Impact of prolonged infection on SARS-CoV-2 evolution

Immunocompromised patients with prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections may serve as reservoirs for viral evolution, with suboptimal immune responses facilitating the accumulation of adaptive mutations. This study aimed to characterize the drivers of SARS-CoV-2 adaptive evolution in such hosts through genomic surveillance. We retrospectively analyzed 24 patients with long-term positive nasopharyngeal reverse transcription-polymerase chain reaction results (symptom onset duration: 7-14 days on average, 1 HIV-positive patient with >20 days of infection). Most infections (April-May 2022) were caused by Omicron variants (predominantly BA.2). Phylogenetic analysis revealed accelerated viral evolution in patients with diverse underlying diseases (e.g., HIV and esophageal cancer). A total of 78 intrahost single-nucleotide variants were identified, with ORF1ab (53.8%) and the Spike protein coding region (20.5%) being hotspots. Notably, the HIV-positive patient's virus developed unique mutations: NSP3-T779I, NSP15-A94T, and Spike double mutations N440K and I794T. Functional assays showed that the N440K/I794T double mutation significantly enhanced infectivity in Hela-hACE2 cells (<i>P</i> < 0.05) but reduced immune evasion (50% neutralizing titer increased ~2-fold vs BA.2, <i>P</i> < 0.001). The I794T mutation was later detected in the JN.1.16 strain, suggesting potential evolutionary persistence. Prolonged SARS-CoV-2 replication in immunocompromised hosts, particularly HIV-positive individuals, drives adaptive mutations with altered infectivity and immune evasion. These findings emphasize the need for monitoring such hosts to prevent the spread of potentially transmissible variants.IMPORTANCEThis study reveals the characteristics of adaptive mutations and their biological functions of SARS-CoV-2 during long-term infection in immunocompromised patients, emphasizes the importance of close monitoring of the long-term replication of SARS-CoV-2, aiming to timely detect and prevent the spread of newly emerging neutralization-resistant variants in susceptible populations, and provides a key scientific basis for formulating effective public health prevention and control strategies.