CpG dinucleotides are underrepresented in the genomes of most RNA viruses. Synonymously increasing CpG content of a range of RNA virus genomes reliably causes replication defects due to the recognition of CpG motifs in RNA by cellular zinc-finger antiviral protein (ZAP). Prior to the discovery of ZAP as a CpG sensor, we described an engineered influenza A virus (IAV) enriched for CpGs in segment 5 that displays the expected replication defects. However, we report here that this CpG-high ("CpGH") mutant is not attenuated by ZAP. Instead, a pair of compensatory nucleotide changes, resulting in a stretch of eight consecutive adenosines (8A), were found to be responsible. Viral polymerase slippage occurs at this site, resulting in the production of aberrant peptides and type I interferon induction. When the nucleotides in either one of these two positions were restored to wild-type sequence, no viral attenuation was seen, despite the 86 extra CpGs encoded by this virus. Introduction of these two adenosines into wild-type virus (thereby introducing the 8A tract) resulted in viral attenuation, polymerase slippage, aberrant peptide production and type I interferon induction. That a single nucleotide change can offset the growth defects in a virus designed to have a formidable barrier to wild-type reversion highlights the importance of understanding the processes underlying viral attenuation. Poly(A) tracts are a correlate for the emergence of polybasic cleavage sites in avian IAV hemagglutinins to produce highly pathogenic strains. These results thereby uncover possible insights into the intermediary events of this important evolutionary process.

Hybridization capture approaches can significantly increase the sensitivity of metagenomics. However, their implementation with Oxford Nanopore Technologies (ONT) sequencing has been limited due to the lack of dedicated workflows and the need for an extra ligation-based library preparation step. We developed a universal four-primer PCR approach that allows the implementation of hybridization capture workflows with ONT without additional ligation-based steps. Our method allowed the fast conversion of a targeted metagenomics workflow, increasing ONT sensitivity by 10 to 100-fold compared to untargeted approaches and providing both rapid detection and whole-genome sequences where the pathogen was abundant and identification where viral loads were low. ### Competing Interest Statement The Conversion method described in this study is patent pending (GB2505119.4) ### Funding Statement SB, SM and OETM are funded by the NIHR Blood and Transplant Research Unit in Genomics to Enhance Microbiology Screening (NIHR203338). LF is funded by UCL Genomics. JB receives funding from an NIHR senior investigator award (NIHR203728) and a personal award from the NIHR UCLH Biomedical Research Centre. TG is supported by an Investigator Grant (GNT2025445) from the National Health and Medical Research Council, Australia (NHMRC). Part of this work was supported by the NIHR GOSH Biomedical Research Centre (Award 23BM06). All research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. ### 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: The study used only commercially available human genetic material. The products used were Human genomic DNA - G1471 | Promega https://www.promega.co.uk/products/biochemicals-and-labware/nucleic-acids/human-genomic-dna/?catNum=G1521 and Human Brain Total RNA - AM7962 | Invitrogen https://www.thermofisher.com/order/catalog/product/AM7962 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 raw data produced in this study is available in the European Nucleotide Archive (ENA) with accession PRJEB87418 (data will be made available after publication).

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.