An Oxford Nanopore Technology-Based Hepatitis B Virus Sequencing Protocol Suitable For Genomic Surveillance Within Clinical Diagnostic Settings.

Chronic hepatitis B virus (HBV) infection remains a significant public health concern, particularly in Africa, where there is a substantial burden. HBV is an enveloped virus, with isolates being classified into ten phylogenetically distinct genotypes (A - J) determined based on full-genome sequence data or reverse hybridization-based diagnostic tests. In practice, limitations are noted in that diagnostic sequencing, generally using Sanger sequencing, tends to focus only on the S-gene, yielding little or no information on intra-patient HBV genetic diversity with very low-frequency variants and reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV genotyping protocol suitable for clinical virology, yielding complete HBV genome sequences and extensive data on intra-patient HBV diversity. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit, ONT GridION sequencing, genotyping using Genome Detective software, recombination analysis using jpHMM and RDP5 software, and drug resistance profiling using Geno2pheno software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 left-over diagnostic Hepatitis B patient samples obtained in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.

An emerging clade of Chikungunya West African genotype discovered in real-time during 2023 outbreak in Senegal.

Chikungunya (CHIKV) is a re-emerging endemic arbovirus in West Africa. Since July 2023, Senegal and Burkina Faso have been experiencing an ongoing outbreak, with over 300 confirmed cases detected so far in the regions of Kédougou and Tambacounda in Senegal, the largest recorded outbreak yet. CHIKV is typically maintained in a sylvatic cycle in Senegal but its evolution and factors contributing to re-emergence are so far unknown in West Africa, leaving a gap in understanding and responding to recurrent epidemics. We produced, in real-time, the first locally-generated and publicly available CHIKV whole genomes in West Africa, to characterize the genetic diversity of circulating strains, along with phylodynamic analysis to estimate time of emergence and population growth dynamics. A novel strain of the West African genotype, phylogenetically distinct from strains circulating in previous outbreaks, was identified. This suggests a likely new spillover from sylvatic cycles in rural Senegal and potential of seeding larger epidemics in urban settings in Senegal and elsewhere.

Insights into SARS-CoV-2 in Angola during the COVID-19 peak: Molecular epidemiology and genome surveillance.

Background: In Angola, COVID-19 cases have been reported in all provinces, resulting in >105,000 cases and >1900 deaths. However, no detailed genomic surveillance into the introduction and spread of the SARS-CoV-2 virus has been conducted in Angola. We aimed to investigate the emergence and epidemic progression during the peak of the COVID-19 pandemic in Angola. Methods: We generated 1210 whole-genome SARS-CoV-2 sequences, contributing West African data to the global context, that were phylogenetically compared against global strains. Virus movement events were inferred using ancestral state reconstruction. Results: The epidemic in Angola was marked by four distinct waves of infection, dominated by 12 virus lineages, including VOCs, VOIs, and the VUM C.16, which was unique to South-Western Africa and circulated for an extended period within the region. Virus exchanges occurred between Angola and its neighboring countries, and strong links with Brazil and Portugal reflected the historical and cultural ties shared between these countries. The first case likely originated from southern Africa. Conclusion: A lack of a robust genome surveillance network and strong dependence on out-of-country sequencing limit real-time data generation to achieve timely disease outbreak responses, which remains of the utmost importance to mitigate future disease outbreaks in Angola.

Correction: Subramoney et al. Molecular Epidemiology of SARS-CoV-2 during Five COVID-19 Waves and the Significance of Low-Frequency Lineages. Viruses 2023, 15, 1194.

In the original publication [...].

Molecular Epidemiology of SARS-CoV-2 during Five COVID-19 Waves and the Significance of Low-Frequency Lineages.

SARS-CoV-2 lineages and variants of concern (VOC) have gained more efficient transmission and immune evasion properties with time. We describe the circulation of VOCs in South Africa and the potential role of low-frequency lineages on the emergence of future lineages. Whole genome sequencing was performed on SARS-CoV-2 samples from South Africa. Sequences were analysed with Nextstrain pangolin tools and Stanford University Coronavirus Antiviral & Resistance Database. In 2020, 24 lineages were detected, with B.1 (3%; 8/278), B.1.1 (16%; 45/278), B.1.1.348 (3%; 8/278), B.1.1.52 (5%; 13/278), C.1 (13%; 37/278) and C.2 (2%; 6/278) circulating during the first wave. Beta emerged late in 2020, dominating the second wave of infection. B.1 and B.1.1 continued to circulate at low frequencies in 2021 and B.1.1 re-emerged in 2022. Beta was outcompeted by Delta in 2021, which was thereafter outcompeted by Omicron sub-lineages during the 4th and 5th waves in 2022. Several significant mutations identified in VOCs were also detected in low-frequency lineages, including S68F (E protein); I82T (M protein); P13L, R203K and G204R/K (N protein); R126S (ORF3a); P323L (RdRp); and N501Y, E484K, D614G, H655Y and N679K (S protein). Low-frequency variants, together with VOCs circulating, may lead to convergence and the emergence of future lineages that may increase transmissibility, infectivity and escape vaccine-induced or natural host immunity.

SARS-CoV-2 spike protein diversity at an intra-host level, among SARS-CoV-2 infected individuals in South Africa, 2020 to 2022.

Intra-host diversity studies are used to characterise the mutational heterogeneity of SARS-CoV-2 infections in order 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. The study included SARS-CoV-2 respiratory samples, from individuals of all ages, received at the National Health Laboratory Service at Charlotte Maxeke Johannesburg Academic hospital, Gauteng, South Africa, from June 2020 to May 2022. Single nucleotide polymorphism (SNP) assays and whole genome sequencing were performed on a random selection of SARS-CoV-2 positive samples. The allele frequency (AF) was determined using TaqMan Genotyper software for SNP PCR analysis and galaxy.eu for analysis of FASTQ reads from sequencing. The SNP assays identified 5.3% (50/948) of Delta cases with heterogeneity at delY144 (4%; 2/50), E484Q (6%; 3/50), N501Y (2%; 1/50) and P681H (88%; 44/50), however only heterogeneity for E484Q and delY144 were confirmed by sequencing. From sequencing we identified 9% (210/2381) of cases with Beta, Delta, Omicron BA.1, BA.2.15, and BA.4 lineages that had 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). Mutations at heterozygous amino acid positions 19, 371 and 484 are known antibody escape mutations, however the impact of the combination of multiple substitutions identified 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.

Efficacy of primary series AZD1222 (ChAdOx1 nCoV-19) vaccination against SARS-CoV-2 variants of concern: Final analysis of a randomized, placebo-controlled, phase 1b/2 study in South African adults (COV005).

COVID-19 vaccine efficacy (VE) has been observed to vary against antigenically distinct SARS-CoV-2 variants of concern (VoC). Here we report the final analysis of VE and safety from COV005: a phase 1b/2, multicenter, double-blind, randomized, placebo-controlled study of primary series AZD1222 (ChAdOx1 nCoV-19) vaccination in South African adults aged 18-65 years. South Africa's first, second, and third waves of SARS-CoV-2 infections were respectively driven by the ancestral SARS-CoV-2 virus (wild type, WT), and SARS-CoV-2 Beta and Delta VoCs. VE against asymptomatic and symptomatic infection was 90.6% for WT, 6.7% for Beta and 77.1% for Delta. No cases of severe COVID-19 were documented ahead of unblinding. Safety was consistent with the interim analysis, with no new safety concerns identified. Notably, South Africa's Delta wave occurred ≥ 9 months after primary series vaccination, suggesting that primary series AZD1222 vaccination offers a good durability of protection, potentially due to an anamnestic response. Clinical trial identifier: CT.gov NCT04444674.

Evaluation of miniaturized Illumina DNA preparation protocols for SARS-CoV-2 whole genome sequencing.

The global pandemic caused by SARS-CoV-2 has increased the demand for scalable sequencing and diagnostic methods, especially for genomic surveillance. Although next-generation sequencing has enabled large-scale genomic surveillance, the ability to sequence SARS-CoV-2 in some settings has been limited by the cost of sequencing kits and the time-consuming preparations of sequencing libraries. We compared the sequencing outcomes, cost and turn-around times obtained using the standard Illumina DNA Prep kit protocol to three modified protocols with fewer clean-up steps and different reagent volumes (full volume, half volume, one-tenth volume). We processed a single run of 47 samples under each protocol and compared the yield and mean sequence coverage. The sequencing success rate and quality for the four different reactions were as follows: the full reaction was 98.2%, the one-tenth reaction was 98.0%, the full rapid reaction was 97.5% and the half-reaction, was 97.1%. As a result, uniformity of sequence quality indicated that libraries were not affected by the change in protocol. The cost of sequencing was reduced approximately seven-fold and the time taken to prepare the library was reduced from 6.5 hours to 3 hours. The sequencing results obtained using the miniaturised volumes showed comparability to the results obtained using full volumes as described by the manufacturer. The adaptation of the protocol represents a lower-cost, streamlined approach for SARS-CoV-2 sequencing, which can be used to produce genomic data quickly and more affordably, especially in resource-constrained settings.

Molecular Epidemiology and Diversity of SARS-CoV-2 in Ethiopia, 2020-2022.

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 ~7500 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 outcompeted by the Delta variant. Notably, Ethiopia's 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's role in SARS-CoV-2 dissemination and the urgent need for balanced, near-real-time genomic sequencing.

Genomic epidemiology of SARS-CoV-2 during the first four waves in Mozambique.

Mozambique reported the first case of coronavirus disease 2019 (COVID-19) in March 2020 and it has since spread to all provinces in the country. To investigate the introductions and spread of SARS-CoV-2 in Mozambique, 1 142 whole genome sequences sampled within Mozambique were phylogenetically analyzed against a globally representative set, reflecting the first 25 months of the epidemic. The epidemic in the country was marked by four waves of infection, the first associated with B.1 ancestral lineages, while the Beta, Delta, and Omicron Variants of Concern (VOCs) were responsible for most infections and deaths during the second, third, and fourth waves. Large-scale viral exchanges occurred during the latter three waves and were largely attributed to southern African origins. Not only did the country remain vulnerable to the introductions of new variants but these variants continued to evolve within the borders of the country. Due to the Mozambican health system already under constraint, and paucity of data in Mozambique, there is a need to continue to strengthen and support genomic surveillance in the country as VOCs and Variants of interests (VOIs) are often reported from the southern African region.

Metabarcoding analysis of marine zooplankton confirms the ecological role of a sheltered bight along an exposed continental shelf.

Zooplankton plays an essential role in marine ecosystems as the link between primary producers (phytoplankton) and higher trophic levels in food webs, and as a dynamic pool of recruits for invertebrates and fish. Zooplankton communities are diverse with a patchy distribution at different spatial scales, influenced by oceanographic processes. The continental shelf of eastern South Africa is narrow and exposed to the western-boundary Agulhas Current, with some shelter against strong directional flow provided by the broader KwaZulu-Natal Bight, a coastal offset adjacent to an estuary. We compared zooplankton species richness, diversity and relative abundance of key taxa among sheltered and exposed shelf areas using metabarcoding and community analysis, to explore the ecological role of the bight in a highly dynamic ocean region. Metabarcoding recovered higher richness and diversity at a finer resolution than could previously be achieved with traditional microscopy. Of 271 operational taxonomic units (OTUs) recovered through metabarcoding, 63% could be matched with >95% sequence similarity to reference barcodes. OTUs were dominated by malacostracan crustaceans (161 spp.), ray-finned fishes (45 spp.) and copepods (28 spp.). Species richness, diversity and the relative abundance of key taxa differed between sheltered and exposed shelf areas. Lower species richness in the bight was partly attributed to structurally homogeneous benthic habitats, and an associated reduction of meroplanktonic species originating from local benthic-pelagic exchange. High relative abundance of a ray-finned fish in the bight, as observed based on fish eggs and read counts, confirmed that the bight is an important fish spawning area. Overall, zooplankton metabarcoding outputs were congruent with findings of previous ecological research using more traditional methods of observation.

Antiretroviral therapy adherence patterns, virological suppression, and emergence of drug resistance: A nested case-control study from Uganda and South Africa.

BACKGROUND: Relationships between distinct antiretroviral therapy (ART) adherence patterns and risk of drug resistance are not well understood. METHODS: We conducted a nested case-control analysis within a longitudinal cohort study of individuals initiating efavirenz-based ART. Primary outcomes of interest, measured at 6 and 12 months after treatment initiation, were: 1) virologic suppression, 2) virologic failure with resistance, and 3) virologic failure without resistance. Our primary exposure of interest was ART adherence, measured over the 6 months before each visit with electronic pill monitors, and categorized in three ways: 1) 6 months average adherence; 2) running adherence, defined as the proportion of days with average adherence over 9 days of less than or equal to 10%, 20%, and 30%; and 3) number of 3-, 7-, and 28-day treatment gaps in the prior 6 months. RESULTS: We analyzed data from 166 individuals (107 had virologic failure during observation and 59 had virologic suppression at 6 and 12 months). Average adherence was higher among those with virologic suppression (median 83%, IQR 58-96%) versus those with virologic failure with resistance (median 35%, IQR 20-77%, pairwise P < 0.01) and those with virologic failure without resistance (median 21%, IQR 2-54%, pairwise P < 0.01). Although treatment gaps generally predicted virologic failure (P < 0.01), they did not differentiate failure with and without drug resistance (P > 0.6). CONCLUSIONS: Average adherence patterns, but not the assessed frequency of treatment gaps, differentiated failure with versus without drug resistance among individuals initiating efavirenz-based ART. Future work should explore adherence-resistance relationships for integrase inhibitor-based regimens.

Omicron BA.4/BA.5 escape neutralizing immunity elicited by BA.1 infection.

SARS-CoV-2 Omicron (B.1.1.529) BA.4 and BA.5 sub-lineages, first detected in South Africa, have changes relative to Omicron BA.1 including substitutions in the spike receptor binding domain. Here we isolated live BA.4 and BA.5 viruses and measured BA.4/BA.5 neutralization elicited by BA.1 infection either in the absence or presence of previous vaccination as well as from vaccination without BA.1 infection. In BA.1-infected unvaccinated individuals, neutralization relative to BA.1 declines 7.6-fold for BA.4 and 7.5-fold for BA.5. In vaccinated individuals with subsequent BA.1 infection, neutralization relative to BA.1 decreases 3.2-fold for BA.4 and 2.6-fold for BA.5. The fold-drop versus ancestral virus neutralization in this group is 4.0-fold for BA.1, 12.9-fold for BA.4, and 10.3-fold for BA.5. In contrast, BA.4/BA.5 escape is similar to BA.1 in the absence of BA.1 elicited immunity: fold-drop relative to ancestral virus neutralization is 19.8-fold for BA.1, 19.6-fold for BA.4, and 20.9-fold for BA.5. These results show considerable escape of BA.4/BA.5 from BA.1 elicited immunity which is moderated with vaccination and may indicate that BA.4/BA.5 may have the strongest selective advantage in evading neutralization relative to BA.1 in unvaccinated, BA.1 infected individuals.

Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa.

Three lineages (BA.1, BA.2 and BA.3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern predominantly drove South Africa's fourth Coronavirus Disease 2019 (COVID-19) wave. We have now identified two new lineages, BA.4 and BA.5, responsible for a fifth wave of infections. The spike proteins of BA.4 and BA.5 are identical, and similar to BA.2 except for the addition of 69-70 deletion (present in the Alpha variant and the BA.1 lineage), L452R (present in the Delta variant), F486V and the wild-type amino acid at Q493. The two lineages differ only outside of the spike region. The 69-70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure, on the background of variants not possessing this feature. BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa by the first week of April 2022. Using a multinomial logistic regression model, we estimated growth advantages for BA.4 and BA.5 of 0.08 (95% confidence interval (CI): 0.08-0.09) and 0.10 (95% CI: 0.09-0.11) per day, respectively, over BA.2 in South Africa. The continued discovery of genetically diverse Omicron lineages points to the hypothesis that a discrete reservoir, such as human chronic infections and/or animal hosts, is potentially contributing to further evolution and dispersal of the virus.

Comparison of SARS-CoV-2 sequencing using the ONT GridION and the Illumina MiSeq.

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 SARS-CoV-2 consensus genomes obtained from 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 consensus genomes classified by Nextclade as good and mediocre compared to the GridION. The MiSeq also had a significantly higher genome coverage and mutation counts than the GridION. CONCLUSION: Due to the low genome coverage, high number of indels, and sensitivity to SARS-CoV-2 viral load noted with the GridION when compared to MiSeq, we can conclude that the MiSeq is more favourable for SARS-CoV-2 genomic surveillance, as successful genomic surveillance is dependent on high quality, near-whole consensus genomes.

Identification of SARS-CoV-2 Omicron variant using spike gene target failure and genotyping assays, Gauteng, South Africa, 2021.

The circulation of Omicron BA.1 led to the rapid increase in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases in South Africa in November 2021, which warranted the use of more rapid detection methods. We, therefore, assessed the ability to detect Omicron BA.1 using genotyping assays to identify specific mutations in SARS-CoV-2 positive samples, Gauteng province, South Africa. The TaqPath™ COVID-19 real-time polymerase chain reaction assay was performed on all samples selected to identify spike gene target failure (SGTF). SARS-CoV-2 genotyping assays were used for the detection of del69/70 and K417N mutation. Whole-genome sequencing was performed on a subset of genotyped samples to confirm these findings. Of the positive samples received, 11.0% (175/1589) were randomly selected to assess if SGTF and genotyping assays, that detect del69/70 and K417N mutations, could identify Omicron BA.1. We identified SGTF in 98.9% (173/175) of samples, of which 88.0% (154/175) had both the del69/70 and K417N mutation. The genotyped samples (45.7%; 80/175) that were sequenced confirmed Omicron BA.1 (97.5%; 78/80). Our data show that genotyping for the detection of the del69/70 and K417N coupled with SGTF is efficient to exclude Alpha and Beta variants and rapidly detect Omicron BA.1. However, we still require assays for the detection of unique mutations that will allow for the differentiation between other Omicron sublineages. Therefore, the use of genotyping assays to detect new dominant or emerging lineages of SARS-CoV-2 will be beneficial in limited-resource settings.

Emergence and phenotypic characterization of the global SARS-CoV-2 C.1.2 lineage.

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 initial C.1.2 detection is associated with a high substitution rate, and includes changes within the spike protein that have been associated with increased transmissibility or reduced neutralization sensitivity in SARS-CoV-2 variants of concern or variants of interest. 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 show 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.

Rapid Replacement of SARS-CoV-2 Variants by Delta and Subsequent Arrival of Omicron, Uganda, 2021.

Genomic surveillance in Uganda showed rapid replacement of severe acute respiratory syndrome coronavirus 2 over time by variants, dominated by Delta. However, detection of the more transmissible Omicron variant among travelers and increasing community transmission highlight the need for near-real-time genomic surveillance and adherence to infection control measures to prevent future pandemic waves.