Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines.

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

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.

Signatures of VH1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins.

An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the VH1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred VH1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.

Viral, Bacterial, Metabolic, and Autoimmune Causes of Severe Acute Encephalopathy in Sub-Saharan Africa: A Multicenter Cohort Study.

OBJECTIVES: To assess whether viral, bacterial, metabolic, and autoimmune diseases are missed by conventional diagnostics among children with severe acute encephalopathy in sub-Saharan Africa. STUDY DESIGN: One hundred thirty-four children (6 months to 18 years) presenting with nontraumatic coma or convulsive status epilepticus to 1 of 4 medical referral centers in Uganda, Malawi, and Rwanda were enrolled between 2015 and 2016. Locally available diagnostic tests could be supplemented in 117 patients by viral, bacterial, and 16s quantitative polymerase chain reaction testing, metagenomics, untargeted metabolomics, and autoimmune immunohistochemistry screening. RESULTS: Fourteen (12%) cases of viral encephalopathies, 8 (7%) cases of bacterial central nervous system (CNS) infections, and 4 (4%) cases of inherited metabolic disorders (IMDs) were newly identified by additional diagnostic testing as the most likely cause of encephalopathy. No confirmed cases of autoimmune encephalitis were found. Patients for whom additional diagnostic testing aided causal evaluation (aOR 3.59, 90% CI 1.57-8.36), patients with a viral CNS infection (aOR 7.91, 90% CI 2.49-30.07), and patients with an IMD (aOR 9.10, 90% CI 1.37-110.45) were at increased risk for poor outcome of disease. CONCLUSIONS: Viral and bacterial CNS infections and IMDs are prevalent causes of severe acute encephalopathy in children in Uganda, Malawi, and Rwanda that are missed by conventional diagnostics and are associated with poor outcome of disease. Improved diagnostic capacity may increase diagnostic yield and might improve outcome of disease.

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.

Induction of cross-neutralizing antibodies by a permuted hepatitis C virus glycoprotein nanoparticle vaccine candidate.

Hepatitis C virus (HCV) infection affects approximately 58 million people and causes ~300,000 deaths yearly. The only target for HCV neutralizing antibodies is the highly sequence diverse E1E2 glycoprotein. Eliciting broadly neutralizing antibodies that recognize conserved cross-neutralizing epitopes is important for an effective HCV vaccine. However, most recombinant HCV glycoprotein vaccines, which usually include only E2, induce only weak neutralizing antibody responses. Here, we describe recombinant soluble E1E2 immunogens that were generated by permutation of the E1 and E2 subunits. We displayed the E2E1 immunogens on two-component nanoparticles and these nanoparticles induce significantly more potent neutralizing antibody responses than E2. Next, we generated mosaic nanoparticles co-displaying six different E2E1 immunogens. These mosaic E2E1 nanoparticles elicit significantly improved neutralization compared to monovalent E2E1 nanoparticles. These results provide a roadmap for the generation of an HCV vaccine that induces potent and broad neutralization.

Structure of the hepatitis C virus E1E2 glycoprotein complex.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo-electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies-AR4A, AT1209, and IGH505-at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.

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.

Cross-genotype AR3-specific neutralizing antibodies confer long-term protection in injecting drug users after HCV clearance.

BACKGROUND & AIMS: In order to design an effective vaccine against hepatitis C virus (HCV) infection, it is necessary to understand immune protection. A number of broadly reactive neutralizing antibodies have been isolated from B cells of HCV-infected patients. However, it remains unclear whether B cells producing such antibodies contribute to HCV clearance and long-term immune protection against HCV. METHODS: We analysed the B cell repertoire of 13 injecting drug users from the Amsterdam Cohort Study, who were followed up for a median of 17.5 years after primary infection. Individuals were classified into 2 groups based on the outcome of HCV infection: 5 who became chronically infected either after primary infection or after reinfection, and 8 who were HCV RNA negative following spontaneous clearance of ≥1 HCV infection(s). From each individual, 10,000 CD27+IgG+B cells, collected 0.75 year after HCV infection, were cultured to characterize the antibody repertoire. RESULTS: Using a multiplex flow cytometry-based assay to study the antibody binding to E1E2 from genotype 1 to 6, we found that a high frequency of cross-genotype antibodies was associated with spontaneous clearance of 1 or multiple infections (p = 0.03). Epitope specificity of these cross-genotype antibodies was determined by alanine mutant scanning in 4 individuals who were HCV RNA negative following spontaneous clearance of 1 or multiple infections. Interestingly, the cross-genotype antibodies were mainly antigenic region 3 (AR3)-specific and showed cross-neutralizing activity against HCV. In addition to AR3 antibodies, 3 individuals developed antibodies recognizing antigenic region 4, of which 1 monoclonal antibody showed cross-neutralizing capacity. CONCLUSIONS: Together, these data suggest that a strong B cell response producing cross-genotype and neutralizing antibodies, especially targeting AR3, contributes to HCV clearance and long-term immune protection against HCV. LAY SUMMARY: Although effective treatments against hepatitis C virus (HCV) are available, 500,000 people die from liver disease caused by HCV each year and approximately 1.75 million people are newly infected. This could be prevented by a vaccine. To design a vaccine against HCV, more insight into the role of antibodies in the protection against HCV infection is needed. In a cohort of injecting drug users, we found that antibodies interfering with virus cell entry, and recognizing multiple HCV genotypes, conferred long-term protection against chronic HCV infection.

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.

Polarized Entry of Human Parechoviruses in the Airway Epithelium.

Human parechoviruses (HPeVs), a poorly studied genus within the Picornaviridae family, are classified into 19 genotypes of which HPeV1 and HPeV3 are the most often detected. HPeV1 VP1 C terminus contains an arginine-glycine-aspartic acid (RGD) motif and has been shown to depend on the host cell surface αV integrins (αV ITGs) and heparan sulfate (HS) for entry. HPeV3 lacks this motif and the receptors remain unknown. HPeVs can be detected in patient nasopharyngeal and stool samples, and infection is presumed to occur after respiratory or gastro-intestinal transmission. HPeV pathogenesis is poorly understood as there are no animal models and previous studies have been conducted in immortalized monolayer cell cultures which do not adequately represent the characteristics of human tissues. To bridge this gap, we determined the polarity of infection, replication kinetics, and cell tropism of HPeV1 and HPeV3 in the well-differentiated human airway epithelial (HAE) model. We found the HAE cultures to be permissive for HPeVs. Both HPeV genotypes infected the HAE preferentially from the basolateral surface while the progeny virus was shed toward the apical side. Confocal microscopy revealed the target cell type to be the p63+ basal cells for both viruses, αV ITG and HS blocking had no effect on the replication of either virus, and transcriptional profiling suggested that HPeV3 infection induced stronger immune activation than HPeV1. Genotype-specific host responses may contribute to the differences in pathogenesis and clinical outcomes associated with HPeV1 and HPeV3.

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.

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing.

In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottleneck as well as fluctuations in viral genetic diversity over time, accompanied by dynamic shifts in viral subpopulations.