Intra-host evolution during SARS-CoV-2 prolonged infection

Long-term infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a challenge to virus dispersion and the control of coronavirus disease 2019 (COVID-19) pandemic. The reason why some people have prolonged infection and how the virus persists for so long are still not fully understood. Recent studies suggested that the accumulation of intra-host single nucleotide variants (iSNVs) over the course of the infection might play an important role in persistence as well as emergence of mutations of concern. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during prolonged infection. Thirty-three patients who remained reverse transcription polymerase chain reaction (RT-PCR) positive in the nasopharynx for on average 18 days from the symptoms onset were included in this study. Whole-genome sequences were obtained for each patient at two different time points. Phylogenetic, populational, and computational analyses of viral sequences were consistent with prolonged infection without evidence of coinfection in our cohort. We observed an elevated within-host genomic diversity at the second time point samples positively correlated with cycle threshold (Ct) values (lower viral load). Direct transmission was also confirmed in a small cluster of healthcare professionals that shared the same workplace by the presence of common iSNVs. A differential accumulation of missense variants between the time points was detected targeting crucial structural and non-structural proteins such as Spike and helicase. Interestingly, longitudinal acquisition of iSNVs in Spike protein coincided in many cases with SARS-CoV-2 reactive and predicted T cell epitopes. We observed a distinguishing pattern of mutations over the course of the infection mainly driven by increasing A→U and decreasing G→A signatures. G→A mutations may be associated with RNA-editing enzyme activities; therefore, the mutational profiles observed in our analysis were suggestive of innate immune mechanisms of the host cell defense. Therefore, we unveiled a dynamic and complex landscape of host and pathogen interaction during prolonged infection of SARS-CoV-2, suggesting that the host's innate immunity shapes the increase of intra-host diversity. Our findings may also shed light on possible mechanisms underlying the emergence and spread of new variants resistant to the host immune response as recently observed in COVID-19 pandemic.