Comparison of multiple whole-genome and Spike-only sequencing protocols for estimating variant frequencies via wastewater-based epidemiology (preprint)

Sequencing of SARS-CoV-2 in wastewater provides a key opportunity to monitor the prevalence of variants spatiotemporally, potentially facilitating their detection simultaneously with, or even prior to, observation through clinical testing. However, there are multiple sequencing methodologies available. This study aimed to evaluate the performance of alternative protocols for detecting SARS-CoV-2 variants. We tested the detection of two synthetic RNA SARS-CoV-2 genomes in a wide range of ratios and at two concentrations representative of those found in wastewater using whole-genome and Spike-gene-only protocols utilising Illumina and Oxford Nanopore platforms. We developed a Bayesian hierarchical model to determine the predicted frequencies of variants and the error surrounding our predictions. We found that most of the sequencing protocols detected polymorphic nucleotide frequencies at a level that would allow accurate determination of the variants present at higher concentrations. Most methodologies, including the Spike-only approach, could also predict variant frequencies with a degree of accuracy in low-concentration samples but, as expected, with higher error around the estimates. All methods were additionally confirmed to detect the same prevalent variants in a set of wastewater samples. Our results provide the first quantitative statistical comparison of a range of alternative methods that can be used successfully in the surveillance of SARS-CoV-2 variant frequencies from wastewater.

Genomic Investigations of Acute Hepatitis of Unknown Aetiology in Children (preprint)

Since the first reports of hepatitis of unknown aetiology occurring in UK children, over 1000 cases have been reported worldwide, including 268 cases in the UK, with the majority younger than 6 years old. Using genomic, proteomic and immunohistochemical methods, we undertook extensive investigation of 28 cases and 136 control subjects. In five cases who underwent liver transplantation, we detected high levels of adeno-associated virus 2 (AAV2) in the explanted livers. AAV2 was also detected at high levels in blood from 10/11 non-transplanted cases. Low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), both of which enable AAV2 lytic replication, were also found in the five explanted livers and blood from 15/17 and 6/9 respectively, of the 23 non-transplant cases tested. In contrast, AAV2 was detected at low titre in 6/100 whole bloods from child controls from cohorts with presence or absence of hepatitis and/or adenovirus infection. Our data show an association of AAV2 at high titre in blood or liver tissue, with unexplained hepatitis in children infected in the recent HAdV-F41 outbreak. We were unable to find evidence by electron microscopy, immunohistochemistry or proteomics of HAdV or AAV2 viral particles or proteins in explanted livers, suggesting that hepatic pathology is not due to direct lytic infection by either virus. The potential that AAV2, although not previously associated with disease, may, together with HAdV-F41 and/or HHV-6, be causally implicated in the outbreak of unexplained hepatitis, requires further investigation.

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks (preprint)

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

Evaluating the effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control teams: the COG-UK hospital-onset COVID-19 infection study (preprint)

Introduction Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings. Methods We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data-collection period, followed by intervention periods comprising 8 weeks of 'rapid' (<48h) and 4 weeks of 'longer-turnaround' (5-10 day) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital onset COVID-19 infections (HOCIs; detected [≥]48h from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on incidence of probable/definite hospital-acquired infections (HAIs) was evaluated. Results A total of 2170 HOCI cases were recorded from October 2020-April 2021, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (IRR 1.60, 95%CI 0.85-3.01; P=0.14) or rapid (0.85, 0.48-1.50; P=0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8% and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2% and 11.6% of cases where the report was returned. In a per-protocol sensitivity analysis there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days. Conclusion While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days.

Genetic architecture of host proteins interacting with SARS-CoV-2

Strategies to develop therapeutics for SARS-CoV-2 infection may be informed by experimental identification of viral-host protein interactions in cellular assays and measurement of host response proteins in COVID-19 patients. Identification of genetic variants that influence the level or activity of these proteins in the host could enable rapid 'in silico' assessment in human genetic studies of their causal relevance as molecular targets for new or repurposed drugs to treat COVID-19. We integrated large-scale genomic and aptamer-based plasma proteomic data from 10,708 individuals to characterize the genetic architecture of 179 host proteins reported to interact with SARS-CoV-2 proteins or to participate in the host response to COVID-19. We identified 220 host DNA sequence variants acting in cis (MAF 0.01-49.9%) and explaining 0.3-70.9% of the variance of 97 of these proteins, including 45 with no previously known protein quantitative trait loci (pQTL) and 38 encoding current drug targets. Systematic characterization of pQTLs across the phenome identified protein-drug-disease links, evidence that putative viral interaction partners such as MARK3 affect immune response, and establish the first link between a recently reported variant for respiratory failure of COVID-19 patients at the ABO locus and hypercoagulation, i.e. maladaptive host response. Our results accelerate the evaluation and prioritization of new drug development programmes and repurposing of trials to prevent, treat or reduce adverse outcomes. Rapid sharing and dynamic and detailed interrogation of results is facilitated through an interactive webserver ( https://omicscience.org/apps/covidpgwas/ ).

The Safety and Immunogenicity of Concomitant Administration of COVID-19 Vaccines (ChAdOx1 or BNT162b2) with Seasonal Influenza Vaccines in Adults: A Phase IV, Multicentre Randomised Controlled Trial with Blinding (ComFluCOV) (preprint)

Background: Concomitant administration of COVID-19 and influenza vaccines would reduce burden on healthcare systems. We assess the safety of concomitant administration. Methods: Adults in receipt of a single dose of ChAdOx1 or BNT162b2 were enrolled at 12 UK sites and randomised 1:1 to receive concomitant administration of either age-appropriate influenza or placebo alongside second COVID-19 vaccine. Three weeks later the group who received placebo received the influenza vaccine, and vice versa. Participants were followed to six weeks. The influenza vaccines were three seasonal, inactivated vaccines (trivalent, MF59C adjuvanted (aTIV) or a cellular or recombinant quadrivalent vaccine (QIVc/QIVr)). Participants and investigators were masked to the allocation. The primary endpoint was one or more participant-reported solicited systemic reaction in the seven days after first trial vaccination(s), with a difference of <25% considered non-inferior. Local and unsolicited systemic reactions and humoral responses were also assessed (ISRCTN14391248). Findings: Between 1st April and 26th June 2021, 679 participants were recruited to one of six cohorts: (129 ChAdOx1/QIVc; 139 BNT162b2/QIVc; 146 ChAdOx1/aTIV; 79 BNT162b2/aTIV; 128 ChAdOx1/QIVr; 58 BNT162b2/QIVr). Overall, 340 participants were randomised to concomitant administration of influenza and COVID-19 vaccine and 339 were randomised to placebo and COVID-19 vaccine. Non-inferiority was indicated in four cohorts; ChAdOx1/QIVc: risk difference (influenza vaccine minus placebo) -1·29% (95% confidence interval (CI) ‑14·7%, 12·1%); BNT162b2/QIVc: 6·17% (‑6·27%, 18·6%); BNT162b2/aTIV: -12·9% (‑34·2%, 8·37%); ChAdOx1/QIVr: 2·53% (‑13·3%, 18·3%). In two cohorts the upper limit of the 95%CI exceeded 25%; ChAdOx1/aTIV: 10·3% (‑5·44%, 26·0%) and BNT162b2/QIVr: 6·75% (‑11·8%, 25·3%). Most reactions were mild or moderate. Rates of local and unsolicited systemic reactions were similar between randomised groups. One serious adverse event, hospitalisation with severe headache, was considered related to the trial intervention. Immune responses were not adversely affected. Interpretation: Concomitant vaccination raises no safety concerns and preserves the immune response to both vaccines.Clinical Trial Registration Details: The trial is registered (ISRCTN14391248)Funding Information: The trial is commissioned and funded by the Department of Health and Social Care (DHSC) through the National Institute for Health Research (NIHR). This research was supported by the Vaccine Task Force (VTF) and NIHR Policy Research Programme (PR-R17-0916-22001, NIHR203243).Declaration of Interest: RL reports grants from National Institute for Health Research during the conduct of the trial, and grants from Elizabeth Blackwell Institute, AstraZeneca, Janssen and Valneva outside the submitted work. CR reports grants from National Institute for Health Research, during the conduct of the trial. JSN-V-T reports he is seconded to the Department of Health and Social Care, England. AF reports grants from Pfizer during the conduct of the trial, and grants from Elizabeth Blackwell Institute, Gates Foundation, Sanofi Pasteur, VBI Vaccines, Pfizer, Janssen, GSK, MedImmune, Novavax and Valneva outside the submitted work. Between May 2015 and May 2019 AF was President of the European Society for Paediatric Infectious Diseases which, during this period, received sponsorship from GSK for its annual congress. He currently serves as chief investigator on the Valneva (Covid-19) vaccine phase 1/2 and 2/3 studies .He also serves as co-investigator on the Janssen (Covid-19) vaccine 2 dose phase 3 study. He does advisory work related to vaccines for the UK government, the World Health Organisation and several companies developing vaccines. He also leads clinical trials of vaccines funded by the UK government, charities and vaccine manufacturers. He receives no personal remuneration or benefits in kind for any of this work apart from his salary via the University of Bristol from the Higher Education Funding Council and the NHS. He is a member of the UK Department of Health’s Joint Committee on Vaccination, Chair of the WHO European Technical Advisory Group of Experts in which capacity he attends SAGE. AM reports grants from National Institute for Health Research during the conduct of the trial, and grants from AstraZeneca, Janssen and Valneva outside the submitted work. MDS acts on behalf of the University of Oxford as an investigator on studies funded or sponsored by vaccine manufacturers, including AstraZeneca, GlaxoSmithKline, Pfizer, Novavax, Pfizer, Janssen, Medimmune and MCM. The views in this paper are those of its authors and not necessarily those of the DHSC.Ethical Approval Statement: Approvals were received from the Medicines and Healthcare products Regulatory Agency (MHRA) (EudraCT number 2021-001124-18) and the South-Central Berkshire Research Ethics Committee (21/SC/0100).

SARS-CoV-2 lineage B.1.1.7 is associated with greater disease severity among hospitalised women but not men (preprint)

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.1.7 has been associated with an increased rate of transmission and disease severity among subjects testing positive in the community. Its impact on hospitalised patients is less well documented. Methods We collected viral sequences and clinical data of patients admitted with SARS-CoV-2 and hospital-onset COVID-19 infections (HOCIs), sampled 16/11/2020 - 10/01/2021, from eight hospitals participating in the COG-UK-HOCI study. Associations between the variant and the outcomes of all-cause mortality and intensive therapy unit (ITU) admission were evaluated using mixed effects Cox models adjusted by age, sex, comorbidities, care home residence, pregnancy and ethnicity. Results Sequences were obtained from 2341 inpatients (HOCI cases = 786) and analysis of clinical outcomes was carried out in 2147 inpatients with all data available. The hazard ratio (HR) for mortality of B.1.1.7 compared to other lineages was 1.01 (95% CI 0.79-1.28, P=0.94) and for ITU admission was 1.01 (95% CI 0.75-1.37, P=0.96). Analysis of sex-specific effects of B.1.1.7 identified increased risk of mortality (HR 1.30, 95% CI 0.95-1.78) and ITU admission (HR 1.82, 95% CI 1.15-2.90) in females infected with the variant but not males (mortality HR 0.82, 95% CI 0.61-1.10; ITU HR 0.74, 95% CI 0.52-1.04). Conclusions In common with smaller studies of patients hospitalised with SARS-CoV-2 we did not find an overall increase in mortality or ITU admission associated with B.1.1.7 compared to other lineages. However, women with B.1.1.7 may be at an increased risk of admission to intensive care and at modestly increased risk of mortality.

Remdesivir induced viral RNA and subgenomic RNA suppression, and evolution of viral variants in SARS-CoV-2 infected patients. (preprint)

While changes in SARS-CoV-2 viral load over time have been documented, detailed information on the impact of remdesivir and how it might alter intra-host viral evolution is limited. Sequential viral loads and deep sequencing of SARS-CoV-2 recovered from the upper respiratory tract of hospitalised children revealed that remdesivir treatment suppressed viral RNA levels in one patient but not in a second infected with an identical strain. Evidence of drug resistance to explain this difference was not found. Reduced levels of subgenomic (sg) RNA during treatment of the second patient, suggest an additional effect of remdesivir on viral replication that is independent of viral RNA levels. Haplotype reconstruction uncovered persistent SARS-CoV-2 variant genotypes in four patients. We conclude that these are likely to have arisen from within-host evolution, and not co-transmission, although superinfection cannot be excluded in one case. Sample-to-sample heterogeneity in the abundances of variant genotypes is best explained by the presence of discrete viral populations in the lung with incomplete population sampling in diagnostic swabs. Such compartmentalisation is well described in serious lung infections caused by influenza and Mycobacterium tuberculosis and has been associated with poor drug penetration, suboptimal treatment and drug resistance. Our data provide evidence that remdesivir is able to suppress SARS-CoV-2 replication in vivo but that its efficacy may be compromised by factors reducing penetration into the lung. Based on data from influenza and Mycobacterium tuberculosis lung infections we conclude that early use of remdesivir combined with other agents should now be evaluated. Summary SentenceDeep sequencing of longitudinal samples from SARS-CoV-2 infected paediatric patients identifies evidence of remdesivir-associated inhibition of viral replication in vivo and uncovers evidence of within host evolution of distinct viral genotypes.

SARS-CoV-2 genomes recovered by long amplicon tiling multiplex approach using nanopore sequencing and applicable to other sequencing platforms (preprint)

Genomic surveillance has become a useful tool for better understanding virus pathogenicity, origin and spread. Obtaining accurately assembled, complete viral genomes directly from clinical samples is still a challenging. Here, we describe three protocols using a unique primer set designed to recover long reads of SARS-CoV-2 directly from total RNA extracted from clinical samples. This protocol is useful, accessible and adaptable to laboratories with varying resources and access to distinct sequencing methods: Nanopore, Illumina and/or Sanger.