Background
Persistent infection with SARS-CoV-2 is hypothesised to give rise to new variants of concern (VoCs). However, sequencing methods to investigate intrahost variation are limited: short-read technology precludes haplotyping, and long-read technology has high error rates. We describe a new method for investigating SARS-CoV-2 quasispecies and apply it to identify divergent spike haplotypes arising during persistent infection.
Methods
A long-read nanopore sequencing workflow was developed to generate high-quality reads (99.9% accuracy) covering the entire spike gene sequence, along with a bioinformatic tool (HaploVar) to identify and quantify unique spike haplotypes at ≥1% frequency in samples. Respiratory samples were processed from patients with acute and persistent infection lasting longer than 4 weeks. Molecular-clock and divergence phylogenies were constructed using TreeTime v0.11.3 and IQTree v2.3.0, then displayed in ete v3.1.3. Haplotypes were screened for recombination using GARD v0.2.
Results
Sixteen patients with persistent infection were identified, ranging from 31-505 days and with a median of 7 longitudinal samples per patient. Infecting variants spanned the pandemic from B.1 until JN.1. Eighty-one samples from persistent infection and 75 samples from acute infection were successfully processed. Number of spike haplotypes identified in each sample was related to days post onset of symptoms (p<0.001) and being a persistent infection (p<0.001), but not viral load (Ct value) nor sequencing data yielded. During acute infection the median number of spike haplotypes was 1 (IQR 1-2). In persistent infection after 28 days the median number of spike haplotypes was 3 (IQR 2-7), compared to 7 (IQR 2-11) after 100 days. Many mutations were identical or at the same position to those seen in subsequent VoCs, including L18K and E484Q (Beta); K417T (Gamma); T19S, L452R (Delta); A570T (Alpha); H655Y (Omicron); G446S, Q954K (BA.1); S477N, D796H (BA.2); N460K (BA.2.75); Q493E (KP.3); and Y453F (Mink). Individuals treated with sotrovimab showed evidence of quasispecies with resistance mutations. Putative intra-spike recombination between haplotypes was identified in 3 persistent infections.
Conclusions
This novel workflow identifies intrahost spike haplotypes, and provides strong evidence for the emergence of variants of concern from persistent infection. We provide the first evidence of intrahost recombination from within a single individual as a mechanism of generating diversity of SARS-CoV-2.