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

Readthrough-mediated functional suppression of homozygous nonsense mutations accounts for variable bleeding phenotypes in factor VII deficiency

BACKGROUND. Nonsense mutations cause premature stop codons that lead to the potential absence of the gene product. Whereas the rare homozygous nonsense mutations causing factor (F)VII deficiency may predict null conditions almost completely incompatible with life, they are associated with appreciable differences in hemorrhagic symptoms, possibly due to residual levels of full-length FVII arising from spontaneous readthrough. This process, based on misrecognition of premature stop codons, may induce the synthesis of variable levels of functional full-length proteins. The sequence context (namely, stop codon and downstream nucleotide) predicts a variable degree of readthrough efficiency in terms of amount of full-length protein produced. The protein outcome is also strictly dependent on the most probable substituted residues, ranging from the wild-type amino acid reinsertion to missense changes impairing secretion and function. The aim of the study was to experimentally evaluate the basal and drug-induced levels of FVII resulting from the homozygous p.Cys132X and p.Ser112X nonsense mutations that are associated with moderate or life-threatening symptoms, respectively, and are predicted to undergo readthrough with (132X) or without (112X) production of wild-type FVII. METHODS. We transiently expressed recombinant FVII (rFVII) nonsense and missense variants in human embryonic kidney 293 cells. Secreted FVII protein levels were evaluated by ELISA while functional levels were assessed through activated FX generation and coagulation assays. RESULTS. The levels of functional FVII produced by p.Cys132X and p.Ser112X mutants (rFVII-132X, 1.1%±0.2% of wild-type rFVII; rFVII-112X, 0.5%±0.1% of wild-type rFVII) were compatible with the occurrence of spontaneous readthrough. Treatment with G418, an aminoglycoside known to increase misrecognition of stop codons, allowed to magnify the occurrence of drug-induced readthrough, up to 12% of the wild-type value for the rFVII-132X nonsense variant. Previous studies indicated that TGA is suppressed by the incorporation of Trp>Cys>Arg, in order of frequency. To dissect the mechanism underlying the production of a functional full-length protein we expressed all the predicted missense variants arising from readthrough, which showed abolished (rFVII-132Trp/Arg) or reduced (rFVII-112Trp/Cys/Arg, 22–45% of wild-type levels) secretion and function. These data suggest that the appreciable rescue of p.Cys132X function was driven by reinsertion of the wild-type residue, whereas the minimal p.Ser112X function was explained by missense changes permitting FVII secretion and function. CONCLUSIONS. Our experimental findings support the notion that the extent of functional readthrough contributes to the variable bleeding phenotype in patients homozygous for F7 nonsense mutations, possibly preventing null conditions even for the most readthrough-unfavorable mutations

Differential functional readthrough over homozygous nonsense mutations contributes to the bleeding phenotype in coagulation factor VII deficiency

Background Whereas the rare homozygous nonsense mutations causing factor (F)VII deficiency may predict null conditions that are almost completely incompatible with life, they are associated with appreciable differences in hemorrhagic symptoms. The misrecognition of premature stop codons (readthrough) may account for variable levels of functional full-length proteins. Objectives To experimentally evaluate the basal and drug-induced levels of FVII resulting from the homozygous p.Cys132X and p.Ser112X nonsense mutations that are associated with moderate (132X) or life-threatening (112X) symptoms, and that are predicted to undergo readthrough with (132X) or without (112X) production of wild-type FVII. Methods We transiently expressed recombinant FVII (rFVII) nonsense and missense variants in human embryonic kidney 293 cells, and evaluated secreted FVII protein and functional levels by ELISA, activated FX generation, and coagulation assays. Results The levels of functional FVII produced by p.Cys132X and p.Ser112X mutants (rFVII-132X, 1.1% 0.2% of wild-type rFVII; rFVII-112X, 0.5% +/- 0.1% of wild-type rFVII) were compatible with the occurrence of spontaneous readthrough, which was magnified by the addition of G418 - up to 12% of the wild-type value for the rFVII-132X nonsense variant. The predicted missense variants arising from readthrough abolished (rFVII-132Trp/Arg) or reduced (rFVII-112Trp/Cys/Arg, 22-45% of wild-type levels) secretion and function. These data suggest that the appreciable rescue of p.Cys132X function was driven by reinsertion of the wild-type residue, whereas the minimal p.Ser112X function was explained by missense changes permitting FVII secretion and function. Conclusions The extent of functional readthrough might explain differences in the bleeding phenotype of patients homozygous for F7 nonsense mutations, and prevent null conditions even for the most readthrough-unfavorable mutations

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ≃100 seconds of untreated mice to ≃80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients

Rescue of coagulation factor VII mRNA processing and protein function by engineered U1+5A snRNA

Changes affecting mRNA processing represent a frequent cause of severe coagulation factor defects and of all inherited human diseases. We extensively investigated the IVS7+5G/A mutation (9726+5A) in the coagulation factor VII (FVII) gene, occurring in the IVS7 donor splice site (5ss) in the first of six highly homologous 37bp repeats containing several cryptic splice sites. This mutation is the most frequent cause of severe FVII deficiency in Central Italy. A cellular model of this deficiency was created by producing a full length FVII splicing competent construct (pSCFVII-wt). This minigene drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 ±4 ng/mL), which were virtually undetectable upon introduction of the mutation (pSCFVII-9726+5A). At the mRNA level the mutation caused exon 7 skipping and, to a less extent, activation of the 37-bp downstream cryptic site. To attempt rescue of FVII expression, we have engineered the U1-snRNA, the spliceosome component selectively recognizing donor splice sites (5'ss), to re-direct recognition of the mutated donor splice site. Upon cotrasfection of the engineered U1- snRNA (pU1+5A) with pSCFVII-9726+5A, the splicing pattern and protein level were evaluated. At RNA level, the expression of U1+5A reduced from 80% to 40% the exon 7 skipping and increased recognition of the correct 5'ss, resulting in appreciable synthesis of normal transcripts (from hardly detectable to 20%). At protein level, we observed an increase of secreted protein levels in medium (5.0±2.8 ng/mL) and of the FVII coagulant activity, which reached 9.5±3.2% of pSCFVII-wt. The effects of engineered U1-snRNA were dose-dependent. Altogether these results demonstrate for the first time the U1- snRNAmediated rescue at the mRNA and protein levels, thus highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels

Coagulation Factor XIIIA (F13A1): novel perspectives in treatment and pharmacogenetics.

Factor XIII (FXIII) is a key molecule in the field of blood coagulation and in the last decades it has weakened attention within the field of angiogenesis and tissue repair. FXIII positively influences wound healing in several tissues by exerting multiple plasma and cellular functions. In the field of haemostasis, FXIII cross-links the neo formed fibrin fibers and supports platelet adhesion to the damaged sub-endothelium warranting a solid architecture. In addition, the pro-angiogenic functions of FXIII are directed by the interaction of vascular endothelial growth factor receptor 2 (VEGFR2) and the integrin αVβ3, on the cell membrane, favouring an important step in the formation of granulation tissue at the wound site for optimal tissue healing. Conversely, the same mechanisms could lead to undesired increased neovascularisation, for example in inflammatory bowel disease or in the retinal degenerative pathologies. The classical symptoms of FXIII deficiency span from intracranial haemorrhage to delay bleeding or the staying of chronic wounds in the skin including impaired mucosal healing. In this view, FXIII bridges primary haemostasis, coagulation and definite tissue healing. Another important recently discovered function ascribed to FXIII is its ability to limit bacterial spreading from the lesion by incorporating specific macromolecules addressed to cellular infiltration, favouring in turn cell migration and survival, as observed also in fibrin-heart cultures for stem cell recruitment. In the field of the novel prognostic biomarkers, the monitoring of the residual circulating FXIII level during acute myocardial infarction has been considered predictive of the post-myocardial infarction healing. Accordingly, adequate FXIII levels can drive and predict the prognosis of complex diseases and the outcome of the associated therapies or interventions. In addition, peculiar pharmacogenetics aspects of the FXIII gene are of extraordinary interest. The present review accounts for the recognized role of FXIII in the healing process and gives some examples on how to use it as prognostic biological/molecular marker or as potential tailored therapeutic molecule in complex diseases

Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency

We report 2 asymptomatic homozygotes for the nonsense p.R462X mutation affecting the carboxy-terminus of coagulation factor VII (FVII, 466 aminoacids). FVII levels of 3–5% and 2.7±0.4% were found in prothrombin time-based and activated factor X (FXa) generation assays with human thromboplastins. Noticeably, FVII antigen levels were barely detectable (0.7±0.2%) which suggested a gain-of-function effect. This effect was more pronounced with bovine thromboplastin (4.8±0.9%) and disappeared with rabbit thromboplastin (0.7±0.2%). This suggests that the mutation influences tissue factor/FVII interactions