Intrahost evolution leading to distinct lineages in the upper and lower respiratory tracts during SARS-CoV-2 prolonged infection
Accumulating evidence points to persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in immunocompromised individuals as the source of genetically divergent novel lineages characterized by potentially heightened transmissibility and immune escape. While intrahost evolutionary dynamics of the virus in chronically infected patients have been documented, existing knowledge is primarily confined to samples obtained from the nasopharyngeal compartment. In this study, we investigate the intrahost evolution and genetic diversity that accumulated during a prolonged SARS-CoV-2 infection with the BF.7 sublineage, presumed to have persisted for over one year in an immunosuppressed patient. Intrahost diversity analysis, based on sequencing of all samples (n=8) at 6 time points, identified 86 intrahost single-nucleotide variants (iSNVs), 2 indels, and a 362 bp deletion, with allele frequencies ranging from 18 to 99% depending on sample origin. Our analysis of intrahost evolution revealed the early emergence of distinct viral genotypes observed in nasopharyngeal (NP), endotracheal aspirate (ETA), and bronchoalveolar (BAL) samples. Notably, while significant divergence was observed between NP and BAL samples, most of the iSNVs found in ETA samples were also detected in NP or BAL samples. This suggests that NP samples may not offer a comprehensive representation of the overall intrahost viral diversity. Enrichment for nonsynonymous mutations, particularly in the S and Env genes, was observed, along with loss-of-function mutations in ORF8, generated by a frameshift mutation and a large deletion detected in the BAL and NP samples, respectively. Using long-range PCR on routine samples, we validated that similar deletions causing ORF8 loss of function can be carried by SARS-CoV-2 during acute infection. Our findings not only demonstrate that the Omicron BF.7 lineage can further diverge from its already exceptionally mutated state but also highlight that patients chronically infected with SARS-CoV-2 can develop genetically specific viral populations across distinct anatomical compartments. This provides novel insights into the intricate nature of viral diversity and evolution dynamics in persistent infection.