Intra-Host Diversity of SARS-Cov-2 Should Not Be Neglected: Case of the State of Victoria, Australia

Abstract

Since the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the etiological agent of the current COVID-19 pandemic, a rapid and massive effort has been made to obtain the genomic sequences of this virus to monitor (in near real time) the phylodynamic and diversity of this new pathogen. However, less attention has been given to the assessment of intra-host diversity. RNA viruses such as SARS-CoV-2 inhabit the host as a population of variants called quasispecies. We studied the quasispecies diversity in four of the main SARS-CoV-2 genes (ORF1a, ORF1b, S and N genes), using a dataset consisting of 210 next-generation sequencing (NGS) samples collected between January and early April of 2020 in the State of Victoria, Australia. We found evidence of quasispecies diversity in 68% of the samples, 76% of which was nonsynonymous variants with a higher density in the spike (S) glycoprotein and ORF1a genes. About one-third of the nonsynonymous intra-host variants were shared among the samples, suggesting host-to-host transmission. Quasispecies diversity changed over time. Phylogenetic analysis showed that some of the intra-host single-nucleotide variants (iSNVs) were restricted to specific lineages, highlighting their potential importance in the epidemiology of this virus. A greater effort must be made to determine the magnitude of the genetic bottleneck during transmission and the epidemiological and/or evolutionary factors that may play a role in the changes in the diversity of quasispecies over time.

Keywords: SARS-CoV-2; genetic bottleneck; nonsynonymous iSNVs; phylogenetic; quasispecies; transmission.

Figure 1

Temporal patterns of the diversity of nonsynonymous intra-host single-nucleotide variants (iSNVs) in four SARS-CoV-2 genes. Each fish plot represents the proportion of a specific nonsynonymous iSNV in the time interval covered by the samples. The dates indicate the days of sample collection.

Figure 2

Genomic distribution of nonsynonymous iSNVs of two temporal groups in the S gene. Each dot represents the occurrence and frequency of a specific variant from Early and Late groups. The majority of iSNVs of the Early group are concentrated in a small genomic region starting at position 21,650; they have low intra-host frequencies but are shared by a significant number of samples. The variants of the “Late” group are observed at three positions, one of them localized in the receptor-binding domain (RBD); these mutations have higher frequencies but are shared by a small number of samples. The bottom of the figure shows a schematic representation of the S gene and its main domains: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat (HR).

Figure 3

Phylogenetic tree of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic sequences collected in the State of Victoria, Australia. The clusters are colored according to Pangolin annotation. The black tips represent the consensus sequences derived from the next-generation sequencing (NGS) data.