In a recent study posted to the medRxiv* preprint server, researchers investigated the intrahost evolution and genetic diversity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.517 variant in an immunosuppressed person with chronic coronavirus disease 2019 (COVID-19).

Study: Accelerated SARS-CoV-2 intrahost evolution leading to distinct genotypes during chronic infection. Image Credit: Ninc Vienna/Shutterstock
Study: Accelerated SARS-CoV-2 intrahost evolution leading to distinct genotypes during chronic infection. Image Credit: Ninc Vienna/Shutterstock


Intrahost SARS-CoV-2 replication could cause faster SARS-CoV-2 evolution than interhost SARS-CoV-2 replication since SARS-CoV-2 abundance in the host would be subject to lesser genetic hindrances, and this could increase SARS-CoV-2 recombination chances. Studies have reported on viral presence in chronically ill community residents; however, detailed genetic analyses exploring intrahost SARS-CoV-2 evolution dynamics among persons with chronic SARS-CoV-2 infections are lacking.

About the study

In the present study, researchers explored the intrahost B.1.517 evolution and genomic diversity during a chronic SARS-CoV-2 infection lasting 471 days (and counting) with high viral loads to investigate if chronic SARS-CoV-2 infections could drive SARS-CoV-2 variant emergence.

The team detected the B.1.517 variant in Connecticut till March 2022 via a dataset of SARS-CoV-2 genomic surveillance. B.1.517 genetic sequences were traced to an immunosuppressed person with chronic COVID-19 for >1 year, from whom 30 nasopharyngeal swabs were obtained to sequence the SARS-CoV-2 genome. Whole-genome sequencing (WGS) was used for sequencing between day 79 and day 471 to assess SARS-CoV-2 infectivity, and 12 swabs were also subjected to in vitro testing for viable SARS-CoV-2.

SARS-CoV-2 ribonucleic acid (RNA) titers were measured using reverse transcription-polymerase chain reaction (RT-PCR), and the intrahost genomic diversities, recombination, and the spectrum and frequency of mutations were characterized. Phylogenetic analyses were performed to explore SARS-CoV-2 genetic diversification in chronic infections and to assess the intrahost SARS-CoV-2 evolution. To evaluate the increase in intrahost SARS-CoV-2 genetic diversity with time, deep RNA sequencing was performed, and intrahost single nucleotide variants (iSNVs) frequencies were quantified and validated by SARS-CoV-2 spike (S) gene sequencing using unique molecular identifiers (UMIs).

The individual was in his 60s and suffered from B-lymphocyte lymphoma, and had received a stem cell transplant in 2019. The disease relapsed in early 2020 and November 2022, after which chemotherapy and palliation radiotherapy were initiated, respectively. The individual’s immunoglobulin G (IgG) titers were close to or within the normal ranges during intravenous Ig (IVIG) infusion therapy till day 205, after which they declined. The IgA titers and T lymphocyte counts were low prior to and post-infection, reflective of the immunocompromised status. In the later asymptomatic stage, only bamlanivimab infusion was administered to the patient on day 90.


In the RT-PCR analysis, swab samples obtained between day 79 and day 471 post-COVID-19 diagnosis had an average cycle threshold (Ct) value of 25.5 reflective of ~ 3.1 × 108 SARS-CoV-2 copies in every mL; however, SARS-CoV-2 copies reduced with time. Among the 12 samples tested for viable SARS-CoV-2 presence, the virus was detected at 10 time points of sampling between day 79 and day 401 except on day 394 and day 471, that corresponded with greater Ct values of 34 and 31, respectively.

During the chronic SARS-CoV-2 infection, an accelerated rate of SARS-CoV-2 intrahost evolution was observed (35.6 mutations each year or 1.2 ×10-3 nt mutations for each site for each year (s/s/y), nearly double the global SARS-CoV-2 evolutionary rate (5.8 × 10-04 s/s/y). The intrahost evolution gave rise to >3 genetically distinct genotypes.

The reseeded genotypes could be considered new variants if transmitted to the community. The first genotype had 24 nucleotide (nt) mutations [13 mutations of amino acids (aa)] till day 379. The second genotype had 40 nt mutations (28 aa mutations) between day 281 and day 471. The third genotype diverged from the first genotype into two sub-genotypes between day 394 and day 401. Sub-genotype 1 had 37 nt mutations (30 aa mutations) and sub-genotype 2 had 29 nt substitutions (27 aa mutations).

Despite fluctuations, the iSNV frequencies obtained on WGS analysis largely correlated with those of the UMI-sequenced S gene. The iSNVs increased with time among the genotypes identified with average iSNVs numbers of 32.1. The second genotype had more iSNVs than the first one, and the number of iSNVs positively correlated with the sampling periods.

The mean iSNV persistence from different SARS-CoV-2 genes was comparable during the infection course, irrespective of their occurrence frequency. The three most commonly observed iSNVs (in 88% samples) were S: Q493K, S: T1027I, and ORF1ab: L2144P. Further, three S gene iSNVs were detected related to bamlanivimab resistance viz. Q493R, L452R, and E484K.

The estimated effective SARS-CoV-2 population size (Ne) reflected iSNVs numbers, particularly in the initial stage of infection. The estimated SARS-CoV-2 accrual rate was 37.5 iSNVs each year, in accordance with SARS-CoV-2 evolutionary rates estimated based on the nt mutations. Most mutations were detected at the first codon (22% non-synonymous alterations) and second codon (38% non-synonymous alterations) positions.

Non-synonymous changes were more abundant than synonymous changes in SARS-CoV-2 S (88%), nucleocapsid (66%), and envelope (100%) structural genes. Likewise, non-synonymous aa changes were more abundant in nonstructural genes such as the open reading frame 10 (ORF10, 100%) and ORF1ab (58%).

To conclude, based on the study findings, chronic infections could accelerate SARS-CoV-2 evolution and thereby give rise to genetically divergent and potentially more transmissible and immune-evasive SARS-CoV-2 variants.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.


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