) titres against HPeV3 were found. Additionally, very low nAb titres were observed in sera of two HPeV3-infected donors, while high nAb titres against HPeV1 could be detected. Our data suggest that the mild clinical course of HPeV1 infection is primarily influenced by strong nAb responses, while HPeV3 might be difficult to neutralize in vivo and therefore the course of infection will mainly be determined by in vivo cell tropism.

Human parechovirus seroprevalence in Finland and the Netherlands

BACKGROUND Human parechoviruses (HPeVs) are RNA viruses associated with mild gastrointestinal and respiratory infections in children, but may also cause neonatal sepsis and CNS infections in infants. While the prevalence of HPeVs is known mostly among hospitalized populations, the knowledge of HPeV seroprevalence in the general population is poor. OBJECTIVES The aim of this study was to identify and compare the HPeV1-6 seroprevalence in Finnish and Dutch populations. STUDY DESIGN A type specific microneutralization assay was set up for detecting neutralizing antibodies (nABs) against HPeV types 1-6. Altogether 616 serum samples from Finnish and Dutch population were analyzed for antibodies against HPeVs. The samples were collected from Finnish children aged 1, 5 or 10 years, Finnish adults, 0- to 5-year-old Dutch children, Dutch women of childbearing age and Dutch HIV-positive men. RESULTS In both adult populations, seropositivity was high against HPeV1 (99% in Finnish and 92% in Dutch samples) and HPeV2 (86% and 95%). Against HPeV4, the seropositivity was similar (62% and 60%). In Dutch adults, nABs against HPeV5 and 6 (75% and 74%) were detected more often than in Finnish adults (35% and 57%, respectively). In contrast, seropositivity against HPeV3 was as low as 13% in the Finnish and 10% in the Dutch adults. The seroprevalence of all HPeV types increased with age. CONCLUSIONS The seroprevalence of HPeVs is high in Finnish and Dutch populations and HPeV type 2 and types 4-6 are significantly more prevalent compared to earlier reports. The seroprevalence of antibodies observed against HPeV3 was low.

Pleconaril revisited: clinical course of chronic enteroviral meningoencephalitis after treatment correlates with in vitro susceptibility

BACKGROUND Human enteroviruses (HEVs) can cause severe infections, especially in patients with a deficient humoral immune response, such as X-linked agammaglobulinemia. In this patient group, chronic enteroviral meningitis (CEMA) is feared because of extensive morbidity and high fatality rate. Treatment options consist of intravenous immunoglobulin (IVIG), with various outcomes. Pleconaril is an antiviral agent with in vitro activity against HEVs that has been used in the treatment of HEV infections. METHODS The efficacy of pleconaril and IVIG against HEV isolated from the patients was assessed in vitro in two patients with CEMA. RESULTS Echovirus 11 was found in the cerebrospinal fluid (CSF) of case 1. Treatment with high-dose IVIG and pleconaril did not provide any clinical improvement and HEV PCR in CSF remained positive. Case 2 (echovirus 13 positive in CSF) was also treated with IVIG and pleconaril. The patient recovered completely and HEV PCR in CSF became negative. Recent IVIG batches contained low titres of neutralizing antibodies against the patient strains. Echovirus 11 (case 1) was resistant to pleconaril in vitro, whereas echovirus 13 (case 2) was susceptible, in accordance with virological response after treatment and subsequent clinical results. CONCLUSIONS This is the first report that evaluates efficacy of antiviral treatment in CEMA patients in relation to in vitro susceptibility of clinical virus isolates. Since pleconaril is no longer available for compassionate use we strongly propagate that new drugs should be developed against these potential life threatening HEV infections.

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

ABSTRACT Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.

Presence of human non-polio enterovirus and parechovirus genotypes in an Amsterdam hospital in 2007 to 2011 compared to national and international published surveillance data: a comprehensive review.

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.

Evaluation of a rapid antigen detection point-of-care test for respiratory syncytial virus and influenza in a pediatric hospitalized population in the Netherlands

This pilot study evaluates the diagnostic performance of Sofia RSV Fluorescent Immunoassay Analyzer (FIA) and Sofia Influenza A + B FIA for rapid detection of respiratory syncytial virus and influenza A and B. Sofia had a lower-than-expected sensitivity for all viruses and a high rate of false-positive results for influenza B virus.

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

ABSTRACT 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.