Genomics and evolutionary analysis of Chlorella variabilis-infecting viruses demarcate criteria for defining species of giant viruses.

Chloroviruses exhibit a close relationship with their hosts with the phenotypic aspect of their ability to form lytic plaques having primarily guided the taxonomy. However, with the isolation of viruses that are only able to complete their replication cycle in one strain of Chlorella variabilis, systematic challenges emerged. In this study, we described the genomic features of 53 new chlorovirus isolates and used them to elucidate part of the evolutionary history and taxonomy of this clade. Our analysis revealed new chloroviruses with the largest genomes to date (>400 kbp) and indicated that four genomic features are statistically different in the viruses that only infect the Syngen 2-3 strain of C. variabilis (OSy viruses). We found large regions of dissimilarity in the genomes of viruses PBCV-1 and OSy-NE5 when compared with the other genomes. These regions contained genes related to the interaction with the host cell machinery and viral capsid proteins, which provided insights into the evolution of the replicative and structural modules in these giant viruses. Phylogenetic analysis using hallmark genes of Nucleocytoviricota revealed that OSy-viruses evolved from the NC64A-viruses, possibly emerging as a result of the strict relationship with their hosts. Merging phylogenetics and nucleotide identity analyses, we propose strategies to demarcate viral species, resulting in seven new species of chloroviruses. Collectively, our results show how genomic data can be used as lines of evidence to demarcate viral species. Using the chloroviruses as a case study, we expect that similar initiatives will emerge using the basis exhibited here.IMPORTANCEChloroviruses are a group of giant viruses with long dsDNA genomes that infect different species of Chlorella-like green algae. They are host-specific, and some isolates can only replicate within a single strain of Chlorella variabilis. The genomics of these viruses is still poorly explored, and the characterization of new isolates provides important data on their genetic diversity and evolution. In this work, we describe 53 new chlorovirus genomes, including many isolated from alkaline lakes for the first time. Through comparative genomics and molecular phylogeny, we provide evidence of genomic gigantism in chloroviruses and show that a subset of viruses became highly specific for their hosts at a particular point in evolutionary history. We propose criteria to demarcate species of chloroviruses, paving the way for an update in the taxonomy of other groups of viruses. This study is a new and important piece in the complex puzzle of giant algal viruses.

Lessons from Chloroviruses: the Complex and Diverse Roles of Viruses in Food Webs.

Viruses can have large effects on the ecological communities in which they occur. Much of this impact comes from the mortality of host cells, which simultaneously alters microbial community composition and causes the release of matter that can be used by other organisms. However, recent studies indicate that viruses may be even more deeply integrated into the functioning of ecological communities than their effect on nutrient cycling suggests. In particular, chloroviruses, which infect chlorella-like green algae that typically occur as endosymbionts, participate in three types of interactions with other species. Chlororviruses (i) can lure ciliates from a distance, using them as a vector; (ii) depend on predators for access to their hosts; and (iii) get consumed as a food source by, at least, a variety of protists. Therefore, chloroviruses both depend on and influence the spatial structures of communities as well as the flows of energy through those communities, driven by predator-prey interactions. The emergence of these interactions are an eco-evolutionary puzzle, given the interdependence of these species and the many costs and benefits that these interactions generate.

Viral Chemotaxis of Paramecium Bursaria Altered by Algal Endosymbionts.

Chemotaxis is widespread across many taxa and often aids resource acquisition or predator avoidance. Species interactions can modify the degree of movement facilitated by chemotaxis. In this study, we investigated the influence of symbionts on Paramecium bursaria's chemotactic behavior toward chloroviruses. To achieve this, we performed choice experiments using chlorovirus and control candidate attractors (virus stabilization buffer and pond water). We quantified the movement of Paramecia grown with or without algal and viral symbionts toward each attractor. All Paramecia showed some chemotaxis toward viruses, but cells without algae and viruses showed the most movement toward viruses. Thus, the endosymbiotic algae (zoochlorellae) appeared to alter the movement of Paramecia toward chloroviruses, but it was not clear that ectosymbiotic viruses (chlorovirus) also had this effect. The change in behavior was consistent with a change in swimming speed, but a change in attraction remains possible. The potential costs and benefits of chemotactic movement toward chloroviruses for either the Paramecia hosts or its symbionts remain unclear.

Structural basis of a multi-functional deaminase in chlorovirus PBCV-1.

2-Deoxycytidylate deaminase (dCD) is a member of the zinc-dependent cytidine deaminase family features in its allosterically regulated mechanism by dCTP and dTTP. The large double-stranded DNA-containing chlorovirus PBCV-1 encodes a dCD family enzyme PBCV1dCD that was reported to be able to deaminize both dCMP and dCTP, which makes PBCV1dCD unique in the dCD family proteins. In this study, we report the crystal structure of PBCV1dCD in complex with dCTP/dCMP and dTTP/dTMP, respectively. We further proved the ability of PBCV1dCD in the deamination of dCDP, which makes PBCV1dCD a multi-functional deaminase. The structural basis for the versatility of PBCV1dCD is analyzed and discussed, with the finding of a unique Trp121 residue key to the deamination and substrate binding ability. Our findings may broaden the understanding of dCD family proteins and provide novel insights into the multi-functional enzyme.

Chloroviruses Lure Hosts through Long-Distance Chemical Signaling.

Chloroviruses exist in aquatic systems around the planet and they infect certain eukaryotic green algae that are mutualistic endosymbionts in a variety of protists and metazoans. Natural chlorovirus populations are seasonally dynamic, but the precise temporal changes in these populations and the mechanisms that underlie them have heretofore been unclear. We recently reported the novel concept that predator/prey-mediated virus activation regulates chlorovirus population dynamics, and in the current study, we demonstrate virus-packaged chemotactic modulation of prey behavior.IMPORTANCE Viruses have not previously been reported to act as chemotactic/chemoattractive agents. Rather, viruses as extracellular entities are generally viewed as non-metabolically active spore-like agents that await further infection events upon collision with appropriate host cells. That a virus might actively contribute to its fate via chemotaxis and change the behavior of an organism independent of infection is unprecedented.

Structure of the N-glycans from the chlorovirus NE-JV-1.

Results from recent studies are breaking the paradigm that all viruses depend on their host machinery to glycosylate their proteins. Chloroviruses encode several genes involved in glycan biosynthesis and some of their capsid proteins are decorated with N-linked oligosaccharides with unique features. Here we describe the elucidation of the N-glycan structure of an unusual chlorovirus, NE-JV-1, that belongs to the Pbi group. The host for NE-JV-1 is the zoochlorella Micractinium conductrix. Spectroscopic analyses established that this N-glycan consists of a core region that is conserved in all of the chloroviruses. The one difference is that the residue 3OMe-L-rhamnose is acetylated at the O-2 position in a non-stoichiometric fashion.

Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice.

Chloroviruses (family Phycodnaviridae) are large DNA viruses known to infect certain eukaryotic green algae and have not been previously shown to infect humans or to be part of the human virome. We unexpectedly found sequences homologous to the chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1) in a metagenomic analysis of DNA extracted from human oropharyngeal samples. These samples were obtained by throat swabs of adults without a psychiatric disorder or serious physical illness who were participating in a study that included measures of cognitive functioning. The presence of ATCV-1 DNA was confirmed by quantitative PCR with ATCV-1 DNA being documented in oropharyngeal samples obtained from 40 (43.5%) of 92 individuals. The presence of ATCV-1 DNA was not associated with demographic variables but was associated with a modest but statistically significant decrease in the performance on cognitive assessments of visual processing and visual motor speed. We further explored the effects of ATCV-1 in a mouse model. The inoculation of ATCV-1 into the intestinal tract of 9-11-wk-old mice resulted in a subsequent decrease in performance in several cognitive domains, including ones involving recognition memory and sensory-motor gating. ATCV-1 exposure in mice also resulted in the altered expression of genes within the hippocampus. These genes comprised pathways related to synaptic plasticity, learning, memory formation, and the immune response to viral exposure.

Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes.

Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

The Role of Chloroviruses as Possible Infectious Agents for Human Health: Putative Mechanisms of ATCV-1 Infection and Potential Routes of Transmission.

The Chlorovirus genus of the Phycodnaviridae family includes large viruses with a double-stranded DNA genome. Chloroviruses are widely distributed in freshwater bodies around the world and have been isolated from freshwater sources in Europe, Asia, Australia, and North and South America. One representative of chloroviruses is Acanthocystis turfacea chlorella virus 1 (ATCV-1), which is hosted by Chlorella heliozoae. A few publications in the last ten years about the potential effects of ATCV-1 on the human brain sparked interest among specialists in the field of human infectious pathology. The goal of our viewpoint was to compile the scant research on the effects of ATCV-1 on the human body, to demonstrate the role of chloroviruses as new possible infectious agents for human health, and to indicate potential routes of virus transmission. We believe that ATCV-1 transmission routes remain unexplored. We also question whether chlorella-based nutritional supplements are dangerous for ATCV-1 infections. Further research will help to identify the routes of infection, the cell types in which ATCV-1 can persist, and the pathological mechanisms of the virus's effect on the human body.

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.

Early-Phase Drive to the Precursor Pool: Chloroviruses Dive into the Deep End of Nucleotide Metabolism.

Viruses face many challenges on their road to successful replication, and they meet those challenges by reprogramming the intracellular environment. Two major issues challenging Paramecium bursaria chlorella virus 1 (PBCV-1, genus Chlorovirus, family Phycodnaviridae) at the level of DNA replication are (i) the host cell has a DNA G+C content of 66%, while the virus is 40%; and (ii) the initial quantity of DNA in the haploid host cell is approximately 50 fg, yet the virus will make approximately 350 fg of DNA within hours of infection to produce approximately 1000 virions per cell. Thus, the quality and quantity of DNA (and RNA) would seem to restrict replication efficiency, with the looming problem of viral DNA synthesis beginning in only 60-90 min. Our analysis includes (i) genomics and functional annotation to determine gene augmentation and complementation of the nucleotide biosynthesis pathway by the virus, (ii) transcriptional profiling of these genes, and (iii) metabolomics of nucleotide intermediates. The studies indicate that PBCV-1 reprograms the pyrimidine biosynthesis pathway to rebalance the intracellular nucleotide pools both qualitatively and quantitatively, prior to viral DNA amplification, and reflects the genomes of the progeny virus, providing a successful road to virus infection.

Strong selection and high mutation supply characterize experimental Chlorovirus evolution.

Characterizing how viruses evolve expands our understanding of the underlying fundamental processes, such as mutation, selection and drift. One group of viruses whose evolution has not yet been extensively studied is the Phycodnaviridae, a globally abundant family of aquatic large double-stranded (ds)DNA (dsDNA) viruses. Here we studied the evolutionary change of Paramecium bursaria chlorella virus 1 during experimental coevolution with its algal host. We used pooled genome sequencing of six independently evolved populations to characterize genomic change over five time points. Across six experimental replicates involving either strong or weak demographic fluctuations, we found single nucleotide polymorphisms (SNPs) at sixty-seven sites. The occurrence of genetic variants was highly repeatable, with just two of the SNPs found in only a single experimental replicate. Three genes A122/123R, A140/145R and A540L showed an excess of variable sites, providing new information about potential targets of selection during Chlorella-Chlorovirus coevolution. Our data indicated that the studied populations were not mutation-limited and experienced strong positive selection. Our investigation highlighted relevant processes governing the evolution of aquatic large dsDNA viruses, which ultimately contributes to a better understanding of the functioning of natural aquatic ecosystems.

Chlorovirus ATCV-1 Accelerates Motor Deterioration in SOD1-G93A Transgenic Mice and Its SOD1 Augments Induction of Inflammatory Factors From Murine Macrophages.

Background: Genetically polymorphic Superoxide Dismutase 1 G93A (SOD1-G93A) underlies one form of familial Amyotrophic Lateral Sclerosis (ALS). Exposures from viruses may also contribute to ALS, possibly by stimulating immune factors, such as IL-6, Interferon Stimulated Genes, and Nitric Oxide. Recently, chlorovirus ATCV-1, which encodes a SOD1, was shown to replicate in macrophages and induce inflammatory factors. Objective: This study aimed to determine if ATCV-1 influences development of motor degeneration in an ALS mouse model and to assess whether SOD1 of ATCV-1 influences production of inflammatory factors from macrophages. Methods: Sera from sporadic ALS patients were screened for antibody to ATCV-1. Active or inactivated ATCV-1, saline, or a viral mimetic, polyinosinic:polycytidylic acid (poly I:C) were injected intracranially into transgenic mice expressing human SOD1-G93A- or C57Bl/6 mice. RAW264.7 mouse macrophage cells were transfected with a plasmid vector expressing ATCV-1 SOD1 or an empty vector prior to stimulation with poly I:C with or without Interferon-gamma (IFN-γ). Results: Serum from sporadic ALS patients had significantly more IgG1 antibody directed against ATCV-1 than healthy controls. Infection of SOD1-G93A mice with active ATCV-1 significantly accelerated onset of motor loss, as measured by tail paralysis, hind limb tucking, righting reflex, and latency to fall in a hanging cage-lid test, but did not significantly affect mortality when compared to saline-treated transgenics. By contrast, poly I:C treatment significantly lengthened survival time but only minimally slowed onset of motor loss, while heat-inactivated ATCV-1 did not affect motor loss or survival. ATCV-1 SOD1 significantly increased expression of IL-6, IL-10, ISG promoter activity, and production of Nitric Oxide from RAW264.7 cells. Conclusion: ATCV-1 chlorovirus encoding an endogenous SOD1 accelerates pathogenesis but not mortality, while poly I:C that stimulates antiviral immune responses delays mortality in an ALS mouse model. ATCV-1 SOD1 enhances induction of inflammatory factors from macrophages.

Identification of a Chlorovirus PBCV-1 Protein Involved in Degrading the Host Cell Wall during Virus Infection.

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561LD4, with cell wall degrading activity. An A561LD4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561LD4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561LD4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561LD4 increased the specific infectivity of PBCV-1 from about 25-30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates.

Inter-vendor variance of enteric eukaryotic DNA viruses in specific pathogen free C57BL/6N mice.

The laboratory mouse strain C57BL/6 is widely used as an animal model for various applications. It is becoming increasingly clear that the bacterial enteric community highly influences the phenotype. Eukaryotic viruses represent a sparsely investigated member of the enteric microbiome that might also affect the phenotype. We here investigated the presence of enteric eukaryotic DNA viruses (EDVs) in specific pathogen-free (SPF) C57BL/6N mice purchased from three vendors upon arrival and after being fed a low-fat diet (LFD) or high-fat diet (HFD). We detected genetic fragments of EDVs belonging to the viral families of Herpes-, Mimi-, Baculo- and Phycodnaviridae represented by two genera; Chlorovirus and Prasinovirus. The EDVs were detected in the mice upon arrival and persisted for 13 weeks. However, these signals of EDVs were only detected at notable levels in mice fed LFD from 2 out of 3 vendors, which suggested that the enteric composition of these EDVs were affected by both vendor (p < 0.003) and different dietary regimes (p < 0.013). This highlights the need of additional studies assessing the potential function of these EDVs that may influence the mouse phenotype and the reproducibility of animal studies using this C57BL/6N substrain.

Gene Gangs of the Chloroviruses: Conserved Clusters of Collinear Monocistronic Genes.

Chloroviruses (family Phycodnaviridae) are dsDNA viruses found throughout the world's inland waters. The open reading frames in the genomes of 41 sequenced chloroviruses (330 ± 40 kbp each) representing three virus types were analyzed for evidence of evolutionarily conserved local genomic "contexts", the organization of biological information into units of a scale larger than a gene. Despite a general loss of synteny between virus types, we informatically detected a highly conserved genomic context defined by groups of three or more genes that we have termed "gene gangs". Unlike previously described local genomic contexts, the definition of gene gangs requires only that member genes be consistently co-localized and are not constrained by strand, regulatory sites, or intervening sequences (and therefore represent a new type of conserved structural genomic element). An analysis of functional annotations and transcriptomic data suggests that some of the gene gangs may organize genes involved in specific biochemical processes, but that this organization does not involve their coordinated expression.