Detection and Characterisation of SARS-CoV-2 in Eastern Province of Zambia: A Retrospective Genomic Surveillance Study

Abstract

Mutations have driven the evolution and development of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with potential implications for increased transmissibility, disease severity and vaccine escape among others. Genome sequencing is a technique that allows scientists to read the genetic code of an organism and has become a powerful tool for studying emerging infectious diseases. Here, we conducted a cross-sectional study in selected districts of the Eastern Province of Zambia, from November 2021 to February 2022. We analyzed SARS-CoV-2 samples (n = 76) using high-throughput sequencing. A total of 4097 mutations were identified in 69 SARS-CoV-2 genomes with 47% (1925/4097) of the mutations occurring in the spike protein. We identified 83 unique amino acid mutations in the spike protein of the seven Omicron sublineages (BA.1, BA.1.1, BA.1.14, BA.1.18, BA.1.21, BA.2, BA.2.23 and XT). Of these, 43.4% (36/83) were present in the receptor binding domain, while 14.5% (12/83) were in the receptor binding motif. While we identified a potential recombinant XT strain, the highly transmissible BA.2 sublineage was more predominant (40.8%). We observed the substitution of other variants with the Omicron strain in the Eastern Province. This work shows the importance of pandemic preparedness and the need to monitor disease in the general population.

Keywords: Eastern Province; Omicron variant; SAR-CoV-2 mutations; SARS-CoV-2; Zambia; genomic surveillance.

Figure 1

Genetic distance plot. Nucleotide diversity of SARS-CoV sequences from this study was calculated using the Wuhan HU-1 reference sequence (accession no. NC_045512).

Figure 2

Pairwise genetic identity matrices of nucleotide sequences of the complete open reading frame of the SARS-CoV-2 spike protein. Viruses from this study (1 November 2021 to 28 February 2022). Reference sequences detected in Zambia between 1 January 2021 to 31 October 2021 are in black text denoted by blue right brackets. The colour indicates the homology level between sequences. Pairwise matrices were generated using the Sequence Demarcation Tool v.1.2 [38].

Figure 3

Mutation analysis of SARS-CoV-2 genomes in the present study. (A) Ten of the most mutated samples. (B) Number of overall mutations per sample. (C) Most frequently observed variant classifications. (D) Most frequently encountered substitution type. (E) Frequently observed nucleotide substitutions. (F) Most frequently observed amino acid mutations. Analysis was performed on 69 complete unique sequences using the Coronapp [39].

Figure 4

Frequency of observed variants in the spike protein of SARS-CoV-2 strains from the present study. The dotted blue line denotes the total number of analyzed samples (n = 69). Analysis was performed on the complete S protein of 69 unique sequences using the Coronapp [39].

Figure 5

Pairwise nucleotide alignment of complete SARS-CoV-2 genomes from the present study and those downloaded from the GISAID database at https://www.epicov.org/epi3/ (accessed on 5 May 2024). Viruses from this study are in blue text and yellow highlight. Reference sequences are denoted in blue text. The horizontal bar denotes the percent similarity between sequences.

Figure 6

SARS-CoV-2 lineages identified in this study. (A) Frequency of SARS-CoV-2 Pango lineages identified in Eastern Province. (B) Distribution of SARS-CoV-2 Pango lineages in Eastern Province.

Figure 7

SARS-CoV-2 Lineages identified in this study. (A) Frequency of SARS-CoV-2 Pango lineages identified in Eastern Province. (B) Distribution of SARS-CoV-2 Pango lineages by age groups. (C) Distribution of Pango lineages by sex. (D) Detected Pango lineages by District.

Figure 8

Detection of recombination hot/cold spots. Analysis was performed in RPD4 using a 200-base pair (bp) window at a 20-bp step and the Kimura two-parameter model on a nucleotide alignment generated by MAFFT. Recombination hotspots are denoted by red horizontal bars.

Figure 9

Maximum likelihood phylogenetic tree of SARS-CoV-2 genomes from Zambia and reference sequences retrieved from the GISAID database. The tree was implemented in IQ TREE [41] based on the best nucleotide substitution model (GTR + F+I + G4) in ModelFinder [42].

Figure 10

Maximum likelihood phylogenetic tree of SARS-CoV-2 genomes from the Eastern Province of Zambia collected between September 2021 and October 2022. The tree was implemented in IQ TREE [41] based on the best nucleotide substitution model (GTR + F+I + G4) in ModelFinder [42]. Phylogenetic tree reliability was evaluated by 10,000 ultrafast bootstrap replicates [43]. Coloured strips represent SARS-CoV-2 variants. Bar, number of substitutions per site.

Figure 11

Map of Eastern Province showing the study sites of Chadiza, Nyimba, Katete, Chipangali, Mambwe and Lundazi Districts.