Enteric adenovirus F41 genetic diversity comparable to pre-COVID-19 era: validation of a multiplex amplicon-MinION sequencing method

Abstract

Human adenovirus F41 causes acute gastroenteritis in children, and has recently been associated with an apparent increase in paediatric hepatitis of unknown aetiology in the UK, with further cases reported in multiple countries. Relatively little is known about the genetic diversity of adenovirus F41 in otherwise-healthy individuals; and it is unclear what, if any, impact the COVID-19 pandemic has had on viral diversity in the UK. Methods that allow F41 to be sequenced from clinical samples without the need for viral culture are required to provide the genomic data to address these questions.We therefore evaluated an overlapping-amplicon method of sequencing adenovirus genomes from clinical samples using Oxford Nanopore technology. We applied this method to a small sample of adenovirus species F-positive extracts collected as part of standard care in the East of England region in January-May 2022. This method produced genomes with >75% coverage in 13/22 samples and >50% coverage in 19/22 samples.We identified two F41 lineages present in paediatric patients in East of England in 2022. Where F41 genomes from paediatric hepatitis cases were available (n=2), these genomes fell within the diversity of F41 from the UK and continental Europe sequenced before and after the 2020-21 phase of the COVID-19 pandemic.Our analyses suggest that overlapping amplicon sequencing is an appropriate method for generating F41 genomic data from high virus load clinical samples, and currently circulating F41 viral lineages were present in the UK and Europe before the COVID-19 pandemic.

Detection of human adenovirus F41 in wastewater and its relationship to clinical cases of acute hepatitis of unknown aetiology

“…To delineate between HAdV-F40 and HAdV-F41 within the wastewater, five positive samples from May 2022 were selected for overlappingamplicon whole-genome sequencing using the recently developed HAdV-F41 ARTIC primers (Maes et al, 2022); https://github.com/quick-lab/ HAdV) following the Mini-XT SARS-CoV-2 protocol (Fuchs et al, 2022). Sequencing was performed with a MiSeq reagent kit v2 (500 cycles).…”

Multi-species co-circulation of adenoviruses identified by next generation sequencing during an outbreak in coastal Kenya in 2023

“…This is likely due to the relaSvely short ~300 base pair hexon region that is amplified and the highly recombinatorial nature of species D HAdVs within the hexon locus 21 . Within HAdV F, there is a high divergence in the strains that have been reported to be circulaSng in Kenya and other regions across the globe 17,[22][23][24] . If this divergence occurs in primer and probe binding sites, nucleic acid amplificaSon methods used for detecSon may be affected.…”

“…Within HAdV F, there is a high divergence in the strains that have been reported to be circulating in Kenya and other regions across the globe 17,22–24 . If this divergence occurs in primer and probe binding sites, nucleic acid amplification methods used for detection may be affected.…”

‘Vivaldi’: An amplicon-based whole genome sequencing method for the four seasonal human coronaviruses 229E, NL63, OC43 & HKU1, alongside SARS-CoV-2’

“…To reproducibly detect whole genomes from seasonal coronavirus strains (45-47) in unenriched clinical samples, demanded not only an amplicon approach, but also a fine balance between minimising priming sites and maximising the sensitivity of detecting smaller RNA fragments. We thus opted for a 1200bp amplicon scheme as used previously for SARS-CoV-2 (20), rather than the commonly adopted 400bp amplicon scheme (19,21), larger amplicon schemes (18,23,24) or an unbiased metagenomic approach (28,48). Initial attempts on even a small batch of clinical samples can quickly identify amplicon drop-out, whilst careful continued evaluation is required to guard against evolving dropout such as seen previously in SARS-CoV-2 (25), and similarly seen in our cohort in postpandemic 229E infections.…”

“…Amplicon-based Whole Genome Sequencing (WGS), utilising contiguous overlapping PCR products of various size, has previously been described for a wide range of viruses (18)(19)(20)(21)(22)(23)(24), yielding both population-level epidemiological insight and outbreak management with near-realtime capability (19). There is a trade off in the priming strategy between the increased sensitivity of smaller amplicon tiling versus the heightened chance of primer mismatch where greater number of primers are used.…”