Mpox outbreak among men who have sex with men in Amsterdam and Rotterdam, the Netherlands: no evidence for undetected transmission prior to May 2022, a retrospective study.

Since May 2022, over 21,000 mpox cases have been reported from 29 EU/EEA countries, predominantly among men who have sex with men (MSM). The Netherlands was the fourth most affected country in Europe, with more than 1,200 cases and a crude notification rate of 70.7 per million population. The first national case was reported on 10 May, yet potential prior transmission remains unknown. Insight into prolonged undetected transmission can help to understand the current outbreak dynamics and aid future public health interventions. We performed a retrospective study and phylogenetic analysis to elucidate whether undetected transmission of human mpox virus (hMPXV) occurred before the first reported cases in Amsterdam and Rotterdam. In 401 anorectal and ulcer samples from visitors to centres for sexual health in Amsterdam or Rotterdam dating back to 14 February 2022, we identified two new cases, the earliest from 6 May. This coincides with the first cases reported in the United Kingdom, Spain and Portugal. We found no evidence of widespread hMPXV transmission in Dutch sexual networks of MSM before May 2022. Likely, the mpox outbreak expanded across Europe within a short period in the spring of 2022 through an international highly intertwined network of sexually active MSM.

Reduced Seasonal Coronavirus Antibody Responses in Children Following COVID-19 Mitigation Measures, The Netherlands.

SARS-CoV-2 prevention and control measures did not only impact SARS-CoV-2 circulation, but also the timing and prevalence of other seasonal respiratory viruses. Especially in children, information on exposure and infections to seasonal coronaviruses as well as SARS-CoV-2 in the first year of the pandemic is largely lacking. Therefore, we set up a one-year serological survey in a large tertiary hospital in the Netherlands. We show that seasonal coronavirus seroprevalence significantly decreased in 2021 in children less than one year, most likely due to COVID-19 control measures. The SARS-CoV-2 seroprevalence in children and adolescents increased from 0.4% to 11.3%, the highest in adolescents. This implies higher exposure rates in adolescents as compared to the general population (>18 years old). It is clear that there have been significant changes in the circulation and subsequent immunity against most respiratory pathogens as a result of the mitigation measures. The implications on shorter as well as longer term are still largely unknown, but the impact of the SARS-CoV-2 pandemic and subsequent control measures will continue to affect the dynamics of other pathogens.

Profiling of humoral immune responses to norovirus in children across Europe.

Norovirus is a leading cause of epidemic acute gastroenteritis. More than 30 genotypes circulate in humans, some are common, and others are only sporadically detected. Here, we investigated whether serology can be used to determine which genotypes infect children. We established a multiplex protein microarray with structural and non-structural norovirus antigens that allowed simultaneous antibody testing against 30 human GI and GII genotypes. Antibody responses of sera obtained from 287 children aged < 1 month to 5.5 years were profiled. Most specific IgG and IgA responses were directed against the GII.2, GII.3, GII.4, and GII.6 capsid genotypes. While we detected antibody responses against rare genotypes, we found no evidence for wide circulation. We also detected genotype-specific antibodies against the non-structural proteins p48 and p22 in sera of older children. In this study, we show the age-dependent antibody responses to a broad range of norovirus capsid and polymerase genotypes, which will aid in the development of vaccines.

Seasonal coronavirus-specific B cells with limited SARS-CoV-2 cross-reactivity dominate the IgG response in severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Little is known about the interplay between preexisting immunity to endemic seasonal coronaviruses and the development of a SARS-CoV-2-specific IgG response. We investigated the kinetics, breadth, magnitude, and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in patients with mild or severe COVID-19 as well as in disease control patients. We assessed antibody reactivity to nucleocapsid and spike antigens and correlated this IgG response to SARS-CoV-2 neutralization. Patients with COVID-19 mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against a seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings indicate a boost of poorly protective CoV-specific antibodies in patients with COVID-19 that correlated with disease severity, revealing "original antigenic sin."

Advances on Antiviral Activity of Morus spp. Plant Extracts: Human Coronavirus and Virus-Related Respiratory Tract Infections in the Spotlight.

(1) Background: Viral respiratory infections cause life-threatening diseases in millions of people worldwide every year. Human coronavirus and several picornaviruses are responsible for worldwide epidemic outbreaks, thus representing a heavy burden to their hosts. In the absence of specific treatments for human viral infections, natural products offer an alternative in terms of innovative drug therapies. (2) Methods: We analyzed the antiviral properties of the leaves and stem bark of the mulberry tree (Morus spp.). We compared the antiviral activity of Morus spp. on enveloped and nonenveloped viral pathogens, such as human coronavirus (HCoV 229E) and different members of the Picornaviridae family-human poliovirus 1, human parechovirus 1 and 3, and human echovirus 11. The antiviral activity of 12 water and water-alcohol plant extracts of the leaves and stem bark of three different species of mulberry-Morus alba var. alba, Morus alba var. rosa, and Morus rubra-were evaluated. We also evaluated the antiviral activities of kuwanon G against HCoV-229E. (3) Results: Our results showed that several extracts reduced the viral titer and cytopathogenic effects (CPE). Leaves' water-alcohol extracts exhibited maximum antiviral activity on human coronavirus, while stem bark and leaves' water and water-alcohol extracts were the most effective on picornaviruses. (4) Conclusions: The analysis of the antiviral activities of Morus spp. offer promising applications in antiviral strategies.

Publisher Correction: Specific memory B cell response in humans upon infection with highly pathogenic H7N7 avian influenza virus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Specific memory B cell response in humans upon infection with highly pathogenic H7N7 avian influenza virus.

H7 avian influenza viruses represent a major public health concern, and worldwide outbreaks raise the risk of a potential pandemic. Understanding the memory B cell response to avian (H7) influenza virus infection in humans could provide insights in the potential key to human infection risks. We investigated an epizootic of the highly pathogenic A(H7N7) in the Netherlands, which in 2003 led to infection of 89 persons and one fatal case. Subtype-specificity of antibodies were determined for confirmed H7N7 infected individuals (cases) (n = 19), contacts of these cases (n = 21) and a comparison group controls (n = 16), by microarray, using recombinant hemagglutinin (HA)1 proteins. The frequency and specificity of memory B cells was determined by detecting subtype-specific antibodies in the culture supernatants from in vitro stimulated oligoclonal B cell cultures, from peripheral blood of cases and controls. All cases (100%) had high antibody titers specific for A(H7N7)2003 (GMT > 100), whereas H7-HA1 antigen binding was detected in 29% of contacts and 31% of controls, suggesting that some of the H7 reactivity stems from cross reactive antibodies. To unravel homotypic and heterotypic responses, the frequency and specificity of memory B cells were determined in 2 cases. Ten of 123 HA1 reactive clones isolated from the cases bound to only H7- HA1, whereas 5 bound both H7 and other HA1 antigens. We recovered at least four different epitopal reactivities, though none of the H7 reactive antibodies were able to neutralize H7 infections in vitro. Our study serologically confirms the infection with H7 avian influenza viruses, and shows that H7 infection triggers a mixture of strain -specific and cross-reactive antibodies.

High frequency and diversity of parechovirus A in a cohort of Malawian children.

Parechoviruses (PeVs) are highly prevalent viruses worldwide. Over the last decades, several studies have been published on PeV epidemiology in Europe, Asia and North America, while information on other continents is lacking. The aim of this study was to describe PeV circulation in a cohort of children in Malawi, Africa. A total of 749 stool samples obtained from Malawian children aged 6 to 60 months were tested for the presence of PeV by real-time PCR. We performed typing by phylogenetic and Basic Local Alignment Search Tool (BLAST) analysis. PeV was found in 57% of stool samples. Age was significantly associated with PeV positivity (p = 0.01). Typing by phylogenetic analysis resulted in 15 different types, while BLAST typing resulted in 14 different types and several indeterminate strains. In total, six strains showed inconsistencies in typing between the two methods. One strain, P02-4058, remained untypable by all methods, but appeared to belong to the recently reclassified PeV-A19 genotype. PeV-A1, -A2 and -A3 were the most prevalent types (26.8%, 13.8% and 9.8%, respectively). Both the prevalence and genetic diversity found in our study were remarkably high. Our data provide an important contribution to the scarce data available on PeV epidemiology in Africa.

Human Parechovirus 1, 3 and 4 Neutralizing Antibodies in Dutch Mothers and Infants and Their Role in Protection Against Disease.

BACKGROUND: Human parechoviruses (HPeVs) are common pathogens in young children, and in the Netherlands, HPeV1, HPeV3 and HPeV4 are the most frequently detected genotypes. HPeV3 in particular has been associated with severe disease in young infants below 3 months of age while the other genotypes more often infect older children and elicit mild symptoms. We investigated if maternal neutralizing antibodies (nAbs) against HPeV1, HPeV3 and HPeV4 protect young Dutch infants from severe disease related to HPeV infection. METHODS: We conducted a prospective case-control study of Dutch mother-infant pairs. Thirty-eight HPeV-infected infants and their mothers were included as cases, and 65 HPeV-negative children and their mothers as controls. RESULTS: In control infants, we observed nAb seropositivity rates of 41.4%, 33.3% and 27.6%, with median nAb titers of 1:16, 1:12 and 1:8, against HPeV1, HPeV3 and HPeV4, respectively. In control mothers, nAb seropositivity rates were 84.6%, 55.4% and 60.0% with median nAb titers of 1:128, 1:32 and 1:45 against HPeV1, HPeV3 and HPeV4, respectively. The HPeV3 nAb seroprevalence was significantly lower in HPeV3-infected infants and their mothers (0.0% with P < 0.05 and 10.0% with P < 0.001, respectively). In contrast, no differences in nAb seroprevalence against HPeV1 or HPeV4 could be detected between case and control infants or mothers. CONCLUSIONS: Our results suggest that young Dutch infants are protected against severe disease related to HPeV1 and HPeV4 by maternal nAbs, but less so against HPeV3 explaining the distinct age distributions and disease severity profiles of children infected with these HPeV genotypes.

High frequency of Polio-like Enterovirus C strains with differential clustering of CVA-13 and EV-C99 subgenotypes in a cohort of Malawian children.

Enteroviruses (EVs) are among the most commonly detected viruses infecting humans worldwide. Although the prevalence of EVs is widely studied, the status of EV prevalence in sub-Saharan Africa remains largely unknown. The objective of our present study was therefore to increase our knowledge on EV circulation in sub-Saharan Africa. We obtained 749 fecal samples from a cross-sectional study conducted on Malawian children aged 6 to 60 months. We tested the samples for the presence of EVs using real time PCR, and typed the positive samples based on partial viral protein 1 (VP1) sequences. A large proportion of the samples was EV positive (89.9%). 12.9% of the typed samples belonged to EV species A (EV-A), 48.6% to species B (EV-B) and 38.5% to species C (EV-C). More than half of the EV-C strains (53%) belonged to subgroup C containing, among others, Poliovirus (PV) 1-3. The serotype most frequently isolated in our study was CVA-13, followed by EV-C99. The strains of CVA-13 showed a vast genetic diversity, possibly representing a new cluster, 'F'. The majority of the EV-C99 strains grouped together as cluster B. In conclusion, this study showed a vast circulation of EVs among Malawian children, with an EV prevalence of 89.9%. Identification of prevalences for species EV-C comparable to our study (38.5%) have only previously been reported in sub-Saharan Africa, and EV-C is rarely found outside of this region. The data found in this study are an important contribution to our current knowledge of EV epidemiology within sub-Saharan Africa.

A chip-based rapid genotyping assay to discriminate between rhinovirus species A, B and C.

BACKGROUND: Human rhinoviruses (RVs) are increasingly associated with severe disease of the respiratory tract. Multiple studies highlighted the clinical significance of different RV species; RV-C is linked to asthma exacerbations and increased disease severity in children, whereas RV-B seems to correlate with milder disease. OBJECTIVES: Current typing strategies for differentiation of RV species are time consuming and require extensive equipment. Here we present a novel genotyping tool to discriminate RV species A, B and C. STUDY DESIGN: The method encompasses a VP4/VP2 polymerase chain reaction (PCR), followed by hybridization of the product on a macro array with probes covering RV-A, B, and C, produced by Chipron as custom array. Validation was performed with respiratory specimens submitted for diagnostic evaluation to the Academic Medical Center. A selection of RV PCR-positive samples genotyped based on VP4/VP2 sequencing was evaluated. Diagnostic performance was tested on respiratory samples positive for RV in an in-house multiplex respiratory PCR from January 2016 to January 2017. In-house primers and additional genotype-specific primers were used for sequencing to investigate array-negative and array-double-positive samples. RESULTS: The majority of samples pretyped RVs (n = 135) were classified correctly, except for one that was assigned RV-C instead of RV-A, and 3 samples tested negative. The array gave four double-positive results; the presence of more than one genotype was confirmed in two samples. In 173/187 (92.5%) RV-positive tested patient samples from 2016, the test resulted in a designated species. RV species A was identified in 109 specimens (58.3%), RV-B in 26 (13.9%), and RV-C in 56 (29.9%) samples. Sequencing of the probe region of 14 (7.6%) negative samples revealed up to 3 mismatches to the probes for 12 samples; in 2 cases no PCR product was generated. Notably, in 18 samples the chip detected more than one species, of which 16 were confirmed by sequencing. DISCUSSION: The Chipron LCD RV array provides a fast and highly sensitive method for discrimination between rhinovirus species, and has the power to detect dual infections.

Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis.

The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA-protein interactions and dissection of virus assembly on the basis of RNA nucleation.

Influenza virus A(H1N1)2009 antibody-dependent cellular cytotoxicity in young children prior to the H1N1 pandemic.

Pre-existing immunity played a significant role in protection during the latest influenza A virus H1N1 pandemic, especially in older age groups. Structural similarities were found between A(H1N1)2009 and older H1N1 virus strains to which humans had already been exposed. Broadly cross-reactive antibodies capable of neutralizing the A(H1N1)2009 virus have been implicated in this immune protection in adults. We investigated the serological profile of a group of young children aged 9 years (n=55), from whom paired blood samples were available, just prior to the pandemic wave (March 2009) and shortly thereafter (March 2010). On the basis of A(H1N1)2009 seroconversion, 27 of the 55 children (49 %) were confirmed to be infected between these two time points. Within the non-infected group of 28 children (51 %), high levels of seasonal antibodies to H1 and H3 HA1 antigens were detected prior to pandemic exposure, reflecting past infection with H1N1 and H3N2, both of which had circulated in The Netherlands prior to the pandemic. In some children, this reactivity coincided with specific antibody reactivity against A(H1N1)2009. While these antibodies were not able to neutralize the A(H1N1)2009 virus, they were able to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro upon interaction with the A(H1N1)2009 virus. This finding suggests that cross-reactive antibodies could contribute to immune protection in children via ADCC.

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies.

UNLABELLED: 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.

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.

Growth characteristics of human parechovirus 1 to 6 on different cell lines and cross- neutralization of human parechovirus antibodies: a comparison of the cytopathic effect and real time PCR.

BACKGROUND: Human parechoviruses (HPeVs) are among the most frequently detected picornaviruses in humans. HPeVs are usually associated with mild gastrointestinal and respiratory symptoms with the exception of HPeV3 which causes neonatal sepsis and CNS infection. Previous studies showed various results in culturing different HPeV genotypes, inducing only a low cytopathic effect (CPE). METHODS: In vitro growth characteristics of the different HPeV genotypes in a range of 10 different cell lines are scored with CPE and measured in the supernatant by real time PCR. In the optimal cell line for each genotype a standard neutralization assay with the available HPeV antibodies (Abs) was performed and scored by CPE and measured by real time PCR. RESULTS: All six HPeV types were able to replicate on the RD99, A549, and Vero cell lines. HPeV1 was the only genotype able to replicate on all cell lines. Most efficient growth of HPeV1, 2, 4, 5, and 6 was shown on the HT29 cell line, while HPeV3 was unable to replicate on HT29. In all cases viral replication could be measured by real time PCR before CPE appeared. The polyclonal Abs available against HPeV1, 2, 4 and 5 all showed neutralization of their respective genotype after 7 days with inhibition of >60% in real time PCR and full inhibition of CPE, although cross-neutralization is shown. Replication of HPeV3 could only be inhibited by 12% by the anti-HPeV3 (aHPeV3) Ab and no inhibition of CPE was shown after 7 days. CONCLUSION: When replication is monitored by PCR, growth of HPeV genotypes 1 to 6 is supported by most of the cell lines tested, where viral replication is measured before appearance of CPE. A combination of HT29 and Vero cells would therefore support replication of all culturable HPeV types, so viral replication could be detected by PCR within 3 days for all genotypes.In addition, we showed efficient neutralization for HPeV1, 2, 4, 5, while cross- neutralization was shown between these types, indicating possible common neutralizing epitopes. For HPeV3 no efficient (cross-) neutralization was shown, indicating different neutralizing epitopes for HPeV3 compared to the other HPeV genotypes.

Prevalence and clinical course in invasive infections with meningococcal endotoxin variants.

BACKGROUND: Meningococci produce a penta-acylated instead of hexa-acylated lipid A when their lpxL1 gene is inactivated. Meningococcal strains with such lipid A endotoxin variants have been found previously in adult meningitis patients, where they caused less blood coagulopathy because of decreased TLR4 activation. METHODS: A cohort of 448 isolates from patients with invasive meningococcal disease in the Netherlands were screened for the ability to induce IL-6 in monocytic cell Mono Mac 6 cells. The lpxL1 gene was sequenced of isolates, which show poor capacity to induce IL-6.. Clinical characteristics of patients were retrieved from hospital records. RESULTS: Of 448 patients, 29 (6.5%) were infected with meningococci expressing a lipid A variant strain. Lipid A variation was not associated with a specific serogroup or genotype. Infections with lipid A variants were associated with older age (19.3 vs. 5.9 (median) years, p = 0.007) and higher prevalence of underlying comorbidities (39% vs. 17%; p = 0.004) compared to wild-type strains. Patients infected with lipid A variant strains had less severe infections like meningitis or shock (OR 0.23; 95%CI 0.09-0.58) and were less often admitted to intensive care (OR 0.21; 95%CI 0.07-0.60) compared to wild-type strains, independent of age, underlying comorbidities or strain characteristics. CONCLUSIONS: In adults with meningococcal disease lipid A variation is rather common. Infection with penta-acylated lipid A variant meningococci is associated with a less severe disease course.

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.

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.

Clinical characteristics of human parechoviruses 4-6 infections in young children.

BACKGROUND: Human parechoviruses (HPeVs) and enteroviruses (EVs) belong to the family Picornaviridae. EVs are known to cause a wide range of disease such as meningitis, encephalitis, and sepsis. HPeV1 and 2 have been associated with mild gastrointestinal or respiratory symptoms in young children. HPeV3 is associated with more severe neonatal infection. Little is known about the epidemiology and pathology of HPeV4-6 in children. METHODS: We evaluated the clinical symptoms of the children with an HPeV 4, 5, or 6 infection. The patients with positive HPeV4-6 in stool samples were selected and available plasma or cerebrospinal fluid samples from these patients were tested for HPeV. Data on clinical symptoms, diagnosis, presence and duration of fever, medical history, mean age, use of antibiotics of the children infected with HPeV4-6 were retrospectively documented. RESULTS: HPeV4-6 were found in 31 of the 277 HPeV positive children (11%). Coinfection with EV was seen in 8 patients. Fever was seen in 13 (42%) patients. Of the HPeV4-6 positive patients, 20 of the 31 children (64%) presented with gastrointestinal complaints and 18 of 31 (58%) patients had respiratory symptoms. The mean age was 14 months, 58% of the patients had an underlying disorder such as bronchomalacia or a cardiac disorder. CONCLUSIONS: Symptomatic HPeV4-6 infections are seen in relative young children and are associated with respiratory and/or gastrointestinal symptoms. HPeV type 4 was detected more frequently than HPeV types 5 and 6.