A panel of hepatitis C virus glycoproteins for the characterization of antibody responses using antibodies with diverse recognition and neutralization patterns.

A vaccine against Hepatitis C virus (HCV) is urgently needed to limit the spread of HCV. The large antigenic diversity of the HCV glycoprotein E1E2 makes it difficult to design a vaccine but also to fully understand the antibody response after infection or vaccination. Here we designed a panel of HCV pseudoparticles (HCVpps) that cover a wide range of genetically and antigenically diverse E1E2s. We validate our panel using neutralization and a binding antibody multiplex assay (BAMA). The panel of HCVpps includes E1E2 glycoproteins from acute and chronically infected cases in the Netherlands, as well as E1E2 glycoproteins from previously reported HCVs. Using eight monoclonal antibodies targeting multiple antigenic regions on E1E2, we could categorize four groups of neutralization sensitive viruses with viruses showing neutralization titers over a 100-fold range. One HCVpp (AMS0230) was extremely neutralization resistant and only neutralized by AR4-targeting antibodies. In addition, using binding antibody multiplex competition assay, we delineated mAb epitopes and their interactions. The binding and neutralization sensitivity of the HCVpps were confirmed using patient sera. At the end, eleven HCVpps with unique antibody binding and neutralization profiles were selected as the final panel for standardized HCV antibody assessments. In conclusion, this HCVpp panel can be used to evaluate antibody binding and neutralization breadth and potency as well as delineate the epitopes targeted in sera from patients or candidate vaccine trials. The HCVpp panel in combination with the established antibody competition assay present highly valuable tools for HCV vaccine development and evaluation.

Optimization of the pseudoparticle system for standardized assessments of neutralizing antibodies against hepatitis C virus.

A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.

Metagenomic DNA sequencing for semi-quantitative pathogen detection from urine: a prospective, laboratory-based, proof-of-concept study.

BACKGROUND: Semi-quantitative bacterial culture is the reference standard to diagnose urinary tract infection, but culture is time-consuming and can be unreliable if patients are receiving antibiotics. Metagenomics could increase diagnostic accuracy and speed by sequencing the microbiota and resistome directly from urine. We aimed to compare metagenomics to culture for semi-quantitative pathogen and resistome detection from urine. METHODS: In this proof-of-concept study, we prospectively included consecutive urine samples from a clinical diagnostic laboratory in Amsterdam. Urine samples were screened by DNA concentration, followed by PCR-free metagenomic sequencing of randomly selected samples with a high concentration of DNA (culture positive and negative). A diagnostic index was calculated as the product of DNA concentration and fraction of pathogen reads. We compared results with semi-quantitative culture using area under the receiver operating characteristic curve (AUROC) analyses. We used ResFinder and PointFinder for resistance gene detection and compared results to phenotypic antimicrobial susceptibility testing for six antibiotics commonly used for urinary tract infection treatment: nitrofurantoin, ciprofloxacin, fosfomycin, cotrimoxazole, ceftazidime, and ceftriaxone. FINDINGS: We screened 529 urine samples of which 86 were sequenced (43 culture positive and 43 culture negative). The AUROC of the DNA concentration-based screening was 0·85 (95% CI 0·81-0·89). At a cutoff value of 6·0 ng/mL, culture positivity was ruled out with a negative predictive value of 91% (95% CI 87-93; 26 of 297 samples), reducing the number of samples requiring sequencing by 56% (297 of 529 samples). The AUROC of the diagnostic index was 0·87 (95% CI 0·79-0·95). A diagnostic index cutoff value of 17·2 yielded a positive predictive value of 93% (95% CI 85-97) and a negative predictive value of 69% (55-80), correcting for a culture-positive prevalence of 66%. Gram-positive pathogens explained eight (89%) of the nine false-negative metagenomic test results. Agreement of phenotypic and genotypic antimicrobial susceptibility testing varied between 71% (22 of 31 samples) and 100% (six of six samples), depending on the antibiotic tested. INTERPRETATION: This study provides proof-of-concept of metagenomic semi-quantitative pathogen and resistome detection for the diagnosis of urinary tract infection. The findings warrant prospective clinical validation of the value of this approach in informing patient management and care. FUNDING: EU Horizon 2020 Research and Innovation Programme.

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV.

Hepatitis C virus (HCV) represents a major global health challenge and an efficient vaccine is urgently needed. Many HCV vaccination strategies employ recombinant versions of the viral E2 glycoprotein. However, recombinant E2 readily forms disulfide-bonded aggregates that might not be optimally suited for vaccines. Therefore, we have designed an E2 protein in which we strategically changed eight cysteines to alanines (E2.C8A). E2.C8A formed predominantly monomers and virtually no aggregates. Furthermore, E2.C8A also interacted more efficiently with broadly neutralizing antibodies than conventional E2. We used mice to evaluate different prime/boost immunization strategies involving a modified vaccinia virus Ankara (MVA) expressing the nearly full-length genome of HCV (MVA-HCV) in combination with either the E2 aggregates or the E2.C8A monomers. The combined MVA-HCV/E2 aggregates prime/boost strategy markedly enhanced HCV-specific effector memory CD4+ T cell responses and antibody levels compared to MVA-HCV/MVA-HCV. Moreover, the aggregated form of E2 induced higher levels of anti-E2 antibodies in vaccinated mice than E2.C8A monomers. These antibodies were cross-reactive and mainly of the IgG1 isotype. Our findings revealed how two E2 viral proteins that differ in their capacity to form aggregates are able to enhance to different extent the HCV-specific cellular and humoral immune responses, either alone or in combination with MVA-HCV. These combined protocols of MVA-HCV/E2 could serve as a basis for the development of a more effective HCV vaccine.

Bile-salt stimulated lipase polymorphisms do not associate with HCV susceptibility.

Bile-salt stimulate lipase (BSSL) is a glycoprotein found in human milk and blood that can potently bind DC-SIGN. The BSSL gene is highly polymorphic with a variant number of O-linked glycosylated 11 amino acid repeats at the C-terminus of the protein, encoded in exon 11 of the gene. It has been shown that certain BSSL genotypes associate with decreased HIV-1 transmission in vitro and decreased HIV-1 disease progression. The protein forms dimers and individuals possessing one high (typically 14-21) and one low (typically 7-11) number of repeat domains has been shown to have stronger binding of BSSL to DC-SIGN and HIV-1 inhibitory activity in vitro. Since we previously demonstrated that SNPs within the DC-SIGN gene can associate with risk of HCV sexual transmission and which can be linked to diminished DC-SIGN gene expression we aimed to identify whether BSSL polymorphisms associated similarly through differential binding to DC-SIGN. DNA was isolated from the HIV-1 infected MSM cohort (MOSAIC) composed of HCV multiple exposed uninfected (MEU) (N = 30) and multiple exposed HCV infected (MEI) (N = 32) individuals and from the Amsterdam cohort studies (ACS) intravenous drug using (IDU) cohort (22 MEI and 40 MEU). The numbers of repeats in exon 11 were determined by PCR with repeat distributions compared between MEI and MEU. No statistical significant difference in the copy number of exon 11 repeats, or combinations of, in the BSSL gene was observed when comparing HCV infected MEI with MEU, thus the exon 11 repeat copy number in the BSSL gene does not affect HCV susceptibility.

HIV-1 exposure and immune activation enhance sexual transmission of Hepatitis C virus by primary Langerhans cells.

INTRODUCTION: The significant rise in incidence of Hepatitis C virus (HCV) infection among men-who-have-sex-with-men (MSM) living with HIV-1 suggests that HCV under specific circumstances is transmitted via sexual contact. During sexual transmission HCV has to cross the epithelial barrier to either directly enter the blood stream or indirectly via mucosal immune cells. However, the mechanisms of sexual transmission of HCV remain unclear. We investigated the role of Langerhans cells (LCs) in HCV susceptibility during sexual contact as LCs are among the first cells in mucosal tissues to encounter invading viruses. METHODS: We investigated the phenotype of primary LCs in anal biopsies from MSM living with HIV-1. To investigate the role of primary LCs in HCV infection and transmission, we have used both isolated primary skin LCs and the ex vivo tissue transmission model. RESULTS: Our data identified an important role for mucosal LCs in facilitating HCV transmission after HIV-1 exposure or immune activation. LCs were detected within mucosal anal tissues obtained from HIV-1 positive MSM biopsies. In order to perform functional studies, we used primary LCs from skin, which have a similar phenotype as mucosal LCs. Immature LCs were neither infected nor transmitted HCV to hepatocytes. Notably, exposure to HIV-1 significantly increased HCV transmission by LCs in the ex vivo transmission model. HIV-1 replication was crucial for the increased HCV transmission as HIV-1 inhibitors significantly reduced HIV-1-induced HCV transmission. Moreover, tissue immune activation of LCs also increased HCV transmission to target cells. CONCLUSIONS: Thus, our data strongly indicate that HIV-1 or immune activation in MSM leads to capture of HCV by mucosal LCs, which might facilitate transmission to other cells or allow entry of HCV into the blood. This novel transmission mechanism by LCs also implicates that the activation state of LCs is an important cellular determinant for HCV susceptibility after sexual contact.

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.

SNP rs688 within the low-density lipoprotein receptor (LDL-R) gene associates with HCV susceptibility.

BACKGROUND & AIMS: Despite high-risk behaviour, 10%-20% of HCV multiple exposed individuals remain uninfected (MEU), whilst the remainder become infected (MEI). We hypothesize that host factors play a role in HCV susceptibility. We aimed to identify polymorphisms in host genes that encode for proteins involved in viral entry: CD81, Scavenger receptor 1 (SR-1), Low-density lipoprotein receptor (LDL-R), Claudin-1 (CLDN1), Occludin (OCLN) and Niemann-Pick C1-like 1 (NPC1L1). METHODS: Multiple exposed infected and MEU from two observational cohorts were selected. From the MSM study of acute infection with HCV (MOSAIC), HIV-1 infected MEU cases (n = 30) and HIV-1 infected MEI controls (n = 32) were selected based on reported high-risk behaviour. From the Amsterdam Cohorts Studies (ACS) injecting drug users (IDU) cohort, MEU cases (n = 40) and MEI controls (n = 22) were selected who injected drugs for ≥2 years, in the nineties, when HCV incidence was high. Selected single nucleotide polymorphisms (SNPs) were determined by sequencing or SNP assays. RESULTS: No associations were found for SNPs within genes coding for CD81, SR-1, Claudin-1 or Occludin between the MEU and MEI individuals from either cohort. We did observe a significant association for rs688 within the LDL-R gene with HCV infection (OR: 0.41 P = 0.001), however, LDL cholesterol levels did not vary between individuals carrying the differential SNPs. Additionally, a marginal significant effect was found for rs217434 and rs2072183 (OR: 2.07 P = 0.032 and OR: 1.76 P = 0.039, respectively) within NPC1L1. CONCLUSIONS: Our results demonstrate that the rs688 SNP within the LDL-R gene associates with HCV susceptibility through mucosal as well as intravenous exposure.

Enterovirus 71 infection of human airway organoids reveals VP1-145 as a viral infectivity determinant.

Human enteroviruses frequently cause severe diseases in children. Human enteroviruses are transmitted via the fecal-oral route and respiratory droplets, and primary replication occurs in the gastro-intestinal and respiratory tracts; however, how enteroviruses infect these sites is largely unknown. Human intestinal organoids have recently proven to be valuable tools for studying enterovirus-host interactions in the intestinal tract. In this study, we demonstrated the susceptibility of a newly developed human airway organoid model for enterovirus 71 (EV71) infection. We showed for the first time in a human physiological model that EV71 replication kinetics are strain-dependent. A glutamine at position 145 of the VP1 capsid protein was identified as a key determinant of infectivity, and residues VP1-98K and VP1-104D were identified as potential infectivity markers. The results from this study provide new insights into EV71 infectivity in the human airway epithelia and demonstrate the value of organoid technology for virus research.

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.

DC-SIGN Polymorphisms Associate with Risk of Hepatitis C Virus Infection Among Men who Have Sex with Men but not Among Injecting Drug Users.

We aimed to identify whether genetic polymorphisms within L-SIGN or DC-SIGN correlate with hepatitis C virus (HCV) susceptibility. A men who have sex with men (MSM) and an injecting drug users (IDU) cohort of HCV cases and multiple-exposed uninfected controls were genotyped for numerous L-SIGN and DC-SIGN polymorphisms. DC-SIGN single nucleotide polymorphisms (SNPs) -139, -871, and -939 correlated with HCV acquisition in the MSM cohort only. When the same SNPs were introduced into a transcription activity assay they demonstrated a reduction in expression with predicted alteration in binding of transcription factors. DC-SIGN promoter SNPs correlated with risk of HCV acquisition via sexual but not IDU exposure, likely through modulation of mRNA expression levels.

The hepatitis C virus nonstructural protein 3 Q80K polymorphism is frequently detected and transmitted among HIV-infected MSM in the Netherlands.

OBJECTIVES: The Q80K polymorphism is a naturally occurring resistance-associated variant in the hepatitis C virus (HCV) nonstructural protein 3 (NS3) region and is likely transmissible between hosts. This study describes the Q80K origin and prevalence among HCV risk groups in the Netherlands and examines whether Q80K is linked to specific transmission networks. DESIGN AND METHODS: Stored blood samples from HCV genotype 1a-infected patients were used for PCR and sequencing to reconstruct the NS3 maximum likelihood phylogeny. The most recent common ancestor was estimated with a coalescent-based model within a Bayesian statistical framework. RESULTS: Study participants (n = 150) were either MSM (39%), people who inject drugs (17%), or patients with other (15%) or unknown/unreported (29%) risk behavior. Overall 45% was coinfected with HIV. Q80K was present in 36% (95% confidence interval 28-44%) of patients throughout the sample collection period (2000-2015) and was most prevalent in MSM (52%, 95% confidence interval 38-65%). Five MSM-specific transmission clusters were identified, of which three exclusively contained sequences with Q80K. The HCV-1a most recent common ancestor in the Netherlands was estimated in 1914 (95% higher posterior density 1879-1944) and Q80K originated in 1957 (95% higher posterior density 1942-1970) within HCV-1a clade I. All Q80K lineages could be traced back to this single origin. CONCLUSION: Q80K is a highly stable and transmissible resistance-associated variant and was present in a large part of Dutch HIV-coinfected MSM. The introduction and expansion of Q80K variants in this key population suggest a founder effect, potentially jeopardizing future treatment with simeprevir.

Hepatitis C virus Broadly Neutralizing Monoclonal Antibodies Isolated 25 Years after Spontaneous Clearance.

Hepatitis C virus (HCV) is world-wide a major cause of liver related morbidity and mortality. No vaccine is available to prevent HCV infection. To design an effective vaccine, understanding immunity against HCV is necessary. The memory B cell repertoire was characterized from an intravenous drug user who spontaneously cleared HCV infection 25 years ago. CD27+IgG+ memory B cells were immortalized using BCL6 and Bcl-xL. These immortalized B cells were used to study antibody-mediated immunity against the HCV E1E2 glycoproteins. Five E1E2 broadly reactive antibodies were isolated: 3 antibodies showed potent neutralization of genotype 1 to 4 using HCV pseudotyped particles, whereas the other 2 antibodies neutralized genotype 1, 2 and 3 or 1 and 2 only. All antibodies recognized non-linear epitopes on E2. Finally, except for antibody AT12-011, which recognized an epitope consisting of antigenic domain C /AR2 and AR5, all other four antibodies recognized epitope II and domain B. These data show that a subject, who spontaneously cleared HCV infection 25 years ago, still has circulating memory B cells that are able to secrete broadly neutralizing antibodies. Presence of such memory B cells strengthens the argument for undertaking the development of an HCV vaccine.

Prediction of Protection against Asian Enterovirus 71 Outbreak Strains by Cross-neutralizing Capacity of Serum from Dutch Donors, The Netherlands.

Outbreaks of human enterovirus 71 (EV-71) in Asia are related to high illness and death rates among children. To gain insight into the potential threat for the population of Europe, we determined the neutralizing activity in intravenous immunoglobulin (IVIg) batches and individual serum samples from donors in the Netherlands against EV-71 strains isolated in Europe and in Asia. All IVIg batches and 41%, 79%, and 65% of serum samples from children ≤5 years of age, women of childbearing age, and HIV-positive men, respectively, showed high neutralizing activity against a Dutch C1 strain, confirming widespread circulation of EV-71 in the Netherlands. Asian B3-4 and C4 strains were efficiently cross-neutralized, predicting possible protection against extensive circulation and associated outbreaks of those types in Europe. However, C2 and C5 strains that had few mutations in the capsid region consistently escaped neutralization, emphasizing the importance of monitoring antigenic diversity among circulating EV-71 strains.

A method for near full-length amplification and sequencing for six hepatitis C virus genotypes.

BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient. RESULTS: In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90%, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1-3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies. CONCLUSIONS: The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications.

Febrile neutropenia: significance of elaborated screening for respiratory viruses, and the comparison of different sampling methods, in neutropenic patients with hematological malignancies.

BACKGROUND: During febrile neutropenia in only 30 to 60 percent an infectious agent is identified. This diagnostic gap could hypothetically be reduced with the broad implementation of molecular detection techniques like PCR, which has revolutionized the detection of infectious diseases during the last two decades. FINDINGS: We performed a longitudinal prospective study (N = 81) of neutropenic patients to assess the role of respiratory viruses in neutropenic fever and to determine the clinical relevance of blind screening for these viruses. Respiratory viruses were recovered in 14% of the patients prior to neutropenia. In 13% of neutropenic patients without fever and in 19% of those with fever, a respiratory virus was detected. Comparing different sample types; nasal swabs performed significantly better (16/117 = 43%), than throat swabs (6/106 = 6%). Throat gurgles did not show significant differences from the latter sample types. CONCLUSIONS: Blind diagnostic screening for respiratory viruses before or during neutropenia is not useful. Nasal swabs are sensitive and practical option for screening on respiratory viruses.

Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism.

The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains.

Performance of VIDISCA-454 in feces-suspensions and serum.

Virus discovery combining sequence unbiased amplification with next generation sequencing is now state-of-the-art. We have previously determined that the performance of the unbiased amplification technique which is operational at our institute, VIDISCA-454, is efficient when respiratory samples are used as input. The performance of the assay is, however, not known for other clinical materials like blood or stool samples. Here, we investigated the sensitivity of VIDISCA-454 with feces-suspensions and serum samples that are positive and that have been quantified for norovirus and human immunodeficiency virus type 1, respectively. The performance of VIDISCA-454 in serum samples was equal to its performance in respiratory material, with an estimated lower threshold of 1,000 viral genome copies. The estimated threshold in feces-suspension is around 200,000 viral genome copies. The decreased sensitivity in feces suspension is mainly due to sequences that share no recognizable identity with known sequences. Most likely these sequences originate from bacteria and phages which are not completely sequenced.

Origin and evolution of the unique hepatitis C virus circulating recombinant form 2k/1b.

Since its initial identification in St. Petersburg, Russia, the recombinant hepatitis C virus (HCV) 2k/1b has been isolated from several countries throughout Eurasia. The 2k/1b strain is the only recombinant HCV to have spread widely, raising questions about the epidemiological background in which it first appeared. In order to further understand the circumstances by which HCV recombinants might be formed and spread, we estimated the date of the recombination event that generated the 2k/1b strain using a Bayesian phylogenetic approach. Our study incorporates newly isolated 2k/1b strains from Amsterdam, The Netherlands, and has employed a hierarchical Bayesian framework to combine information from different genomic regions. We estimate that 2k/1b originated sometime between 1923 and 1956, substantially before the first detection of the strain in 1999. The timescale and the geographic spread of 2k/1b suggest that it originated in the former Soviet Union at about the time that the world's first centralized national blood transfusion and storage service was being established. We also reconstructed the epidemic history of 2k/1b using coalescent theory-based methods, matching patterns previously reported for other epidemic HCV subtypes. This study demonstrates the practicality of jointly estimating dates of recombination from flanking regions of the breakpoint and further illustrates that rare genetic-exchange events can be particularly informative about the underlying epidemiological processes.

Frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values.

Highly sensitive techniques, such as PCR, have greatly improved the detection of respiratory viruses. However, the sensitivity of PCR tests also complicates clinical interpretation, as the presence of small amounts of viral targets may not necessarily have clinical relevance. We performed a prospective case-control study in asymptomatic and symptomatic young children. PCR detection of 14 respiratory viruses was performed in nasal washes, and results were quantified in copies per milliliter. A total of 141 cases and 157 controls were included. In 72% of the cases and 28% of the controls, at least one virus was identified. When stratified for age, at least one virus was identified in 47% of the controls younger than 1 year old. Rhinovirus (RV) was frequently detected in both symptomatic and asymptomatic individuals. Receiver operating characteristic analysis for quantitative rhinovirus detection showed that cutoff values for clinical relevance are feasible for RV. In contrast to rhinovirus, respiratory syncytial virus (RSV) was rarely detected in controls, suggesting that a positive RSV test result is almost always of clinical relevance, independent of viral quantity. In conclusion, our study shows that asymptomatic carriage of a respiratory virus occurs frequently in young children. However, significant differences in the amount of virus present were observed between cases and controls. This suggests that defining cutoff levels should be feasible and represents the next necessary step for diagnosing viral respiratory infections using molecular tests.