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.

The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma.

SARS-CoV-2 variants of concern (VOC) have arisen independently at multiple locations1,2 and may reduce the efficacy of current vaccines that target the spike glycoprotein of SARS-CoV-23. Here, using a live-virus neutralization assay, we compared the neutralization of a non-VOC variant with the 501Y.V2 VOC (also known as B.1.351) using plasma collected from adults who were hospitalized with COVID-19 during the two waves of infection in South Africa, the second wave of which was dominated by infections with the 501Y.V2 variant. Sequencing demonstrated that infections of plasma donors from the first wave were with viruses that did not contain the mutations associated with 501Y.V2, except for one infection that contained the E484K substitution in the receptor-binding domain. The 501Y.V2 virus variant was effectively neutralized by plasma from individuals who were infected during the second wave. The first-wave virus variant was effectively neutralized by plasma from first-wave infections. However, the 501Y.V2 variant was poorly cross-neutralized by plasma from individuals with first-wave infections; the efficacy was reduced by 15.1-fold relative to neutralization of 501Y.V2 by plasma from individuals infected in the second wave. By contrast, cross-neutralization of first-wave virus variants using plasma from individuals with second-wave infections was more effective, showing only a 2.3-fold decrease relative to neutralization of first-wave virus variants by plasma from individuals infected in the first wave. Although we tested only one plasma sample from an individual infected with a SARS-CoV-2 variant with only the E484K substitution, this plasma sample potently neutralized both variants. The observed effective neutralization of first-wave virus by plasma from individuals infected with 501Y.V2 provides preliminary evidence that vaccines based on VOC sequences could retain activity against other circulating SARS-CoV-2 lineages.

Detection of a SARS-CoV-2 variant of concern in South Africa.

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus1,2. Here we describe a newly arisen lineage of SARS-CoV-2 (designated 501Y.V2; also known as B.1.351 or 20H) that is defined by eight mutations in the spike protein, including three substitutions (K417N, E484K and N501Y) at residues in its receptor-binding domain that may have functional importance3-5. This lineage was identified in South Africa after the first wave of the epidemic in a severely affected metropolitan area (Nelson Mandela Bay) that is located on the coast of the Eastern Cape province. This lineage spread rapidly, and became dominant in Eastern Cape, Western Cape and KwaZulu-Natal provinces within weeks. Although the full import of the mutations is yet to be determined, the genomic data-which show rapid expansion and displacement of other lineages in several regions-suggest that this lineage is associated with a selection advantage that most plausibly results from increased transmissibility or immune escape6-8.

Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.

We propose a novel, non-discriminatory classification of monkeypox virus diversity. Together with the World Health Organization, we named three clades (I, IIa and IIb) in order of detection. Within IIb, the cause of the current global outbreak, we identified multiple lineages (A.1, A.2, A.1.1 and B.1) to support real-time genomic surveillance.

Characterization of HIV variants from paired Cerebrospinal fluid and Plasma samples in primary microglia and CD4+ T-cells.

Despite antiretroviral therapy (ART), HIV persistence in the central nervous system (CNS) continues to cause a range of cognitive impairments in people living with HIV (PLWH). Upon disease progression, transmigrating CCR5-using T-cell tropic viruses are hypothesized to evolve into macrophage-tropic viruses in the CNS that can efficiently infect low CD4-expressing cells, such as microglia. We examined HIV-1 RNA concentration, co-receptor usage, and CSF compartmentalization in paired CSF and blood samples from 19 adults not on treatment. Full-length envelope CSF- and plasma-derived reporter viruses were generated from 3 subjects and phenotypically characterized in human primary CD4+ T-cells and primary microglia. Median HIV RNA levels were higher in plasma than in CSF (5.01 vs. 4.12 log10 cp/mL; p = 0.004), and coreceptor usage was mostly concordant for CCR5 across the paired samples (n = 17). Genetically compartmentalized CSF viral populations were detected in 2 subjects, one with and one without neurological symptoms. All viral clones could replicate in T-cells (R5 T cell-tropic). In addition, 3 CSF and 1 plasma patient-derived viral clones also had the capacity to replicate in microglia/macrophages and, therefore have an intermediate macrophage tropic phenotype. Overall, with this study, we demonstrate that in a subset of PLWH, plasma-derived viruses undergo genetic and phenotypic evolution within the CNS, indicating viral infection and replication in CNS cells. It remains to be studied whether the intermediate macrophage-tropic phenotype observed in primary microglia represents a midpoint in the evolution towards a macrophage-tropic phenotype that can efficiently replicate in microglial cells and propagate viral infection in the CNS.

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.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous 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 (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) 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 overall 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.

Subregional origins of emerging SARS-CoV-2 variants during the second pandemic wave in Côte d'Ivoire.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility, virulence and immune escape abilities have heavily altered the COVID-19 pandemic's course. Deciphering local and global transmission patterns of those variants is thus key in building a profound understanding of the virus' spread around the globe. In the present study, we investigate SARS-CoV-2 variant epidemiology in Côte d'Ivoire, Western sub-Saharan Africa. We therefore generated 234 full SARS-CoV-2 genomes stemming from Central and Northern Côte d'Ivoire. Covering the first and second pandemic wave the country had been facing, we identified 20 viral lineages and showed that in Côte d'Ivoire the second pandemic wave in 2021 was driven by the spread of the Alpha (B.1.1.7) and Eta (B.1.525) variant. Our analyses are consistent with a limited number of international introductions of Alpha and Eta into Côte d'Ivoire, and those introduction events mostly stemmed from within the West African subregion. This suggests that subregional travel to Côte d'Ivoire had more impact on local pandemic waves than direct intercontinental travel.

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.

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.

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.

High rate of occult hepatitis B virus infection in hemodialysis units of KwaZulu-Natal, South Africa.

Occult hepatitis B virus (HBV) infection (OBI) is defined as the presence of HBV DNA in the liver with or without detectable HBV DNA in the serum of individuals testing HBV surface antigen (HBsAg) negative using currently available assays. The prevalence of OBI among patients receiving hemodialysis (HD) treatment remains poorly characterized in South Africa despite the high prevalence of HBV. We sought to determine the prevalence of OBI in HD units in tertiary hospitals of KwaZulu-Natal and to characterize the HBV S gene mutations potentially responsible for OBI. A cross-sectional descriptive study of residual diagnostic plasma samples from 85 HBsAg-negative patients receiving HD treatment was included. The PreS/S gene was amplified with a nested HBV polymerase chain reaction for downstream next-generation sequencing, to determine the viral genotype and identify S gene mutations associated with OBI. Nine of the 85 samples had OBI, based on detectable HBV DNA. The point prevalence of OBI was 10.6% (95% control interval: 5.5%-19.1%). Phylogenetic analysis of the samples with OBI showed that all belonged to genotype A. Three (~33%) samples had mutations in the major hydrophilic region (MHR) within the S gene, three (~33%) had mutations within the S gene but outside the MHR, whilst the remaining three had no mutations observed. The prevalence of OBI in HBsAg-negative patients undergoing HD was 10.6%, suggesting that OBI is a clinically significant problem in patients with HD in this region. The screening methods for HBV infection need to be revised to include nucleic acid testing.

Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo.

As the epidemiological epicenter of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. The env gp41 region was sequenced and characterized for 172 HIV-positive specimens. The env sequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs; 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next-generation sequencing (NGS) by a modified HIV-switching mechanism at the 5' end of the RNA template (SMART) method to obtain full-genome sequences. NGS produced 14 new complete genomes, which included pure subtype C (n = 2), D (n = 1), F1 (n = 1), H (n = 3), and J (n = 1) genomes. The two subtype C genomes and one of the subtype H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including subtypes A1, F, G, H, J, and K and unclassified fragments, including one subtype CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant subtype H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full-genome and subgenomic sequences identified in our study population significantly increase the documented diversity of the strains involved in the continually evolving HIV-1 pandemic.IMPORTANCE Very little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin, where HIV-1 expanded early in the global pandemic. By sequencing a subgenomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences that we recovered suggests that these strains are more closely related to ancestral HIV-1 strains than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic.

High Rates of Transmission of Drug-resistant HIV in Aruba Resulting in Reduced Susceptibility to the WHO Recommended First-line Regimen in Nearly Half of Newly Diagnosed HIV-infected Patients.

Background: In Western countries emergence of human immunodeficiency virus (HIV) drug resistance has tremendously decreased, and transmission of drug resistance has merely stabilized in recent years. However, in many endemic settings with limited resources rates of emerging and transmitted drug resistance are not regularly assessed. Methods: We performed a survey including all HIV-infected individuals who received resistance testing in 2010-2015 in Aruba, a highly endemic HIV area in the Caribbean. Transmitted HIV drug resistance was determined using World Health Organization (WHO) criteria. Transmission dynamics were investigated using phylogenetic analyses. In a subset, baseline samples were re-analyzed using next generation sequencing (NGS). Results: Baseline resistance testing was performed in 104 newly diagnosed untreated individuals (54% of all newly diagnosed individuals in 2010-2015): 86% were men, 39% were foreign-born, and 22% had AIDS at diagnosis. And 33% (95% CI: 24-42%) was infected with a drug-resistant HIV variant. The prevalence of resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) reached 45% (95% CI: 27-64%) in 2015, all based on the prevalence of mutation K103N. NGS did not demonstrate additional minority K103N-variants compared to routine resistance testing. K103N-harboring strains were introduced into the therapy-unexposed population via at least 6 independent transmissions epidemiologically linked to the surrounding countries. Virological failure of the WHO-recommended first-line NNRTI-based regimen was higher in the presence of K103N. Conclusions: The prevalence of resistant HIV in Aruba has increased to alarming levels, compromising the WHO-recommended first-line regimen. As adequate surveillance as advocated by the WHO is limited, the Caribbean region could face an unidentified rise of NNRTI-resistant HIV.

Virologic Nonsuppression and HIV Drug Resistance Among People Who Inject Drugs and Their Sexual and Injecting Partners in Kenya.

We evaluated the prevalence and correlates of HIV viral nonsuppression and HIV drug resistance (HIV-DR) in a cohort of people who inject drugs living with HIV (PWID-LH) and their sexual and injecting partners living with HIV in Kenya. HIV-DR testing was performed on participants with viral nonsuppression. Of 859 PWID-LH and their partners, 623 (72.5%) were on antiretroviral therapy (ART) ≥4 months and 148/623 (23.8%) were not virally suppressed. Viral nonsuppression was more common among younger participants and those on ART for a shorter duration. Among 122/148 (82.4%) successfully sequenced samples, 55 (45.1%) had detectable major HIV-DR mutations, mainly to non-nucleoside and nucleotide reverse transcriptase inhibitors (NNRTI and NRTI). High levels of HIV-DR among those with viral nonsuppression suggests need for viral load monitoring, adherence counseling, and timely switching to alternate ART regimens in this key population.

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.

Consequences of rpoB mutations missed by the GenoType MTBDRplus assay in a programmatic setting in South Africa.

Background: Rifampicin resistance missed by commercial rapid molecular assays but detected by phenotypic assays may lead to discordant susceptibility results and affect patient management. Objective: This study was conducted to evaluate the causes of rifampicin resistance missed by the GenoType MTBDRplus and its impact on the programmatic management of tuberculosis in KwaZulu-Natal, South Africa. Methods: We analysed routine tuberculosis programme data from January 2014 to December 2014 on isolates showing rifampicin susceptibility on the GenoType MTBDRplus assay but resistance on the phenotypic agar proportion method. Whole-genome sequencing was performed on a subset of these isolates. Results: Out of 505 patients with isoniazid mono-resistant tuberculosis on the MTBDRplus, 145 (28.7%) isolates showed both isoniazid and rifampicin resistance on the phenotypic assay. The mean time from MTBDRplus results to initiation of drug-resistant tuberculosis therapy was 93.7 days. 65.7% of the patients had received previous tuberculosis treatment. The most common mutations detected in the 36 sequenced isolates were I491F (16; 44.4%) and L452P (12; 33.3%). Among the 36 isolates, resistance to other anti-tuberculosis drugs was 69.4% for pyrazinamide, 83.3% for ethambutol, 69.4% for streptomycin, and 50% for ethionamide. Conclusion: Missed rifampicin resistance was mostly due to the I491F mutation located outside the MTBDRplus detection area and the L452P mutation, which was not included in the initial version 2 of the MTBDRplus. This led to substantial delays in the initiation of appropriate therapy. The previous tuberculosis treatment history and the high level of resistance to other anti-tuberculosis drugs suggest an accumulation of resistance.