Emergence of the B.1.214.2 SARS-CoV-2 lineage with an Omicron-like spike insertion and a unique upper airway immune signature.

We investigate the emergence, mutation profile, and dissemination of SARS-CoV-2 lineage B.1.214.2, first identified in Belgium in January 2021. This variant, featuring a 3-amino acid insertion in the spike protein similar to the Omicron variant, was speculated to enhance transmissibility or immune evasion. Initially detected in international travelers, it substantially transmitted in Central Africa, Belgium, Switzerland, and France, peaking in April 2021. Our travel-aware phylogeographic analysis, incorporating travel history, estimated the origin to the Republic of the Congo, with primary European entry through France and Belgium, and multiple smaller introductions during the epidemic. We correlate its spread with human travel patterns and air passenger data. Further, upon reviewing national reports of SARS-CoV-2 outbreaks in Belgian nursing homes, we found this strain caused moderately severe outcomes (8.7% case fatality ratio). A distinct nasopharyngeal immune response was observed in elderly patients, characterized by 80% unique signatures, higher B- and T-cell activation, increased type I IFN signaling, and reduced NK, Th17, and complement system activation, compared to similar outbreaks. This unique immune response may explain the variant's epidemiological behavior and underscores the need for nasal vaccine strategies against emerging variants.

Clade I mpox virus genomic diversity in the Democratic Republic of the Congo, 2018-2024: Predominance of zoonotic transmission.

Recent reports raise concerns on the changing epidemiology of mpox in the Democratic Republic of the Congo (DRC). High-quality genomes were generated for 337 patients from 14/26 provinces to document whether the increase in number of cases is due to zoonotic spillover events or viral evolution, with enrichment of APOBEC3 mutations linked to human adaptation. Our study highlights two patterns of transmission contributing to the source of human cases. All new sequences from the eastern South Kivu province (n = 17; 4.8%) corresponded to the recently described clade Ib, associated with sexual contact and sustained human-to-human transmission. By contrast, all other genomes are clade Ia, which exhibits high genetic diversity with low numbers of APOBEC3 mutations compared with clade Ib, suggesting multiple zoonotic introductions. The presence of multiple clade I variants in urban areas highlights the need for coordinated international response efforts and more studies on the transmission and the reservoir of mpox.

First imported Cases of MPXV Clade Ib in Goma, Democratic Republic of the Congo: Implications for Global Surveillance and Transmission Dynamics.

The ongoing national mpox outbreak in the Democratic Republic of the Congo has resulted in more >30,000 suspected cases in the country from January 2023 to August 2024. While these historic case totals have been driven by primarily by zoonosis, the emergence of Clade Ib monkeypox virus (MPXV), which is connected to more sustained human-to-human transmission, has been associated with increasing public health impacts in eastern DRC. First identified in South Kivu province, Clade Ib MPXV has been identified in multiple non-endemic East African countries for the first time. In DRC, there have been concerns over broader Clade Ib expansion in the country that could further complicate containment and mitigation responses. Here, we report the first introductions of Clade Ib into North Kivu province, including within internal displacement camps, with suspected close contact transmission that includes non-intimate contacts and children. These findings demonstrate that mpox case investigations and community messaging campaigns should include considerations for non-sexual contact-mediated transmission of Clade Ib that includes children <15 years.

Co-circulation of monkeypox virus subclades Ia and Ib in Kinshasa Province, Democratic Republic of the Congo, July to August 2024.

Between January and August 2024, mpox cases have been reported in nearly all provinces of the Democratic Republic of the Congo (DRC). Monkeypox virus genome sequences were obtained from 11 mpox cases' samples, collected in July-August 2024 in several health zones of Kinshasa. Characterisation of the sequences showed subclades Ia and Ib co-circulating in the Limete health zone, while phylogenetic analyses suggested multiple introductions of the two subclades in Kinshasa. This illustrates the growing complexity of Clade I mpox outbreaks in DRC.

Publication-ready single nucleotide polymorphism visualization with snipit.

SUMMARY: Snipit is an analysis and visualization tool designed for summarizing single nucleotide polymorphisms in sequences in comparison to a reference sequence. This tool efficiently catalogues nucleotide and amino acid differences, enabling clear comparisons through customizable, publication-ready figures. With features such as configurable colour palettes, customizable record sorting, and the ability to output figures in multiple formats, snipit offers a user-friendly interface for researchers across diverse disciplines. In addition, snipit includes a specialized recombi-mode for illustrating recombination patterns, which can highlight otherwise often difficult-to-detect relationships between sequences. AVAILABILITY AND IMPLEMENTATION: Snipit is an open-source python-based tool that is hosted on GitHub under a GNU-GPL 3.0 licence (https://github.com/aineniamh/snipit). It can be installed from PyPi using pip. Source code and additional documentation can be found on the GitHub repository.

Molecular epidemiology of recurrent zoonotic transmission of mpox virus in West Africa.

Nigeria and Cameroon reported their first mpox cases in over three decades in 2017 and 2018 respectively. The outbreak in Nigeria is recognised as an ongoing human epidemic. However, owing to sparse surveillance and genomic data, it is not known whether the increase in cases in Cameroon is driven by zoonotic or sustained human transmission. Notably, the frequency of zoonotic transmission remains unknown in both Cameroon and Nigeria. To address these uncertainties, we investigated the zoonotic transmission dynamics of the mpox virus (MPXV) in Cameroon and Nigeria, with a particular focus on the border regions. We show that in these regions mpox cases are still driven by zoonotic transmission of a newly identified Clade IIb.1. We identify two distinct zoonotic lineages that circulate across the Nigeria-Cameroon border, with evidence of recent and historic cross border dissemination. Our findings support that the complex cross-border forest ecosystems likely hosts shared animal populations that drive cross-border viral spread, which is likely where extant Clade IIb originated. We identify that the closest zoonotic outgroup to the human epidemic circulated in southern Nigeria in October 2013. We also show that the zoonotic precursor lineage circulated in an animal population in southern Nigeria for more than 45 years. This supports findings that southern Nigeria was the origin of the human epidemic. Our study highlights the ongoing MPXV zoonotic transmission in Cameroon and Nigeria, underscoring the continuous risk of MPXV (re)emergence.

Genomic epidemiology uncovers the timing and origin of the emergence of mpox in humans.

Five years before the 2022-2023 global mpox outbreak Nigeria reported its first cases in nearly 40 years, with the ongoing epidemic since driven by sustained human-to-human transmission. However, limited genomic data has left questions about the timing and origin of the mpox virus' (MPXV) emergence. Here we generated 112 MPXV genomes from Nigeria from 2021-2023. We identify the closest zoonotic outgroup to the human epidemic in southern Nigeria, and estimate that the lineage transmitting from human-to-human emerged around July 2014, circulating cryptically until detected in September 2017. The epidemic originated in Southern Nigeria, particularly Rivers State, which also acted as a persistent and dominant source of viral dissemination to other states. We show that APOBEC3 activity increased MPXV's evolutionary rate twenty-fold during human-to-human transmission. We also show how Delphy, a tool for near-real-time Bayesian phylogenetics, can aid rapid outbreak analytics. Our study sheds light on MPXV's establishment in West Africa before the 2022-2023 global outbreak and highlights the need for improved pathogen surveillance and response.

Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo.

Outbreaks of monkeypox (mpox) have historically resulted from zoonotic spillover of clade I monkeypox virus (MPXV) in Central Africa and clade II MPXV in West Africa. In 2022, subclade IIb caused a global epidemic linked to transmission through sexual contact. Here we describe the epidemiological and genomic features of an mpox outbreak in a mining region in eastern Democratic Republic of the Congo, caused by clade I MPXV. Surveillance data collected between September 2023 and January 2024 identified 241 suspected cases. Genomic analysis demonstrates a distinct clade I lineage divergent from previously circulating strains in the Democratic Republic of the Congo. Of the 108 polymerase chain reaction-confirmed mpox cases, the median age of individuals was 22 years, 51.9% were female and 29% were sex workers, suggesting a potential role for sexual transmission. The predominance of APOBEC3-type mutations and the estimated emergence time around mid-September 2023 imply recent sustained human-to-human transmission.

Epigenetic age estimation of wild mice using faecal samples.

Age is a key parameter in population ecology, with a myriad of biological processes changing with age as organisms develop in early life then later senesce. As age is often hard to accurately measure with non-lethal methods, epigenetic methods of age estimation (epigenetic clocks) have become a popular tool in animal ecology and are often developed or calibrated using captive animals of known age. However, studies typically rely on invasive blood or tissue samples, which limit their application in more sensitive or elusive species. Moreover, few studies have directly assessed how methylation patterns and epigenetic age estimates compare across environmental contexts (e.g. captive or laboratory-based vs. wild animals). Here, we built a targeted epigenetic clock from laboratory house mice (strain C57BL/6, Mus musculus) using DNA from non-invasive faecal samples, and then used it to estimate age in a population of wild mice (Mus musculus domesticus) of unknown age. This laboratory mouse-derived epigenetic clock accurately predicted adult wild mice to be older than juveniles and showed that wild mice typically increased in epigenetic age over time, but with wide variation in epigenetic ageing rate among individuals. Our results also suggested that, for a given body mass, wild mice had higher methylation across targeted CpG sites than laboratory mice (and consistently higher epigenetic age estimates as a result), even among the smallest, juvenile mice. This suggests wild and laboratory mice may display different CpG methylation levels from very early in life and indicates caution is needed when developing epigenetic clocks on laboratory animals and applying them in the wild.

Automated detection and classification of polioviruses from nanopore sequencing reads using piranha.

Widespread surveillance, rapid detection, and appropriate intervention will be critical for successful eradication of poliovirus. Using deployable next-generation sequencing (NGS) approaches, such as Oxford Nanopore Technologies' MinION, the time from sample to result can be significantly reduced compared to cell culture and Sanger sequencing. We developed piranha (poliovirus investigation resource automating nanopore haplotype analysis), a 'sequencing reads-to-report' solution to aid routine poliovirus testing of both stool and environmental samples and alleviate the bioinformatic bottleneck that often exists for laboratories adopting novel NGS approaches. Piranha can be used for efficient intratypic differentiation of poliovirus serotypes, for classification of Sabin-like polioviruses, and for detection of wild-type and vaccine-derived polioviruses. It produces interactive, distributable reports, as well as summary comma-separated values files and consensus poliovirus FASTA sequences. Piranha optionally provides phylogenetic analysis, with the ability to incorporate a local database, processing from raw sequencing reads to an interactive, annotated phylogeny in a single step. The reports describe each nanopore sequencing run with interpretable plots, enabling researchers to easily detect the presence of poliovirus in samples and quickly disseminate their results. Poliovirus eradication efforts are hindered by the lack of real-time detection and reporting, and piranha can be used to complement direct detection sequencing approaches.

SARS-CoV-2 lineage assignments using phylogenetic placement/UShER are superior to pangoLEARN machine-learning method.

With the rapid spread and evolution of SARS-CoV-2, the ability to monitor its transmission and distinguish among viral lineages is critical for pandemic response efforts. The most commonly used software for the lineage assignment of newly isolated SARS-CoV-2 genomes is pangolin, which offers two methods of assignment, pangoLEARN and pUShER. PangoLEARN rapidly assigns lineages using a machine-learning algorithm, while pUShER performs a phylogenetic placement to identify the lineage corresponding to a newly sequenced genome. In a preliminary study, we observed that pangoLEARN (decision tree model), while substantially faster than pUShER, offered less consistency across different versions of pangolin v3. Here, we expand upon this analysis to include v3 and v4 of pangolin, which moved the default algorithm for lineage assignment from pangoLEARN in v3 to pUShER in v4, and perform a thorough analysis confirming that pUShER is not only more stable across versions but also more accurate. Our findings suggest that future lineage assignment algorithms for various pathogens should consider the value of phylogenetic placement.

A framework for automated scalable designation of viral pathogen lineages from genomic data.

Pathogen lineage nomenclature systems are a key component of effective communication and collaboration for researchers and public health workers. Since February 2021, the Pango dynamic lineage nomenclature for SARS-CoV-2 has been sustained by crowdsourced lineage proposals as new isolates were sequenced. This approach is vulnerable to time-critical delays as well as regional and personal bias. Here we developed a simple heuristic approach for dividing phylogenetic trees into lineages, including the prioritization of key mutations or genes. Our implementation is efficient on extremely large phylogenetic trees consisting of millions of sequences and produces similar results to existing manually curated lineage designations when applied to SARS-CoV-2 and other viruses including chikungunya virus, Venezuelan equine encephalitis virus complex and Zika virus. This method offers a simple, automated and consistent approach to pathogen nomenclature that can assist researchers in developing and maintaining phylogeny-based classifications in the face of ever-increasing genomic datasets.

APOBEC3 deaminase editing in mpox virus as evidence for sustained human transmission since at least 2016.

Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.

SARS-CoV-2 evolution in the Omicron era.

Since SARS-CoV-2 BA.5 (Omicron) emerged and spread in 2022, Omicron lineages have markedly diversified. Here we review the evolutionary trajectories and processes that underpin the emergence of these lineages, and identify the most prevalent sublineages. We discuss the potential origins of second-generation BA.2 lineages. Simple and complex recombination, antigenic drift and convergent evolution have enabled SARS-CoV-2 to accumulate mutations that alter its antigenicity. We also discuss the potential evolutionary trajectories of SARS-CoV-2 in the future.

Correction: Implementation of Genomic Surveillance of SARS-CoV-2 in the Caribbean: Lessons learned for sustainability in resource-limited settings.

[This corrects the article DOI: 10.1371/journal.pgph.0001455.].

SCORPIO: a utility for defining and classifying mutation constellations of virus genomes.

SUMMARY: Scorpio provides a set of command line utilities for classifying, haplotyping, and defining constellations of mutations for an aligned set of genome sequences. It was developed to enable exploration and classification of variants of concern within the SARS-CoV-2 pandemic, but can be applied more generally to other species. AVAILABILITY AND IMPLEMENTATION: Scorpio is an open-source project distributed under the GNU GPL version 3 license. Source code and binaries are available at https://github.com/cov-lineages/scorpio, and binaries are also available from Bioconda. SARS-CoV-2 specific definitions can be installed as a separate dependency from https://github.com/cov-lineages/constellations.

Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study.

Timely detection of outbreaks is needed for poliovirus eradication, but gold standard detection in the Democratic Republic of the Congo takes 30 days (median). Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method. Here we report prospective testing of stool samples from suspected polio cases, and their contacts, in the Democratic Republic of the Congo between 10 August 2021 and 4 February 2022. DDNS detected polioviruses in 62/2,339 (2.7%) of samples, while gold standard combination of cell culture, quantitative PCR and Sanger sequencing detected polioviruses in 51/2,339 (2.2%) of the same samples. DDNS provided case confirmation in 7 days (median) in routine surveillance conditions. DDNS enabled confirmation of three serotype 2 circulating vaccine-derived poliovirus outbreaks 23 days (mean) earlier (range 6-30 days) than the gold standard method. The mean sequence similarity between sequences obtained by the two methods was 99.98%. Our data confirm the feasibility of implementing DDNS in a national poliovirus laboratory.

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.

Implementation of genomic surveillance of SARS-CoV-2 in the Caribbean: Lessons learned for sustainability in resource-limited settings.

The COVID-19 pandemic highlighted the importance of global genomic surveillance to monitor the emergence and spread of SARS-CoV-2 variants and inform public health decision-making. Until December 2020 there was minimal capacity for viral genomic surveillance in most Caribbean countries. To overcome this constraint, the COVID-19: Infectious disease Molecular epidemiology for PAthogen Control & Tracking (COVID-19 IMPACT) project was implemented to establish rapid SARS-CoV-2 whole genome nanopore sequencing at The University of the West Indies (UWI) in Trinidad and Tobago (T&T) and provide needed SARS-CoV-2 sequencing services for T&T and other Caribbean Public Health Agency Member States (CMS). Using the Oxford Nanopore Technologies MinION sequencing platform and ARTIC network sequencing protocols and bioinformatics pipeline, a total of 3610 SARS-CoV-2 positive RNA samples, received from 17 CMS, were sequenced in-situ during the period December 5th 2020 to December 31st 2021. Ninety-one Pango lineages, including those of five variants of concern (VOC), were identified. Genetic analysis revealed at least 260 introductions to the CMS from other global regions. For each of the 17 CMS, the percentage of reported COVID-19 cases sequenced by the COVID-19 IMPACT laboratory ranged from 0·02% to 3·80% (median = 1·12%). Sequences submitted to GISAID by our study represented 73·3% of all SARS-CoV-2 sequences from the 17 CMS available on the database up to December 31st 2021. Increased staffing, process and infrastructural improvement over the course of the project helped reduce turnaround times for reporting to originating institutions and sequence uploads to GISAID. Insights from our genomic surveillance network in the Caribbean region directly influenced non-pharmaceutical countermeasures in the CMS countries. However, limited availability of associated surveillance and clinical data made it challenging to contextualise the observed SARS-CoV-2 diversity and evolution, highlighting the need for development of infrastructure for collecting and integrating genomic sequencing data and sample-associated metadata.

Comparison of Eleven RNA Extraction Methods for Poliovirus Direct Molecular Detection in Stool Samples.

Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods (7 manual and 4 semiautomated) for poliovirus molecular detection using stool samples (n = 59) that had been previously identified as poliovirus positive by cell culture. To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant (P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time. IMPORTANCE Poliovirus, the causative agent of poliomyelitis, is a target of global eradication led by the World Health Organization since 1988. Direct molecular detection and genomic sequencing without virus propagation in cell culture is arguably a critical tool in the final stages of polio eradication. Efficient recovery of good-quality viral RNA from stool samples is a prerequisite for direct detection by nucleic acid amplification. We tested 11 nucleic acid extraction methods to identify those facilitating sensitive, fast, simple, and cost-effective extraction, with flexibility for manual and automated protocols considered. Several different PCR assays were used to compare the recovered viral RNA to test suitability for poliovirus direct molecular detection. Our findings highlight the importance of choosing a suitable RNA extraction protocol and provide useful information to diagnostic laboratories and researchers facing the choice of RNA extraction method for direct molecular virus detection from stool.