The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC50 of 1.25 mg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC50 10 mM (2.3 mg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-a, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer

SDGE induces apoptosis in endometrial cancer cells.

<p>Ishikawa cells were treated with 250 ng/ml of SDGE for 30 min, 2 h, 4 h, and 16 h. After incubation with SDGE, cell survival was determined by labeling the cells with FITC-conjugated FITC-Annexin V and propidium iodide (A). The cells were analyzed by flow cytometry and cell death and apoptosis were identified as the events that were single positive for FITC-Annexin V (lower right quadrant) or double positive for both FITC-Annexin V and proidium iodide (upper right quadrant). SDGE-induced apoptotic cell death in the endometrial cancer cells was confirmed by detecting cleaved caspase 3 in western blot analysis (B). An increase in cleaved caspase 3 levels was observed when the Ishikawa cells were treated with SDGE (250 ng/ml) for 0, 24, 48, and 72 h. Data in A and B is representative of results obtained in three separate experiments,</p

Citral inhibits proliferation of endometrial cancer cells.

<p>MTT assays were conducted to determine the effect of 6-gingerol (A and B) and citral (C and D) on the proliferation of Ishikawa and ECC-1 cells. Each data point is a mean of 16 individual readings. Based on this data the IC₅₀ of citral was calculated to be 15–25 µM.</p

SDGE mediates endometrial cancer cell apoptosis through the activation of p53.

<p>After treating Ishikawa cells with SDGE (250 ng/ml) for the designated time points, the cells were harvested and lysates were analyzed by Western blotting for expression of Bcl-2 and Bax (A). Phosphorylation of Ser-15 of p53 was monitored in Ishikawa cells treated with SDGE (250 ng/ml) (B). Ishikawa cells were incubated with SDGE in the presence or absence of pifithrin-α for 18 h. Control Ishikawa cells were not exposed to either SDGE or pifithrin-α. (C) Cells were harvested and apoptosis was measured by flow cytometry after staining with FITC-Annexin V and propidium iodide. Apoptosis in the p53^neg SKOV-3 cells after treatment with SDGE (250 ng/ml) was also measured by the FITC-Annexin V assay (D) or by monitoring the levels of cleaved Caspase3 by Western blotting (E) at the designated time intervals.</p

SDGE increases intracellular calcium and decreases the mitochondrial membrane potential of endometrial cancer cells.

<p>Effect of SDGE on the intracellular calcium flux was determined by treating Indo-1 loaded Ishikawa cells (A). Immediately after addition of SDGE to the cell suspensions, the Ishikawa cells were flowed through the cytometer and increase in fluorescence was measured to detect calcium flux. Decrease in mitochondrial membrane potential was detected by loading Ishikawa cells with DiOC6 (B). SDGE or vehicle control was added to the cells. After 24 h of treatment, the cells were harvested and incubated with DiOC6 for 15 min. Fluorescence was measured to determine changes in mitochondrial membrane potential.</p