Tracking SARS-CoV-2 variants of concern in wastewater: an assessment of nine computational tools using simulated genomic data.

Wastewater-based surveillance (WBS) is an important epidemiological and public health tool for tracking pathogens across the scale of a building, neighbourhood, city, or region. WBS gained widespread adoption globally during the SARS-CoV-2 pandemic for estimating community infection levels by qPCR. Sequencing pathogen genes or genomes from wastewater adds information about pathogen genetic diversity, which can be used to identify viral lineages (including variants of concern) that are circulating in a local population. Capturing the genetic diversity by WBS sequencing is not trivial, as wastewater samples often contain a diverse mixture of viral lineages with real mutations and sequencing errors, which must be deconvoluted computationally from short sequencing reads. In this study we assess nine different computational tools that have recently been developed to address this challenge. We simulated 100 wastewater sequence samples consisting of SARS-CoV-2 BA.1, BA.2, and Delta lineages, in various mixtures, as well as a Delta-Omicron recombinant and a synthetic 'novel' lineage. Most tools performed well in identifying the true lineages present and estimating their relative abundances and were generally robust to variation in sequencing depth and read length. While many tools identified lineages present down to 1 % frequency, results were more reliable above a 5 % threshold. The presence of an unknown synthetic lineage, which represents an unclassified SARS-CoV-2 lineage, increases the error in relative abundance estimates of other lineages, but the magnitude of this effect was small for most tools. The tools also varied in how they labelled novel synthetic lineages and recombinants. While our simulated dataset represents just one of many possible use cases for these methods, we hope it helps users understand potential sources of error or bias in wastewater sequencing analysis and to appreciate the commonalities and differences across methods.

A Novel Tiled Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus (ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada.

Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important Solanaceae crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5' and 3' ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples.

Antiviral discovery in toxic cyanobacteria: Low hanging fruit in the age of pandemics.

The power of novel vaccination technologies and their rapid development were elucidated clearly during the COVID-19 pandemic. At the same time, it also became clear that there is an urgent need to discover and manufacture new antivirals that target emerging viral threats. Toxic species of cyanobacteria produce a range of bioactive compounds that makes them good candidates for drug discovery. Nevertheless, few studies demonstrate the antiviral potential of cyanobacteria. This is partly due to the lack of specific and simple protocols designed for the rapid detection of antiviral activity in cyanobacteria and partly because specialized facilities for work with pathogenic viruses are few and far between. We therefore developed an easy method for the screening of cyanobacterial cultures for antiviral activity and used our private culture collection of non-pathogenic virus isolates to show that antiviral activity is a prominent feature in the cyanobacterium Microcystis aeruginosa. In this proof-of-concept study, we show that M. aeruginosa extracts from three different cyanobacterial strains delay infection of diatom-infecting single-stranded DNA and single-stranded RNA viruses by up to 2 days. Our work shows the ease with which cyanobacteria from culture collections can be screened for antiviral activity and highlights the potential of cyanobacteria as an excellent source for the discovery of novel antiviral compounds, warranting further investigation.

Ubiquitous, B12-dependent virioplankton utilizing ribonucleotide-triphosphate reductase demonstrate interseasonal dynamics and associate with a diverse range of bacterial hosts in the pelagic ocean.

Through infection and lysis of their coexisting bacterial hosts, viruses impact the biogeochemical cycles sustaining globally significant pelagic oceanic ecosystems. Currently, little is known of the ecological interactions between lytic viruses and their bacterial hosts underlying these biogeochemical impacts at ecosystem scales. This study focused on populations of lytic viruses carrying the B12-dependent Class II monomeric ribonucleotide reductase (RNR) gene, ribonucleotide-triphosphate reductase (Class II RTPR), documenting seasonal changes in pelagic virioplankton and bacterioplankton using amplicon sequences of Class II RTPR and the 16S rRNA gene, respectively. Amplicon sequence libraries were analyzed using compositional data analysis tools that account for the compositional nature of these data. Both virio- and bacterioplankton communities responded to environmental changes typically seen across seasonal cycles as well as shorter term upwelling-downwelling events. Defining Class II RTPR-carrying viral populations according to major phylogenetic clades proved a more robust means of exploring virioplankton ecology than operational taxonomic units defined by percent sequence homology. Virioplankton Class II RTPR populations showed positive associations with a broad phylogenetic diversity of bacterioplankton including dominant taxa within pelagic oceanic ecosystems such as Prochlorococcus and SAR11. Temporal changes in Class II RTPR virioplankton, occurring as both free viruses and within infected cells, indicated possible viral-host pairs undergoing sustained infection and lysis cycles throughout the seasonal study. Phylogenetic relationships inferred from Class II RTPR sequences mirrored ecological patterns in virio- and bacterioplankton populations demonstrating possible genome to phenome associations for an essential viral replication gene.

Functional Profiling and Evolutionary Analysis of a Marine Microalgal Virus Pangenome.

Phycodnaviridae are large double-stranded DNA viruses, which facilitate studies of host-virus interactions and co-evolution due to their prominence in algal infection and their role in the life cycle of algal blooms. However, the genomic interpretation of these viruses is hampered by a lack of functional information, stemming from the surprising number of hypothetical genes of unknown function. It is also unclear how many of these genes are widely shared within the clade. Using one of the most extensively characterized genera, Coccolithovirus, as a case study, we combined pangenome analysis, multiple functional annotation tools, AlphaFold structural modeling, and literature analysis to compare the core and accessory pangenome and assess support for novel functional predictions. We determined that the Coccolithovirus pangenome shares 30% of its genes with all 14 strains, making up the core. Notably, 34% of its genes were found in at most three strains. Core genes were enriched in early expression based on a transcriptomic dataset of Coccolithovirus EhV-201 algal infection, were more likely to be similar to host proteins than the non-core set, and were more likely to be involved in vital functions such as replication, recombination, and repair. In addition, we generated and collated annotations for the EhV representative EhV-86 from 12 different annotation sources, building up information for 142 previously hypothetical and putative membrane proteins. AlphaFold was further able to predict structures for 204 EhV-86 proteins with a modelling accuracy of good-high. These functional clues, combined with generated AlphaFold structures, provide a foundational framework for the future characterization of this model genus (and other giant viruses) and a further look into the evolution of the Coccolithovirus proteome.

Temperate infection in a virus-host system previously known for virulent dynamics.

The blooming cosmopolitan coccolithophore Emiliania huxleyi and its viruses (EhVs) are a model for density-dependent virulent dynamics. EhVs commonly exhibit rapid viral reproduction and drive host death in high-density laboratory cultures and mesocosms that simulate blooms. Here we show that this system exhibits physiology-dependent temperate dynamics at environmentally relevant E. huxleyi host densities rather than virulent dynamics, with viruses switching from a long-term non-lethal temperate phase in healthy hosts to a lethal lytic stage as host cells become physiologically stressed. Using this system as a model for temperate infection dynamics, we present a template to diagnose temperate infection in other virus-host systems by integrating experimental, theoretical, and environmental approaches. Finding temperate dynamics in such an established virulent host-virus model system indicates that temperateness may be more pervasive than previously considered, and that the role of viruses in bloom formation and decline may be governed by host physiology rather than by host-virus densities.

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology.

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi-EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range.

The mutual interplay between calcification and coccolithovirus infection.

Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non-calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV-free, 'induced media'. Our findings show that calcification is a key component of the E. huxleyi-EhV arms race and an aspect that is critical both to the modelling of these host-virus interactions in the ocean and interpreting their impact on the global carbon cycle.

Dynamics of transparent exopolymer particle production and aggregation during viral infection of the coccolithophore, Emiliania huxleyi.

Emiliania huxleyi produces calcium carbonate (CaCO3 ) coccoliths and transparent exopolymer particles (TEP), sticky, acidic carbohydrates that facilitate aggregation. E. huxleyi's extensive oceanic blooms are often terminated by coccolithoviruses (EhVs) with the transport of cellular debris and associated particulate organic carbon (POC) to depth being facilitated by TEP-bound 'marine snow' aggregates. The dynamics of TEP production and particle aggregation in response to EhV infection are poorly understood. Using flow cytometry, spectrophotometry and FlowCam visualization of alcian blue (AB)-stained aggregates, we assessed TEP production and the size spectrum of aggregates for E. huxleyi possessing different degrees of calcification and cellular CaCO3 :POC mass ratios, when challenged with two EhVs (EhV207 and EhV99B1). FlowCam imaging also qualitatively assessed the relative amount of AB-stainable TEP (i.e., blue:red ratio of each particle). We show significant increases in TEP during early phase EhV207-infection (∼ 24 h) of calcifying strains and a shift towards large aggregates following EhV99B1-infection. We also observed the formation of large aggregates with low blue:red ratios, suggesting that other exopolymer substances contribute towards aggregation. Our findings show the potential for virus infection and the associated response of their hosts to impact carbon flux dynamics and provide incentive to explore these dynamics in natural populations.

Coccolithovirus facilitation of carbon export in the North Atlantic.

Marine phytoplankton account for approximately half of global primary productivity 1 , making their fate an important driver of the marine carbon cycle. Viruses are thought to recycle more than one-quarter of oceanic photosynthetically fixed organic carbon 2 , which can stimulate nutrient regeneration, primary production and upper ocean respiration 2 via lytic infection and the 'virus shunt'. Ultimately, this limits the trophic transfer of carbon and energy to both higher food webs and the deep ocean 2 . Using imagery taken by the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the Aqua satellite, along with a suite of diagnostic lipid- and gene-based molecular biomarkers, in situ optical sensors and sediment traps, we show that Coccolithovirus infections of mesoscale (~100 km) Emiliania huxleyi blooms in the North Atlantic are coupled with particle aggregation, high zooplankton grazing and greater downward vertical fluxes of both particulate organic and particulate inorganic carbon from the upper mixed layer. Our analyses captured blooms in different phases of infection (early, late and post) and revealed the highest export flux in 'early-infected blooms' with sinking particles being disproportionately enriched with infected cells and subsequently remineralized at depth in the mesopelagic. Our findings reveal viral infection as a previously unrecognized ecosystem process enhancing biological pump efficiency.

Coccolithoviruses: A Review of Cross-Kingdom Genomic Thievery and Metabolic Thuggery.

Coccolithoviruses (Phycodnaviridae) infect and lyse the most ubiquitous and successful coccolithophorid in modern oceans, Emiliania huxleyi. So far, the genomes of 13 of these giant lytic viruses (i.e., Emiliania huxleyi viruses-EhVs) have been sequenced, assembled, and annotated. Here, we performed an in-depth comparison of their genomes to try and contextualize the ecological and evolutionary traits of these viruses. The genomes of these EhVs have from 444 to 548 coding sequences (CDSs). Presence/absence analysis of CDSs identified putative genes with particular ecological significance, namely sialidase, phosphate permease, and sphingolipid biosynthesis. The viruses clustered into distinct clades, based on their DNA polymerase gene as well as full genome comparisons. We discuss the use of such clustering and suggest that a gene-by-gene investigation approach may be more useful when the goal is to reveal differences related to functionally important genes. A multi domain "Best BLAST hit" analysis revealed that 84% of the EhV genes have closer similarities to the domain Eukarya. However, 16% of the EhV CDSs were very similar to bacterial genes, contributing to the idea that a significant portion of the gene flow in the planktonic world inter-crosses the domains of life.

Intragenus competition between coccolithoviruses: an insight on how a select few can come to dominate many.

Viruses are a major cause of coccolithophore bloom demise in both temperate and sub-temperate oceanic regions. Most infection studies on coccolithoviruses have been conducted with a single virus strain, and the effect of intragenus competition by closely related coccolithoviruses has been ignored. Here we conducted combined infection experiments, infecting Emiliania huxleyi CCMP 2090 with two coccolithoviruses: EhV-86 and EhV-207 both simultaneously and independently. EhV-207 displayed a shorter lytic cycle and increased production potential than EhV-86 and was remarkably superior under competitive conditions. Although the viruses displayed identical adsorption kinetics in the first 2 h post infection, EhV-207 gained a numerical advantage as early as 8 h post infection. Quantitative polymerase chain reaction (PCR) revealed that when infecting in combination, EhV-207 was not affected by the presence of EhV-86, whereas EhV-86 was quickly out-competed, and a significant reduction in free and cell-associated EhV-86 was seen as early as 2 days after the initial infection. The observation of such clear phenotypic differences between genetically distinct, yet similar, coccolithovirus strains, by flow cytometry and quantitative real-time PCR allowed tentative links to the burgeoning genomic, transcriptomic and metabolic data to be made and the factors driving their selection, in particular to the de novo coccolithovirus-encoded sphingolipid biosynthesis pathway. This work illustrates that, even within a family, not all viruses are created equally, and the potential exists for relatively small genetic changes to infer disproportionately large competitive advantages for one coccolithovirus over another, ultimately leading to a few viruses dominating the many.

Permanent draft genomes of four new coccolithoviruses: EhV-18, EhV-145, EhV-156 and EhV-164.

Coccolithoviruses infect the marine coccolithophorid microalga Emiliania huxleyi. Here, we describe the genomes of four new coccolithoviruses isolated from UK coastal locations. Of particular interest, EhV-18 and EhV-145 encode serine palmitoyltransferase function via two distinct genes, whereas all other coccolithoviruses have SPT as a gene fusion of LCB1/LCB2 domains.

Functional inferences of environmental coccolithovirus biodiversity.

The cosmopolitan calcifying alga Emiliania huxleyi is one of the most abundant bloom forming coccolithophore species in the oceans and plays an important role in global biogeochemical cycling. Coccolithoviruses are a major cause of coccolithophore bloom termination and have been studied in laboratory, mesocosm and open ocean studies. However, little is known about the dynamic interactions between the host and its viruses, and less is known about the natural diversity and role of functionally important genes within natural coccolithovirus communities. Here, we investigate the temporal and spatial distribution of coccolithoviruses by the use of molecular fingerprinting techniques PCR, DGGE and genomic sequencing. The natural biodiversity of the virus genes encoding the major capsid protein (MCP) and serine palmitoyltransferase (SPT) were analysed in samples obtained from the Atlantic Meridional Transect (AMT), the North Sea and the L4 site in the Western Channel Observatory. We discovered nine new coccolithovirus genotypes across the AMT and L4 site, with the majority of MCP sequences observed at the deep chlorophyll maximum layer of the sampled sites on the transect. We also found four new SPT gene variations in the North Sea and at L4. Their translated fragments and the full protein sequence of SPT from laboratory strains EhV-86 and EhV-99B1 were modelled and revealed that the theoretical fold differs among strains. Variation identified in the structural distance between the two domains of the SPT protein may have an impact on the catalytic capabilities of its active site. In summary, the combined use of 'standard' markers (i.e. MCP), in combination with metabolically relevant markers (i.e. SPT) are useful in the study of the phylogeny and functional biodiversity of coccolithoviruses, and can provide an interesting intracellular insight into the evolution of these viruses and their ability to infect and replicate within their algal hosts.

Genome sequence of Stenotrophomonas maltophilia PML168, which displays Baeyer-Villiger monooxygenase activity.

Stenotrophomonas maltophilia PML168 was isolated from Wembury Beach on the English Coast from a rock pool following growth and selection on agar plates. Here we present the permanent draft genome sequence, which has allowed prediction of function for several genes encoding enzymes relevant to industrial biotechnology, including a novel flavoprotein monooxygenase.

Draft genome sequence of four coccolithoviruses: Emiliania huxleyi virus EhV-88, EhV-201, EhV-207, and EhV-208.

The Coccolithoviridae are a group of viruses which infect the marine coccolithophorid microalga Emiliania huxleyi. The Emiliania huxleyi viruses (known as EhVs) described herein have 160- to 180-nm diameter icosahedral structures, have genomes of approximately 400 kbp, and consist of more than 450 predicted coding sequences (CDSs). Here, we describe the genomic features of four newly sequenced coccolithoviruses (EhV-88, EhV-201, EhV-207, and EhV-208) together with their draft genome sequences and their annotations, highlighting the homology and heterogeneity of these genomes to the EhV-86 model reference genome.

Draft genome sequence of the coccolithovirus Emiliania huxleyi virus 202.

Emiliania huxleyi virus 202 (EhV-202) is a member of the Coccolithoviridae, a group of viruses that infect the marine coccolithophorid Emiliania huxleyi. EhV-202 has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 407 kbp, consisting of 485 coding sequences (CDSs). Here we describe the genomic features of EhV-202, together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome.

Draft genome sequence of the coccolithovirus EhV-84.

The Coccolithoviridae is a recently discovered group of viruses that infect the marine coccolithophorid Emiliania huxleyi. Emiliania huxleyi virus 84 (EhV-84) has a 160 -180 nm diameter icosahedral structure and a genome of approximately 400 kbp. Here we describe the structural and genomic features of this virus, together with a near complete draft genome sequence (~99%) and its annotation. This is the fourth genome sequence of a member of the coccolithovirus family.

Draft genome sequence of the Coccolithovirus Emiliania huxleyi virus 203.

The Coccolithoviridae are a recently discovered group of viruses that infect the marine coccolithophorid Emiliania huxleyi. Emiliania huxleyi virus 203 (EhV-203) has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 400 kbp, consisting of 464 coding sequences (CDSs). Here we describe the genomic features of EhV-203 together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome.