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

Genome-wide regulatory dynamics of translation in the Plasmodium falciparum asexual blood stages

The characterization of the transcriptome and proteome of Plasmodium falciparum has been a tremendous resource for the understanding of the molecular physiology of this parasite. However, the translational dynamics that link steady-state mRNA with protein levels are not well understood. In this study, we bridge this disconnect by measuring genome-wide translation using ribosome profiling, through five stages of the P. falciparum blood phase developmental cycle. Our findings show that transcription and translation are tightly coupled, with overt translational control occurring for less than 10% of the transcriptome. Translationally regulated genes are predominantly associated with merozoite egress functions. We systematically define mRNA 5' leader sequences, and 3' UTRs, as well as antisense transcripts, along with ribosome occupancy for each, and establish that accumulation of ribosomes on 5' leaders is a common transcript feature. This work represents the highest resolution and broadest portrait of gene expression and translation to date for this medically important parasite

PoMeLo: a systematic computational approach to predicting metabolic loss in pathogen genomes

Abstract Background Genome streamlining, the process by which genomes become smaller and encode fewer genes over time, is a common phenomenon among pathogenic bacteria. This reduction is driven by selection for minimized energy expenditure in a nutrient-rich environment. As pathogens evolve to become more reliant on the host, metabolic genes and resulting capabilities are lost in favor of siphoning metabolites from the host. Characterizing genome streamlining, gene loss, and metabolic pathway degradation can be useful in assessing pathogen dependency on host metabolism and identifying potential targets for host-directed therapeutics. Results PoMeLo (Predictor of M etabolic L oss) is a novel evolutionary genomics-guided computational approach for identifying metabolic gaps in the genomes of pathogenic bacteria. PoMeLo leverages a centralized public database of high-quality genomes and annotations and allows the user to compare an unlimited number of genomes across individual genes and pathways. PoMeLo runs locally using user-friendly prompts in a matter of minutes and generates tabular and visual outputs for users to compare predicted metabolic capacity between groups of bacteria and individual species. Each pathway is assigned a Predicted Metabolic Loss (PML) score to assess the magnitude of genome streamlining. Optionally, PoMeLo places the results in an evolutionary context by including phylogenetic relationships in visual outputs. It can also initially compute phylogenetically-weighted mean genome sizes to identify genome streamlining events. Here, we describe PoMeLo and demonstrate its use in identifying metabolic gaps in genomes of pathogenic Treponema species. Conclusions PoMeLo represents an advance over existing methods for identifying metabolic gaps in genomic data, allowing comparison across large numbers of genomes and placing the resulting data in a phylogenetic context. PoMeLo is freely available for academic and non-academic use at https://github.com/czbiohub-sf/pomelo

Metagenomic sequencing of post-mortem tissue samples for the identification of pathogens associated with neonatal deaths

Abstract Postmortem minimally invasive tissue sampling together with the detailed review of clinical records has been shown to be highly successful in determining the cause of neonatal deaths. However, conventional tests including traditional culture methods and nucleic acid amplification tests have periodically proven to be insufficient to detect the causative agent in the infectious deaths. In this study, metagenomic next generation sequencing was used to explore for putative pathogens associated with neonatal deaths in post-mortem blood and lung tissue samples, in Soweto, South Africa. Here we show that the metagenomic sequencing results corroborate the findings using conventional methods of culture and nucleic acid amplifications tests on post-mortem samples in detecting the pathogens attributed in the causal pathway of death in 90% (18/20) of the decedents. Furthermore, metagenomic sequencing detected a putative pathogen, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Serratia marcescens, in a further nine of 11 (81%) cases where no causative pathogen was identified. The antimicrobial susceptibility profile was also determined by the metagenomic sequencing for all pathogens with numerous multi drug resistant organism identified. In conclusion, metagenomic sequencing is able to successfully identify pathogens contributing to infection associated deaths on postmortem blood and tissue samples

The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved anti-malarials.

BackgroundThe continued spectre of resistance to existing anti-malarials necessitates the pursuit of novel targets and mechanisms of action for drug development. One class of promising targets consists of the 80S ribosome and its associated components comprising the parasite translational apparatus. Development of translation-targeting therapeutics requires a greater understanding of protein synthesis and its regulation in the malaria parasite. Research in this area has been limited by the lack of appropriate experimental methods, particularly a direct measure of parasite translation.MethodsAn in vitro method directly measuring translation in whole-cell extracts from the malaria parasite Plasmodium falciparum, the PfIVT assay, and a historically-utilized indirect measure of translation, S35-radiolabel incorporation, were compared utilizing a large panel of known translation inhibitors as well as anti-malarial drugs.ResultsHere, an extensive pharmacologic assessment of the PfIVT assay is presented, using a wide range of known inhibitors demonstrating its utility for studying activity of both ribosomal and non-ribosomal elements directly involved in translation. Further, the superiority of this assay over a historically utilized indirect measure of translation, S35-radiolabel incorporation, is demonstrated. Additionally, the PfIVT assay is utilized to investigate a panel of clinically approved anti-malarial drugs, many with unknown or unclear mechanisms of action, and show that none inhibit translation, reaffirming Plasmodium translation to be a viable alternative drug target. Within this set, mefloquine is unambiguously found to lack translation inhibition activity, despite having been recently mischaracterized as a ribosomal inhibitor.ConclusionsThis work exploits a direct and reproducible assay for measuring P. falciparum translation, demonstrating its value in the continued study of protein synthesis in malaria and its inhibition as a drug target

The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved anti-malarials

Abstract Background The continued spectre of resistance to existing anti-malarials necessitates the pursuit of novel targets and mechanisms of action for drug development. One class of promising targets consists of the 80S ribosome and its associated components comprising the parasite translational apparatus. Development of translation-targeting therapeutics requires a greater understanding of protein synthesis and its regulation in the malaria parasite. Research in this area has been limited by the lack of appropriate experimental methods, particularly a direct measure of parasite translation. Methods An in vitro method directly measuring translation in whole-cell extracts from the malaria parasite Plasmodium falciparum, the PfIVT assay, and a historically-utilized indirect measure of translation, S35-radiolabel incorporation, were compared utilizing a large panel of known translation inhibitors as well as anti-malarial drugs. Results Here, an extensive pharmacologic assessment of the PfIVT assay is presented, using a wide range of known inhibitors demonstrating its utility for studying activity of both ribosomal and non-ribosomal elements directly involved in translation. Further, the superiority of this assay over a historically utilized indirect measure of translation, S35-radiolabel incorporation, is demonstrated. Additionally, the PfIVT assay is utilized to investigate a panel of clinically approved anti-malarial drugs, many with unknown or unclear mechanisms of action, and show that none inhibit translation, reaffirming Plasmodium translation to be a viable alternative drug target. Within this set, mefloquine is unambiguously found to lack translation inhibition activity, despite having been recently mischaracterized as a ribosomal inhibitor. Conclusions This work exploits a direct and reproducible assay for measuring P. falciparum translation, demonstrating its value in the continued study of protein synthesis in malaria and its inhibition as a drug target

Ribosome Profiling read coverage plots

Ribosome footprint read coverage plots (normalized by millions of reads sequenced). mRNA read coverage plots (normalized by millions of reads sequenced). Timepoints are referred to as 1-5 (Rings, Early Trophs, Late Trophs, Schizont, Merozoites, respectively