Comprehensive profiling of the human intestinal DNA virome and prediction of disease-associated bacterial hosts in severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling disorder of unknown etiology with severely affected patients being house- and/or bedbound. A historical association with chronic virus infection and subsequent recent reports correlating intestinal microbial dysbiosis with disease pathology prompted us to analyze the intestinal virome in a small cohort of severely-affected ME/CFS patients and same household healthy controls (SHHC). Datasets from whole metagenomic sequencing (WMS) and sequencing of virus-like particles (VLP)-enriched metagenomes from the same fecal sample yielded diverse, high-quality vOTUs with high read coverage and high genome completeness. The core intestinal virome was largely composed of tailed phages in the class Caudoviricetes with no significant differences in alpha diversity between ME/CFS and SHHC groups. However, the WMS dataset had a higher Shannon measure than the VLP dataset ( p < 0.0001), with VLP- and WMS-derived sequences indicating differential abundances within several viral families and different viral compositions in beta diversity. This confirms that combining different isolation methodologies identifies a greater diversity of viruses including extracellular phages and integrated prophages. DNA viromes and bacteriomes from ME/CFS and SHHC groups were comparable with no differences in any alpha or beta diversity measures. One vOTU derived from the VLP-derived dataset was assigned to ssDNA human virus smacovirus 1. Using an in-silico approach to predict cohort-based bacterial hosts, we identified members of the Anaerotruncus genus interacting with unique viruses present in ME/CFS microbiomes; this may contribute to the GI microbial dysbiosis described in ME/CFS patients. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The Biotechnology and Biological Sciences Research Council (BBSRC); this research was supported by the BBSRC Institute Strategic Programme Grant Gut Microbes and Health BB/R012490/1 and its constituent projects BBS/E/F/000PR10353, BBS/E/F/000PR10355 and BBS/E/F/000PR10356 (EMA, SRC). GMS was funded by the BBSRC Core Capability Grant BB/CCG1860/1. KAS and FN were supported by PhD studentships jointly funded by Invest in ME Research (UK Charity number 1153730) and the University of East Anglia. FN was funded by a Ramsey Award from SOLVE M.E. ### 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: Ethical approval was obtained from the University of East Anglia Faculty of Medicine and Health Sciences Research Ethics Committee in 2014 (reference FMH20142015.28) and the Health Research Authority NRES Committee London Hampstead (reference 17/LO/1102; IRAS project ID: 218545). 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The sequences derived from the VLP-enriched metagenomes have been deposited to ENA () under the study accession no. PRJEB57952 and the sequences from the whole metagenomes (WMS) have also been deposited to ENA under the project accession no. PRJEB57953.