Untargeted metagenomics protocol for the diagnosis of infection from CSF and tissue from sterile sites.

Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from sterile sites. We use this method routinely in our clinical service, processing 178 specimens over the past 2.5 years in a laboratory that adheres to ISO:15189 standards. We have successfully used this protocol to diagnose multiple cases of encephalitis and hepatitis.

Postpandemic rebound of adeno-associated virus type 2 (AAV2) infections temporally associated with an outbreak of unexplained severe acute hepatitis in children in the United Kingdom.

Over 1000 cases of unexplained severe acute hepatitis in children have been reported to date worldwide. An association with adeno-associated virus type 2 (AAV2) infection, a human parvovirus, prompted us to investigate the epidemiology of AAV in the United Kingdom. Three hundred pediatric respiratory samples collected before (April 03, 2009-April 03, 2013) and during (April 03, 2022) the COVID-19 pandemic were obtained. Wastewater samples were collected from 50 locations in London (August 2021-March 2022). Samples were tested for AAV using real-time polymerase chain reaction followed by sequencing. Selected adenovirus (AdV)-positive samples were also sequenced. The detection frequency of AAV2 was a sevenfold higher in 2022 samples compared with 2009-2013 samples (10% vs. 1.4%) and highest in AdV-positive samples compared with negatives (10/37, 27% vs. 5/94, 5.3%, respectively). AAV2-positive samples displayed high genetic diversity. AAV2 sequences were either very low or absent in wastewater collected in 2021 but increased in January 2022 and peaked in March 2022. AAV2 was detected in children in association with AdV of species C, with a highest frequency in 2022. Our findings are consistent with the expansion of the population of children unexposed to AAV2, leading to greater spread of the virus once distancing restrictions were lifted.

Modeling antibody dynamics following herpes zoster indicates that higher varicella-zoster virus viremia generates more VZV-specific antibodies.

Introduction: Studying antibody dynamics following re-exposure to infection and/or vaccination is crucial for a better understanding of fundamental immunological processes, vaccine development, and health policy research. Methods: We adopted a nonlinear mixed modeling approach based on ordinary differential equations (ODE) to characterize varicella-zoster virus specific antibody dynamics during and after clinical herpes zoster. Our ODEs models convert underlying immunological processes into mathematical formulations, allowing for testable data analysis. In order to cope with inter- and intra-individual variability, mixed models include population-averaged parameters (fixed effects) and individual-specific parameters (random effects). We explored the use of various ODE-based nonlinear mixed models to describe longitudinally collected markers of immunological response in 61 herpes zoster patients. Results: Starting from a general formulation of such models, we study different plausible processes underlying observed antibody titer concentrations over time, including various individual-specific parameters. Among the converged models, the best fitting and most parsimonious model implies that once Varicella-zoster virus (VZV) reactivation is clinically apparent (i.e., Herpes-zoster (HZ) can be diagnosed), short-living and long-living antibody secreting cells (SASC and LASC, respectively) will not expand anymore. Additionally, we investigated the relationship between age and viral load on SASC using a covariate model to gain a deeper understanding of the population's characteristics. Conclusion: The results of this study provide crucial and unique insights that can aid in improving our understanding of VZV antibody dynamics and in making more accurate projections regarding the potential impact of vaccines.

Absence of detectable monkeypox virus DNA in 11,000 English blood donations during the 2022 outbreak.

BACKGROUND: A large, worldwide outbreak of mpox (formerly referred to as monkeypox) involving mainly men who have sex with men commenced in May 2022. We evaluated the frequency of positivity for the causative agent, monkeypox virus (MPXV), in blood donations collected in August 2022, during the outbreak period in Southern England. METHODS/MATERIALS: The sensitivity and specificity of an MPXV-specific PCR and a generic non-variola orthopoxvirus (NVO) PCR were evaluated using samples from mpox cases and synthetic DNA standards. Residual minipools from nucleic acid testing were obtained from 10,896 blood donors in Southern England, with 21% from London. RESULTS: MPXV and NVO PCRs were both capable of detection of single copies of target sequence with calculated limits of detection (LOD)90 s of 2.3 and 2.1 DNA copies and analytical sample sensitivities of 46 and 42 MPXV DNA copies/ml, respectively. 454 minipools produced from 10,896 unique donors were assayed for MPXV DNA by both methods. No positive minipools were detected by either PCR. CONCLUSIONS: Although blood donors are unrepresentative of the UK population in terms of MPXV infection risk, the uniformly negative MPXV DNA testing results provide reassurance that MPXV viraemia and potential transmission risk were rare or absent in donors during the outbreak period. Minipools from blood donors allow rapid implementation of large-scale population-based screening for emerging pathogens and represent an important resource for pandemic preparedness.

Comparative assessment of viral dynamic models for SARS-CoV-2 for pharmacodynamic assessment in early treatment trials.

Pharmacometric analyses of time series viral load data may detect drug effects with greater power than approaches using single time points. Because SARS-CoV-2 viral load rapidly rises and then falls, viral dynamic models have been used. We compared different modelling approaches when analysing Phase II-type viral dynamic data. Using two SARS-CoV-2 datasets of viral load starting within 7 days of symptoms, we fitted the slope-intercept exponential decay (SI), reduced target cell limited (rTCL), target cell limited (TCL) and TCL with eclipse phase (TCLE) models using nlmixr. Model performance was assessed via Bayesian information criterion (BIC), visual predictive checks (VPCs), goodness-of-fit plots, and parameter precision. The most complex (TCLE) model had the highest BIC for both datasets. The estimated viral decline rate was similar for all models except the TCL model for dataset A with a higher rate (median [range] day-1 : dataset A; 0.63 [0.56-1.84]; dataset B: 0.81 [0.74-0.85]). Our findings suggest simple models should be considered during pharmacodynamic model development.

Early antiviral treatment in outpatients with COVID-19 (FLARE): a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The objective of this trial is to assess whether early antiviral therapy in outpatients with COVID-19 with either favipiravir plus lopinavir/ritonavir, lopinavir/ritonavir alone, or favipiravir alone, is associated with a decrease in viral load of SARS-CoV-2 compared with placebo. TRIAL DESIGN: FLARE is a phase IIA randomised, double-blind, 2x2 factorial placebo-controlled, interventional trial. PARTICIPANTS: This trial is being conducted in the United Kingdom, with Royal Free Hospital, London as the lead site. Participants are non-hospitalised adults with highly suspected COVID-19 within the first 5 days of symptom onset, or who have tested positive with SARS-CoV-2 causing COVID-19 within the first 7 days of symptom onset, or who are asymptomatic but tested positive for SARS-CoV-2 for the first time within the last 48 hours. Inclusion criteria are as follows: 1. Any adult with the following: Symptoms compatible with COVID-19 disease (Fever >37.8°C on at least one occasion AND either cough and/ or anosmia) within the first 5 days of symptom onset (date/time of enrolment must be within the first 5 days of symptom onset) OR ANY symptoms compatible with COVID-19 disease (may include, but are not limited to fever, cough, shortness of breath, malaise, myalgia, headache, coryza) and tested positive for SARS-CoV-2 within the first 7 days of symptom onset) (date/time of enrolment must be within the first 7 days of symptom onset) OR no symptoms but tested positive for SARS-CoV-2 within the last 48 hours (date/time of test must be within 48 hours of enrolment) 2. Male or female aged 18 years to 70 years old inclusive at screening 3. Willing and able to take daily saliva samples 4. Able to provide full informed consent and willing to comply with trial-related procedures Exclusion criteria are as follows: 1. Known hypersensitivity to any of the active ingredients or excipients in favipiravir and matched placebo, and in lopinavir/ritonavir and matched placebo (See Appendix 2) 2. Chronic liver disease at screening (known cirrhosis of any aetiology, chronic hepatitis (e.g. autoimmune, viral, steatohepatitis), cholangitis or any known elevation of liver aminotransferases with AST or ALT > 3 X ULN)* 3. Chronic kidney disease (stage 3 or beyond) at screening: eGFR < 60 ml/min/1.73m2 * 4. HIV infection, if untreated, detectable viral load or on protease inhibitor therapy 5. Any clinical condition which the investigator considers would make the participant unsuitable for the trial 6. Concomitant medications known to interact with favipiravir and matched placebo, and with lopinavir/ritonavir and matched placebo, and carry risk of toxicity for the participant 7. Current severe illness requiring hospitalisation 8. Pregnancy and/ or breastfeeding 9. Eligible female participants of childbearing potential and male participants with a partner of childbearing potential not willing to use highly effective contraceptive measures during the trial and within the time point specified following last trial treatment dose. 10. Participants enrolled in any other interventional drug or vaccine trial (co-enrolment in observational studies is acceptable) 11. Participants who have received the COVID-19 vaccine *Considering the importance of early treatment of COVID-19 to impact viral load, the absence of known chronic liver/ kidney disease will be confirmed verbally by the participant during pre-screening and Screening/Baseline visit. Safety blood samples will be collected at Screening/Baseline visit (Day 1) and test results will be examined as soon as they become available and within 24 hours. INTERVENTION AND COMPARATOR: Participants will be randomised 1:1:1:1 using a concealed online minimisation process into one of the following four arms: Arm 1: Favipiravir + Lopinavir/ritonavir Oral favipiravir at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. Arm 2: Favipiravir + Lopinavir/ritonavir placebo Oral favipiravir at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir matched placebo at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. Arm 3: Favipiravir placebo + Lopinavir/ritonavir Oral favipiravir matched placebo at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. Arm 4: Favipiravir placebo + Lopinavir/ritonavir placebo Oral favipiravir matched placebo at 1800mg twice daily on Day 1, followed by 400mg four (4) times daily from Day 2 to Day 7 PLUS lopinavir/ritonavir matched placebo at 400mg/100mg twice daily on Day 1, followed by 200mg/50mg four (4) times daily from Day 2 to Day 7. MAIN OUTCOMES: The primary outcome is upper respiratory tract viral load at Day 5. SECONDARY OUTCOMES: Percentage of participants with undetectable upper respiratory tract viral load after 5 days of therapy Proportion of participants with undetectable stool viral load after 7 days of therapy Rate of decrease in upper respiratory tract viral load during 7 days of therapy Duration of fever following commencement of trial medications Proportion of participants with hepatotoxicity after 7 days of therapy Proportion of participants with other medication-related toxicity after 7 days of therapy and 14 days post-randomisation Proportion of participants admitted to hospital with COVID-19 related illness Proportion of participants admitted to ICU with COVID-19 related illness Proportion of participants who have died with COVID-19 related illness Pharmacokinetic and pharmacodynamic analysis of favipiravir Exploratory: Proportion of participants with deleterious or resistance-conferring mutations in SARS-CoV-2 RANDOMISATION: Participants will be randomised 1:1:1:1 using a concealed online minimisation process, with the following factors: trial site, age (≤ 55 vs > 55 years old), gender, obesity (BMI <30 vs ≥30), symptomatic or asymptomatic, current smoking status (Yes = current smoker, No = ex-smoker, never smoker), ethnicity (Caucasian, other) and presence or absence of comorbidity (defined as diabetes, hypertension, ischaemic heart disease (including previous myocardial infarction), other heart disease (arrhythmia and valvular heart disease), asthma, COPD, other chronic respiratory disease). BLINDING (MASKING): Participants and investigators will both be blinded to treatment allocation (double-blind). NUMBERS TO BE RANDOMISED (SAMPLE SIZE): 240 participants, 60 in each arm. TRIAL STATUS: Protocol version 4.0 dated 7th January 2021. Date of first enrolment: October 2020. Recruitment is ongoing, with anticipated finish date of 31st March 2021. TRIAL REGISTRATION: The FLARE trial is registered with Clinicaltrials.gov, trial identifying number NCT04499677 , date of registration 4th August 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Systematic Review and Patient-Level Meta-Analysis of SARS-CoV-2 Viral Dynamics to Model Response to Antiviral Therapies.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) viral loads change rapidly following symptom onset, so to assess antivirals it is important to understand the natural history and patient factors influencing this. We undertook an individual patient-level meta-analysis of SARS-CoV-2 viral dynamics in humans to describe viral dynamics and estimate the effects of antivirals used to date. This systematic review identified case reports, case series, and clinical trial data from publications between January 1, 2020, and May 31, 2020, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A multivariable Cox proportional hazards (Cox-PH) regression model of time to viral clearance was fitted to respiratory and stool samples. A simplified four parameter nonlinear mixed-effects (NLME) model was fitted to viral load trajectories in all sampling sites and covariate modeling of respiratory viral dynamics was performed to quantify time-dependent drug effects. Patient-level data from 645 individuals (age 1 month to 100 years) with 6,316 viral loads were extracted. Model-based simulations of viral load trajectories in samples from the upper and lower respiratory tract, stool, blood, urine, ocular secretions, and breast milk were generated. Cox-PH modeling showed longer time to viral clearance in older patients, men, and those with more severe disease. Remdesivir was associated with faster viral clearance (adjusted hazard ratio (AHR) = 9.19, P < 0.001), as well as interferon, particularly when combined with ribavirin (AHR = 2.2, P = 0.015; AHR = 6.04, P = 0.006). Combination therapy should be further investigated. A viral dynamic dataset and NLME model for designing and analyzing antiviral trials has been established.

Cluster of Symptomatic Graft-to-Host Transmission of Herpes Simplex Virus Type 1 in an Endothelial Keratoplasty Setting.

Purpose: Descemet's membrane endothelial keratoplasty (DMEK) is becoming the gold standard to treat corneal endothelial dysfunctions worldwide. Compared with conventional penetrating keratoplasty, infectious complications after DMEK are ill defined. We describe the clinical picture of 2 DMEK recipients, operated on the same day and in the same clinic, who developed atypical herpes simplex virus type 1 (HSV-1) infection in the transplant recipient eye within days post-DMEK. Because recipients received cornea tissue from 2 different donors prepared by the same eye bank, the likelihood of a common HSV-1 source was determined. Design: Case series. Participants: Two DMEK recipients who developed atypical intraocular HSV-1 disease shortly after surgery and surplus cornea specimens of 6 donors. Methods: Surplus cornea donor (pre-DMEK cornea remnants and conditioned cornea storage and transport media) and recipient samples (post-DMEK aqueous humor) were assayed for HSV-1 DNA and infectious virus by real-time polymerase chain reaction (RT-PCR) and cell culture, respectively. Target-enriched whole viral genome sequencing was performed on HSV-1 DNA-positive ocular specimens. Main Outcome Measures: Clinical picture of atypical intraocular HSV-1 infection post-DMEK and presence and homology of HSV-1 genomes between ocular specimens of DMEK donors and recipients. Results: Herpes simplex virus type 1 DNA was detected in aqueous humor and donor cornea specimens of both DMEK cases, but not in the cornea remnants of 6 randomly selected donors processed by the same eye bank. Infectious HSV-1 was isolated from the cornea remnant and corresponding culture medium of 1 cornea donor. Notably, whole-genome sequencing of virus DNA-positive specimens demonstrated exceptionally high genetic similarity between HSV-1 strains in recipient and donor specimens of both DMEK cases. Conclusions: Data indicate cross-contamination of cornea grafts during DMEK preparation with subsequent graft-to-host HSV-1 transmission that caused atypical sight-threatening herpetic eye disease shortly after DMEK. Ophthalmologists should be aware that HSV-1 transmission by DMEK is possible and can lead to atypical ocular disease, a condition that can easily be prevented by taking appropriate technical and clinical measures at both eye bank and surgical levels.

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure.

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.

In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix.

Protease inhibitors (PIs) are the second- and last-line therapy for the majority of HIV-infected patients worldwide. Only around 20% of individuals who fail PI regimens develop major resistance mutations in protease. We sought to explore the role of mutations in gag-pro genotypic and phenotypic changes in viruses from six Nigerian patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. Target enrichment and next-generation sequencing (NGS) with the Illumina MiSeq system were followed by haplotype reconstruction. Full-length Gag-protease gene regions were amplified from baseline (pre-PI) and virologic failure (VF) samples, sequenced, and used to construct gag-pro-pseudotyped viruses. Phylogenetic analysis was performed using maximum-likelihood methods. Susceptibility to lopinavir (LPV) and darunavir (DRV) was measured using a single-cycle replication assay. Western blotting was used to analyze Gag cleavage. In one of six participants (subtype CRF02_AG), we found 4-fold-lower LPV susceptibility in viral clones during failure of second-line treatment. A combination of four mutations (S126del, H127del, T122A, and G123E) in the p17 matrix of baseline virus generated a similar 4-fold decrease in susceptibility to LPV but not darunavir. These four amino acid changes were also able to confer LPV resistance to a subtype B Gag-protease backbone. Western blotting demonstrated significant Gag cleavage differences between sensitive and resistant isolates in the presence of drug. Resistant viruses had around 2-fold-lower infectivity than sensitive clones in the absence of drug. NGS combined with haplotype reconstruction revealed that resistant, less fit clones emerged from a minority population at baseline and thereafter persisted alongside sensitive fitter viruses. We used a multipronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV-1 matrix, revealing the interplay between Gag-associated resistance and fitness.

Favipiravir and Zanamivir Cleared Infection with Influenza B in a Severely Immunocompromised Child.

A combination of favipiravir and zanamivir successfully cleared influenza B infection in a child who had undergone bone marrow transplant for X-linked severe combined immunodeficiency, with no recovery of T lymphocytes. Deep sequencing of viral samples illuminated the within-host dynamics of infection, demonstrating the effectiveness of favipiravir in this case.

Norovirus Transmission Dynamics in a Pediatric Hospital Using Full Genome Sequences.

Background: Norovirus is a leading cause of worldwide and nosocomial gastroenteritis. The study aim was to assess the utility of molecular epidemiology using full genome sequences compared to routine infection prevention and control (IPC) investigations. Methods: Norovirus genomes were generated from new episodes of norovirus at a pediatric tertiary referral hospital over a 19-month period (n = 182). Phylogeny identified clusters of related sequences that were verified using epidemiological and clinical data. Results: Twenty-four clusters of related norovirus sequences ("sequence clusters") were observed, including 8 previously identified by IPC investigations ("IPC outbreaks"). Seventeen sequence clusters (involving 77/182 patients) were corroborated by epidemiological data ("epidemiologically supported clusters"), suggesting transmission between patients. Linked infections were identified among 44 patients who were missed by IPC investigations. Thirty-three percent of norovirus sequences were linked, suggesting nosocomial transmission; 24% of patients had nosocomial infections from an unknown source; and 43% were norovirus positive on admission. Conclusions: We show there are frequent introductions of multiple norovirus strains with extensive onward nosocomial transmission of norovirus in a pediatric hospital with a high proportion of immunosuppressed patients nursed in isolation. Phylogenetic analysis using full genome sequences is more sensitive than classic IPC investigations for identifying linked cases and should be considered when investigating norovirus nosocomial transmission. Sampling of staff, visitors, and the environment may be required for complete understanding of infection sources and transmission routes in patients with nosocomial infections not linked to other patients and among patients with phylogenetically linked cases but no evidence of direct contact.

Validation of Multiplex Serology detecting human herpesviruses 1-5.

Human herpesviruses (HHV) cause a variety of clinically relevant conditions upon primary infection of typically young and immunocompetent hosts. Both primary infection and reactivation after latency can lead to more severe disease, such as encephalitis, congenital defects and cancer. Infections with HHV are also associated with cardiovascular and neurodegenerative disease. However, most of the associations are based on retrospective case-control analyses and well-powered prospective cohort studies are needed for assessing temporality and causality. To enable comprehensive investigations of HHV-related disease etiology in large prospective population-based cohort studies, we developed HHV Multiplex Serology. This methodology represents a low-cost, high-throughput technology that allows simultaneous measurement of specific antibodies against five HHV species: Herpes simplex viruses 1 and 2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus. The newly developed HHV species-specific ('Monoplex') assays were validated against established gold-standard reference assays. The specificity and sensitivity of the HHV species-specific Monoplex Serology assays ranged from 92.3% to 100.0% (median 97.4%) and 91.8% to 98.7% (median 96.6%), respectively. Concordance with reference assays was very high with kappa values ranging from 0.86 to 0.96 (median kappa 0.93). Multiplexing the Monoplex Serology assays resulted in no loss of performance and allows simultaneous detection of antibodies against the 5 HHV species in a high-throughput manner.

Correction to: Chronic Aichi Virus Infection in a Patient with X-Linked Agammaglobulinemia.

The original version of this article unfortunately did not display the appropriate captions in the figure. The correct version is displayed below.

The association between human herpesvirus infections and stroke: a systematic review protocol.

INTRODUCTION: After primary infection, human herpesviruses establish latency and persist lifelong. Periodic virus reactivation can lead to serious inflammatory complications. Recent research suggests that herpesvirus reactivation may also be linked to acute stroke. An improved understanding of this relationship is vital to inform public health prevention strategies. We will review the evidence regarding the role of human herpesviruses in triggering stroke. METHODS AND ANALYSIS: A systematic literature review of published and grey literature studies with a human herpesvirus (infection or reactivation) as an exposure and stroke as an outcome will be carried out. Randomised controlled trials, cohort, case-control, case crossover and self-controlled case series designs will be eligible; no restrictions will be placed on publication status, language and geographical or healthcare setting. The Cochrane Central Register of Controlled Trials, Embase, Global Health, Medline, Scopus and Web of Science will be searched from dates of inception to January 2017. A prespecified search strategy of medical subject headings and free text terms (in the title and abstract) for human herpesviruses AND stroke will be used. Two reviewers will independently screen titles and abstracts for eligible studies, followed by full-text screening. The reviewers will then extract data from the eligible studies using standardised, pilot-tested tables and assess risk of bias in individual studies, in line with the Cochrane Collaboration approach. The data will be synthesised in a narrative format, and meta-analyses considered where there are sufficient data. Quality of evidence will be assessed in line with theGrading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. ETHICS AND DISSEMINATION: As this is a systematic review, ethical approval is not required. The results will be submitted for peer-review publication and presented at national conferences. A lay and short summary will be disseminated on appropriate webpages. PROSPERO REGISTRATION NUMBER: CRD42017054502.

Viral gastrointestinal infections and norovirus genotypes in a paediatric UK hospital, 2014-2015.

BACKGROUND: Diarrhoea in children is a common disease; understanding the incidence of causative viruses can aid infection control and vaccine development. OBJECTIVES: Describe the incidence and characteristics of gastroenteric viruses including norovirus genotypes in a paediatric hospital cohort. STUDY DESIGN: Norovirus, adenovirus, sapovirus, astrovirus, rotavirus qPCR and norovirus genotyping results for all stool specimens (n=4786; 1393 patients) at a UK paediatric tertiary referral hospital June 2014-July 2015. RESULTS AND DISCUSSION: 24% (329/1393) of patients were positive for a GI virus; the majority were positive for norovirus (44%, 144/329) or adenovirus (44%, 146/329). The overall incidence of rotavirus (2%) is reduced compared to pre-vaccination studies; however the incidence of other GI viruses has not increased. Norovirus infections had a significantly higher virus burden compared to other GI viruses (P ≤0.03); sapovirus infections had the lowest viral burden. The number of norovirus cases per month did not follow the typical winter seasonal trend of nationally reported outbreaks. The number of cases per month correlates with the number of hospital admissions (R=0.703, P=0.011); the number of admissions accounts for 50% of the variability in number of cases per month. The breadth of genotypes seen (48% non-GII.4), suggests a community source for many norovirus infections and has implications for vaccine development. All GI viruses caused chronic infections, with the majority (50-100%) in immunocompromised patients. Incidence or duration of infection in chronic norovirus infections did not differ between genotypes, suggesting host-mediated susceptibility.

Norovirus Whole-Genome Sequencing by SureSelect Target Enrichment: a Robust and Sensitive Method.

Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription-real-time PCR cycle threshold (CT) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless of CT values. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.

Enrichment by hybridisation of long DNA fragments for Nanopore sequencing.

Enrichment of DNA by hybridisation is an important tool which enables users to gather target-focused next-generation sequence data in an economical fashion. Current in-solution methods capture short fragments of around 200-300 nt, potentially missing key structural information such as recombination or translocations often found in viral or bacterial pathogens. The increasing use of long-read third-generation sequencers requires methods and protocols to be adapted for their specific requirements. Here, we present a variation of the traditional bait-capture approach which can selectively enrich large fragments of DNA or cDNA from specific bacterial and viral pathogens, for sequencing on long-read sequencers. We enriched cDNA from cultured influenza virus A, human cytomegalovirus (HCMV) and genomic DNA from two strains of Mycobacterium tuberculosis (M. tb) from a background of cell line or spiked human DNA. We sequenced the enriched samples on the Oxford Nanopore MinION™ and the Illumina MiSeq platform and present an evaluation of the method, together with analysis of the sequence data. We found that unenriched influenza A and HCMV samples had no reads matching the target organism due to the high background of DNA from the cell line used to culture the pathogen. In contrast, enriched samples sequenced on the MinION™ platform had 57 % and 99 % best-quality on-target reads respectively.