Molecular Epidemiology and Diversity of SARS-CoV-2 in Ethiopia, 2020-2022 (preprint)

Ethiopia is the second most populous country in Africa and the sixth most affected by COVID-19 on the continent. Despite having experienced five infection waves, >499 000 cases, and ~7 500 COVID-19-related deaths as of January 2023, there is still no detailed genomic epidemiological report on the introduction and spread of SARS-CoV-2 in Ethiopia. In this study, we reconstructed and elucidated the COVID-19 epidemic dynamics. Specifically, we investigated the introduction, local transmission, ongoing evolution, and spread of SARS-CoV-2 during the first four infection waves using 353 high-quality near-whole genomes sampled in Ethiopia. Our results show that whereas viral introductions seeded the first wave, subsequent waves were seeded by local transmission. The B.1.480 lineage emerged in the first wave and notably remained in circulation even after the emergence of the Alpha variant. The B.1.480 was out-competed by the Delta variant. Notably, Ethiopia lack of local sequencing capacity was further limited by sporadic, uneven, and insufficient sampling that limited the incorporation of genomic epidemiology in the epidemic public health response in Ethiopia. These results highlight Ethiopia role in SARS-CoV-2 dissemination and the urgent need for balanced, near-real-time genomic sequencing.

Genomic assessment of invasion dynamics of SARS-CoV-2 Omicron BA.1 (preprint)

SARS-CoV-2 variants of concern (VOCs) arise against the backdrop of increasingly heterogeneous human connectivity and population immunity. Through a large-scale phylodynamic analysis of 115,622 Omicron genomes, we identified >6,000 independent introductions of the antigenically distinct virus into England and reconstructed the dispersal history of resulting local transmission. Travel restrictions on southern Africa did not reduce BA.1 importation intensity as secondary hubs became major exporters. We explored potential drivers of BA.1 spread across England and discovered an early period during which viral lineage movements mainly occurred between larger cities, followed by a multi-focal spatial expansion shaped by shorter distance mobility patterns. We also found evidence that disease incidence impacted human commuting behaviours around major travel hubs. Our results offer a detailed characterisation of processes that drive the invasion of an emerging VOC across multiple spatial scales and provide unique insights on the interplay between disease spread and human mobility.

Global Expansion of SARS-CoV-2 Variants of Concern: Dispersal Patterns and Influence of Air Travel (preprint)

In many regions of the world, the Alpha, Beta and Gamma SARS-CoV-2 Variants of Concern (VOCs) co-circulated during 2020-21 and fueled waves of infections. During 2021, these variants were almost completely displaced by the Delta variant, causing a third wave of infections worldwide. This phenomenon of global viral lineage displacement was observed again in late 2021, when the Omicron variant disseminated globally. In this study, we use phylogenetic and phylogeographic methods to reconstruct the dispersal patterns of SARS-CoV-2 VOCs worldwide. We find that the source-sink dynamics of SARS-CoV-2 varied substantially by VOC, and identify countries that acted as global hubs of variant dissemination, while other countries became regional contributors to the export of specific variants. We demonstrate a declining role of presumed origin countries of VOCs to their global dispersal: we estimate that India contributed <15% of all global exports of Delta to other countries and South Africa <1-2% of all global Omicron exports globally. We further estimate that >80 countries had received introductions of Omicron BA.1 100 days after its inferred date of emergence, compared to just over 25 countries for the Alpha variant. This increased speed of global dissemination was associated with a rebound in air travel volume prior to Omicron emergence in addition to the higher transmissibility of Omicron relative to Alpha. Our study highlights the importance of global and regional hubs in VOC dispersal, and the speed at which highly transmissible variants disseminate through these hubs, even before their detection and characterization through genomic surveillance.

Comparing SARS-CoV-2 sequencing methodologies during early phase detection of the Delta variant in South Africa (preprint)

Background: Genomic surveillance, with the aid of next-generation sequencing (NGS) technologies, revolutionized the SARS-CoV-2 pandemic. Coupled with high-performance analysis software, methodologies such as the Ion Torrent S5 and Illumina MiSeq dramatically improved the genomic surveillance capacity within South Africa during the height of the pandemic. Using de-identified remnant samples collected from Eastern Cape and analysis software, Genome Detective and NextClade, we compared the sequencing process, genomic coverage, quantification of mutations, and clade classification from sequence data generated by these two common “benchtop” NGS platforms. Results: Sequence data analysis revealed success rates of 175/183 (96%) and 172/183 (94%) on the Ion Torrent S5 and Illumina MiSeq, respectively. Internal quality metrics were assessed in terms of genomic coverage (>80%) and the number of mutations identified (<100). A greater number of higher-genomic coverage sequences were generated on the Ion Torrent S5 (99%) than on the Illumina MiSeq (80%) and <100 mutations was obtained by both platforms. Ion Torrent S5 generated high coverage sequences from samples having a broader range of viral loads (VL) compared to the Illumina MiSeq, which was less successful in sequencing samples with lower viral loads. Clade assignments were comparable across platforms which accurately differentiated between Beta (<45%) and Delta (≤30%) VOCs. A disparity in clade assignment was observed in <10% of sequences due to poor coverage obtained on the Illumina MiSeq, followed by a failure rate of ≤6% across the two platforms. Manual library preparation found both methods similar in terms of sample processing, handling of larger sample quantities, and clade assignment for SARS-CoV-2. Variability between the Ion Torrent S5 and Illumina MiSeq was observed in sequencing run duration (3,5 hrs vs 36 hrs), sequencing process (semi-automation vs manual), genomic coverage (99% vs 80%), and viral load requirements (broad range vs high VL). Conclusion: The Illumina MiSeq and Ion Torrent S5 are both reliable platforms capable of performing WGS with the use of amplicons and providing specific, accurate, and high throughput analysis of the SARS-CoV-2 whole viral genomes. Both sequencing platforms are feasible platforms for the genomic surveillance of SARS-CoV-2, each with its specific advantages and trade-offs.

Impact of intra-host immune adaptations on the evolution of SARS-CoV-2 S protein among individuals with SARS-CoV-2 infections in South Africa, 2020 to 2022 (preprint)

Background: Intra-host diversity studies are used to characterise mutational heterogeneity of SARS-CoV-2 infections to understand the impact of virus-host adaptations. This study investigated the frequency and diversity of the spike (S) protein mutations within SARS-CoV-2 infected South African individuals. Methods Single nucleotide polymorphism (SNP) assays and whole genome sequencing were performed on SARS-CoV-2 positive samples. Allele frequency (AF) was determined using TaqMan Genotyper software for SNP analysis and galaxy.eu for analysis of FASTQ reads. Results The SNP assays identified 5.3% (50/948) Delta cases with heterogeneity at delY144 (4%; 2/50), E484Q (6%; 3/50), N501Y (2%; 1/50) and P681H (88%; 44/50). Sequencing identified 9% (210/2381) cases with Beta, Delta, Omicron BA.1, BA.2.15, and BA.4 lineages with heterogeneity in the S protein. Heterogeneity was primarily identified at positions 19 (1.4%) with T19IR (AF 0.2-0.7), 371 (92.3%) with S371FP (AF 0.1-1.0), and 484 (1.9%) with E484AK (0.2-0.7), E484AQ (AF 0.4-0.5) and E484KQ (AF 0.1-0.4). Conclusion Mutations at heterozygous amino acid positions 19, 371 and 484 reduce recognition of neutralising antibodies, however the impact of the multiple substitutions at the same position is unknown. Therefore, we hypothesise that intra-host SARS-CoV-2 quasispecies with heterogeneity in the S protein facilitate competitive advantage of variants that can completely/partially evade host’s natural and vaccine-induced immune responses.

Continued Emergence and Evolution of Omicron in South Africa: New BA.4 and BA.5 lineages (preprint)

South Africa's fourth COVID-19 wave was driven predominantly by three lineages (BA.1, BA.2 and BA.3) of the SARS-CoV-2 Omicron variant of concern. We have now identified two new lineages, BA.4 and BA.5. The spike proteins of BA.4 and BA.5 are identical, and comparable to BA.2 except for the addition of 69-70del, L452R, F486V and the wild type amino acid at Q493. The 69-70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure with the TaqPath COVID-19 qPCR assay. BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa from the first week of April 2022 onwards. Using a multinomial logistic regression model, we estimate growth advantages for BA.4 and BA.5 of 0.08 (95% CI: 0.07 - 0.09) and 0.12 (95% CI: 0.09 - 0.15) per day respectively over BA.2 in South Africa.

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

Investment in Africa over the past year with regards to SARS-CoV-2 genotyping has led to a massive increase in the number of sequences, exceeding 100,000 genomes generated to track the pandemic on the continent. Our results show an increase in the number of African countries able to sequence within their own borders, coupled with a decrease in sequencing turnaround time. Findings from this genomic surveillance underscores the heterogeneous nature of the pandemic but we observe repeated dissemination of SARS-CoV-2 variants within the continent. Sustained investment for genomic surveillance in Africa is needed as the virus continues to evolve, particularly in the low vaccination landscape. These investments are very crucial for preparedness and response for future pathogen outbreaks.

Selection analysis identifies unusual clustered mutational changes in Omicron lineage BA.1 that likely impact Spike function (preprint)

Among the 30 non-synonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (i) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (ii) interactions of Spike with ACE2 receptors, and (iii) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron over all previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Comparison of SARS-CoV-2 sequencing using the ONT GridION and the Illumina MiSeq (preprint)

Background: Over 4 million SARS-CoV-2 genomes have been sequenced globally in the past 2 years. This has been crucial in elucidating transmission chains within communities, the development of new diagnostic methods, vaccines, and antivirals. Although several sequencing technologies have been employed, Illumina and Oxford Nanopore remain the two most commonly used platforms. The sequence quality between these two platforms warrants a comparison of the genomes produced by the two technologies. Here, we compared the sequence quality produced by the Oxford Nanopore Technology GridION and the Illumina MiSeq for 28 sequencing runs. Results: Our results show that the MiSeq had a significantly higher number of sequences classified by Nextclade as good and mediocre compared to the GridION. The MiSeq also had a significantly higher sequence coverage and mutation counts than the GridION. Conclusion: Due to the low sequence coverage, high number of indels, and sensitivity to viral load noted with the GridION when compared to MiSeq, we can conclude that the MiSeq is more favourable for genomic surveillance, as successful genomic surveillance is dependent on high quality, near-whole genome sequences.

SARS-CoV-2 Genetic diversity and lineage dynamics of in Egypt (preprint)

COVID-19 was first diagnosed in Egypt on 14 February 2020. By the end of November 2021, over 333,840 cases and 18,832 deaths had been reported. As part of national genomic surveillance, 1,027 SARS-CoV-2 near whole-genomes had been generated and published by the end of May 2021. Here we describe the genomic epidemiology of SARS-CoV-2 in Egypt over this period using a subset of 976 high-quality Egyptian genomes analysed together with a representative set of global sequences within a phylogenetic framework. We show that a single lineage, C.36, introduced early in the pandemic was responsible for most cases in Egypt. Furthermore, we show that to remain dominant in the face of mounting immunity from previous infection and vaccination, this lineage evolved into various sub-lineages acquiring several mutations known to confer adaptive advantage and pathogenic properties. These results highlight the value of continuous genomic surveillance in regions where VOCs are not predominant and enforcement of public health measures to prevent expansion of existing lineages.

Replacement of the Gamma by the Delta variant in Brazil: impact of lineage displacement on the ongoing pandemic (preprint)

The COVID-19 epidemic in Brazil was driven mainly by the spread of Gamma (P.1), a locally emerged Variant of Concern (VOC) that was first detected in early January 2021. This variant was estimated to be responsible for more than 96% of cases reported between January and June 2021, being associated with increased transmissibility and disease severity, a reduction in neutralization antibodies and effectiveness of treatments or vaccines, as well as diagnostic detection failure. Here we show that, following several importations predominantly from the USA, the Delta variant rapidly replaced Gamma after July 2021. However, in contrast to what was seen in other countries, the rapid spread of Delta did not lead to a large increase in the number of cases and deaths reported in Brazil. We suggest that this was likely due to the relatively successful early vaccination campaign coupled with natural immunity acquired following prior infection with Gamma. Our data reinforces reports of the increased transmissibility of the Delta variant and, considering the increasing concern due to the recently identified Omicron variant, argues for the necessity to strengthen genomic monitoring on a national level to quickly detect and curb the emergence and spread of other VOCs that might threaten global health.

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in southern Africa has been characterised by three distinct waves. The first was associated with a mix of SARS-CoV-2 lineages, whilst the second and third waves were driven by the Beta and Delta variants respectively. In November 2021, genomic surveillance teams in South Africa and Botswana detected a new SARS-CoV-2 variant associated with a rapid resurgence of infections in Gauteng Province, South Africa. Within three days of the first genome being uploaded, it was designated a variant of concern (Omicron) by the World Health Organization and, within three weeks, had been identified in 87 countries. The Omicron variant is exceptional for carrying over 30 mutations in the spike glycoprotein, predicted to influence antibody neutralization and spike function4. Here, we describe the genomic profile and early transmission dynamics of Omicron, highlighting the rapid spread in regions with high levels of population immunity.

Management and containment of a SARS-CoV-2 Beta variant outbreak at the Malawi University of Science and Technology (preprint)

Outbreaks of COVID at university campuses can spread rapidly and threaten the broader community. We describe the management of an outbreak at a Malawian university in April 2021 during Malawi's second wave. Classes were suspended following detection of infections by routine testing and campus-wide PCR mass testing was conducted. Fifty seven cases were recorded, 55 among students, two among staff. Classes resumed 28 days after suspension following two weeks without cases. Just 6.3% of full-time staff and 87.4% of outsourced staff tested while 65% of students at the main campus and 74% at the extension campus were tested. Final year students had significantly higher positivity and lower testing coverage compared to freshmen. All viruses sequenced were beta variant and at least four separate virus introductions onto campus were observed. These findings are useful for development of campus outbreak responses and indicate the need to emphasize staff, males and senior students in testing.

Genomic epidemiology reveals how restriction measures shaped the SARS-CoV-2 epidemic in Brazil (preprint)

Brazil has experienced some of the highest numbers of COVID-19 infections and deaths globally and made Latin America a pandemic epicenter from May 2021. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of local virus transmission dynamics. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and an adjacent country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed, the absence of effective restriction measures led to the local emergence and international spread of Variants of Concern (VOC) and under monitoring (VUM), including the Gamma (P.1) and Zeta (P.2) variants. In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring and providing a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies.

Rapid replacement of the Beta variant by the Delta variant in South Africa (preprint)

The Beta variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in South Africa in late 2020 and rapidly became the dominant variant, causing over 95% of infections in the country during and after the second epidemic wave. Here we show rapid replacement of the Beta variant by the Delta variant, a highly transmissible variant of concern (VOC) that emerged in India and subsequently spread around the world. The Delta variant was imported to South Africa primarily from India, spread rapidly in large monophyletic clusters to all provinces, and became dominant within three months of introduction. This was associated with a resurgence in community transmission, leading to a third wave which was associated with a high number of deaths. We estimated a growth advantage for the Delta variant in South Africa of 0.089 (95% confidence interval [CI] 0.084-0.093) per day which corresponds to a transmission advantage of 46% (95% CI 44-48) compared to the Beta variant. These data provide additional support for the increased transmissibility of the Delta variant relative to other VOC and highlight how dynamic shifts in the distribution of variants contribute to the ongoing public health threat.

Divergence of delta and beta variants and SARS-CoV-2 evolved in prolonged infection into distinct serological phenotypes (preprint)

Characterizing SARS-CoV-2 evolution in specific geographies may help predict the properties of variants coming from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from the ancestral virus in a person with advanced HIV disease. Infection was before the emergence of the Beta variant first identified in South Africa, and the Delta variant. We compared early and late evolved virus to the ancestral, Beta, Alpha, and Delta viruses and tested against convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral, whereas late virus was similar to Beta, exhibiting vaccine escape and, despite pre-dating Delta, strong escape of Delta-elicited neutralization. This example is consistent with the notion that variants arising in immune-compromised hosts, including those with advanced HIV disease, may evolve immune escape of vaccines and enhanced escape of Delta immunity, with implications for vaccine breakthrough and reinfections. HighlightsO_LIA prolonged ancestral SARS-CoV-2 infection pre-dating the emergence of Beta and Delta resulted in evolution of a Beta-like serological phenotype C_LIO_LISerological phenotype includes strong escape from Delta infection elicited immunity, intermediate escape from ancestral virus immunity, and weak escape from Beta immunity C_LIO_LIEvolved virus showed substantial but incomplete escape from antibodies elicited by BNT162b2 vaccination C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/21263564v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1194bfdorg.highwire.dtl.DTLVardef@1cbe318org.highwire.dtl.DTLVardef@aa74f8org.highwire.dtl.DTLVardef@e57969_HPS_FORMAT_FIGEXP M_FIG C_FIG

The continuous evolution of SARS-CoV-2 in South Africa: a new lineage with rapid accumulation of mutations of concern and global detection (preprint)

Global genomic surveillance of SARS-CoV-2 has identified variants associated with increased transmissibility, neutralization resistance and disease severity. Here we report the emergence of the PANGO lineage C.1.2, detected at low prevalence in South Africa and eleven other countries. The emergence of C.1.2, associated with a high substitution rate, includes changes within the spike protein that have been associated with increased transmissibility or reduced neutralization sensitivity in SARS-CoV-2 VOC/VOIs. Like Beta and Delta, C.1.2 shows significantly reduced neutralization sensitivity to plasma from vaccinees and individuals infected with the ancestral D614G virus. In contrast, convalescent donors infected with either Beta or Delta showed high plasma neutralization against C.1.2. These functional data suggest that vaccine efficacy against C.1.2 will be equivalent to Beta and Delta, and that prior infection with either Beta or Delta will likely offer protection against C.1.2.

A Novel and Expanding SARS-CoV-2 Variant, B.1.1.318, dominates infections in Mauritius (preprint)

Mauritius, a small island in the Indian Ocean, has had a unique experience of the SARS-CoV-2 pandemic. In March 2020, Mauritius endured a small first wave and quickly implemented control measures which allowed elimination of local transmission of SARS-CoV-2. When borders to the island reopened, it was accompanied by mandatory quarantine and testing of incoming passengers to avoid reintroduction of the virus into the community. As variants of concern (VOCs) emerged elsewhere in the world, Mauritius began using genomic surveillance to keep track of quarantined cases of these variants. In March 2021, another local outbreak occurred, and sequencing was used to investigate this new wave of local infections. Here, we analyze 154 SARS-CoV-2 viral genomes from Mauritius, which represent 12% of all the infections seem in Mauritius, these were both from specimens of incoming passengers before March 2021 and those of cases during the second wave. Our findings indicate that despite the presence of known VOCs Beta (B.1.351) and Alpha (B.1.1.7) among quarantined passengers, the second wave of local SARS-CoV-2 infections in Mauritius was caused by a single introduction and dominant circulation of the B.1.1.318 virus. The B.1.1.318 variant is characterized by fourteen non-synonymous mutations in the S-gene, with five encoded amino acid substitutions (T95I, E484K, D614G, P681H, D796H) and one deletion (Y144del) in the Spike glycoprotein. This variant seems to be increasing in prevalence and it is now present in 34 countries. This study highlights that despite having stopped the introduction of more transmissible VOCs by travel quarantines, a single undetected introduction of a B.1.1.318 lineage virus was enough to initiate a large local outbreak in Mauritius and demonstrated the need for continuous genomic surveillance to fully inform public health decisions.

Persistent SARS-CoV-2 infection and intra-host evolution in association with advanced HIV infection (preprint)

While most people effectively clear SARS-CoV-2, there are several reports of prolonged infection in immunosuppressed individuals. Here we present a case of prolonged infection of greater than 6 months with the shedding of high titter SARS-CoV-2 in an individual with advanced HIV and antiretroviral treatment failure. Through whole-genome sequencing at multiple time points, we demonstrate the early emergence of the E484K substitution associated with escape from neutralizing antibodies, followed by other escape mutations and the N501Y substitution found in most variants of concern. This provides support to the hypothesis of intra-host evolution as one mechanism for the emergence of SARS-CoV-2 variants with immune evasion properties.