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Vibrio parahaemolyticus(VP) is a bacterial pathogen found in brackish and marine water that infects many marine organisms, such as oysters and shrimp. Consumption of raw or undercooked seafood contaminated withV. parahaemolyticusis a primary cause of seafood-borne gastroenteritis in humans. Due to increasing ocean temperatures,V. parahaemolyticuscontamination of oyster beds in the United States has spread up the east and west coasts to the northern-most states. Promising new research is exploring the isolation of bacteriophages againstV. parahaemolyticuswith a long-term goal to possibly decontaminate oyster beds, thereby expanding the harvest season and allowing for safer consumption of seafood. In this study, store-bought oysters harvested from the Chesapeake Bay in Virginia were used to isolate four bacteriophages with activity against a specificV. parahaemolyticusstrain. A standard double agar overlay plaque assay was used to identify phage activity. After phage isolation, the genomes were sequenced, and transmission electron microscopy (TEM) was performed to visualize the virions. The genomes and TEM images revealed four distinct phages. Three of the phages are distinct isolates that exhibit podovirus-like morphology with short tails and genome sizes of approximately 43 kbp. One phage has siphovirus-like morphology and is a mid-sized tailed phage with a genome size of 80 kbp. Although spot tests performed with the oyster homogenates on up to 10 differentV. parahaemolyticusstrains recovered activity across a wide range of hosts, plaque assays with the isolated phages showed limited host range. Future work will be necessary to determine the viability of using the bacteriophages for elimination ofV. parahaemolyticusin harvested oysters, treatment of aquaculture seed and spat, and/or the environment. 

ABSTRACT Members of the phylumNucleocytoviricota, which include “giant viruses” known for their large physical dimensions and genome lengths, are a diverse group of dsDNA viruses that infect a wide range of eukaryotic hosts. The genomes of nucleocytoviruses frequently encode eukaryotic signature proteins (ESPs) such as RNA- and DNA-processing proteins, vesicular trafficking factors, cytoskeletal components, and proteins involved in ubiquitin signaling. Despite the prevalence of these genes in many nucleocytoviruses, the timing and number of gene acquisitions remains unclear. While the presence of DNA- and RNA-processing proteins in nucleocytoviruses likely reflects ancient gene transfers, the origins and evolutionary history of other proteins are largely unknown. In this study, we examined the distribution and evolutionary history of a subset of viral-encoded ESPs (vESPs) that are widespread in nucleocytoviruses. Our results demonstrate that most vESPs involved in vesicular trafficking were acquired multiple times independently by nucleocytoviruses at different time points after the emergence of the eukaryotic supergroups, while viral proteins associated with cytoskeletal and ubiquitin system proteins exhibited a more complex evolutionary pattern exhibited by both shallow and deep branching viral clades. This pattern reveals a dynamic interplay between the co-evoluton of eukaryotes and their viruses, suggesting that the viral acquisition of many genes involved in cellular processes has occurred both through ancient and more recent horizontal gene transfers. The timing and frequency of these gene acquisitions may provide insight into their role and functional significance during viral infection.IMPORTANCEThis research is pertinent for understanding the evolution of nucleocytoviruses and their interactions with eukaryotic hosts. By investigating the distribution and evolutionary history of viral-encoded eukaryotic signature proteins, the study reveals gene transfer patterns, highlighting how viruses acquire genes that allow them to manipulate host cellular processes. Identifying the timing and frequency of gene acquisitions related to essential cellular functions provides insights into their roles during viral infections. This work expands our understanding of viral diversity and adaptability, contributing valuable knowledge to virology and evolutionary biology, while offering new perspectives on the relationship between viruses and their hosts. 

Classifying viruses systematically has remained a key challenge of virology due to the absence of universal genes and vast genetic diversity of viruses. In particular, the most dominant and diverse group of viruses, the tailed double-stranded DNA viruses of prokaryotes belonging to the classCaudoviricetes, lack sufficient similarity in the genetic machinery that unifies them to reconstruct an inclusive, stable phylogeny of these genes. While previous approaches to organize tailed phage diversity have managed to distinguish various taxonomic levels, these methods are limited in scalability, reproducibility, and the inclusion of modes of evolution, like gene gains and losses, remain key challenges. Here, we present a novel, comprehensive, and reproducible framework for examining evolutionary relationships of tailed phages. In this framework, we compare phage genomes based on the presence and absence of a fixed set of gene families which are used as binary trait data that is input into maximum likelihood models. Our resulting phylogeny stably recovers known taxonomic families of tailed phages, with and without the inclusion of metagenome-derived phages. We also quantify the mosaicism of replication and structural genes among known families, and our results suggest that these exchanges likely underpin the emergence of new families. Additionally, we apply this framework to large phages (>100 kilobases) to map emergences of traits associated with genome expansion. Taken together, this evolutionary framework for charting and organizing tailed phage diversity improves the systemization of phage taxonomy, which can unify phage studies and advance our understanding of their evolution. 

Despite the recent surge of viral metagenomic studies, it remains a significant challenge to recover complete virus genomes from metagenomic data. The majority of viral contigs generated from de novo assembly programs are highly fragmented, presenting significant challenges to downstream analysis and inference. To address this issue, we have developed Virseqimprover, a computational pipeline that can extend assembled contigs to complete or nearly complete genomes while maintaining extension quality. Virseqimprover first examines whether there is any chimeric sequence based on read coverage, breaks the sequence into segments if there is, then extends the longest segment with uniform depth of coverage, and repeats these procedures until the sequence cannot be extended. Finally, Virseqimprover annotates the gene content of the resulting sequence. Results show that Virseqimprover has good performances on correcting and extending viral contigs to their full lengths, hence can be a useful tool to improve the completeness and minimize the assembly errors of viral contigs. Both a web server and a conda package for Virseqimprover are provided to the research community free of charge. 

Abstract Viruses of the phylumNucleocytoviricota, often referred to as “giant viruses,” are prevalent in various environments around the globe and play significant roles in shaping eukaryotic diversity and activities in global ecosystems. Given the extensive phylogenetic diversity within this viral group and the highly complex composition of their genomes, taxonomic classification of giant viruses, particularly incomplete metagenome-assembled genomes (MAGs) can present a considerable challenge. Here we developed TIGTOG (TaxonomicInformation ofGiant viruses usingTrademarkOrthologousGroups), a machine learning-based approach to predict the taxonomic classification of novel giant virus MAGs based on profiles of protein family content. We applied a random forest algorithm to a training set of 1531 quality-checked, phylogenetically diverseNucleocytoviricotagenomes using pre-selected sets of giant virus orthologous groups (GVOGs). The classification models were predictive of viral taxonomic assignments with a cross-validation accuracy of 99.6% at the order level and 97.3% at the family level. We found that no individual GVOGs or genome features significantly influenced the algorithm’s performance or the models’ predictions, indicating that classification predictions were based on a comprehensive genomic signature, which reduced the necessity of a fixed set of marker genes for taxonomic assigning purposes. Our classification models were validated with an independent test set of 823 giant virus genomes with varied genomic completeness and taxonomy and demonstrated an accuracy of 98.6% and 95.9% at the order and family level, respectively. Our results indicate that protein family profiles can be used to accurately classify large DNA viruses at different taxonomic levels and provide a fast and accurate method for the classification of giant viruses. This approach could easily be adapted to other viral groups. 

Abstract Phages (viruses of bacteria and archaea) are a ubiquitous top-down control on microbial communities by selectively infecting and killing cells. As obligate parasites, phages are inherently linked to processes that impact their hosts’ distribution and physiology, but phages can also be impacted by external, environmental factors, such as UV radiation degrading their virions. To better understand these complex links of phages to their hosts and the environment, we leverage the unique ecological context of the Isthmus of Panama, which narrowly disconnects the productive Tropical Eastern Pacific (EP) and nutrient-poor Tropical Western Atlantic (WA) provinces. We could thus compare patterns of phage and prokaryotic communities at both global scales (between oceans) and local-scales (between habitats within an ocean). Although both phage and prokaryotic communities differed sharply between the oceans, phage community composition did not significantly differ between mangroves and reefs of the WA, while prokaryotic communities were distinct. These results suggest phages are more shaped by dispersal processes than local conditions regardless of spatial scale, while prokaryotes tend to be shaped by local conditions at smaller spatial scales. Collectively, we provide a framework for addressing the co-variability between phages and prokaryotes in marine systems and identifying factors that drive consistent versus disparate trends in community shifts, essential to informing models of biogeochemical cycles that include these interactions. 

Since the discovery of the first “giant virus,” particular attention has been paid toward isolating and culturing these large DNA viruses throughAcanthamoebaspp. bait systems. While this method has allowed for the discovery of plenty novel viruses in theNucleocytoviricota, environmental -omics-based analyses have shown that there is a wealth of diversity among this phylum, particularly in marine datasets. The prevalence of these viruses in metatranscriptomes points toward their ecological importance in nutrient turnover in our oceans and as such, in depth study into non-amoebalNucleocytoviricotashould be considered a focal point in viral ecology. In this review, we report onKratosvirus quantuckense(née Aureococcus anophagefferens Virus), an algae-infecting virus of theImitervirales. Current systems for study in theNucleocytoviricotadiffer significantly from this virus and its relatives, and a litany of trade-offs within physiology, coding potential, and ecology compared to these other viruses reveal the importance ofK. quantuckense. Herein, we review the research that has been performed on this virus as well as its potential as a model system for algal-virus interactions. 

Abstract Viruses of the phylum Nucleocytoviricota are ubiquitous in ocean waters and play important roles in shaping the dynamics of marine ecosystems. In this study, we leveraged the bioGEOTRACES metagenomic dataset collected across the Atlantic and Pacific Oceans to investigate the biogeography of these viruses in marine environments. We identified 330 viral genomes, including 212 in the order Imitervirales and 54 in the order Algavirales. We found that most viruses appeared to be prevalent in shallow waters (<150 m), and that viruses of the Mesomimiviridae (Imitervirales) and Prasinoviridae (Algavirales) are by far the most abundant and diverse groups in our survey. Five mesomimiviruses and one prasinovirus are particularly widespread in oligotrophic waters; annotation of these genomes revealed common stress response systems, photosynthesis-associated genes, and oxidative stress modulation genes that may be key to their broad distribution in the pelagic ocean. We identified a latitudinal pattern in viral diversity in one cruise that traversed the North and South Atlantic Ocean, with viral diversity peaking at high latitudes of the northern hemisphere. Community analyses revealed three distinct Nucleocytoviricota communities across latitudes, categorized by latitudinal distance towards the equator. Our results contribute to the understanding of the biogeography of these viruses in marine systems.