Intra-host variation and evolutionary dynamics of SARS-CoV-2 populations in COVID-19 patients

Abstract

Background: Since early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown.

Methods: Using high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT).

Results: The 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks.

Conclusions: Our findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.

Keywords: COVID-19; Dynamics; Intra-host; SARS-CoV-2; Variation.

Fig. 1

Sequence data from various sample types of patients with COVID-19. a SARS-CoV-2 RPM of metatranscriptomic data plotted against RT-qPCR cycle threshold (Ct) value for the clinical samples. b Frequency distribution of samples based on SARS-CoV-2 reads per million (SARS-CoV-2 RPM). c Maximum likelihood tree of consensus SARS-CoV-2 genomes using IQ-TREE. Colors of dotted tips represent the geographic locations of samples. Nucleotide mutations that define the branch were labeled outside the tree. d Distribution of consensus variants (in round circles) detected in the GZMU cohort across the SARS-CoV-2 genome. Colors represent the biological effect of mutations. Non-synonymous variants are denoted by green, synonymous variants by red, and frameshift by blue. EPI_ISL_402125 was used as the reference sequence

Fig. 2

Characteristics of iSNVs. a Heatmap showing the alternative allele frequencies (AAFs) of intra-host single nucleotide variants (iSNVs) and consensus variants among samples. The sample (e.g., P01N0129) name indicates patient number P01, sample type (N nasal swab, T throat swab, A anal swab, F feces, S sputum) and collection date (January 29). Common iSNVs were marked by star symbols. Variant type and sample type were marked in different colors, and consensus variants were indicated in red. b The number of detected iSNVs per patient. c Number of iSNV sites among protein-encoding genes. d Box plot showing the distribution of alternative allele frequencies (AAFs) of non-synonymous and synonymous iSNVs. Each dot indicates the median AAF among all the detected iSNVs of samples from the same patient. e Box plot showing the distribution of AAFs of common and rare iSNVs. Each dot indicates the median AAF among all the detected iSNVs of samples from the same patient

Fig. 3

Genetic diversity and genetic distance. a Box plot showing the distribution of genetic diversity among samples from the gastrointestinal tract (GIT) and respiratory tract (RT). For the patients with more than one GIT/RT samples, only the median value was selected to represent the genetic diversity in GIT/RT. b Box plot showing the distribution of L1-norm distances among samples from the gastrointestinal tract (GIT) and respiratory tract (RT). Each dot represents the genetic distance between a unique pair

Fig. 4

Temporal dynamics of intra-host populations in patients P01 and P08. a, b Alternative allele frequencies (AAFs) among sampling dates in patients P01 and P08. Days post the first symptom date are shown in the bracket. The sample (e.g., P01A0129) name indicates patient number P01, sample type (N nasal swab, T throat swab, A anal swab, F feces, S sputum), and collection date (January 29, 2020). Combined iSNVs are the average frequency of four similar iSNVs (A391T, A2275G, C25163A, and T27817G). Colors represent different iSNVs. Underlines represent common iSNVs