Genome sequence diversity of SARS-CoV-2 obtained from clinical samples in Uzbekistan

Abstract

Tracking temporal and spatial genomic changes and evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are among the most urgent research topics worldwide, which help to elucidate the coronavirus disease 2019 (COVID-19) pathogenesis and the effect of deleterious variants. Our current study concentrates genetic diversity of SARS-CoV-2 variants in Uzbekistan and their associations with COVID-19 severity. Thirty-nine whole genome sequences (WGS) of SARS-CoV-2 isolated from PCR-positive patients from Tashkent, Uzbekistan for the period of July-August 2021, were generated and further subjected to further genomic analysis. Genome-wide annotations of clinical isolates from our study have revealed a total of 223 nucleotide-level variations including SNPs and 34 deletions at different positions throughout the entire genome of SARS-CoV-2. These changes included two novel mutations at the Nonstructural protein (Nsp) 13: A85P and Nsp12: Y479N, which were unreported previously. There were two groups of co-occurred substitution patterns: the missense mutations in the Spike (S): D614G, Open Reading Frame (ORF) 1b: P314L, Nsp3: F924, 5`UTR:C241T; Nsp3:P2046L and Nsp3:P2287S, and the synonymous mutations in the Nsp4:D2907 (C8986T), Nsp6:T3646A and Nsp14:A1918V regions, respectively. The "Nextstrain" clustered the largest number of SARS-CoV-2 strains into the Delta clade (n = 32; 82%), followed by two Alpha-originated (n = 4; 10,3%) and 20A (n = 3; 7,7%) clades. Geographically the Delta clade sample sequences were grouped into several clusters with the SARS-CoV genotypes from Russia, Denmark, USA, Egypt and Bangladesh. Phylogenetically, the Delta isolates in our study belong to the two main subclades 21A (56%) and 21J (44%). We found that females were more affected by 21A, whereas males by 21J variant (χ2 = 4.57; p ≤ 0.05, n = 32). The amino acid substitution ORF7a:P45L in the Delta isolates found to be significantly associated with disease severity. In conclusion, this study evidenced that Identified novel substitutions Nsp13: A85P and Nsp12: Y479N, have a destabilizing effect, while missense substitution ORF7a: P45L significantly associated with disease severity.

Fig 1. COVID-19 cases in Uzbekistan since March 2020 to September 2021 (according to Worldometer, https://www.worldometers.info).

Fig 2. Deaths caused by COVID-19 in Uzbekistan since March 2020 to September 2021 (according to Worldometer, https://www.worldometers.info).

Fig 3. Violin plot of nucleotide and amino acid substitutions, and deletions per the SARS-CoV-2 genome among 39 samples identified in Uzbekistan (with respect to the reference genome NC_045512.2).

The dots mark the median values, the bold bar marks interquartile range; nt–nucleotide; aa–amino acid.

Fig 4. Box plot of distribution of single nucleotide substitutions per genome among the SARS-CoV-2 isolates (n = 39) in Uzbekistan.

The cross marks the mean values, the bold line marks median.

Fig 5. Box plot of distribution of transitions and transversions per genome among the SARS-CoV-2 isolates (n = 39) in Uzbekistan.

The cross marks the mean values, the bold line marks median.

Fig 6. The Nextclade based phylogenetic tree of the SARS-CoV-2 variants isolated in Uzbekistan.

Sequences are placed on a reference tree, clades were assigned to the nearest neighbor, and private mutations analyzed. Brunches with colored circle represents variants from Uzbekistan.

Fig 7. Neighbor-joining phylogenetic tree of the SARS-CoV-2 isolates from Uzbekistan.

Samples are colored by taxonomic affiliation to clades or subclades. Clades are assigned according to Nextclade nomenclature. Delta variant divided to subclades 21A (green) and 21J (orange). Blue color—clade 20A, Red color–clade 20I (Alpha variant). Bootstrap values were shown.

Fig 8. Distribution of mutation types in the Delta variant subclades 21A (n = 18) and 21J (n = 14).

The dots mark the median values, the bold bar marks interquartile range. nt–nucleotide; aa–amino acid.

Fig 9. Age and gender distribution among COVID-19 patients.

Fig 10. Distribution of SARS-CoV-2 Delta subclades between male and female patients in Uzbekistan.

Fig 11. Analysis of different prediction models of the NSP12: Y479N, NSP13: A85P and ORF7a: P45L amino-acid substitution on protein stability.

ΔΔG—free energy of unfolding (kcal/mol). The negative and positive predicted ΔΔG values mean the destabilizing and stabilizing effect, respectively [24, 26].

Fig 12. 3D structure of the ORF7a: P45L substitution effect on protein stability.

Dotted line represents hydrogen bonds. Substitution of Pro to Leu (left) results in destabilizing effect due to loss of hydrogen bond.

Fig 13. Phylogenetic tree of the SARS-CoV-2 isolates distributed in Uzbekistan during the 1-st (2020) and 2-nd (2021) coronavirus pandemic waves.

Samples with blue circles are SARS-CoV-2 isolates from the first wave (November 2020), sample with red triangles are from the second wave (mid 2021).