SARS-CoV-2 infection in immunosuppression evolves sub-lineages which independently accumulate neutralization escape mutations.

One mechanism of variant formation may be evolution during long-term infection in immunosuppressed people. To understand the viral phenotypes evolved during such infection, we tested SARS-CoV-2 viruses evolved from an ancestral B.1 lineage infection lasting over 190 days post-diagnosis in an advanced HIV disease immunosuppressed individual. Sequence and phylogenetic analysis showed two evolving sub-lineages, with the second sub-lineage replacing the first sub-lineage in a seeming evolutionary sweep. Each sub-lineage independently evolved escape from neutralizing antibodies. The most evolved virus for the first sub-lineage (isolated day 34) and the second sub-lineage (isolated day 190) showed similar escape from ancestral SARS-CoV-2 and Delta-variant infection elicited neutralizing immunity despite having no spike mutations in common relative to the B.1 lineage. The day 190 isolate also evolved higher cell-cell fusion and faster viral replication and caused more cell death relative to virus isolated soon after diagnosis, though cell death was similar to day 34 first sub-lineage virus. These data show that SARS-CoV-2 strains in prolonged infection in a single individual can follow independent evolutionary trajectories which lead to neutralization escape and other changes in viral properties.

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.

Will climate change amplify epidemics and give rise to pandemics?

While the world recovers from the COVID-19 pandemic, another crisis continues to spiral at a much faster speed than was expected. Climate change is dominating our lives and causing a high level of distress. Countries all over the world are struggling to survive the damage caused by extreme events. They are trying to control wildfires, rebuild roads and houses damaged by floods, and learn to survive in a hotter and more dangerous world. However, there is also a new threat that is being overlooked-the interaction between climate change and infectious diseases. A comprehensive meta-analysis revealed that climate change could aggravate more than 50% of known human pathogens. Unfortunately, this is happening now.

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 [...].

Rapid Epidemic Expansion of Chikungunya Virus East/Central/South African Lineage, Paraguay.

The spread of Chikungunya virus is a major public health concern in the Americas. There were >120,000 cases and 51 deaths in 2023, of which 46 occurred in Paraguay. Using a suite of genomic, phylodynamic, and epidemiologic techniques, we characterized the ongoing large chikungunya epidemic in Paraguay.

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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) now arise in the context of heterogeneous human connectivity and population immunity. Through a large-scale phylodynamic analysis of 115,622 Omicron BA.1 genomes, we identified >6,000 introductions of the antigenically distinct VOC into England and analyzed their local transmission and dispersal history. We find that six of the eight largest English Omicron lineages were already transmitting when Omicron was first reported in southern Africa (22 November 2021). Multiple datasets show that importation of Omicron continued despite subsequent restrictions on travel from southern Africa as a result of export from well-connected secondary locations. Initiation and dispersal of Omicron transmission lineages in England was a two-stage process that can be explained by models of the country's human geography and hierarchical travel network. Our results enable a comparison of the processes that drive the invasion of Omicron and other VOCs across multiple spatial scales.

Dispersal patterns and influence of air travel during the global expansion of SARS-CoV-2 variants of concern.

The Alpha, Beta, and Gamma SARS-CoV-2 variants of concern (VOCs) co-circulated globally during 2020 and 2021, fueling waves of infections. They were displaced by Delta during a third wave worldwide in 2021, which, in turn, was displaced by Omicron in late 2021. In this study, we use phylogenetic and phylogeographic methods to reconstruct the dispersal patterns of VOCs worldwide. We find that source-sink dynamics varied substantially by VOC and identify countries that acted as global and regional hubs of dissemination. We demonstrate the declining role of presumed origin countries of VOCs in their global dispersal, estimating that India contributed <15% of Delta exports and South Africa <1%-2% of Omicron dispersal. We estimate that >80 countries had received introductions of Omicron within 100 days of its emergence, associated with accelerated passenger air travel and higher transmissibility. Our study highlights the rapid dispersal of highly transmissible variants, with implications for genomic surveillance along the hierarchical airline network.

Rapid epidemic expansion of chikungunya virus-ECSA lineage in Paraguay.

The spread of vector-borne viruses, such as CHIKV, is a significant public health concern in the Americas, with over 120,000 cases and 51 deaths in 2023, of which 46 occurred in Paraguay. Using a suite of genomic, phylodynamic, and epidemiological techniques, we characterized the ongoing large CHIKV epidemic in Paraguay. Article Summary Line: Genomic and epidemiological characterization of the ongoing Chikungunya virus epidemic in Paraguay.

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.

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.

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.

Persistent Severe Acute Respiratory Syndrome Coronavirus 2 Infection With accumulation of mutations in a patient with poorly controlled Human Immunodeficiency Virus infection.

A 22-year-old woman with uncontrolled advanced human immunodeficiency virus (HIV) infection was persistently infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beta variant for 9 months, the virus accumulating >20 additional mutations. Antiretroviral therapy suppressed HIV and cleared SARS-CoV-2 within 6 to 9 weeks. Increased vigilance is warranted to benefit affected individuals and prevent the emergence of novel SARS-CoV-2 variants.