{"paper_id":"270a773c-4b13-4095-8d87-cb2c1e95a8cf","body_text":"Strain-level diversity of giant viruses infecting chlorarachniophyte algae in the\nsubtropical North Pacific\nAuthors:\n1\n*Max Emil Schön,\n2\nChristopher R. Schvarcz,\n1\nSilja V. Malkewitz,\n1\nFanny C. Hinner,\n3\nAnna\nKoslova,\n1\nUlrike Mersdorf,\n1\nFiona Schimm,\n1\nSebastian Rickert,\n4\nNadiia Pozhydaieva,\n2\nKelsey\nMcBeain,\n5\nThomas Hackl,\n1\nAlina Cosima Schneider,\n1\nKarina Barenhoff,\n4,6,7\nKatharina Höfer,\n2\nKyle F. Edwards,\n2\nGrieg F. Steward,\n1‡\n*Matthias G. Fischer\n1\nMax Planck Institute for Medical Research, Heidelberg, Germany\n2\nUniversity of Hawaiʻi at Mānoa, Honolulu, HI, USA\n3\nInstitute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic\n4\nMax Planck Institute for Terrestrial Microbiology, Marburg, Germany\n5\nGroningen University, Groningen, The Netherlands\n6\nDepartment of Pharmacy, Institute of Pharmaceutical Biology and Biotechnology, Philipps\nUniversität Marburg, Marburg, Germany\n7\nCenter for Synthetic Microbiology (SYNMIKRO), Philipps Universität Marburg, Marburg,\nGermany\n‡\nCurrent affiliation: Max Planck Institute for Marine Microbiology, Bremen, Germany\n*Corresponding authors: mschoen@mr.mpg.de; mfischer@mpi-bremen.de\nAbstract:\nGiant DNA viruses are ubiquitous among unicellular eukaryotes and occur in marine,\nfreshwater, and terrestrial environments. Despite intense metagenomic data mining, their\nstrain-level diversity remains largely unexplored. Here we introduce a model system\ncomprising four isolates of a giant virus called ChlorV, which infects marine microalgae of the\nclass Chlorarachniophyceae (Rhizaria) from station ALOHA, Hawai’i. The ChlorV genomes\nare 469 kbp to 493 kbp long and encode approximately 400 proteins, at least 106 of which\nare present in purified virions. Although the four viral genomes are highly syntenic, they differ\nby several insertions and deletions that often encode methyltransferases. Interestingly, we\nfound that some of these methyltransferase genes correlated with specific DNA methylation\npatterns in the same ChlorV strain. Our study describes the first giant viruses infecting the\neukaryotic supergroup Rhizaria and demonstrates how viral strain-level variation in gene\ncontent and epigenetic features may affect eco-evolutionary processes in marine microalgae.\nIntroduction:\nDouble-stranded DNA viruses of the phylum Nucleocytoviricota are the giants of the viral\nworld, with particle sizes of up to 1.5 µm and genome lengths of up to 2.5 megabases\n1–5\n.\nWhen the first such virus, Acanthamoeba polyphaga mimivirus, was isolated and analyzed, it\nshowed a remarkable genetic make-up previously unknown from any other virus\n6\n. Since\nthen, a large number of additional giant virus isolates and thousands of\nmetagenome-assembled genomes have been published. The phylum Nucleocytoviricota\ncurrently comprises five orders and 15 families\n7\n; among them, the family Mimiviridae (order\nImitervirales ) is the best-studied. The Mimiviridae comprises three subfamilies:\nMegamimivirinae , Klosneuvirinae , and Aliimimivirinae . The Megamimivirinae include the\n1\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\n28\n29\n30\n31\n32\n33\n34\n35\n36\n37\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\noriginal mimivirus and relatives such as tupanvirus, moumouvirus, megavirus etc., all of\nwhich have been isolated on Amoebozoa hosts such as Acanthamoeba or Vermamoeba\n1,8–11\n.\nThe Klosneuvirinae subfamily contains the isolates Bodo saltans virus, Fadolivirus and\nYasminevirus\n12–15\n. The third subfamily, Aliimimivirinae , contains the well-characterised\nCafeteria roenbergensis virus (CroV) that infects the marine heterotrophic stramenopile\nCafeteria burkhardae , and Chlorella Virus XW01 that was isolated recently on the freshwater\ngreen alga Chlorella sp\n16,17\n. Nevertheless, the abundance of mimivirid-like sequence\nfragments found in eukaryotic genomes as well as the inferred co-evolutionary history of\ngiant viruses and protists suggests that most microbial eukaryotes are potential hosts for\ngiant viruses\n18,19\n.\nThe marine alga Bigelowiella natans belongs to the chlorarachniophytes, a mixotrophic\nlineage within the otherwise heterotrophic Rhizaria whose members acquired their plastid\nfrom a green alga by secondary endosymbiosis\n20,21\n. In addition to the nuclear and plastid\ngenomes, they retain a remnant of the green algal nucleus, called the nucleomorph\n22,23\n. B.\nnatans has been studied in the context of plastid evolution and was the first sequenced\ngenome of any rhizarian species\n24\n. Blanc et al.\n25\nidentified several endogenous viral\nelements in the B. natans genome assembly, suggesting that this alga frequently interacts\nwith both giant viruses and virophages, the latter being smaller dsDNA viruses that depend\non giant viruses for their own replication\n26\n. However, no virus infecting a chlorarachniophyte\nhas been described to date, limiting insight into virus–host interactions in this clade.\nHere, we characterize four isolated strains of Chlorarachniophyte virus (ChlorV), a new\nspecies of virulent giant virus isolated from Pacific waters of Hawai’i. Phylogenetically,\nChlorVs cluster with members of the subfamily Aliimimivirinae , thus expanding the confirmed\nhost range of giant viruses to include chlorarachniophytes (Rhizaria). We describe the\ninfection dynamics of ChlorV infection in its algal host and present a comparative genome\nanalysis of the four ChlorV strains including methylation pattern and associated\nmethyltransferase genes.\nResults & Discussion\nIn search of new algal viruses from tropical marine environments, we collected samples from\nsurface waters at Station ALOHA, a long term oceanographic monitoring site located 100 km\nnorth of the Hawaiian Island of Oʻahu (Fig. S1). Several strains of chlorarachniophyte algae\n(Fig. S2) and viruses infecting them were isolated between 2010 and 2012 (Table S1). As\nthese viruses replicate in members of the Chlorarachniophyceae, we refer to them\ncollectively as ‘ChlorVs’ (for chlor arachniophyte v iruses).\nAll viruses replicated in chlorarachniophyte host strains, albeit with notable differences in\nstrain specificity. Whereas host strains AL-TEMP06, AL-TEMP07 and AL-DI01 were\npermissive to all four ChlorV strains, host strain AL-FL05 supported only ChlorV-1 replication\nand was resistant to the remaining three ChlorV strains. In contrast, host strain AL-FL10\ncould only be infected by ChlorV-2, but not by the other virus strains. All host strains belong\nto Bigelowiella natans , except for strain AL-FL05, which appears to represent a distinct\nchlorarachniophyte genus (Fig. S2). Because algal strains AL-FL05 and AL-TEMP06 grew\nmost favorably in our culture conditions, we used them as production strains for ChlorV-1\nand ChlorV-2 to ChlorV-4, respectively.\n2\n49\n50\n51\n52\n53\n54\n55\n56\n57\n58\n59\n60\n61\n62\n63\n64\n65\n66\n67\n68\n69\n70\n71\n72\n73\n74\n75\n76\n77\n78\n79\n80\n81\n82\n83\n84\n85\n86\n87\n88\n89\n90\n91\n92\n93\n94\n95\n96\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nExtracellular ChlorV particles were readily detectable by flow cytometry after DNA staining\nwith SYBR Gold (Fig. S3). By infecting several liters of algal cultures, we collected and\nconcentrated approximately 1E+11 virus particles for each of the four ChlorV strains.\nImaging virus particles by negative staining transmission electron microscopy revealed\nprojected pentagonal or hexagonal outlines typical of icosahedral capsids (Fig. 1A-D).\nSimilar to the two other isolated viruses of the subfamily Aliimimivirinae (CroV and Chlorella\nvirus XW01), their capsids appear to be lacking external fibrils. The capsid diameter varied\nfrom 200 nm to 230 nm across the four virus strains. Occasionally, small protrusions (13 nm\nx 5 nm) at one of the capsid corners were visible, which suggests a modified vertex that\ncould function in host recognition or genome release (Fig. 1A,C)\n27,28\n.\nFigure 1: ChlorV particle morphology and infection dynamics. A-D: Negative stain transmission\nelectron micrographs of ChlorV particles. A) ChlorV-1. B) ChlorV-2. C) ChlorV-3. D) ChlorV-4. Arrows\nindicate potential unique capsid structures such as vertex portals. E) ChlorV-1 infection dynamics in\nhost strain AL-FL05. Densities of host cells and extracellular virus-like particles (VLPs) of uninfected\nand ChlorV-1 infected AL-FL05 cultures were measured by flow cytometry. Quantitative PCR was\nused to monitor viral DNA replication. VLP concentrations and Cq values of uninfected cultures are\n3\n97\n98\n99\n100\n101\n102\n103\n104\n105\n106\n107\n108\n109\n110\n111\n112\n113\n114\n115\n116\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nnot plotted, as these were without exception below the limit of detection. Error bars show standard\ndeviations between the three biological replicates and may be smaller than the data symbols.\nInfection dynamics\nWe analyzed the infection dynamics of ChlorV-1 in host strain AL-FL05, because this\ncombination resulted consistently in the highest virus titers when compared to any other\nChlorV – host strain combinations. We exposed the algal cultures to a 12 h light - 12 h dark\ncycle and infected AL-FL05 with ChlorV-1 at the onset of the light cycle. Triplicate cultures of\nAL-FL05 were infected with freshly prepared ChlorV-1 at a virus-to-host ratio of two and\nmonitored hourly for the first 36 hours by flow cytometry and quantitative PCR using\nChlorV-specific primers (Fig. 1E). The qPCR signal remained constant from the time of\ninfection until 4 h post infection (hpi); thereafter, the quantification cycle (Cq) decreased from\n22.5 to 16.0 by 19 hpi, indicating the onset of viral DNA replication around 3-4 hpi and a\npeak in viral DNA by ~19 hpi. Using flow cytometry, we observed a continuous decrease in\nextracellular VLPs from 0 hours post infection (hpi) to 8 hpi, most likely due to the uptake of\nChlorV-1 particles by host cells. Following this eclipse phase, VLP concentrations in the\nculture medium increased steadily from 9 hpi to 36 hpi. The slope of VLP increase was\nsteeper towards the end of the first light cycle (8 - 12 hpi) than during the following dark and\nlight cycles (13 - 36 hpi). Furthermore, we observed a delayed decline of host cells in the\ninfected cultures, which occurs only after extracellular VLP concentrations have reached\ntheir maximum at 36 hpi. These data are not compatible with a one-step growth curve and\ninstead suggest a constant release of virus particles. In summary, our infection experiments\nshow that ChlorV-1 is a lytic virus; however, the release of progeny virus particles is not\nimmediately linked to host lysis, suggesting a continuous and, at least initially,\nnon-destructive exit strategy, such as budding from host membranes or exocytosis\n29\n.\nGenome features\nAfter purifying the virus particles through iodixanol density gradients and isolation of viral\nDNA, we generated high-quality genome assemblies using ultra long MinION sequencing\nreads at 31x to 700x coverage (Table 1). For ChlorV-1 and ChlorV-4, we also generated\nshort-read Illumina sequencing data to further polish the assemblies. The genomes appear\nto have a linear topology, as they contained short (1.5 kb to 2.5 kb) terminal inverted repeats\n(TIR) at both ends, and ranged in size from 469 kb (ChlorV-3) to 493 kb (ChlorV-4). While the\ngenomes of ChlorV-2 and ChlorV-4 were virtually identical with an average nucleotide\nidentity (ANI) exceeding 99.8%, ANIs between other pairs of genomes ranged between\n93.2% (ChlorV-1 vs. ChlorV-4) and 97.3% (ChlorV-3 vs. ChlorV-4), see Fig. S4. The\ndifferences (insertions/deletions) between genomes were distributed over their complete\nlengths, and none of the observed differences were longer than 10,000 bp. In comparison,\nthe ANI between any of the four ChlorV genomes and their most closely related giant virus\nisolates, Cafeteria roenbergensis virus (CroV) and Chlorella virus XW01, was ~70%.\nHowever, the alignments between Chlorella Virus XW01 and ChlorVs covered about 10-12%\nof their respective genomes, whereas those between CroV and the ChlorVs covered only\n~4–6% (within ChlorVs, the coverage ranged from 75% to 100%, Fig. S4).\nTable 1: Genome statistics of ChlorV strains 1-4. In all cases, ONT was used for the initial assembly\nwhich was then polished using Illumina data if available. PacBio data for ChlorV-1 was only used to\nvalidate the ONT-based assembly.\n4\n117\n118\n119\n120\n121\n122\n123\n124\n125\n126\n127\n128\n129\n130\n131\n132\n133\n134\n135\n136\n137\n138\n139\n140\n141\n142\n143\n144\n145\n146\n147\n148\n149\n150\n151\n152\n153\n154\n155\n156\n157\n158\n159\n160\n161\n162\n163\n164\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nStrain Genome\nlength (bp)\n% GC # ORFs Coverage\n(ONT)\nData source Accession\nChlorV-1 487,585 19.05 402 263 ONT+PacBio\n+Illumina\nGCA_974392635\nChlorV-2 490,841 18.54 409 639 ONT GCA_975145565\nChlorV-3 468,809 18.82 391 700 ONT GCA_976281805\nChlorV-4 493,487 18.59 394 31 ONT+Illumina GCA_975141545\nIn each of the four ChlorV genomes, we identified ≈400 open reading frames (ORFs)\nencoding proteins >100 amino acids, with 56 to 72 sequences per genome not matching any\nknown cellular or viral genes (ORFans). Accounting for ORFs that were split in some\nassemblies, 396 non-redundant genes were identified across all virus strains. A core\ngenome of 307 genes was shared across all strains, while 381 were shared between\nChlorV-2 and ChlorV-4, 336 between ChlorV-2, ChlorV-3 and ChlorV-4 and 316 between\nChlorV-1 and ChlorV-3.\nChlorV phylogenetic position\nTo establish the phylogenetic position of these viruses, we identified putative homologs of\nestablished marker genes specific for viruses of the phylum Nucleocytoviricota\n30\n. Each\nChlorV genome contained a full complement of these conserved genes, further validating the\nquality of the assemblies. For each marker gene, we reconstructed a phylogenetic tree with\nbroad taxonomic sampling across the Nucleocytoviricota . In each of these trees, the four\nChlorV genomes formed a tight cluster in the Mimiviridae family, with CroV and Chlorella\nVirus XW01 being the closest isolated relatives (Supplementary File 1).\nWe then reconstructed a phylogenetic tree based on a concatenated alignment of seven\nmarker proteins, focusing on genomes of Mimiviridae members (isolates and environmental\ngenomes) from the giant virus database (GVDB). Selected representatives of other\nImitervirales lineages were chosen as an outgroup. The ChlorVs formed a well-supported\nmonophyletic group with Chlorella Virus XW01, CroV and several environmental genomes in\nthe subfamily Aliimimivirinae (Fig. 2).\nThe addition of ChlorVs makes the Aliimimivirinae one of the most diverse groups of\nNucleocytoviricota with respect to host range. Three different eukaryotic supergroups\n(Rhizaria: Chlorarachniophytes; Stramenopiles: Cafeteria ; Archaeplastida: Chlorella ) are\ninfected by members of this subfamily. In fact, the ChlorVs, CroV and Chlorella Virus XW01\ncluster in one group within the Aliimimivirinae ( Aliimimivirinae I), whereas the lake\nsturgeon-associated Namao virus and various metagenome-assembled genomes form a\nsecond group ( Aliimimivirinae II), indicating two well-supported clades within Aliimimivirinae\n(here referred to as clades I and II; Fig. 2, Fig. S5).\n5\n165\n166\n167\n168\n169\n170\n171\n172\n173\n174\n175\n176\n177\n178\n179\n180\n181\n182\n183\n184\n185\n186\n187\n188\n189\n190\n191\n192\n193\n194\n195\n196\n197\n198\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure 2: Phylogenetic position of ChlorV isolates. ChlorV 1-4 form a well-supported and closely\nrelated group within the Mimiviridae subfamily Aliimimivirinae . Their closest relative isolates are\nChlorella Virus XW01 and the Cafeteria roenbergensis virus (CroV) in the same subfamily. The tree is\nbased on the concatenated alignment of 7 marker genes and was inferred under the best fitting\n6\n199\n200\n201\n202\n203\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nevolutionary model LG+C40+F+I+R10, while support was estimated using 1000 ultrafast bootstrap\nreplicates. Besides the phylogenetic position, the distribution of marker genes per genome is drawn\nas a coulson plot and the source environment as a colored circle (dark blue: marine, light blue:\nfreshwater, pink: animal host, orange: soil, green: peat moss).\nGenome annotation & Comparative genomics\nWe annotated the predicted protein sequences using multiple strategies and databases,\nincluding EggNOG, InterPro (incorporating PFAM) and the giant virus orthologous groups\n(GVOGs)\n30–33\n. Besides ORFans with no matches to any known sequences, 128-146 ORFs\nper genome only matched poorly characterized reference sequences, often predicted from\nother giant viruses. Of the 173-190 sequences with a robust annotation, most were\ncategorized as ‘hydrolases’ (e.g. peptidases or nucleases), ‘replication, transcription and\nDNA repair’ (e.g. helicases, replication factors, polymerases) or ‘transferases’ (e.g. methyl-\nand glycosyltransferases) (Table S2). The ChlorV genomes encode a full complement of\nDNA replication genes, including a family B DNA polymerase (ChlorV-1..065), a DNA clamp\n(ChlorV-1..016) and clamp loader (replication factor C, e.g. ChlorV-1..164). They further\nencode a DNA ligase (ChlorV-1..107) and a ribonuclease H (ChlorV-1..329) as well as\nmultiple helicases including a D5-like helicase-primase (ChlorV-1..361) and two subunits of a\nribonucleotide reductase (ChlorV-1..059 and ChlorV-1..370). Transcription-related enzymes\nare also encoded in the genomes, including several subunits of the RNA polymerase\n(Rpb1,2,3/11,5,6,7, ChlorV-1..202,039,325,253,359,251), an mRNA capping enzyme\n(ChlorV-1..311), a poly(A) polymerase (ChlorV-1..312) and several transcription factors\nincluding homologs of the Poxvirus VLTF2 (ChlorV-1..152) and VLTF3 (ChlorV-1..384).\nSeveral translation initiation (e.g. eIF4E, ChlorV-1..017) and elongation (EF-Tu,\nChlorV-1..328) factors are also present. A homolog of the DNA polymerase beta\n(ChlorV-1..031) that is involved in base excision repair (BER), a putative DNA mismatch\nrepair protein MutS (ChlorV-1..354) and two DNA photolyases (ChlorV-1..224 and\nChlorV-1..336) that are potentially involved in DNA repair outside of the host are encoded as\nwell. Additionally encoded proteins include many peptidases (e.g. Chlorv-1..086) and several\nankyrin repeat proteins (e.g. Chlorv-1..071). Further noteworthy is the presence of a putative\nYqaJ-like viral recombinase (ChlorV-1..348) which is involved in cleaving genomic\nconcatemers in some bacteriophages\n34\nA putative tubulin–tyrosine ligase (Chlorv-1..043)\ncould be involved in manipulation of host cells\n35\n. Several glycosyltransferases (e.g.\nChlorv-1..212) and glycosylases (e.g. ChlorV-1..397) are encoded in the genomes of all\nstrains and could be involved in capsid modifications and host interactions\n36\n. At least one\nacetyltransferase (ChlorV-1..005) and one aminotransferase (ChlorV-1..145) were also\npresent.\nEach ChlorV genome encoded multiple predicted methyltransferase genes (12 in ChlorV-3,\n11 in the other strains), and these were often located in strain-specific insertions or deletions\n(Fig. 3 A-G). In contrast, most other groups of genes were usually conserved across all\nstrains. Several types of nucleases (both RNases and DNases) were also common\nthroughout the genomes, being either shared among strains or strain specific (e.g. the DNA\nendonuclease ChlorV-1..079, Fig. 3 D, F).\n7\n204\n205\n206\n207\n208\n209\n210\n211\n212\n213\n214\n215\n216\n217\n218\n219\n220\n221\n222\n223\n224\n225\n226\n227\n228\n229\n230\n231\n232\n233\n234\n235\n236\n237\n238\n239\n240\n241\n242\n243\n244\n245\n246\n247\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure 3: ChlorV strain level genomics. C: Overview of the complete genomes from four strains of\nChlorarachniophyte viruses (ChlorVs). Genes are colored according to whether they were identified in\nall strains (orange) or absent in at least one strain (blue). Genes with significant sequence matches\nare connected by dark grey ribbons. A,B,D-G: Details of genomic loci where putative\ninsertions/deletions and an inversion (F) can be observed. DNase: Deoxyribonuclease, SNase:\nStaphylococcal nuclease (DNA and RNA endo-exonuclease)\nWe then predicted the 3D structure of all ChlorV-1 ORFs using AlphaFold 3 and compared\nthem to experimentally derived structures in the protein database to improve the genome\nannotation (Supplementary File 2)\n37,38\n. For most ORFs, the sequence-based and\nstructure-based annotations (where available) agreed well. However, occasionally the\nstructural comparison provided annotations for ORFs with no sequence-based annotation,\ne.g. for the endonuclease ChlorV-1..079 or the putative penton protein Chlorv-1..095 (Table\nS3). In other cases, sequence-based annotation could not be substantiated by the structural\ncomparison, e.g. the predicted homing endonuclease Chlorv-1..154.\nWhereas CroV encodes many FNIP/IP22 repeat proteins, these repeats are absent from\nChlorVs, Chlorella virus XW01, and Aliimimivirinae environmental genomes. Besides the\nprotein-coding genes, ChlorV strains encode up to 7 tRNA genes, in contrast to the high\nnumber of 48 that are encoded in the genome of CroV, whereas Chlorella Virus XW01\nencodes none.\n8\n248\n249\n250\n251\n252\n253\n254\n255\n256\n257\n258\n259\n260\n261\n262\n263\n264\n265\n266\n267\n268\n269\n270\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nWe also investigated putative promoter sequences of ChlorV genes based on the\nphylogenetic position close to CroV and previous work on gene promoters in giant viruses\ngenerally\n39,16,40\n. The early motif (AAAAATTGA) and the late motif (TCTA) appear conserved\nbetween CroV and ChlorVs in both sequence and positional context (Fig. S6). Unlike the\npromoters, gene content seems to be more dynamic over larger phylogenetic distances (Fig.\nS5), which has also been observed in other groups of giant viruses.\nGenome modifications\nFor all ChlorV genomes, nanopore sequencing data was used to analyze DNA base\nmodification patterns. In all assemblies, the dominant modification was N6-methyladenine\n(6mA), followed by C5-methylcytosine (5mC) and N4-methylcytosine (4mC). The latter two\nwere rare, except in ChlorV-1 where 5mC was comparatively frequent (Fig. S7). This pattern\nconfirms previous observations that giant viruses display prokaryotic-like DNA modifications\nwith a majority of 6mA\n41\n. We identified sequence motifs that were highly enriched (all were\nmodified in >90% of occurrences) in methylated bases across the four genomes. We\nidentified three distinct motifs, including two motifs that were shared by two genomes each\n(Fig. 4A). All motifs were palindromic; accordingly, we did not detect strand-specific\ndifferences. ChlorV-1 and ChlorV-3 display a 10 base pair motif (C A NNNNNNTG) that\nresembles a bipartite recognition sequence, consistent with Type I or certain Type II\nsystems\n42,43\n. We identified one gene that is present in ChlorV-1 and ChlorV-3 but not the\nother strains as a candidate methyltransferase targeting this sequence (Fig. 3G). It\npossesses a 6-adenine-specific methyltransferase domain as well as a single target\nspecificity domain similar to other type I modification genes (Fig. 4B). However, this enzyme\ncould also function as a homooligomer, recognizing and modifying the same DNA sequence\non opposite strands, like the type IIB system HaeIV\n44\n. This is supported by the high\nconfidence scores (ipTM=0.8, pTM=0.85) of a protein-DNA complex containing two copies of\nthis protein together with a DNA sequence containing the motif C A NNNNNNTG, but lower\nconfidences (ipTM < 0.8) if using only a single copy or another DNA sequence (Table S4,\nFig. S8). ChlorV-3 (but not ChlorV-1) encodes another candidate methyltransferase with a\nsimilar DNA methyltransferase domain and two specificity domains, more similar to typical\ntype I modification genes\n45\n(Fig. 4E). We could additionally identify a motif for 5mC\nmethylation (R C GY) in ChlorV-1. This motif could be the target of a putative\n5-cytosine-specific methyltransferase encoded only in the ChlorV-1 genome (Fig. 3D, Fig.\n4C). Right next to this methyltransferase an endonuclease (ChlorV-1..079) was encoded,\nsuggesting that this is indeed a functioning restriction modification system. Finally, we\nidentified a different 6mA motif (TGC A ) in ChlorV-2 and ChlorV-4. This could again be\nputatively linked to a 6-adenine-specific methyltransferase gene that resembles type IV\nrestriction modification methyltransferases and was encoded only in these two strains (Fig.\n3C, Fig. 4A, D).\n9\n271\n272\n273\n274\n275\n276\n277\n278\n279\n280\n281\n282\n283\n284\n285\n286\n287\n288\n289\n290\n291\n292\n293\n294\n295\n296\n297\n298\n299\n300\n301\n302\n303\n304\n305\n306\n307\n308\n309\n310\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure 4: DNA modifications in ChlorVs. A: Detected sequence motifs with strong (>99%) modification\nsignal and putative associated methyltransferases. ChlorV-3..102 is a candidate besides\nChlorV-3..365 for methylation of the CANNNNNNTG motif, although no homologue of ChlorV-3..102\ncould be identified in ChlorV-1. For each methyltransferase gene, domain-level annotations are\nshown. Three genes represent 6-adenine-specific DNA methyltransferases (with ChlorV-4..019\nhaving a different domain than e.g. ChlorV-1..374), while ChlorV-1..078 was inferred to modify\ncytosine. B-E: Structural models of the four presented methyltransferases. In each model, the\nsequence domain annotations are visualized using the same colors.\nVirion proteomics\nWe analyzed the protein composition of purified ChlorV-1 virions by mass spectrometry and\nidentified 106 viral proteins, each represented by at least two peptides (Table S5). These\nvirion proteins were encoded by genes that are spread fairly evenly across the viral genome\n(Fig. S9). However, nine of the ten most abundant virion proteins were encoded by genes\nthat are located within a 90 kb central region of the genome, indicating a non-random\ndistribution of important structural genes. The most abundant protein was the double jelly roll\n(DJR) major capsid protein ChlorV-1..153, followed by the major core protein ChlorV-1..167\nwhich is homologous to the core proteins of poxvirus (P4B) and CroV (Crov332). It is\nnoteworthy that the third-most abundant virion protein was ChlorV-1..175, a 709 amino acid\nlong hypothetical protein that lacked homologs in other ChlorV strains. Peptides for all three\n10\n311\n312\n313\n314\n315\n316\n317\n318\n319\n320\n321\n322\n323\n324\n325\n326\n327\n328\n329\n330\n331\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nadditional DJR capsid paralogs, as well as for two of the four single jelly roll (SJR) penton\nprotein candidates, were also found. We then generated structural predictions for three of the\nfour DJR proteins as trimers and for the four SJR proteins as pentamers (Fig. S10, S11). The\nstructures were generally predicted with high confidence, except for some surface\nprotrusions that might interact with other capsid proteins. The peculiarly structured SJR\nprotein ChlorV-1..087 had a lower confidence value, although the core penton-like structure\nwas still resolved with good support (Fig. S11). We did not predict hetero-multimers for\npseudo-hexameric capsomers involving more than one DJR protein, although it is quite\npossible that such capsomers exist in ChlorVs, similar to e.g. Paramecium bursaria Chlorella\nvirus 1 (PBCV-1)\n46\n.\nAmong virion proteins with predicted enzymatic functions, we identified the two predicted\nDNA photolyases ChlorV-1..224 and ChlorV-1..336. As is typical for mimivirids, ChlorV-1 also\npackages its own transcription system that is represented here by five RNA polymerase\nsubunits, mRNA capping enzyme, poly(A) polymerase, and two transcription factors. All of\nthese transcription proteins are also encoded and packaged by CroV\n16,47\n.\nMethods:\nIsolation of hosts and viruses\nStrains of diverse eukaryotic phytoplankton were isolated from seawater samples collected\nfrom an oligotrophic open-ocean site (Station ALOHA, 22°45’ N, 158°00’ W)\n48\n, located\napproximately 100 km north of O‘ahu. For collection dates, see Table S1. Seawater samples\nwere typically enriched with artificial seawater media, F/2 medium or Keller (K) medium\n49,50\nand allowed to incubate in bottles or multiwell plates until cells of interest were identified.\nThese cells were then isolated either by micropipette or by serial dilution. All phytoplankton\ncultures were maintained at 24–26 °C on a 12:12 light:dark cycle with approximately 30–100\nµmol photons m-2 s-1 of photosynthetically active radiation (PAR).\nSmall subunit ribosomal RNA (18S rRNA) gene sequencing was used initially to identify algal\nspecies. For this purpose, cells were pelleted (4000 RCF for 10 min) and DNA extracted and\npurified (MasterPure Complete DNA and RNA Purification Kit; LGC Biosearch Technologies)\nor ZymoBIOMICS DNA Mini Kit; Zymo Research). The near-full length gene was amplified\nby PCR (Expand High Fidelity PCR System; Roche) with forward (5ʻ- ACC TGG TTG ATC\nCTG CC AG -3ʻ) and reverse (5ʻ- TGA TCC TTC YGC AGG TTC AC -3ʻ) primers targeting\nthe 5’ and 3’ ends of the gene\n51\n. PCR products were cloned prior to sequencing (TOPO TA\nCloning Kit for Sequencing; Invitrogen), and the insert was sequenced using four separate\nprimers (M13f, M13r, 502f, and 1174r)\n52\nusing a fluorescence-based Sanger technique\n(BigDye Terminator; Applied Biosystems) and capillary electrophoresis (3730XL DNA\nAnalyzer; Applied Biosystems) at the University of Hawai‘i at Mānoa’s facility for Advanced\nStudies in Genomics, Proteomics, and Bioinformatics (ASGPB).\nViruses were isolated from seawater samples collected from the open-ocean Station ALOHA\nsampling site. Large volumes of seawater were pre-filtered (using either 0.8 µm or 0.45 µm\npore size filters) to partially remove cells, and the virus community was concentrated by\ntangential flow filtration (TFF; Millipore Pellicon 2 Mini System) using 30 kDa nominal\nmolecular weight limit (NMWL) filters. The viral concentrates were amended with media (f/2\nor K) and added to healthy phytoplankton cultures. The virus-challenged cultures were\n11\n332\n333\n334\n335\n336\n337\n338\n339\n340\n341\n342\n343\n344\n345\n346\n347\n348\n349\n350\n351\n352\n353\n354\n355\n356\n357\n358\n359\n360\n361\n362\n363\n364\n365\n366\n367\n368\n369\n370\n371\n372\n373\n374\n375\n376\n377\n378\n379\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nobserved for 1–2 weeks for signs of cell lysis, and lysates that continued to produce a lytic\neffect after multiple transfers to healthy cells (putatively virus-containing) were stored at 4 °C\nand propagated at least once per month by challenging new cells (1–10% v/v of lysate\nadded per challenge).\nInfection experiments\nChlorarachniophyte cultures were grown in K medium in sterile polycarbonate flasks, either\nin 250 mL Erlenmeyer flasks, or in 3 L Fernbach flasks. Cultures were grown in an incubator\nat 24 °C, 50 rpm shaking, and in 12 h light (at 1300 to 1500 lux) - 12 h dark conditions.\nImmediately prior to inoculation, host cultures were diluted with K medium to a density of\n1.0E+06 cells per mL and aliquoted in 50 ml portions in 250 ml PC Erlenmeyer flasks.\nTriplicate cultures were inoculated at the onset of the 12 h light cycle with a fresh (less than 2\nweeks old, 1.2 µm filtered and stored at 4 °C) ChlorV-1 suspension at a virus-to-host ratio of\ntwo. Three flasks with uninfected host cultures were incubated as a negative control. The six\nflasks were sampled every hour for the first 36 hours, and again at 3 dpi and 5 dpi. At each\ntimepoint, the densities of cells and free virus particles were analyzed immediately by flow\ncytometry, and 25 µl of each suspension culture were frozen for later qPCR analysis.\nFlow cytometry\nFor cell counts, 200 µl of suspension culture were transferred to a 1.5 ml microfuge tube and\nanalyzed on an Attune\nTM\nNxT Acoustic Focusing Flow Cytometer (ThermoFisher Scientific,\nWaltham, MA, USA) and proprietary software, with the following instrument settings: Red\nLaser (RL1-H, voltage 320, threshold 1000) for chlorophyll autofluorescence over side\nscatter (SSC-H, voltage 350, threshold 1000); blue laser filter slot 1: 488/10+OD2; 50 µl\nacquisition volume, 25 µl/min flow rate, 25 µl total acquisition. For virus particle counts, 10 µl\nof suspension culture were pipetted in a 1.5 ml microfuge tube containing 2 µl of 25%\nEM-grade glutaraldehyde solution (Sigma Aldrich) and 88 µl of 0.1 µm filtered K medium.\nAfter incubation at 4 ॰ C for 10 min, 860 µl of 0.1 µm filtered Tris-EDTA buffer and 40 µl of\n100x SYBR Gold solution (Life Technologies Corporation, Eugene, OR, USA) were added.\nThe mixture was incubated at 80 ॰ C for 10 min in the dark, allowed to cool to room\ntemperature, and analyzed on an Attune\nTM\nNxT Flow Cytometer with the following settings:\nBlue Laser (BL1-H, voltage 490, threshold 5000) for SYBR fluorescence over side scatter\n(SSC-H, voltage 340, no threshold); blue laser filter slot 1: 488/10 (small particle filter); 50 µl\nacquisition volume, 25 µl/min flow rate, 25 µl total acquisition. Gates for cells and virus\nparticles were set manually, and concentrations derived directly from the Attune\nTM\nsoftware.\nQuantitative PCR analysis\nSample lysis for qPCR use was performed by adding 50 µL of 2x lysis buffer (20 mM\nTris-HCl, pH 8.0, 2 mM EDTA, 0.002% Triton X-100, 0.0002% SDS, 2 mg/ml proteinase K)\nand 25 µL of distilled H ₂ O to 25 µl of sample (stored at -20 °C). The tubes were mixed by\nvortexing and briefly centrifuged, then incubated for 60 minutes at 58 °C and for 10 minutes\nat 95 °C. Each 20 µl qPCR reaction consisted of 10 µl PowerTrack SYBR Green Master Mix\n(ThermoFisher), 7 µl nuclease-free water, 0.5 µl of 100 µM forward primer, 0.5 µl of 100 µM\nreverse primer, and 2 µl of the lysed sample. For ChlorV-1 detection, primers\nMCP_ChlorV-1_F (5’- CAG CAA TAC TCC ATC CGA CGG -3’) and MCP_ChlorV-1_R (5’-\nACT GTT AGC ACC GCC AAT GT -3’) were used, for detection of ChlorV-2, ChlorV-3, and\nChlorV-4 we used primers MCP_ChlorV-2-4_F (5’ GTG CTG CAC CTA CTG TTC CT -3’)\nand MCP_ChlorV-2-4_R (5’ TAG CAG CTT CCC AAT CAC CG- 3’). Samples were analyzed\n12\n380\n381\n382\n383\n384\n385\n386\n387\n388\n389\n390\n391\n392\n393\n394\n395\n396\n397\n398\n399\n400\n401\n402\n403\n404\n405\n406\n407\n408\n409\n410\n411\n412\n413\n414\n415\n416\n417\n418\n419\n420\n421\n422\n423\n424\n425\n426\n427\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nin technical duplicates per 96-well plate, using an Mx3005P Real-Time PCR System\n(Stratagene) with the following cycling conditions: 1 cycle of 7 min at 95 °C, 40 cycles of 15\nsec at 95 °C and 1 min at 60 °C, 1 cycle of 1 min at 72 °C, and a 55-95 °C ramping for\ndissociation curve analysis.\nSequencing of ChlorV genomes\nChlorV-1 samples for Illumina and PacBio sequencing were prepared using 0.45 µm filtration\n(Millipore Durapore 142mm filter, overlaid with Whatman GF/C filter) followed by tangential\nflow filtration (30 kDa NMWL; Millipore Pellicon 2 Mini system). Viral particles were purified\nusing centrifugation onto CsCl density cushions (6.5 mL 1.6 g/mL CsCl solution, overlaid with\n2 mL 1.1 g/mL CsCl solution; 25,000 rpm for 50 min, using SW28 rotor) followed by\ncentrifugation on a CsCl continuous gradient and 0.45 µm filtration (Millipore Sterivex)\nfollowed by another round on a CsCl continuous gradient. CsCl in the virus-containing\nfraction was exchanged with 1X TE buffer by three rounds of concentration and dilution in a\ncentrifugal ultrafilter (30 kDa NMWL; Millipore Amicon Ultra-0.5) followed by DNA extraction\nand purification (MasterPure Complete DNA and RNA Purification Kit). Sequencing runs for\nChlorV-1 were performed on an Illumina MiSeq Instrument (250bp paired-end) using the\nNextera XT library preparation kit at the Georgia Genomics and Bioinformatics Core. PacBio\nsequencing was performed on a PacBio RS II with P6-C4 chemistry at the University of\nWashington PacBio Sequencing Services.\nFor Nanopore MinION sequencing, several liters of ChlorV suspension containing at least\n1E+08 virus particles per millilitre were centrifuged in Nalgene 1 L centrifuge bottles in a\nFiberlite™ F9-6 x 1000 LEX rotor (ThermoFisher) for 20 minutes at 6000 x g, 18 °C. The\nsupernatant was concentrated by tangential flow filtration in a Vivaflow 200 0.2 µm PES unit\n(Sartorius) to 30-50 ml final volume. The concentrate was loaded on a 10-40% (w/v) linear\nOptiprep gradient (in 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM MgCl ₂ ) and centrifuged\nin 14 ml Ultra-Clear SW40 tubes and an SW40 rotor (Beckman) for 2 h at 100,000 x g, 18\n°C. The visible virus band was extracted through the tube wall with a syringe and needle,\ndiluted 3-fold with 50 mM Tris-HCl, pH 8.0, 2 mM MgCl ₂ and the virus particles were pelleted\nby centrifugation (30 min at 20,000 x g, 18 °C) in 1.5 ml microfuge tubes, pellets were\npooled, washed, and pelleted again. The final virus sample was resuspended in 200 µl of 50\nmM Tris-HCl, pH 8.0, 2 mM MgCl ₂ for DNA extraction with the QIAamp DNA Mini Kit\n(Qiagen) following the manufacturer’s instructions, but eluting in 50 µl of nuclease-free water.\nThe genomes of ChlorV-1, ChlorV-2 and ChlorV-3 were sequenced using the SQK-LSK114\nkit and a MinION FLO-MIN114 flow cell; the genome of ChlorV-4 was sequenced with an\nSQK-LSK110 kit and a FLO-MIN106 flow cell (Oxford Nanopore Technologies). For\nsequencing details, see Table S6.\nIn addition, a ChlorV-4 DNA sample was sent to Eurofins Genomics Germany GmbH, where\nit was sequenced on an Illumina NovaSeq 6000 S4 instrument using the TruSeq DNA\nPCR-Free library preparation kit.\nProteomics sample preparation\nA virus suspension with a concentration exceeding 10^10 PFU/ml was used for the\ndetermination of structural proteomics. Virus particles were purified using a linear idioxanol\n(Optiprep) gradient according to the protocol above for Nanopore sequencing. The purified\nvirions were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% SLS, 2 mM TCEP) and\n13\n428\n429\n430\n431\n432\n433\n434\n435\n436\n437\n438\n439\n440\n441\n442\n443\n444\n445\n446\n447\n448\n449\n450\n451\n452\n453\n454\n455\n456\n457\n458\n459\n460\n461\n462\n463\n464\n465\n466\n467\n468\n469\n470\n471\n472\n473\n474\n475\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nheated to 95 °C, 10 min. A sonication step (10 sec, 20% amplitude, 0.5 pulse) was\nperformed to degrade nucleic acids in the samples. Following this, iodoacetamide was added\nto the final concentration of 4 mM, and the samples were incubated for 30 minutes under\nlight protection.\nA total of 20 µl each of Sera-Mag carboxylated magnetic beads (Cytivia) and Sera-Mag\ncarboxylated magnetic beads with 0.05% Azide (Cytivia) were mixed, followed by two rounds\nof washing with 200 µl water and resuspension in 100 µl water. Subsequently, 4 µl of the\nbead mixture was used for protein purification. To this, a protein sample adjusted to a final\nacetonitrile (ACN) concentration of 70% (v/v) was added. The mixture was vortexed before\nthe supernatant was discarded after the magnetic separation of the beads. The beads were\nwashed twice with 300 µl of 70% Ethanol, followed by a wash with 200 µl of ACN, and were\nsubsequently dried. For protein digestion, 100 µl of digestion buffer (50 mM ammonium\nbicarbonate with 1 µg of trypsin) was added to the beads. The mixture was incubated\novernight at 30 °C and 1200 rpm. The supernatant was collected and the beads were further\nwashed with 100 µl of water, which was then combined with the initially collected\nsupernatant. To acidify the final sample, containing purified and digested proteins, 30 µl of\n5% Trifluoroacetic acid (TFA) were added.\nThe resulting supernatant was desalted for mass spectrometric analysis using C18\nsolid-phase columns (Chromabond C18 spin columns, Macherey Nagel, Düren, Germany).\nAfter desalting, the solvent was removed by evaporation and dried peptides were stored at\n-20 °C until further use.\nPeptide analysis using liquid chromatography and mass spectrometry\nDried peptides were reconstituted in 0.1% trifluoroacetic acid and then analyzed using\nliquid-chromatography-mass spectrometry carried out on a Exploris 480 instrument\nconnected to an Ultimate 3000 RSLC nano and a nanospray flex ion source (all Thermo\nScientific). Peptide separation was performed on a reverse phase HPLC column (75 µm x 42\ncm) packed in-house with C18 resin (2.4 µm; Dr. Maisch). The following separating gradient\nwas used: 98% solvent A (0.15% formic acid) and 5% solvent B (99.85% acetonitrile, 0.15%\nformic acid) to 30% solvent B over 45 minutes at a flow rate of 300 nl/min.\nThe data acquisition mode was set to obtain one high-resolution MS scan at a resolution of\n60,000 full width at half maximum (at m/z 200) followed by MS/MS scans of the most intense\nions within 1 s (cycle 1s). To increase the efficiency of MS/MS attempts, the charged state\nscreening modus was enabled to exclude unassigned and singly charged ions. The dynamic\nexclusion duration was set to 14 sec. The ion accumulation time was set to 50 ms (MS) and\n50 ms at 17,500 resolution (MS/MS). The automatic gain control (AGC) was set to 3x106 for\nMS survey scan and 2x105 for MS/MS scans.\nFor spectral-based assessment, MS raw files searches were carried out using MSFragger\nembedded within Scaffold 4 (Proteome Software) with 20 ppm peptide and fragment\ntolerance with Carbamidomethylation (C) as fixed, and oxidation (M) as variable modification\nusing a uniprot protein database.\nHost sequencing and 18S+28S trees\n14\n476\n477\n478\n479\n480\n481\n482\n483\n484\n485\n486\n487\n488\n489\n490\n491\n492\n493\n494\n495\n496\n497\n498\n499\n500\n501\n502\n503\n504\n505\n506\n507\n508\n509\n510\n511\n512\n513\n514\n515\n516\n517\n518\n519\n520\n521\n522\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nWe extracted genomic DNA from uninfected host cultures. Each sample was then\nsequenced on an ONT MinION flow cell and basecalled (for specific flow cell and model\nversions, see Table S7). Sequences were assembled as described below for viral genomes.\nThe minimal read length was 1000 bp, minimal Phred quality score 5 except for AL-FL05\nwhere we used a minimum of 10.\nWe extracted the full 18S and 28S sequences from the initial assemblies using barrnap\n53\n.\nTogether with a selection of chlorarachniophytes and an outgroup from Rhizaria (Table S7),\neach gene was aligned separately using MAFFT-G-INS-i v.7.505\n54\nand trimmed using trimAl\n-gt 0.1 v.1.4.rev15\n55\n. Both trimmed alignments were then concatenated and used to\nreconstruct a phylogenetic tree using IQ-TREE v2.0.3\n56\n(--ufboot 1000 --bnni -m MFP\n57\n),\nselecting TIM3+F+R3 as the best fitting model.\nAssembly\nRaw Nanopore signals were basecalled with Dorado v0.5.0 (ChlorV-1; duplex mode) using\nthe highest-accuracy model dna_r10.4.1_e8.2_400bps_sup@v4.1.0 (ChlorV-1). For a full list\nof all basecalling models and versions of dorado see Table S6.\nThe Nanopore reads were assembled using MetaFlye v.2.9.1-b1780\n58\nwith options\n‘--iterations 3 --nano-hq’ after filtering reads with varying thresholds for length (ChlorV-1:\n10000, ChlorV-2: 1000, ChlorV-3: 1000, ChlorV-4: 3000) and basecalling Phred quality score\n(ChlorV-1: 20, ChlorV-2: 5, ChlorV-3: 15, ChlorV-4: 15). Assemblies were further polished\nusing racon v.1.5.0 with options ‘-m 8 -x -6 -g -8 -w 500’ and medaka v.1.11.3 consensus. If\navailable (i.e. ChlorV-1 and ChlorV-4), Illumina reads were used to polish assemblies using\npolypolish v.0.6.0\n59\n.\nAverage nucleotide identity (ANI) and the corresponding alignment coverage between\nassemblies and related genomes of isolated viruses was assessed with pyani v0.3.0-alpha\n60\n(average_nucleotide_identity.py -m ANIb --fragsize 500).\nOpen reading frames (ORFs) were identified using prodigal v.2.6.3\n61\nand GeneMark v.4.32.\nBoth sets of ORFs were then merged, picking longer ORFs in case of overlapping features\nand disregarding ORFs coding for less than 100 amino acids.\nPhylogenetic analyses\nThe seven marker genes A32 genome packaging ATPase, protein-primed DNA polymerase\nB, large subunit of the RNA polymerase, superfamily II helicase, transcription factor II B,\ntopoisomerase II and the poxvirus late transcription factor VLTF3 were used for phylogenetic\nreconstruction. For each marker, DIAMOND protein similarity searches (v.2.0.15.153\n62\n,\nBLASTp mode) between the predicted protein sequences and the reference sequences from\nthe GVOG database were computed. Alignments were then computed per marker\n(MAFFT-E-INS-i v.7.505\n54\n, trimAl -gt 0.1 v.1.4.rev15\n55\n) for a taxon sampling including all\nMimiviridae sequences and other Imitervirales from the GVDB\n30\nas an outgroup. Single gene\ntrees were reconstructed using IQ-TREE v2.0.3\n56\n(--ufboot 1000 -m MFP\n57\n-mset LG) and\ninspected for duplicates, false positives etc. Curated marker sets were re-aligned as above\nand concatenated. Final tree reconstruction was performed with IQ-TREE (-m MFP --mset\nLG,LG+C10,LG+C20,LG+C30,LG+C40,LG4X --ufboot 1000), selecting LG+C40+F+I+R10\nas the best fitting model.\n15\n523\n524\n525\n526\n527\n528\n529\n530\n531\n532\n533\n534\n535\n536\n537\n538\n539\n540\n541\n542\n543\n544\n545\n546\n547\n548\n549\n550\n551\n552\n553\n554\n555\n556\n557\n558\n559\n560\n561\n562\n563\n564\n565\n566\n567\n568\n569\n570\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nAnnotation & comparative genomics\nPredicted protein sequences were functionally annotated using the eggnog-mapper\nv.2.1.12\n32\nusing the eggNOG database v.5.0.2\n31\n, the GVOG database (using HMMer\nv.3.3.2\n63\n) and InterProScan v.5.62_94.0\n33\n. tRNAs were annotated using tRNAscan-SE\nv.2.0.9\n64\n. Taxonomic annotation of the proteins was performed with diamond v.2.0.15.153\n62\nagainst the NR database. Transposable elements were identified using various tools as\nimplemented in the reasonaTE pipeline v.1.0\n65\n.\nThe MEME tool v.5.5.0\n66\nwas used for predicting candidate promoter motifs. First, the\nupstream sequences of each CDS were partitioned based on the presence of the\nsubsequence ‘TCTA’. The tool was then run on the sequences not containing ‘TCTA’ and\nspanning 50 bases upstream and the sequences that do contain this subsequence and\nspanning 40 bases upstream of a CDS. This partitioning was necessary to improve motif\nsignals, as no transcriptomic data for predicting ‘early’ and ‘late’ genes\n16\nwas present. The\ntwo candidate motifs were then tested on all upstream sequences using the tools\nCENTRIMO and FIMO from the MEME suite v.5.5.0.\nFor each predicted protein sequence of ChlorV-1, we applied AlphaFold v.3.0.1\n37\nto infer its\n3D structure. Each predicted structure was then used as a query to search the PDB\ndatabase using foldseek v.8.ef4e960\n38\n‘easy-search’. Alignments were visualized with\nOpen-Source PyMOL v.3.1.0\n67\n. For the four identified penton proteins and 3 DJR capsid\nproteins we additionally performed pentameric or trimeric structural predictions using\nAlphaFold v.3.0.1, respectively.\nAll predicted proteins from all isolate genomes and MAGs that were classified as\nAliimimivirinae in GVDB\n30\nwere subjected to ortholog identification using OrthoFinder\n68\nv.2.5.4. For the different strains of ChlorVs, CroV and Chlorella Virus XW01, all-vs-all\nproteins searches were performed using MMseqs2\n69\nv.14.7e284. Based on these searches,\ngenes in ChlorV genomes with at least 70% sequence identity were manually grouped\ntogether.\nModifications\nFor ChlorV-2,3 and 4, basecalling of modified bases using dorado v.0.7.0 was performed\nusing the context-free models dna_r10.4.1_e8.2_400bps_sup@v5.0.0_4mC_5mC@v3 for\n4mC and 5mC methylations and dna_r10.4.1_e8.2_400bps_sup@v5.0.0_6mA@v3 for 6mA\nmethylations. For ChlorV-1, the rerio\n70\nmodels\nres_dna_r10.4.1_e8.2_400bps_sup@v4.0.1_6mA@v2 and\nres_dna_r10.4.1_e8.2_400bps_sup@v4.3.0_4mC_5mC@v1 were used. The basecalled\nreads were mapped to the respective genome using minimap2\n71\nv.2.24-r1122 and\nsummarized per-base using modkit\n72\npileup v.0.4.2. Modkit motif was used to find short\nsequence motifs that were enriched in methylations.\nFor each of the four DNA methyltransferases putatively linked to a methylation motif, we\nperformed additionally AlphaFold 3 structural predictions including DNA Sequences either\nincluding the motif or not, and including the protein both as a monomer or as a monodimer.\n16\n571\n572\n573\n574\n575\n576\n577\n578\n579\n580\n581\n582\n583\n584\n585\n586\n587\n588\n589\n590\n591\n592\n593\n594\n595\n596\n597\n598\n599\n600\n601\n602\n603\n604\n605\n606\n607\n608\n609\n610\n611\n612\n613\n614\n615\n616\n617\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nThe genomes were additionally scanned for restriction-modification enzymes and systems\nusing DefenseFinder\n73\nv.2.0.0 and diamond v.2.0.15.153 searches against the Restriction\nEnzyme Database\n43\n.\nData and code availability\nAll sequence data, including raw nanopore, PacBio and Illumina reads, assemblies and host\nrRNA genes (OZ264022-OZ264049) have been deposited in the European Nucleotide\nArchive under project PRJEB90372. Further data such as multiple sequence alignments,\nphylogenetic trees, AlphaFold 3 models and other relevant data have been deposited in\nEdmond (https://doi.org/10.17617/3.CC4JL7). 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Systematic and quantitative view of the antiviral arsenal of prokaryotes.\nNat Commun 13 , 2561 (2022).\n23\n793\n794\n795\n796\n797\n798\n799\n800\n801\n802\n803\n804\n805\n806\n807\n808\n809\n810\n811\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nSupplementary Figures:\nFigure S1: Location of the open-ocean collection point Station ALOHA approximately 100 km\nnorth of O‘ahu.\n24\n812\n813\n814\n815\n816\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure S2: Phylogenetic tree of chlorarachniophyte host strains and references. 18S and\n28S rRNA gene sequence alignments were concatenated and used to reconstruct a\nphylogenetic tree under the TIM3+F+R3 model (selected by automatic model selection) in\nIQ-TREE with 1000 ultrafast bootstraps.\n25\n817\n818\n819\n820\n821\n822\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure S3: Flow cytometric detection of ChlorV-1 particles. Viral particles are clearly\ndetectable after glutaraldehyde fixation and staining with SYBR Gold on an Attune NxT™\nflow cytometer equipped with a small particle SSC filter (488/10). The viral population is the\ncircular cloud of dots with the yellow/red center; diffuse populations of dots at the top\nrepresent bacteria that are present in the cultures.\nFigure S4: ANI and pairwise alignment coverage of ChlorVs and isolated virus genomes in\nAliimimivirinae .\n26\n823\n824\n825\n826\n827\n828\n829\n830\n831\n832\n833\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure S5: Aliimimivirinae gene sharing network and genome sizes. All predicted proteins of\nisolate and cultivation-independently acquired genomes within the subfamily Aliimimivirinae\nwere subjected to orthogroup prediction. The orthogroups were then used to cluster the\ngenomes. Genomes in the cluster Aliimimivirinae I are labelled blue, while members of\nAliimimivirinae II are labelled orange. The genome oPacV_662 was not confidently placed in\neither of these groups and thus labelled green.\n27\n834\n835\n836\n837\n838\n839\n840\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure S6: Promoter motifs in ChlorV-1. Two significant motifs were found, likely representing\nan early (AAAAATTGA) and a late (TCTA) promoter, similar to the related Cafeteria\nroenbergensis virus. The position relative to the transcription start site (TSS) differed\nbetween these two motifs, with AAAAATTGA showing a peak around -34 and TCTA around\n-14.\n28\n841\n842\n843\n844\n845\n846\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure S7: Modification frequency in ChlorVs for N4-methylcytosine (4mC),\nC5-methylcytosine (5mC) and N6-methyladenine (6mA). The ‘fraction modified’ is the ratio of\nreads corroborating a specific modification per site and the total coverage. For ChlorV-2,3\nand 4 slightly different models (dna_r10.4.1_e8.2_400bps_sup@v5.0.0_4mC_5mC@v3 for\n4mC and 5mC methylations and dna_r10.4.1_e8.2_400bps_sup@v5.0.0_6mA@v3 for 6mA)\nwere used to basecall the data than for ChlorV-1 (rerio models\n29\n847\n848\n849\n850\n851\n852\n853\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nres_dna_r10.4.1_e8.2_400bps_sup@v4.0.1_6mA@v2 and\nres_dna_r10.4.1_e8.2_400bps_sup@v4.3.0_4mC_5mC@v1) due to delays between\nsequencing runs. Depth of coverage for ChlorV-1 was 2328, while ChlorV-2 had an average\ndepth of  434, ChlorV-3 983 and ChlorV-4 81.\nFigure S8: AlphaFold 3 prediction of a dimer of ChlorV-1..374 with the DNA sequence\nATTAACAT CAATTGTATG AAAATT. Protein domains are colored as in Figure 4. pTM=0.85,\nipTM=0.8.\n30\n854\n855\n856\n857\n858\n859\n860\n861\n862\n863\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure S9: Genome position plot of ChlorV-1 virion proteins. Genes encoding proteins that\nwere detected in purified virions by mass spectrometry are shown as circles in ascending\norder of their respective gene number. The 10 most abundant virion proteins are marked in\nred and labeled with their gene number.\nFigure S10: Trimeric structure predictions of the putative ChlorV-1 DJR capsid proteins 153,\n160, and 178 with AlphaFold 3. All proteins are experimentally verified virion components.\n31\n864\n865\n866\n867\n868\n869\n870\n871\n872\n873\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint \n\nFigure S11: Pentameric structure predictions of the putative ChlorV-1 penton proteins 087,\n095, 265 and 266 with AlphaFold 3. ChlorV-1..087 and ChlorV-1..266 are experimentally\nverified virion proteins.\nSupplements:\nTable S1 Isolation times and location of hosts and viruses.\nTable S2 Sequence-based annotation for all predicted proteins from all four ChlorV strains\nTable S3 ChlorV gene clusters and sequence/structure-based selected annotations.\nTable S4 Summary of ipTM and pTM values for all structural prediction of dimeric or\nmonomeric methyltransferases and different DNA sequences.\nTable S5 Experimentally detected proteins using mass spectrometry.\nTable S6 Sequencing read accessions and basecalling models.\nTable S7 Chlorarachniophyte hosts 18S and 28S gene accessions.\nSupplementary File 1: Single gene trees for giant virus phylogenetic markers.\nSupplementary File 2: Protein structure predictions using AlphaFold 3 and structure-based\nannotation.\nDatashare: Trees and alignments, AlphaFold 3 models, tabular methylation calls, defense\nfinder result files.\n32\n874\n875\n876\n877\n878\n879\n880\n881\n882\n883\n884\n885\n886\n887\n888\n889\n890\n891\n892\n893\n894\n895\n896\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}