Mutation Analysis of SARS-CoV-2 Variants Isolated from Symptomatic Cases from Andhra Pradesh, India

Abstract

There has been a continuous evolution in the SARS-CoV-2 genome; therefore, it is necessary to monitor the shifts in the SARS-CoV-2 variants. This study aimed to detect various SARS-CoV-2 variants circulating in the state of Andhra Pradesh, India. The study attempted to sequence the complete S-gene of SARS-CoV-2 of 104 clinical samples using Sanger's method to analyze and compare the mutations with the global prevalence. The method standardized in this study was able to amplify the complete length of the S-gene (3822 bp). The resulting nucleotide and amino acid mutations were analyzed and compared with the local and global SARS-CoV-2 databases using Nextclade and GISAID tools. The Delta variant was the most common variant reported in the present study, followed by the Omicron variant. A variant name was not assigned to thirteen samples using the Nextclade tool. There were sixty-nine types of amino acid substitutions reported (excluding private mutations) throughout the spike gene. The T95I mutation was observed predominantly in Delta variants (15/38), followed by Kappa (3/8) and Omicron (1/31). Nearly all Alpha and Omicron lineages had the N501Y substitution; Q493R was observed only in the Omicron lineage; and other mutations (L445, F486, and S494) were not observed in the present study. Most of these mutations found in the Omicron variant are located near the furin cleavage site, which may play a role in the virulence, pathogenicity, and transmission of the virus. Phylogenetic analysis showed that the 104 complete CDS of SARS-CoV-2 belonged to different phylogenetic clades like 20A, 20B, 20I (Alpha), 21A (Delta), 21B (Kappa), 21I (Delta), 21J (Delta), and 21L (Omicron).

Keywords: Andhra Pradesh; India; S-gene sequencing; SARS-CoV-2; amino acid substitutions; circulating clades; mutations.

Figure 1

Trends of COVID-19 peaks (positive percentage) observed in the Rayalaseema districts of Andhra Pradesh state based on the laboratory data The time frame (April 2020 to March 2023) is shown on the “X” axis, and the number of positive cases reported per day is shown as percentages on the “Y” axis.

Figure 2

Amino acid substitutions and their frequency are represented as blue bars (up). The height of the bar corresponds to the number of samples exhibiting the mutation. Some mutations were observed in only one sample each, these mutations are presented as the others and include; L5F, Q14H, L18F, H49Y, A262S, A263P, V382L, L452M, N481K, G496S, V511I, T547K, T604N, Q675H, N856K, and L981F. The genomic diversity chart (down) displaying the nucleotide substitutions for 104 isolates. The scales below the chart (21,600–25,400) represent the corresponding nucleic acid positions, respectively, of the spike gene.

Figure 3

A rectangular phylogeny tree (without roots) was constructed using a local-browser-dependent Nextclade program (updated: 14 December 2022). Sequences (complete spike gene) from the present study were placed (colored dots) on a reference tree (Wuhan-Hu-1/2019 (MN908947)). The Nextstrain and WHO clade names are shown on the respective colored dots.

Figure 4

The distribution of the occurrence of each Alpha (n = 14; blue), Delta (n = 38; orange), Kappa (n = 8; green), Omicron (n = 31; yellow), and others (n = 13; red) over a period of time is shown on the “Y” axis; their occurrence in the time frame is indicated on the “X” axis.

Figure 5

Lineages and sub-lineages reported in the present study are compared with the local (Andhra Pradesh state) and national (India) data. “Unassigned” are sequences that could not be assigned a lineage by Pangolin. Lineages highlighted in the bold text/numbers are major lineages reported from this study and the Country as well. Additional lineages of Delta and Omicron variants reported from the state are shown on the left- and right-side columns, respectively.

Figure 6

Maps representing the peculiar amino acid substitutions observed in Alpha (14), Delta (38), Kappa (8), Omicron (31), and other (13) variants reported in this study. Each row on the “Y” axis represents a specific amino acid change, and squares filled with yellow color indicate a mutation. Sample numbers (corresponding nucleotide sequence GenBank accession numbers and lineage names are provided in the Supplementary Table S1) are represented on the “X” axis. Some substitutions were observed among all lineages of the varietal but are not shown in the map. These substitutions are as follows: Alpha-S:N501Y, S:A570D, and S:D614G; Omicron-S:T19I, S:A27S, G142D, S:V213G, S:G339D, S:S371F, S:S373P, S:S375F, S:T376A, S:D405N, S:R408S, S:K417N, S:N440K, S:S477N, S:T478K, S:E484A, S:Q493R, S:Q498R, S:N501Y505H, and D614G.