Christine N. Palermo

The previous discovery of genomes of Polinton-like viruses (PLVs) associated with viruses of Chrysochromulina parva stimulated this research to determine the biological nature of these putative viral hyperparasites. Purification of C. parva viruses to enable co-infection experiments led to the discovery of a previously unknown virus, CpV-BQ3, which, based on sequence information and electron microscopy, is a species of Tethysvirus, a genus within the Megaviricetes. Purification and TEM imaging of CpV-PLV Moe revealed naked icosahedral particles morphologically similar to other cultivated virophages and PLVs. Mixed-infection experiments with the putative Polinton-like virus CpV-PLV Moe demonstrated that CpV-BQ3 supports its replication, whereas the putative Phycodnavirus CpV-BQ1 does not. Further, experimental infections with differing proportions of the Moe and its helper virus CpV-BQ3 revealed a dose-effect whereby high levels of Moe had a greater negative impact on BQ3 replication compared to lower levels. Conversely, high levels of Moe relative to BQ3 provided greater protection for C. parva, allowing enhanced cell survival, whereas low doses of Moe did not prevent cell lysis. Overall, the results of this study demonstrated the intimate relationship of CpV-PLV Moe with the newly discovered virus, CpV-BQ3, and C. parva, and illustrate the complex ecology of algal viruses.

Hamilton Harbour is an impaired embayment of Lake Ontario that experiences seasonal algal blooms despite decades of remedial efforts. To study the harbour’s cyanobacterial and heterotrophic bacterial communities, we extracted and sequenced community DNA from surface water samples collected biweekly from different sites during summer and fall. Assembled contigs were annotated at the phylum level, and Cyanobacteria were further characterized at order and species levels. Actinobacteria were most abundant in early summer, while Cyanobacteria were dominant in mid-summer. Microcystis aeruginosa and Limnoraphis robusta were most abundant throughout the sampling period, expanding the documented diversity of Cyanobacteria in Hamilton Harbour. Functional annotations were performed using the MG-RAST pipeline and SEED database, revealing that genes for photosynthesis, nitrogen metabolism, and aromatic compound metabolism varied in relative abundances over the season, while phosphorus metabolism was consistent, suggesting that these genes remained essential despite fluctuating environmental conditions and community succession. We observed seasonal shifts from anoxygenic to oxygenic phototrophy, and from ammonia assimilation to nitrogen fixation, coupled with decreasing heterotrophic bacteria and increasing Cyanobacteria relative abundances. Our data contribute important insights into bacterial taxa and functional potentials in Hamilton Harbour, revealing seasonal and spatial dynamics that can be used to inform ongoing remediation efforts.

As primary drivers of organic matter re-mineralization and trophic carbon transfer, bacteria promote biogeochemical cycling and shape ecosystems globally. We studied bacterial communities spatially and seasonally in an impaired harbour of Lake Ontario by extracting and sequencing community DNA from water samples collected biweekly from different sites. Assembled contigs were annotated at the phylum level, and Cyanobacteria were further characterized at order and species levels. Actinobacteria were most abundant in early summer, while Cyanobacteria were dominant in mid-summer. Microcystis aeruginosa and Limnoraphis robusta were most abundant throughout the sampling period, expanding the documented diversity of Cyanobacteria in Hamilton Harbour. Functional annotations were performed using the MG-RAST pipeline and SEED database, revealing that genes for photosynthesis, nitrogen metabolism and aromatic compound metabolism varied in relative abundances over the season, while phosphorus metabolism remained consistent, suggesting that these genes remain essential despite fluctuating environmental conditions and community succession. We observed seasonal shifts from anoxygenic to oxygenic phototrophy, and from ammonia assimilation to nitrogen fixation, coupled with decreasing heterotrophic bacteria and increasing cyanobacteria relative abundances. Our data contribute important insights into bacterial taxa and functional potentials in Hamilton Harbour, revealing seasonal and spatial dynamics that can be used to inform ongoing remediation efforts.

Most studies of aquatic virus communities analyze DNA sequences derived from the smaller-size “free-virus” fraction. Our study demonstrates that analysis of virus communities using only the smaller-size fraction can lead to erroneously low diversity estimates for many of the larger viruses such as Mimiviridae , Phycodnaviridae , Iridoviridae , and Poxviridae , whereas analyzing only the larger->0.45-μm-size fraction can lead to underestimates of Caudovirales diversity and relative abundance.

Metagenomics has enabled rapid increases in virus discovery, in turn permitting revisions of viral taxonomy and our understanding of the ecology of viruses and their hosts. Inspired by recent discoveries of large viruses prevalent in the environment, we re-assessed the longstanding approach of filtering water through small pore-size filters to separate viruses from cells before sequencing. We studied assembled contigs derived from < 0.45 μm and > 0.45 μm size fractions that were annotated as viral to determine the diversity and relative abundances of virus groups from each fraction. Virus communities were vastly different when comparing the size fractions, indicating that analysis of either fraction alone would provide only a partial perspective of environmental viruses. At the level of virus order/family we observed highly diverse and distinct virus communities in the > 0.45 μm size fractions, whereas the < 0.45 μm size fractions were comprised primarily of highly diverse Caudovirales. The relative abundances of Caudovirales for which hosts could be inferred varied widely between size fractions with higher relative abundances of cyanophages in the > 0.45 μm size fractions potentially indicating replication within cells during ongoing infections. Many of the Mimiviridae and Phycodnaviridae , and all Iridoviridae and Poxviridae were detected exclusively in the often disregarded > 0.45 μm size fractions. In addition to observing unique virus communities associated with each size fraction, we detected viruses common to both fractions and argue that these are candidates for further exploration because they may be the product of ongoing or recent lytic events. IMPORTANCE: Most studies of aquatic virus communities analyze DNA sequences derived from the smaller, “free virus” size fraction. Our study demonstrates that analysis of virus communities using only the smaller size fraction can lead to erroneously low diversity estimates for many of the larger viruses such as Mimiviridae, Phycodnaviridae, Iridoviridae , and Poxviridae , whereas analyzing only the larger, > 0.45 μm size fraction can lead to underestimates of Caudovirales diversity and relative abundance. Similarly, our data shows that examining only the smaller size fraction can lead to underestimation of virophage and cyanophage relative abundances that could, in turn, cause researchers to assume their limited ecological importance. Given the considerable differences we observed in this study, we recommend cautious interpretations of environmental virus community assemblages and dynamics when based on metagenomic data derived from different size fractions.

Aquatic viruses have been extensively studied over the past decade, yet fundamental aspects of freshwater virus communities remain poorly described. Our goal was to characterize virus communities captured in the >0.22 µm size-fraction seasonally and spatially in a freshwater harbour. Community DNA was extracted from water samples and sequenced on an Illumina HiSeq platform. Assembled contigs were annotated as belonging to the virus groups (i.e., order or family) Caudovirales, Mimiviridae, Phycodnaviridae, and virophages (Lavidaviridae), or to other groups of undefined viruses. Virophages were often the most abundant group, and discrete virophage taxa were remarkably stable across sites and dates despite fluctuations in Mimiviridae community composition. Diverse Mimiviridae contigs were detected in the samples and the two sites contained distinct Mimiviridae communities, suggesting that Mimiviridae are important algal viruses in this system. Caudovirales and Phycodnaviridae were present at low abundances in most samples. Of the 18 environmental parameters tested, only chlorophyll a explained the variation in the data at the order or family level of classification. Overall, our findings provide insight into freshwater virus community assemblages by expanding the documented diversity of freshwater virus communities, highlighting the potential ecological importance of virophages, and revealing distinct communities over small spatial scales.

Aquatic viruses have been extensively studied over the past decade, yet fundamental aspects of freshwater virus communities remain poorly described. Our goal was to characterize particle-associated virus communities seasonally and spatially in a freshwater harbour. Community DNA was extracted from water samples and sequenced on an Illumina HiSeq platform. Assembled contigs were annotated as belonging to the virus families Caudovirales, Mimiviridae, Phycodnaviridae , and virophages ( Lavidaviridae ), or to other groups of undefined viruses. Diverse Mimiviridae contigs were detected in the samples, but the two sites contained distinct Mimiviridae communities. Virophages were often the most abundant group, and discrete virophage taxa were remarkably stable across sites and dates despite fluctuations in Mimiviridae community composition. Caudovirales were present at low abundances in most samples, contrasting other studies of freshwater environments. Similarly, Phycodnaviridae abundances were surprisingly low in all samples despite the harbour’s capacity to support high algal biomass during the summer and autumn months, suggesting that Mimiviridae are the dominant algae-infecting viruses in this system. Overall, our findings provided insights into freshwater virus community assemblages by expanding the documented diversity of freshwater virus communities, highlighting the potential ecological importance of virophages, and revealing distinct communities over small spatial scales.