Genomic investigations of acute hepatitis of unknown aetiology in children

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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The work was funded by UKHSA, the National Institute for Health Research and Wellcome Trust. S.M is funded by a W.T. Henry Wellcome fellowship (206478/Z/17/Z). J.B receives fundingfrom the National Institute of Health Research UCL/UCLH Biomedical Research Centre DIAMONDS is funded by the European Union (Horizon 2020 grant 848196). PERFORM was funded by the European Union (Horizon 2020 grant 668303) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: National Health Service Research Ethics Service Committee London Fulham, Research Ethics Committee reference 17/LO/1530, gave ethical approval for this work. International Severe Acute Respiratory and Emerging Infections Consortium World Health Organisation Clinical Characterisation Protocol United Kingdom gave ethical approval for this work (RQ3001-0591 RQ301-0594 RQ301-0596 RQ301-0597 RQ301-0598) London Dulwich Research Ethics Committee 20/HRA/1714 and London Central Research Ethics Committee 16/LO/1684 gave ethical approval for this work. Metagenomic analysis and adenovirus sequencing were carried out by the routine diagnostic service at Great Ormond Street Hospital. Additional PCRs, Immunohistochemistry and proteomics on samples received for metagenomics are part of the Great Ormond Street Hospital (GOSH) protocol for confirmation of new and unexpected pathogens. The use for research of anonymised laboratory request data, diagnostic results and residual material from any specimen received in the GOSH diagnostic laboratory, including all cases received from Birmingham's Children Hospital UKHSA, Public Health Wales, Public health Scotland as well as non-case samples from UKHSA, Public Health Scotland and Great Ormond Street Hospital research was approved by UCL Partners Pathogen Biobank under ethical approval granted by the NRES Committee London-Fulham (REC reference: 17/LO/1530). Children undergoing liver transplant were consented for additional research under the International Severe Acute Respiratory and Emerging Infections Con Ethics sortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) [[ISRCTN 66726260][1]] (RQ3001-0591, RQ301-0594, RQ301-0596, RQ301-0597, RQ301-0598). Ethical approval for the ISARIC CCP-UK study was given by the South Central-Oxford C Research Ethics Committee in England (13/SC/0149), the Scotland A Research Ethics Committee (20/SS/0028), and the WHO Ethics Review Committee (RPC571 and RPC572). The United Kingdom Health Security Agency (UKHSA) has legal permission, provided by Regulation 3 of The Health Service (Control of Patient Information) Regulations 2002, to process patient confidential information for national surveillance of communicable diseases and as such, individual patient consent is not required. Control patients for DIAMONDS/PERFORM were recruited according to the approved enrolment procedures of each study, and with the informed consent of parents or guardians: DIAMONDS (London - Dulwich Research Ethics Committee: 20/HRA/1714); PERFORM (London - Central Research Ethics Committee: 16/LO/1684). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: /external-ref?link_type=ISRCTN&access_num=ISRCTN66726260