SARS-CoV-2 variants in the making: Sequential intrahost evolution and forward transmissions in the context of persistent infections

Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been reported in immune-compromised individuals and people undergoing immune-modulatory treatments. Although intrahost evolution has been documented, to our knowledge, no direct evidence of subsequent transmission and stepwise adaptation is available. Here we describe sequential persistent SARS-CoV-2 infections in three individuals that led to the emergence, forward transmission, and continued evolution of a new Omicron sublineage, BA.1.23, over an eight-month period. The initially transmitted BA.1.23 variant encoded seven additional amino acid substitutions within the spike protein (E96D, R346T, L455W, K458M, A484V, H681R, A688V), and displayed substantial resistance to neutralization by sera from boosted and/or Omicron BA.1-infected study participants. Subsequent continued BA.1.23 replication resulted in additional substitutions in the spike protein (S254F, N448S, F456L, M458K, F981L, S982L) as well as in five other virus proteins. Our findings demonstrate that the Omicron BA.1 lineage can diverge further from its already exceptionally mutated genome during persistent infection in more than one host, and also document ongoing transmission of these novel variants. There is an urgent need to implement strategies to prevent prolonged SARS-CoV-2 replication and to limit the spread of newly emerging, neutralization-resistant variants in vulnerable patients. ### Competing Interest Statement The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays (U.S. Provisional Application Numbers: 62/994,252, 63/018,457, 63/020,503 and 63/024,436) and NDV-based SARS-CoV-2 vaccines (U.S. Provisional Application Number: 63/251,020) which list Florian Krammer as co-inventor. Viviana Simon is also listed on the serological assay patent application as co-inventor. Patent applications were submitted by the Icahn School of Medicine at Mount Sinai. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. Florian Krammer has consulted for Merck and Pfizer (before 2020), and is currently consulting for Pfizer, Third Rock Ventures, Seqirus and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. Robert Sebra is VP of Technology Development and a stockholder at Sema4, a Mount Sinai Venture. This work, however, was conducted solely at Icahn School of Medicine at Mount Sinai. ### Funding Statement The work reported was, in part, supported by a contract from the National Institute of Allergy and Infectious Diseases (75N93021C00014, Option 12A) awarded to the Center for Research on Influenza Pathogenesis and Transmission and by an Option to the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 as part of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) Program, a contract from the National Institute of Allergy and Infectious Diseases (HHSN272201400008C, Option 20) awarded to the Center for Research on Influenza Pathogenesis, and awards (S10OD026880 and S10OD030463) from the NIH Office of Research Infrastructure Programs. The Mount Sinai Pathogen Surveillance Program is supported in part by Institutional funds from the Icahn School of Medicine as well as the Mount Sinai Hospital. ### 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 protocols were reviewed and approved by the Mount Sinai Hospital Institutional Review Board for the collection and viral genome sequencing of residual diagnostic specimens by the Pathogen Surveillance Program (protocol HS# 13-00981). The detailed investigation into persistent SARS-CoV-2 infections in patients receiving care at the Mount Sinai Health System was separately reviewed and approved by the MSH IRB (IRB-22-00760). In addition, patient P2 participated in another of our observational study (IRB-16-01215). 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 work are contained in the manuscript or available upon reasonable request to the authors