The presence of cardiotropic viral genomes is not increased in atrial tissue of atrial fibrillation patients.

BACKGROUND: Infections with potentially cardiotropic viruses are associated with the development of atrial fibrillation (AF). However, whether direct viral infection of the atria is involved in the pathogenesis of AF is unclear. We have therefore analysed the presence of cardiotropic viral genomes in AF patients. METHODS: Samples of left atrial tissue were obtained from 50 AF patients (paroxysmal, n = 20; long-standing persistent/permanent, n = 30) during cardiac surgery and from autopsied control patients (n = 14). Herein, the presence of PVB19, EBV, CMV, HHV­6, adenovirus and enterovirus genomes was determined by polymerase chain reaction. The densities of CD45+ and CD3+ cells and fibrosis in the atria were quantified by (immuno)histochemistry. RESULTS: Of the tested viruses only the PVB19 genome was detected in the atria of 10% of patients, paroxysmal AF (2 of 20) and long-standing persistent/permanent AF (3 of 30). Conversely, in 50% of controls (7 of 14) PVB19 genome was found. No significant association was found between PVB19 and CD45+ and CD3+ cells, or between the presence of PVB19 and fibrosis, in either control or AF patients. CONCLUSION: The presence of viral genomes is not increased in the atria of AF patients. These results do not support an important role for viral infection of the atria in the pathogenesis of AF.

Recommendations for the nomenclature of enteroviruses and rhinoviruses.

Enteroviruses (EVs) and rhinoviruses (RVs) are significant pathogens of humans and are the subject of intensive clinical and epidemiological research and public health measures, notably in the eradication of poliovirus and in the investigation and control of emerging pathogenic EV types worldwide. EVs and RVs are highly diverse in their antigenic properties, tissue tropism, disease associations and evolutionary relationships, but the latter often conflict with previously developed biologically defined terms, such as "coxsackieviruses", "polioviruses" and "echoviruses", which were used before their genetic interrelationships were understood. This has created widespread formatting problems and inconsistencies in the nomenclature for EV and RV types and species in the literature and public databases. As members of the International Committee for Taxonomy of Viruses (ICTV) Picornaviridae Study Group, we describe the correct use of taxon names for these viruses and have produced a series of recommendations for the nomenclature of EV and RV types and their abbreviations. We believe their adoption will promote greater clarity and consistency in the terminology used in the scientific and medical literature. The recommendations will additionally provide a useful reference guide for journals, other publications and public databases seeking to use standardised terms for the growing multitude of enteroviruses and rhinoviruses described worldwide.

Correction to: Recommendations for the nomenclature of enteroviruses and rhinoviruses.

Unfortunately, one of the affiliations of author "A. E. Gorbalenya" was missed in original version. The affiliation is updated here.

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.

Changes in microbiota during experimental human Rhinovirus infection.

BACKGROUND: Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbiota. METHODS: Six healthy volunteers were infected with HRV16. We performed 16S ribosomal RNA-targeted pyrosequencing on throat swabs taken prior, during and after infection. We compared overall community diversity, phylogenetic structure of the ecosystem and relative abundances of the different bacteria between time points. RESULTS: During acute infection strong trends towards increases in the relative abundances of Haemophilus parainfluenzae and Neisseria subflava were observed, as well as a weaker trend towards increases of Staphylococcus aureus. No major differences were observed between day-1 and day 60, whereas differences between subjects were very high. CONCLUSIONS: HRV16 infection is associated with the increase of three genera known to be associated with secondary infections following HRV infections. The observed changes of upper respiratory tract microbiota could help explain why HRV infection predisposes to bacterial otitis media, sinusitis and pneumonia.

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.

Laboratory-based surveillance in the molecular era: the TYPENED model, a joint data-sharing platform for clinical and public health laboratories.

Laboratory-based surveillance, one of the pillars of monitoring infectious disease trends, relies on data produced in clinical and/or public health laboratories. Currently, diagnostic laboratories worldwide submit strains or samples to a relatively small number of reference laboratories for characterisation and typing. However, with the introduction of molecular diagnostic methods and sequencing in most of the larger diagnostic and university hospital centres in high-income countries, the distinction between diagnostic and reference/public health laboratory functions has become less clear-cut. Given these developments, new ways of networking and data sharing are needed. Assuming that clinical and public health laboratories may be able to use the same data for their own purposes when sequence-based testing and typing are used, we explored ways to develop a collaborative approach and a jointly owned database (TYPENED) in the Netherlands. The rationale was that sequence data - whether produced to support clinical care or for surveillance -can be aggregated to meet both needs. Here we describe the development of the TYPENED approach and supporting infrastructure, and the implementation of a pilot laboratory network sharing enterovirus sequences and metadata.

Limited CD4+ T-cell renewal in early HIV-1 infection: effect of highly active antiretroviral therapy.

We show that the fraction of proliferating CD4+ lymphocytes is similar in HIV-infected subjects in the early stage of disease and in HIV-negative subjects, whereas the fraction of proliferating CD8+ lymphocytes is increased 6.8-fold in HIV-infected subjects. After initiation of antiviral therapy, there is a late increase in proliferating CD4+ T cells associated with the restoration of CD4+ T-cell counts. These results provide strong support for the idea of limited CD4+ T-cell renewal in the early stage of HIV infection and indicate that after effective suppression of virus replication, the mechanisms of CD4+ T-cell production are still functional in early HIV infection.

Diagnostic accuracy of VIDISCA-NGS in patients with suspected central nervous system infections.

OBJECTIVES: Confirming the diagnosis in viral central nervous system (CNS) infections can be difficult with the currently available diagnostic tools. Virus discovery cDNA-amplified fragment length polymorphism next-generation sequencing (VIDISCA-NGS) is a promising viral metagenomic technique that enables the detection of all viruses in a single assay. We performed a retrospective study on the diagnostic accuracy of VIDISCA-NGS in cerebrospinal fluid (CSF) of individuals with suspected CNS infections. METHODS: Consecutive adult patients presenting to the Emergency Department or inpatients, who underwent a lumbar puncture for the suspicion of a CNS infection, were included if they were diagnosed with a viral CNS infection, or if a viral CNS infection was initially suspected but eventually a different diagnosis was made. A quantitative PCR panel of the most common causative viruses was performed on CSF of these patients as reference standard and compared with the results of VIDISCA-NGS, the index test. RESULTS: We included 38 individuals with viral CNS infections and 35 presenting with suspected CNS infection for whom an alternative aetiology was finally established. Overall sensitivity and specificity were 52% (95% CI 31%-73%) and 100% (95% CI 91%-100%), respectively. One enterovirus, detected by VIDISCA-NGS, was only identified by quantitative PCR upon retesting. Additional viruses identified by VIDISCA-NGS consisted of GB virus C, human papillomavirus, human mastadenovirus C, Merkel cell polyoma virus and anelloviruses. CONCLUSION: In patients for whom routine diagnostics do not yield a causative pathogen, VIDISCA-NGS can be of additional value as it can detect a broader range of viruses, but it does not perform well enough to replace quantitativePCR.

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.

Recombination dynamics of human parechoviruses: investigation of type-specific differences in frequency and epidemiological correlates.

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.

Ongoing transmission of a single hepatitis B virus strain among men having sex with men in Amsterdam.

For the past decade, a specific hepatitis B virus (HBV) genotype A strain has been prevalent among men having sex with men (MSM) in Amsterdam, the Netherlands. At what point in time this strain was introduced in the MSM population, and why only this specific strain continues to be transmitted, remains unclear. Between 1984 and 2003, sera of 1862 MSM were retrospectively screened for anti-HBc in the context of the Amsterdam Cohort studies. After 2003, most MSM participating in this study were vaccinated, making further testing less useful. HBV DNA from anti-HBc seroconverters was amplified and sequenced. Poisson regression was used to test for temporal trends in HBV and HIV incidence. Of the 1042 MSM who were negative for anti-HBc at entry, 64 had seroconverted during follow-up at a median age of 32. At the point of seroconversion, 31 MSM were HIV positive. HBV incidence declined dramatically in the first years and then remained stable throughout the study period. The HBV and HIV incidence ran almost in parallel. With the exception of three MSM, all were infected with genotype A. Fifteen of these (41%) were infected with an identical genotype A strain. For the past two decades, an identical genotype A strain has been circulating among MSM in the Netherlands. Although HBV is generally considered more infectious than HIV, this study shows that the trend and magnitude in HBV and HIV incidence among MSM are similar.

T cell telomere length in HIV-1 infection: no evidence for increased CD4+ T cell turnover.

Progression to acquired immunodeficiency syndrome (AIDS) has been related to exhaustion of the regenerative capacity of the immune system resulting from high T cell turnover. Analysis of telomeric terminal restriction fragment (TRF) length, a marker for cellular replicative history, showed that CD8(+) T cell TRF length decreased but CD4(+) T cell TRF length was stable during the course of human immunodeficiency virus type-1 (HIV-1) infection, which was not explained by differential telomerase activity. This observation provides evidence that turnover in the course of HIV-1 infection can be increased considerably in CD8(+) T cells, but not in CD4(+) T cells. These results are compatible with CD4(+) T cell decline in HIV-1 infection caused by interference with cell renewal.

Blood and cerebrospinal fluid characteristics in neonates with a suspected central nervous system infection.

Clinical signs and symptoms of central nervous system (CNS) infections in neonates are often nonspecific. Therefore, cerebrospinal fluid (CSF) analysis is performed to diagnose CNS infections. Data on combined microbiological results and their correlation with biochemical characteristics in CSF and blood in infants younger than 90 days are limited. This study provides an overview of microbiological test results, CSF- and hematological characteristics among infants with a clinically suspected CNS infection.This retrospective study included infants younger than 90 days, with a clinically suspected CNS infection who underwent a diagnostic lumbar puncture between January 2012 and January 2014. Data on the presence of microbiological pathogens in CSF, CSF inflammation markers (white blood cell [WBC] counts, protein levels and glucose CSF/serum ratio) and blood inflammatory responses (WBC count, C-reactive protein [CRP], neutrophil percentage) were collected by reviewing patient files.We included data from 576 infants (median age 12.5 days, interquartile range, 6-27 days) of whom 383 (66.5%) were born prematurely. In total, 16 bacterial pathogens (3.0%) and 21 viruses (5.5%) were detected in CSF. Escherichia coli was detected in 5 cases (1.0%), Enterovirus was detected in 12 cases (3.1%). Leucocytosis in CSF was associated with identification of a pathogen in CSF. Increased CRP was associated with the identification of a bacterial pathogen in CSF.Bacterial or viral pathogens were only identified in a small proportion of infants with a clinically suspected CNS infection. Leucocytosis in CSF was associated with CNS infection in infants. An increased CRP was indicative of bacterial meningitis.

High prevalence of human Parechovirus (HPeV) genotypes in the Amsterdam region and identification of specific HPeV variants by direct genotyping of stool samples.

Human parechoviruses (HPeV) are widespread pathogens belonging to the Picornavirus family. Six genotypes, which have predominantly been isolated from children, are known. Data on prevalence of HPeV genotypes are generally based on cell culture, which may underestimate the prevalence of certain HPeV strains that are difficult to grow. We studied 1,824 stool samples from 1,379 children (<5 years old) sent to the Academic Medical Center, Amsterdam, The Netherlands, between 2004 and 2006. Samples were screened using specific human enterovirus (HEV) and HPeV real-time PCRs based on the 5' untranslated region. A high percentage of HPeV infections (16.3%), comparable to the percentage of HEV infections (18.4%), were found by PCR in stool samples. HPeV-positive stool samples were directly genotyped based on the VP1 region for the first time to avoid a culture bias. HPeV1 was found to be the most prevalent type. The majority of the HPeV1 strains clustered separately from the prototype strain, Harris, which has not been reported to circulate lately. However, we could identify three strains as HPeV1 Harris. HPeV3 was identified as the second most predominant type during 2004 and 2006 but was not found in 2005. HPeV4 to -6 were found in smaller numbers. One strain could not be associated with a known HPeV type (VP1 gene nucleotide similarity: 71%), possibly indicating a new genotype. Also, we report the first identification of three HPeV5 strains and one HPeV1 strain with a different motif at the C-terminal end of VP1, where the arginine-glycine-aspartic acid (RGD) motif is normally located.

Epidemiology and clinical associations of human parechovirus respiratory infections.

Infections with human parechoviruses (HPeVs) are prevalent in young children and have been associated with mild gastroenteritis and, less frequently, with meningitis and neonatal sepsis. To investigate the involvement of these viruses in respiratory disease, a highly sensitive nested PCR was used to screen a large archive of respiratory specimens, collected between January and December 2007. Respiratory samples had previously been tested for eight respiratory viruses, including respiratory syncytial virus and adenovirus, by PCR. HPeV was detected in 34 of 3,844 specimens, representing 27 of 2,220 study subjects (1.2%). HPeV types were identified by sequencing the VP3/VP1 junction amplified by PCR directly from clinical specimens. The assay could amplify all HPeV types examined with high sensitivity (types 1 and 3 to 6) and also identified HPeV types in all but one of the screen-positive study specimens (25 HPeV1 and eight HPeV6 specimens). Infections with both HPeV1 and HPeV6 were seasonal, with highest frequencies in July and August, and restricted to children aged between 6 months and 5 years. Other respiratory viruses were frequently codetected in HPeV-positive specimens, with significant overrepresentation of adenovirus coinfections (37%). Most HPeV-positive specimens were referred from emergency departments, although no association with specific respiratory symptoms or disease was found. In summary, the low frequency of detection and lack of clear disease associations indicate that HPeV1 and -6 are not major pathogens in individuals presenting with respiratory disease. However, the screening and typing methods developed will be of value in further HPeV testing, including testing for meningitis cases and other suspected HPeV-associated disease presentations.

[An outbreak of measles at an emergency room]. / Een uitbraak van mazelen op de Spoedeisende Hulp.

A small outbreak of measles occurred after a 33-year-old female aircrew (cabin) member presented at an emergency room with fever. Three members of the hospital staff were infected: a 42-year-old man, a 33-year-old woman, and a 26-year-old woman. The first 2 patients had not been immunised, and the third had received 2 immunisations according to the Dutch National Immunisation Programme. Vaccination of the 2 sero-negative patients within 48 h after exposure with the measles-mumps-rubella vaccine (MMR) did not prevent the development of measles. Vaccination was deemed unnecessary in the third patient. No tertiary cases occurred. The same measles virus (genotype D5) was detected by PCR and sequencing in all 4 patients. Measles remains a risk for hospital staff members who have not acquired natural immunity. The current policy of immunising patients within 72 h after exposure to measles may not be sufficient. It also appears that immunisation through the Dutch National Immunisation Programme does not always protect against nosocomial infection. Providing MMR vaccination or boosters to hospital staff in certain departments might be beneficial.

Rapid detection and monitoring of human coronavirus infections.

Human coronaviruses (CoVs) are increasingly recognized as important respiratory pathogens associated with a broad range of clinical diseases. We sought to increase the insight into clinically relevant CoV infections by monitoring antigen concentrations in six confirmed CoV-positive patients using a newly developed assay for rapid detection of CoV OC43 infections. Antigen positivity lasted 3 to 6 days in secondary infections and 13 days in primary infection. CoV infections are clinically diverse, are common, and cannot be diagnosed from clinical symptoms alone.