Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana

Abstract

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12-April 1 2020) and 31 from later time-points ( 25-27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5-99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.

Keywords: COVID-19; SARS-CoV-2; evolution; genomics; novel coronavirus.

Figure 1.

Genomic map showing coverage and homology of Ghanaian SARS-CoV-2 sequences to the Wuhan/Hu-1/2019 genome. The grey colors indicate Ns and gaps, while the rest of the colors indicate differences between sequences without any particular order. We used Nextclade—a webtool that identifies the differences between sequences such as mutations, Ns, and gaps. Nextclade uses an algorithm which estimates the alignment of each genome to the reference genome using short 21-mers along the sequence thereby performing a banded Smith–Waterman alignment with an affine gap-penalty. (A color version of this figure is available in the online journal.)

Figure 2.

Rooted time-resolved maximum likelihood phylogenetic tree. The branch lengths on the phylogenetic tree represent calendar time of sample collection; 15/20 samples were collected March – April (First Batch), while the 31/36 were collected late in May (Second Batch). There is high level of relatedness among genomes sequenced at two different calendar times. Majority of the second batch samples (12/36) branch out from Ghana/1565_S13/2020 and Ghana/2914_S8/2020. Several others (8/36) are phylogenetically related to Ghana/3176_S11/2020. The initial maximum likelihood phylogenetic tree was constructed using a fast and stochastic algorithm (IQ-TREE) and a generalized time reversible (GTR) substitution model, then modified using auger (refine) time tree option. The tree is rooted to the Wuhan reference genome (Wuhan/Hu-1/2019). (A color version of this figure is available in the online journal.)

Figure 3.

Circulating clades of SARS-CoV-2. (a) The tree indicates the clades circulating in Ghana (n = 43/46) whereby majority of the samples belong to clade 19B/S/B4 and clade 20A,B,C/G,GH,GR/A2a. Two samples had mutations that ensure they remained in the 19A/V clade, hence cluster with the Wuhan genomes. The 43 samples can also be clustered into two major legacy clades, the A2a and B4 whereby the B4 clade comprises of the pangolin clade A, while the A2a clade comprises samples in the B.1 and B.1.1 pangolin clades. The first clade to emerge out of Wuhan was 19A which quickly diverged into 19B, which has clearly dominated the samples that were sequenced in Ghana;20A emerged from 19A and dominated the European outbreak in March whereby it spread to Ghana by travelers; 20B and 20C are just genetically distinct sub-clades 20A. (b). Clade composition of sequenced viral isolates shows growing domination of 19B in the samples that were sequenced. (A color version of this figure is available in the online journal.)

Figure 4.

Divergence of the Ghanaian SARS-CoV-2 genomes relative to the Wuhan reference genome. The tree indicates the number of mutations in each of the isolates compared to the Wuhan/Hu-1/2019 reference genome. The viruses circulating in Ghana have 4–12 mutations. The maximum likelihood phylogenetic tree was constructed using IQ-TREE using the GTR substitution model, a clock rate of 0.0008, clock standard deviation of 0.0004, and mutations as the divergence unit. The 20A,B,C/G,GH,GR/A2a clade have accrued more mutations compared to the clade 19B/S/B4. Ghanaian samples have lower-level divergence than global samples but a similar level of D614G dominance. (A color version of this figure is available in the online journal.)

Figure 5.

Maximum likelihood phylogenetic tree of global SARS-CoV-2 genomes depicting the Ghanaian sequence (Ghana/1622_S2/2020) with the highest mutations (n = 12). This sample was collected in March 2020 as an imported case from the United States of America, although the patient transited through the United Kingdom and Dubai. The mutational profile of this case clustered heavily with samples from all three regions: Europe, America, and Asia. The profile of the phylogenetic tree shows that majority of the Asian genomes cluster together and belong to clade 19B, while the African, European, and American genomes cluster together because they have evolved as subclades of 20A. Some genomes from Europe have acquired more than 25 mutations indicating a high level of divergence of isolates. (A color version of this figure is available in the online journal.)

Figure 6.

(a). D614G substitutions segregating to clade 20A,B,C/G,GH,GR/A2a. Relatedness was inferred based on genotypic and phenotypic sequence similarity and sequence of mutational appearance, along with available metadata on residential neighborhoods. Key: Major phenotypes are indicated in the manner “viral protein/locus- amino acid substitution”. Phenotypes are indicated in blue if they have only been reported in this study and show evidence of being locally evolved, green if they have not been reported elsewhere but are detected on an imported virus, and black if they have previously been reported elsewhere. “Major phenotypes/amino acid substitutions” (with the same genotype) are indicated in boxes; when that box is surrounded by a “red glow,” that virus has been transmitted. When silent mutations alone differentiate transmitted viruses, a thick blue arrow is used to link an antecedent virus with its descendent virus. A dotted line with an arrow links a virus with “major phenotype” to their transmitted descendants with additional phenotypic expression (indicated on top or below the dotted line). Sample IDs, indicated in small black ovals are colored yellow to indicate imported viruses and white to indicate locally acquired transmissions. Isolates with identical genotypes are linked using an equal (=) sign. Sample IDs prefaced by GH indicate undetected by deduced genotypes/phenotypes circulating in Ghana. Evolution and transmission analysis of the Ghanaian SARS-CoV-2 genomes harboring and (b). L84S substitutions segregating to clade 19B/S/B4. (A color version of this figure is available in the online journal.)

Figure 7.

Map of Ghana showing the estimated physical residential coordinates of cases. (a) One of the genomes was isolated from northern Ghana (Tamale), while the majority of the cases were from the southern part of Ghana. There was a cluster of cases around Accra (capital city) and its environs. (b) The cases (8/46) that were reported in Ayawaso (Accra) and its surrounding areas. The residential addresses of the cases were used to generate the coordinates (latitude and longitude) using Google maps. The coordinates were plotted on a background world map using leaflet package in R software (Version 4.0.2). (A color version of this figure is available in the online journal.)