Genetic diversity and evolutionary patterns of SARS-CoV-2 among the Bhutanese population during the pandemic

Abstract

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by a dynamic virus, has had a profound global impact. Despite declining global COVID-19 cases and mortality rates, the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains a major concern. This study provides a comprehensive analysis of the genomic sequences of SARS-CoV-2 within the Bhutanese population during the pandemic. The primary aim was to elucidate the molecular epidemiology and evolutionary patterns of SARS-CoV-2 in Bhutan, with a particular focus on genetic variations and lineage dynamics.

Methods: Whole-genome sequences of SARS-CoV-2 collected from Bhutan between May 2020 and February 2023 (n=135) were retrieved from the Global Initiative on Sharing All Influenza Database.

Results: The SARS-CoV-2 variants in Bhutan were predominantly classified within the Nextstrain clade 20A (31.1%), followed by clade 21L (20%) and clade 22D (15.6%). We identified 26 Pangolin lineages with variations in their spatial and temporal distribution. Bayesian time-scaled phylogenetic analysis estimated the time to the most recent common ancestor as February 15, 2020, with a substitution rate of 0.97×10-3 substitutions per site per year. Notably, the spike glycoprotein displayed the highest mutation frequency among major viral proteins, with 116 distinct mutations, including D614G. The Bhutanese isolates also featured mutations such as E484K, K417N, and S477N in the spike protein, which have implications for altered viral properties.

Conclusion: This is the first study to describe the genetic diversity of SARS-CoV-2 circulating in Bhutan during the pandemic, and this data can inform public health policies and strategies for preventing future outbreaks in Bhutan.

Keywords: Coronavirus spike glycoprotein; Molecular epidemiology; Mutation; SARS-CoV-2.

Figure 1.

Temporal variations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral strains. The figure shows the distribution of Pangolin lineages among SARS-CoV-2 genomes from samples collected between May 2020 and February 2023 in Bhutan.

Figure 2.

Maximum likelihood phylogenetic tree of severe acute respiratory syndrome coronavirus 2 genomes from Bhutan. The maximum likelihood tree was inferred using RAxML8. The tree is rooted with the reference sequence NC_045512.2. Bootstrap values more than 70 are indicated at major nodes in the fully resolved tree. The scale bar at the top of the tree indicates the number of nucleotide substitutions per site.

Figure 3.

Distribution of severe acute respiratory syndrome coronavirus 2 clades in Bhutan. Map of Bhutan showing the geographical distribution of Nextstrain clades.

Figure 4.

Maximum clade credibility (MCC) tree of Bhutanese severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. Estimated MCC tree of SARS-CoV-2 genomes using a Hasegawa-Kishino-Yano substitution model and a strict molecular clock with uniform rates across branches. Nodes are labeled with posterior probability values and the 95% highest posterior density of the node heights.

Figure 5.

Frequency of single-nucleotide variations (SNVs). The figure shows the frequency of SNVs in Bhutanese severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates. The locations and frequencies of the nucleotide variations were plotted along genomic sequence of NC_045512.2. The open reading frames (ORFs) of SARS-CoV-2 were shown as rectangles that were aligned with nucleotide positions of SARS-CoV-2. Frequency (n) is the number of samples/sequences with nucleotide variation at the nucleotide position. NSP, non-structural protein.

Figure 6.

Frequency of mutations in various proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The figure shows the frequency of mutations in major SARS-CoV-2 proteins. (A) ORF1ab; NSP1, NSP2, NSP4, NSP5, NSP8, NSP9, NSP10, NSP13, NSP14, NSP15, and NSP16. (B) Structural and accessory proteins; M, E, N, NS3, NS6, NS7a, and NS8. (C) Spike protein. ORF, open reading frame; NSP, non-structural protein.