Strain-level diversity of giant viruses infecting chlorarachniophyte algae in the subtropical North Pacific

Giant DNA viruses are ubiquitous among unicellular eukaryotes and occur in marine, freshwater, and terrestrial environments. Despite intense metagenomic data mining, their strain-level diversity remains largely unexplored. Here we introduce a model system comprising four isolates of a giant virus called ChlorV, which infects marine microalgae of the class Chlorarachniophyceae (Rhizaria) from station ALOHA, Hawai’i. The ChlorV genomes are 469 kbp to 493 kbp long and encode approximately 400 proteins, at least 106 of which are present in purified virions. Although the four viral genomes are highly syntenic, they differ by several insertions and deletions that often encode methyltransferases. Interestingly, we found that some of these methyltransferase genes correlated with specific DNA methylation patterns in the same ChlorV strain. Our study describes the first giant viruses infecting the eukaryotic supergroup Rhizaria and demonstrates how viral strain-level variation in gene content and epigenetic features may affect eco-evolutionary processes in marine microalgae.

Double-stranded DNA viruses of the phylum Nucleocytoviricota are the giants of the viral world, with particle sizes of up to 1.5 µm and genome lengths of up to 2.5 megabases 1–5 . When the first such virus, Acanthamoeba polyphaga mimivirus, was isolated and analyzed, it showed a remarkable genetic make-up previously unknown from any other virus 6 . Since then, a large number of additional giant virus isolates and thousands of metagenome-assembled genomes have been published. The phylum Nucleocytoviricota currently comprises five orders and 15 families 7 ; among them, the family Mimiviridae (order Imitervirales ) is the best-studied. The Mimiviridae comprises three subfamilies: Megamimivirinae , Klosneuvirinae , and Aliimimivirinae . The Megamimivirinae include the 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint original mimivirus and relatives such as tupanvirus, moumouvirus, megavirus etc., all of which have been isolated on Amoebozoa hosts such as Acanthamoeba or Vermamoeba 1,8–11 . The Klosneuvirinae subfamily contains the isolates Bodo saltans virus, Fadolivirus and Yasminevirus 12–15 . The third subfamily, Aliimimivirinae , contains the well-characterised Cafeteria roenbergensis virus (CroV) that infects the marine heterotrophic stramenopile Cafeteria burkhardae , and Chlorella Virus XW01 that was isolated recently on the freshwater green alga Chlorella sp 16,17 . Nevertheless, the abundance of mimivirid-like sequence fragments found in eukaryotic genomes as well as the inferred co-evolutionary history of giant viruses and protists suggests that most microbial eukaryotes are potential hosts for giant viruses 18,19 . The marine alga Bigelowiella natans belongs to the chlorarachniophytes, a mixotrophic lineage within the otherwise heterotrophic Rhizaria whose members acquired their plastid from a green alga by secondary endosymbiosis 20,21 . In addition to the nuclear and plastid genomes, they retain a remnant of the green algal nucleus, called the nucleomorph 22,23 . B. natans has been studied in the context of plastid evolution and was the first sequenced genome of any rhizarian species 24 . Blanc et al. 25 identified several endogenous viral elements in the B. natans genome assembly, suggesting that this alga frequently interacts with both giant viruses and virophages, the latter being smaller dsDNA viruses that depend on giant viruses for their own replication 26 . However, no virus infecting a chlorarachniophyte has been described to date, limiting insight into virus–host interactions in this clade. Here, we characterize four isolated strains of Chlorarachniophyte virus (ChlorV), a new species of virulent giant virus isolated from Pacific waters of Hawai’i. Phylogenetically, ChlorVs cluster with members of the subfamily Aliimimivirinae , thus expanding the confirmed host range of giant viruses to include chlorarachniophytes (Rhizaria). We describe the infection dynamics of ChlorV infection in its algal host and present a comparative genome analysis of the four ChlorV strains including methylation pattern and associated methyltransferase genes.

& Discussion In search of new algal viruses from tropical marine environments, we collected samples from surface waters at Station ALOHA, a long term oceanographic monitoring site located 100 km north of the Hawaiian Island of Oʻahu (Fig. S1). Several strains of chlorarachniophyte algae (Fig. S2) and viruses infecting them were isolated between 2010 and 2012 (Table S1). As these viruses replicate in members of the Chlorarachniophyceae, we refer to them collectively as ‘ChlorVs’ (for chlor arachniophyte v iruses). All viruses replicated in chlorarachniophyte host strains, albeit with notable differences in strain specificity. Whereas host strains AL-TEMP06, AL-TEMP07 and AL-DI01 were permissive to all four ChlorV strains, host strain AL-FL05 supported only ChlorV-1 replication and was resistant to the remaining three ChlorV strains. In contrast, host strain AL-FL10 could only be infected by ChlorV-2, but not by the other virus strains. All host strains belong to Bigelowiella natans , except for strain AL-FL05, which appears to represent a distinct chlorarachniophyte genus (Fig. S2). Because algal strains AL-FL05 and AL-TEMP06 grew most favorably in our culture conditions, we used them as production strains for ChlorV-1 and ChlorV-2 to ChlorV-4, respectively. 2 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Extracellular ChlorV particles were readily detectable by flow cytometry after DNA staining with SYBR Gold (Fig. S3). By infecting several liters of algal cultures, we collected and concentrated approximately 1E+11 virus particles for each of the four ChlorV strains. Imaging virus particles by negative staining transmission electron microscopy revealed projected pentagonal or hexagonal outlines typical of icosahedral capsids (Fig. 1A-D). Similar to the two other isolated viruses of the subfamily Aliimimivirinae (CroV and Chlorella virus XW01), their capsids appear to be lacking external fibrils. The capsid diameter varied from 200 nm to 230 nm across the four virus strains. Occasionally, small protrusions (13 nm x 5 nm) at one of the capsid corners were visible, which suggests a modified vertex that could function in host recognition or genome release (Fig. 1A,C) 27,28 . Figure 1: ChlorV particle morphology and infection dynamics. A-D: Negative stain transmission electron micrographs of ChlorV particles. A) ChlorV-1. B) ChlorV-2. C) ChlorV-3. D) ChlorV-4. Arrows indicate potential unique capsid structures such as vertex portals. E) ChlorV-1 infection dynamics in host strain AL-FL05. Densities of host cells and extracellular virus-like particles (VLPs) of uninfected and ChlorV-1 infected AL-FL05 cultures were measured by flow cytometry. Quantitative PCR was used to monitor viral DNA replication. VLP concentrations and Cq values of uninfected cultures are 3 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint not plotted, as these were without exception below the limit of detection. Error bars show standard deviations between the three biological replicates and may be smaller than the data symbols. Infection dynamics We analyzed the infection dynamics of ChlorV-1 in host strain AL-FL05, because this combination resulted consistently in the highest virus titers when compared to any other ChlorV – host strain combinations. We exposed the algal cultures to a 12 h light - 12 h dark cycle and infected AL-FL05 with ChlorV-1 at the onset of the light cycle. Triplicate cultures of AL-FL05 were infected with freshly prepared ChlorV-1 at a virus-to-host ratio of two and monitored hourly for the first 36 hours by flow cytometry and quantitative PCR using ChlorV-specific primers (Fig. 1E). The qPCR signal remained constant from the time of infection until 4 h post infection (hpi); thereafter, the quantification cycle (Cq) decreased from 22.5 to 16.0 by 19 hpi, indicating the onset of viral DNA replication around 3-4 hpi and a peak in viral DNA by ~19 hpi. Using flow cytometry, we observed a continuous decrease in extracellular VLPs from 0 hours post infection (hpi) to 8 hpi, most likely due to the uptake of ChlorV-1 particles by host cells. Following this eclipse phase, VLP concentrations in the culture medium increased steadily from 9 hpi to 36 hpi. The slope of VLP increase was steeper towards the end of the first light cycle (8 - 12 hpi) than during the following dark and light cycles (13 - 36 hpi). Furthermore, we observed a delayed decline of host cells in the infected cultures, which occurs only after extracellular VLP concentrations have reached their maximum at 36 hpi. These data are not compatible with a one-step growth curve and instead suggest a constant release of virus particles. In summary, our infection experiments show that ChlorV-1 is a lytic virus; however, the release of progeny virus particles is not immediately linked to host lysis, suggesting a continuous and, at least initially, non-destructive exit strategy, such as budding from host membranes or exocytosis 29 . Genome features After purifying the virus particles through iodixanol density gradients and isolation of viral DNA, we generated high-quality genome assemblies using ultra long MinION sequencing reads at 31x to 700x coverage (Table 1). For ChlorV-1 and ChlorV-4, we also generated short-read Illumina sequencing data to further polish the assemblies. The genomes appear to have a linear topology, as they contained short (1.5 kb to 2.5 kb) terminal inverted repeats (TIR) at both ends, and ranged in size from 469 kb (ChlorV-3) to 493 kb (ChlorV-4). While the genomes of ChlorV-2 and ChlorV-4 were virtually identical with an average nucleotide identity (ANI) exceeding 99.8%, ANIs between other pairs of genomes ranged between 93.2% (ChlorV-1 vs. ChlorV-4) and 97.3% (ChlorV-3 vs. ChlorV-4), see Fig. S4. The differences (insertions/deletions) between genomes were distributed over their complete lengths, and none of the observed differences were longer than 10,000 bp. In comparison, the ANI between any of the four ChlorV genomes and their most closely related giant virus isolates, Cafeteria roenbergensis virus (CroV) and Chlorella virus XW01, was ~70%. However, the alignments between Chlorella Virus XW01 and ChlorVs covered about 10-12% of their respective genomes, whereas those between CroV and the ChlorVs covered only ~4–6% (within ChlorVs, the coverage ranged from 75% to 100%, Fig. S4). Table 1: Genome statistics of ChlorV strains 1-4. In all cases, ONT was used for the initial assembly which was then polished using Illumina data if available. PacBio data for ChlorV-1 was only used to validate the ONT-based assembly. 4 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Strain Genome length (bp) % GC # ORFs Coverage (ONT) Data source Accession ChlorV-1 487,585 19.05 402 263 ONT+PacBio +Illumina GCA_974392635 ChlorV-2 490,841 18.54 409 639 ONT GCA_975145565 ChlorV-3 468,809 18.82 391 700 ONT GCA_976281805 ChlorV-4 493,487 18.59 394 31 ONT+Illumina GCA_975141545 In each of the four ChlorV genomes, we identified ≈400 open reading frames (ORFs) encoding proteins >100 amino acids, with 56 to 72 sequences per genome not matching any known cellular or viral genes (ORFans). Accounting for ORFs that were split in some assemblies, 396 non-redundant genes were identified across all virus strains. A core genome of 307 genes was shared across all strains, while 381 were shared between ChlorV-2 and ChlorV-4, 336 between ChlorV-2, ChlorV-3 and ChlorV-4 and 316 between ChlorV-1 and ChlorV-3. ChlorV phylogenetic position To establish the phylogenetic position of these viruses, we identified putative homologs of established marker genes specific for viruses of the phylum Nucleocytoviricota 30 . Each ChlorV genome contained a full complement of these conserved genes, further validating the quality of the assemblies. For each marker gene, we reconstructed a phylogenetic tree with broad taxonomic sampling across the Nucleocytoviricota . In each of these trees, the four ChlorV genomes formed a tight cluster in the Mimiviridae family, with CroV and Chlorella Virus XW01 being the closest isolated relatives (Supplementary File 1). We then reconstructed a phylogenetic tree based on a concatenated alignment of seven marker proteins, focusing on genomes of Mimiviridae members (isolates and environmental genomes) from the giant virus database (GVDB). Selected representatives of other Imitervirales lineages were chosen as an outgroup. The ChlorVs formed a well-supported monophyletic group with Chlorella Virus XW01, CroV and several environmental genomes in the subfamily Aliimimivirinae (Fig. 2). The addition of ChlorVs makes the Aliimimivirinae one of the most diverse groups of Nucleocytoviricota with respect to host range. Three different eukaryotic supergroups (Rhizaria: Chlorarachniophytes; Stramenopiles: Cafeteria ; Archaeplastida: Chlorella ) are infected by members of this subfamily. In fact, the ChlorVs, CroV and Chlorella Virus XW01 cluster in one group within the Aliimimivirinae ( Aliimimivirinae I), whereas the lake sturgeon-associated Namao virus and various metagenome-assembled genomes form a second group ( Aliimimivirinae II), indicating two well-supported clades within Aliimimivirinae (here referred to as clades I and II; Fig. 2, Fig. S5). 5 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure 2: Phylogenetic position of ChlorV isolates. ChlorV 1-4 form a well-supported and closely related group within the Mimiviridae subfamily Aliimimivirinae . Their closest relative isolates are Chlorella Virus XW01 and the Cafeteria roenbergensis virus (CroV) in the same subfamily. The tree is based on the concatenated alignment of 7 marker genes and was inferred under the best fitting 6 199 200 201 202 203 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint evolutionary model LG+C40+F+I+R10, while support was estimated using 1000 ultrafast bootstrap replicates. Besides the phylogenetic position, the distribution of marker genes per genome is drawn as a coulson plot and the source environment as a colored circle (dark blue: marine, light blue: freshwater, pink: animal host, orange: soil, green: peat moss). Genome annotation & Comparative genomics We annotated the predicted protein sequences using multiple strategies and databases, including EggNOG, InterPro (incorporating PFAM) and the giant virus orthologous groups (GVOGs) 30–33 . Besides ORFans with no matches to any known sequences, 128-146 ORFs per genome only matched poorly characterized reference sequences, often predicted from other giant viruses. Of the 173-190 sequences with a robust annotation, most were categorized as ‘hydrolases’ (e.g. peptidases or nucleases), ‘replication, transcription and DNA repair’ (e.g. helicases, replication factors, polymerases) or ‘transferases’ (e.g. methyl- and glycosyltransferases) (Table S2). The ChlorV genomes encode a full complement of DNA replication genes, including a family B DNA polymerase (ChlorV-1..065), a DNA clamp (ChlorV-1..016) and clamp loader (replication factor C, e.g. ChlorV-1..164). They further encode a DNA ligase (ChlorV-1..107) and a ribonuclease H (ChlorV-1..329) as well as multiple helicases including a D5-like helicase-primase (ChlorV-1..361) and two subunits of a ribonucleotide reductase (ChlorV-1..059 and ChlorV-1..370). Transcription-related enzymes are also encoded in the genomes, including several subunits of the RNA polymerase (Rpb1,2,3/11,5,6,7, ChlorV-1..202,039,325,253,359,251), an mRNA capping enzyme (ChlorV-1..311), a poly(A) polymerase (ChlorV-1..312) and several transcription factors including homologs of the Poxvirus VLTF2 (ChlorV-1..152) and VLTF3 (ChlorV-1..384). Several translation initiation (e.g. eIF4E, ChlorV-1..017) and elongation (EF-Tu, ChlorV-1..328) factors are also present. A homolog of the DNA polymerase beta (ChlorV-1..031) that is involved in base excision repair (BER), a putative DNA mismatch repair protein MutS (ChlorV-1..354) and two DNA photolyases (ChlorV-1..224 and ChlorV-1..336) that are potentially involved in DNA repair outside of the host are encoded as well. Additionally encoded proteins include many peptidases (e.g. Chlorv-1..086) and several ankyrin repeat proteins (e.g. Chlorv-1..071). Further noteworthy is the presence of a putative YqaJ-like viral recombinase (ChlorV-1..348) which is involved in cleaving genomic concatemers in some bacteriophages 34 A putative tubulin–tyrosine ligase (Chlorv-1..043) could be involved in manipulation of host cells 35 . Several glycosyltransferases (e.g. Chlorv-1..212) and glycosylases (e.g. ChlorV-1..397) are encoded in the genomes of all strains and could be involved in capsid modifications and host interactions 36 . At least one acetyltransferase (ChlorV-1..005) and one aminotransferase (ChlorV-1..145) were also present. Each ChlorV genome encoded multiple predicted methyltransferase genes (12 in ChlorV-3, 11 in the other strains), and these were often located in strain-specific insertions or deletions (Fig. 3 A-G). In contrast, most other groups of genes were usually conserved across all strains. Several types of nucleases (both RNases and DNases) were also common throughout the genomes, being either shared among strains or strain specific (e.g. the DNA endonuclease ChlorV-1..079, Fig. 3 D, F). 7 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure 3: ChlorV strain level genomics. C: Overview of the complete genomes from four strains of Chlorarachniophyte viruses (ChlorVs). Genes are colored according to whether they were identified in all strains (orange) or absent in at least one strain (blue). Genes with significant sequence matches are connected by dark grey ribbons. A,B,D-G: Details of genomic loci where putative insertions/deletions and an inversion (F) can be observed. DNase: Deoxyribonuclease, SNase: Staphylococcal nuclease (DNA and RNA endo-exonuclease) We then predicted the 3D structure of all ChlorV-1 ORFs using AlphaFold 3 and compared them to experimentally derived structures in the protein database to improve the genome annotation (Supplementary File 2) 37,38 . For most ORFs, the sequence-based and structure-based annotations (where available) agreed well. However, occasionally the structural comparison provided annotations for ORFs with no sequence-based annotation, e.g. for the endonuclease ChlorV-1..079 or the putative penton protein Chlorv-1..095 (Table S3). In other cases, sequence-based annotation could not be substantiated by the structural comparison, e.g. the predicted homing endonuclease Chlorv-1..154. Whereas CroV encodes many FNIP/IP22 repeat proteins, these repeats are absent from ChlorVs, Chlorella virus XW01, and Aliimimivirinae environmental genomes. Besides the protein-coding genes, ChlorV strains encode up to 7 tRNA genes, in contrast to the high number of 48 that are encoded in the genome of CroV, whereas Chlorella Virus XW01 encodes none. 8 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint We also investigated putative promoter sequences of ChlorV genes based on the phylogenetic position close to CroV and previous work on gene promoters in giant viruses generally 39,16,40 . The early motif (AAAAATTGA) and the late motif (TCTA) appear conserved between CroV and ChlorVs in both sequence and positional context (Fig. S6). Unlike the promoters, gene content seems to be more dynamic over larger phylogenetic distances (Fig. S5), which has also been observed in other groups of giant viruses. Genome modifications For all ChlorV genomes, nanopore sequencing data was used to analyze DNA base modification patterns. In all assemblies, the dominant modification was N6-methyladenine (6mA), followed by C5-methylcytosine (5mC) and N4-methylcytosine (4mC). The latter two were rare, except in ChlorV-1 where 5mC was comparatively frequent (Fig. S7). This pattern confirms previous observations that giant viruses display prokaryotic-like DNA modifications with a majority of 6mA 41 . We identified sequence motifs that were highly enriched (all were modified in >90% of occurrences) in methylated bases across the four genomes. We identified three distinct motifs, including two motifs that were shared by two genomes each (Fig. 4A). All motifs were palindromic; accordingly, we did not detect strand-specific differences. ChlorV-1 and ChlorV-3 display a 10 base pair motif (C A NNNNNNTG) that resembles a bipartite recognition sequence, consistent with Type I or certain Type II systems 42,43 . We identified one gene that is present in ChlorV-1 and ChlorV-3 but not the other strains as a candidate methyltransferase targeting this sequence (Fig. 3G). It possesses a 6-adenine-specific methyltransferase domain as well as a single target specificity domain similar to other type I modification genes (Fig. 4B). However, this enzyme could also function as a homooligomer, recognizing and modifying the same DNA sequence on opposite strands, like the type IIB system HaeIV 44 . This is supported by the high confidence scores (ipTM=0.8, pTM=0.85) of a protein-DNA complex containing two copies of this protein together with a DNA sequence containing the motif C A NNNNNNTG, but lower confidences (ipTM < 0.8) if using only a single copy or another DNA sequence (Table S4, Fig. S8). ChlorV-3 (but not ChlorV-1) encodes another candidate methyltransferase with a similar DNA methyltransferase domain and two specificity domains, more similar to typical type I modification genes 45 (Fig. 4E). We could additionally identify a motif for 5mC methylation (R C GY) in ChlorV-1. This motif could be the target of a putative 5-cytosine-specific methyltransferase encoded only in the ChlorV-1 genome (Fig. 3D, Fig. 4C). Right next to this methyltransferase an endonuclease (ChlorV-1..079) was encoded, suggesting that this is indeed a functioning restriction modification system. Finally, we identified a different 6mA motif (TGC A ) in ChlorV-2 and ChlorV-4. This could again be putatively linked to a 6-adenine-specific methyltransferase gene that resembles type IV restriction modification methyltransferases and was encoded only in these two strains (Fig. 3C, Fig. 4A, D). 9 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure 4: DNA modifications in ChlorVs. A: Detected sequence motifs with strong (>99%) modification signal and putative associated methyltransferases. ChlorV-3..102 is a candidate besides ChlorV-3..365 for methylation of the CANNNNNNTG motif, although no homologue of ChlorV-3..102 could be identified in ChlorV-1. For each methyltransferase gene, domain-level annotations are shown. Three genes represent 6-adenine-specific DNA methyltransferases (with ChlorV-4..019 having a different domain than e.g. ChlorV-1..374), while ChlorV-1..078 was inferred to modify cytosine. B-E: Structural models of the four presented methyltransferases. In each model, the sequence domain annotations are visualized using the same colors. Virion proteomics We analyzed the protein composition of purified ChlorV-1 virions by mass spectrometry and identified 106 viral proteins, each represented by at least two peptides (Table S5). These virion proteins were encoded by genes that are spread fairly evenly across the viral genome (Fig. S9). However, nine of the ten most abundant virion proteins were encoded by genes that are located within a 90 kb central region of the genome, indicating a non-random distribution of important structural genes. The most abundant protein was the double jelly roll (DJR) major capsid protein ChlorV-1..153, followed by the major core protein ChlorV-1..167 which is homologous to the core proteins of poxvirus (P4B) and CroV (Crov332). It is noteworthy that the third-most abundant virion protein was ChlorV-1..175, a 709 amino acid long hypothetical protein that lacked homologs in other ChlorV strains. Peptides for all three 10 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint additional DJR capsid paralogs, as well as for two of the four single jelly roll (SJR) penton protein candidates, were also found. We then generated structural predictions for three of the four DJR proteins as trimers and for the four SJR proteins as pentamers (Fig. S10, S11). The structures were generally predicted with high confidence, except for some surface protrusions that might interact with other capsid proteins. The peculiarly structured SJR protein ChlorV-1..087 had a lower confidence value, although the core penton-like structure was still resolved with good support (Fig. S11). We did not predict hetero-multimers for pseudo-hexameric capsomers involving more than one DJR protein, although it is quite possible that such capsomers exist in ChlorVs, similar to e.g. Paramecium bursaria Chlorella virus 1 (PBCV-1) 46 . Among virion proteins with predicted enzymatic functions, we identified the two predicted DNA photolyases ChlorV-1..224 and ChlorV-1..336. As is typical for mimivirids, ChlorV-1 also packages its own transcription system that is represented here by five RNA polymerase subunits, mRNA capping enzyme, poly(A) polymerase, and two transcription factors. All of these transcription proteins are also encoded and packaged by CroV 16,47 .

Isolation of hosts and viruses Strains of diverse eukaryotic phytoplankton were isolated from seawater samples collected from an oligotrophic open-ocean site (Station ALOHA, 22°45’ N, 158°00’ W) 48 , located approximately 100 km north of O‘ahu. For collection dates, see Table S1. Seawater samples were typically enriched with artificial seawater media, F/2 medium or Keller (K) medium 49,50 and allowed to incubate in bottles or multiwell plates until cells of interest were identified. These cells were then isolated either by micropipette or by serial dilution. All phytoplankton cultures were maintained at 24–26 °C on a 12:12 light:dark cycle with approximately 30–100 µmol photons m-2 s-1 of photosynthetically active radiation (PAR). Small subunit ribosomal RNA (18S rRNA) gene sequencing was used initially to identify algal species. For this purpose, cells were pelleted (4000 RCF for 10 min) and DNA extracted and purified (MasterPure Complete DNA and RNA Purification Kit; LGC Biosearch Technologies) or ZymoBIOMICS DNA Mini Kit; Zymo Research). The near-full length gene was amplified by PCR (Expand High Fidelity PCR System; Roche) with forward (5ʻ- ACC TGG TTG ATC CTG CC AG -3ʻ) and reverse (5ʻ- TGA TCC TTC YGC AGG TTC AC -3ʻ) primers targeting the 5’ and 3’ ends of the gene 51 . PCR products were cloned prior to sequencing (TOPO TA Cloning Kit for Sequencing; Invitrogen), and the insert was sequenced using four separate primers (M13f, M13r, 502f, and 1174r) 52 using a fluorescence-based Sanger technique (BigDye Terminator; Applied Biosystems) and capillary electrophoresis (3730XL DNA Analyzer; Applied Biosystems) at the University of Hawai‘i at Mānoa’s facility for Advanced Studies in Genomics, Proteomics, and Bioinformatics (ASGPB). Viruses were isolated from seawater samples collected from the open-ocean Station ALOHA sampling site. Large volumes of seawater were pre-filtered (using either 0.8 µm or 0.45 µm pore size filters) to partially remove cells, and the virus community was concentrated by tangential flow filtration (TFF; Millipore Pellicon 2 Mini System) using 30 kDa nominal molecular weight limit (NMWL) filters. The viral concentrates were amended with media (f/2 or K) and added to healthy phytoplankton cultures. The virus-challenged cultures were 11 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint observed for 1–2 weeks for signs of cell lysis, and lysates that continued to produce a lytic effect after multiple transfers to healthy cells (putatively virus-containing) were stored at 4 °C and propagated at least once per month by challenging new cells (1–10% v/v of lysate added per challenge). Infection experiments Chlorarachniophyte cultures were grown in K medium in sterile polycarbonate flasks, either in 250 mL Erlenmeyer flasks, or in 3 L Fernbach flasks. Cultures were grown in an incubator at 24 °C, 50 rpm shaking, and in 12 h light (at 1300 to 1500 lux) - 12 h dark conditions. Immediately prior to inoculation, host cultures were diluted with K medium to a density of 1.0E+06 cells per mL and aliquoted in 50 ml portions in 250 ml PC Erlenmeyer flasks. Triplicate cultures were inoculated at the onset of the 12 h light cycle with a fresh (less than 2 weeks old, 1.2 µm filtered and stored at 4 °C) ChlorV-1 suspension at a virus-to-host ratio of two. Three flasks with uninfected host cultures were incubated as a negative control. The six flasks were sampled every hour for the first 36 hours, and again at 3 dpi and 5 dpi. At each timepoint, the densities of cells and free virus particles were analyzed immediately by flow cytometry, and 25 µl of each suspension culture were frozen for later qPCR analysis. Flow cytometry For cell counts, 200 µl of suspension culture were transferred to a 1.5 ml microfuge tube and analyzed on an Attune TM NxT Acoustic Focusing Flow Cytometer (ThermoFisher Scientific, Waltham, MA, USA) and proprietary software, with the following instrument settings: Red Laser (RL1-H, voltage 320, threshold 1000) for chlorophyll autofluorescence over side scatter (SSC-H, voltage 350, threshold 1000); blue laser filter slot 1: 488/10+OD2; 50 µl acquisition volume, 25 µl/min flow rate, 25 µl total acquisition. For virus particle counts, 10 µl of suspension culture were pipetted in a 1.5 ml microfuge tube containing 2 µl of 25% EM-grade glutaraldehyde solution (Sigma Aldrich) and 88 µl of 0.1 µm filtered K medium. After incubation at 4 ॰ C for 10 min, 860 µl of 0.1 µm filtered Tris-EDTA buffer and 40 µl of 100x SYBR Gold solution (Life Technologies Corporation, Eugene, OR, USA) were added. The mixture was incubated at 80 ॰ C for 10 min in the dark, allowed to cool to room temperature, and analyzed on an Attune TM NxT Flow Cytometer with the following settings: Blue Laser (BL1-H, voltage 490, threshold 5000) for SYBR fluorescence over side scatter (SSC-H, voltage 340, no threshold); blue laser filter slot 1: 488/10 (small particle filter); 50 µl acquisition volume, 25 µl/min flow rate, 25 µl total acquisition. Gates for cells and virus particles were set manually, and concentrations derived directly from the Attune TM software. Quantitative PCR analysis Sample lysis for qPCR use was performed by adding 50 µL of 2x lysis buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.002% Triton X-100, 0.0002% SDS, 2 mg/ml proteinase K) and 25 µL of distilled H ₂ O to 25 µl of sample (stored at -20 °C). The tubes were mixed by vortexing and briefly centrifuged, then incubated for 60 minutes at 58 °C and for 10 minutes at 95 °C. Each 20 µl qPCR reaction consisted of 10 µl PowerTrack SYBR Green Master Mix (ThermoFisher), 7 µl nuclease-free water, 0.5 µl of 100 µM forward primer, 0.5 µl of 100 µM reverse primer, and 2 µl of the lysed sample. For ChlorV-1 detection, primers MCP_ChlorV-1_F (5’- CAG CAA TAC TCC ATC CGA CGG -3’) and MCP_ChlorV-1_R (5’- ACT GTT AGC ACC GCC AAT GT -3’) were used, for detection of ChlorV-2, ChlorV-3, and ChlorV-4 we used primers MCP_ChlorV-2-4_F (5’ GTG CTG CAC CTA CTG TTC CT -3’) and MCP_ChlorV-2-4_R (5’ TAG CAG CTT CCC AAT CAC CG- 3’). Samples were analyzed 12 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint in technical duplicates per 96-well plate, using an Mx3005P Real-Time PCR System (Stratagene) with the following cycling conditions: 1 cycle of 7 min at 95 °C, 40 cycles of 15 sec at 95 °C and 1 min at 60 °C, 1 cycle of 1 min at 72 °C, and a 55-95 °C ramping for dissociation curve analysis. Sequencing of ChlorV genomes ChlorV-1 samples for Illumina and PacBio sequencing were prepared using 0.45 µm filtration (Millipore Durapore 142mm filter, overlaid with Whatman GF/C filter) followed by tangential flow filtration (30 kDa NMWL; Millipore Pellicon 2 Mini system). Viral particles were purified using centrifugation onto CsCl density cushions (6.5 mL 1.6 g/mL CsCl solution, overlaid with 2 mL 1.1 g/mL CsCl solution; 25,000 rpm for 50 min, using SW28 rotor) followed by centrifugation on a CsCl continuous gradient and 0.45 µm filtration (Millipore Sterivex) followed by another round on a CsCl continuous gradient. CsCl in the virus-containing fraction was exchanged with 1X TE buffer by three rounds of concentration and dilution in a centrifugal ultrafilter (30 kDa NMWL; Millipore Amicon Ultra-0.5) followed by DNA extraction and purification (MasterPure Complete DNA and RNA Purification Kit). Sequencing runs for ChlorV-1 were performed on an Illumina MiSeq Instrument (250bp paired-end) using the Nextera XT library preparation kit at the Georgia Genomics and Bioinformatics Core. PacBio sequencing was performed on a PacBio RS II with P6-C4 chemistry at the University of Washington PacBio Sequencing Services. For Nanopore MinION sequencing, several liters of ChlorV suspension containing at least 1E+08 virus particles per millilitre were centrifuged in Nalgene 1 L centrifuge bottles in a Fiberlite™ F9-6 x 1000 LEX rotor (ThermoFisher) for 20 minutes at 6000 x g, 18 °C. The supernatant was concentrated by tangential flow filtration in a Vivaflow 200 0.2 µm PES unit (Sartorius) to 30-50 ml final volume. The concentrate was loaded on a 10-40% (w/v) linear Optiprep gradient (in 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM MgCl ₂ ) and centrifuged in 14 ml Ultra-Clear SW40 tubes and an SW40 rotor (Beckman) for 2 h at 100,000 x g, 18 °C. The visible virus band was extracted through the tube wall with a syringe and needle, diluted 3-fold with 50 mM Tris-HCl, pH 8.0, 2 mM MgCl ₂ and the virus particles were pelleted by centrifugation (30 min at 20,000 x g, 18 °C) in 1.5 ml microfuge tubes, pellets were pooled, washed, and pelleted again. The final virus sample was resuspended in 200 µl of 50 mM Tris-HCl, pH 8.0, 2 mM MgCl ₂ for DNA extraction with the QIAamp DNA Mini Kit (Qiagen) following the manufacturer’s instructions, but eluting in 50 µl of nuclease-free water. The genomes of ChlorV-1, ChlorV-2 and ChlorV-3 were sequenced using the SQK-LSK114 kit and a MinION FLO-MIN114 flow cell; the genome of ChlorV-4 was sequenced with an SQK-LSK110 kit and a FLO-MIN106 flow cell (Oxford Nanopore Technologies). For sequencing details, see Table S6. In addition, a ChlorV-4 DNA sample was sent to Eurofins Genomics Germany GmbH, where it was sequenced on an Illumina NovaSeq 6000 S4 instrument using the TruSeq DNA PCR-Free library preparation kit. Proteomics sample preparation A virus suspension with a concentration exceeding 10^10 PFU/ml was used for the determination of structural proteomics. Virus particles were purified using a linear idioxanol (Optiprep) gradient according to the protocol above for Nanopore sequencing. The purified virions were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% SLS, 2 mM TCEP) and 13 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint heated to 95 °C, 10 min. A sonication step (10 sec, 20% amplitude, 0.5 pulse) was performed to degrade nucleic acids in the samples. Following this, iodoacetamide was added to the final concentration of 4 mM, and the samples were incubated for 30 minutes under light protection. A total of 20 µl each of Sera-Mag carboxylated magnetic beads (Cytivia) and Sera-Mag carboxylated magnetic beads with 0.05% Azide (Cytivia) were mixed, followed by two rounds of washing with 200 µl water and resuspension in 100 µl water. Subsequently, 4 µl of the bead mixture was used for protein purification. To this, a protein sample adjusted to a final acetonitrile (ACN) concentration of 70% (v/v) was added. The mixture was vortexed before the supernatant was discarded after the magnetic separation of the beads. The beads were washed twice with 300 µl of 70% Ethanol, followed by a wash with 200 µl of ACN, and were subsequently dried. For protein digestion, 100 µl of digestion buffer (50 mM ammonium bicarbonate with 1 µg of trypsin) was added to the beads. The mixture was incubated overnight at 30 °C and 1200 rpm. The supernatant was collected and the beads were further washed with 100 µl of water, which was then combined with the initially collected supernatant. To acidify the final sample, containing purified and digested proteins, 30 µl of 5% Trifluoroacetic acid (TFA) were added. The resulting supernatant was desalted for mass spectrometric analysis using C18 solid-phase columns (Chromabond C18 spin columns, Macherey Nagel, Düren, Germany). After desalting, the solvent was removed by evaporation and dried peptides were stored at -20 °C until further use. Peptide analysis using liquid chromatography and mass spectrometry Dried peptides were reconstituted in 0.1% trifluoroacetic acid and then analyzed using liquid-chromatography-mass spectrometry carried out on a Exploris 480 instrument connected to an Ultimate 3000 RSLC nano and a nanospray flex ion source (all Thermo Scientific). Peptide separation was performed on a reverse phase HPLC column (75 µm x 42 cm) packed in-house with C18 resin (2.4 µm; Dr. Maisch). The following separating gradient was used: 98% solvent A (0.15% formic acid) and 5% solvent B (99.85% acetonitrile, 0.15% formic acid) to 30% solvent B over 45 minutes at a flow rate of 300 nl/min. The data acquisition mode was set to obtain one high-resolution MS scan at a resolution of 60,000 full width at half maximum (at m/z 200) followed by MS/MS scans of the most intense ions within 1 s (cycle 1s). To increase the efficiency of MS/MS attempts, the charged state screening modus was enabled to exclude unassigned and singly charged ions. The dynamic exclusion duration was set to 14 sec. The ion accumulation time was set to 50 ms (MS) and 50 ms at 17,500 resolution (MS/MS). The automatic gain control (AGC) was set to 3x106 for MS survey scan and 2x105 for MS/MS scans. For spectral-based assessment, MS raw files searches were carried out using MSFragger embedded within Scaffold 4 (Proteome Software) with 20 ppm peptide and fragment tolerance with Carbamidomethylation (C) as fixed, and oxidation (M) as variable modification using a uniprot protein database. Host sequencing and 18S+28S trees 14 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint We extracted genomic DNA from uninfected host cultures. Each sample was then sequenced on an ONT MinION flow cell and basecalled (for specific flow cell and model versions, see Table S7). Sequences were assembled as described below for viral genomes. The minimal read length was 1000 bp, minimal Phred quality score 5 except for AL-FL05 where we used a minimum of 10. We extracted the full 18S and 28S sequences from the initial assemblies using barrnap 53 . Together with a selection of chlorarachniophytes and an outgroup from Rhizaria (Table S7), each gene was aligned separately using MAFFT-G-INS-i v.7.505 54 and trimmed using trimAl -gt 0.1 v.1.4.rev15 55 . Both trimmed alignments were then concatenated and used to reconstruct a phylogenetic tree using IQ-TREE v2.0.3 56 (--ufboot 1000 --bnni -m MFP 57 ), selecting TIM3+F+R3 as the best fitting model. Assembly Raw Nanopore signals were basecalled with Dorado v0.5.0 (ChlorV-1; duplex mode) using the highest-accuracy model [email protected] (ChlorV-1). For a full list of all basecalling models and versions of dorado see Table S6. The Nanopore reads were assembled using MetaFlye v.2.9.1-b1780 58 with options ‘--iterations 3 --nano-hq’ after filtering reads with varying thresholds for length (ChlorV-1: 10000, ChlorV-2: 1000, ChlorV-3: 1000, ChlorV-4: 3000) and basecalling Phred quality score (ChlorV-1: 20, ChlorV-2: 5, ChlorV-3: 15, ChlorV-4: 15). Assemblies were further polished using racon v.1.5.0 with options ‘-m 8 -x -6 -g -8 -w 500’ and medaka v.1.11.3 consensus. If available (i.e. ChlorV-1 and ChlorV-4), Illumina reads were used to polish assemblies using polypolish v.0.6.0 59 . Average nucleotide identity (ANI) and the corresponding alignment coverage between assemblies and related genomes of isolated viruses was assessed with pyani v0.3.0-alpha 60 (average_nucleotide_identity.py -m ANIb --fragsize 500). Open reading frames (ORFs) were identified using prodigal v.2.6.3 61 and GeneMark v.4.32. Both sets of ORFs were then merged, picking longer ORFs in case of overlapping features and disregarding ORFs coding for less than 100 amino acids. Phylogenetic analyses The seven marker genes A32 genome packaging ATPase, protein-primed DNA polymerase B, large subunit of the RNA polymerase, superfamily II helicase, transcription factor II B, topoisomerase II and the poxvirus late transcription factor VLTF3 were used for phylogenetic reconstruction. For each marker, DIAMOND protein similarity searches (v.2.0.15.153 62 , BLASTp mode) between the predicted protein sequences and the reference sequences from the GVOG database were computed. Alignments were then computed per marker (MAFFT-E-INS-i v.7.505 54 , trimAl -gt 0.1 v.1.4.rev15 55 ) for a taxon sampling including all Mimiviridae sequences and other Imitervirales from the GVDB 30 as an outgroup. Single gene trees were reconstructed using IQ-TREE v2.0.3 56 (--ufboot 1000 -m MFP 57 -mset LG) and inspected for duplicates, false positives etc. Curated marker sets were re-aligned as above and concatenated. Final tree reconstruction was performed with IQ-TREE (-m MFP --mset LG,LG+C10,LG+C20,LG+C30,LG+C40,LG4X --ufboot 1000), selecting LG+C40+F+I+R10 as the best fitting model. 15 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Annotation & comparative genomics Predicted protein sequences were functionally annotated using the eggnog-mapper v.2.1.12 32 using the eggNOG database v.5.0.2 31 , the GVOG database (using HMMer v.3.3.2 63 ) and InterProScan v.5.62_94.0 33 . tRNAs were annotated using tRNAscan-SE v.2.0.9 64 . Taxonomic annotation of the proteins was performed with diamond v.2.0.15.153 62 against the NR database. Transposable elements were identified using various tools as implemented in the reasonaTE pipeline v.1.0 65 . The MEME tool v.5.5.0 66 was used for predicting candidate promoter motifs. First, the upstream sequences of each CDS were partitioned based on the presence of the subsequence ‘TCTA’. The tool was then run on the sequences not containing ‘TCTA’ and spanning 50 bases upstream and the sequences that do contain this subsequence and spanning 40 bases upstream of a CDS. This partitioning was necessary to improve motif signals, as no transcriptomic data for predicting ‘early’ and ‘late’ genes 16 was present. The two candidate motifs were then tested on all upstream sequences using the tools CENTRIMO and FIMO from the MEME suite v.5.5.0. For each predicted protein sequence of ChlorV-1, we applied AlphaFold v.3.0.1 37 to infer its 3D structure. Each predicted structure was then used as a query to search the PDB database using foldseek v.8.ef4e960 38 ‘easy-search’. Alignments were visualized with Open-Source PyMOL v.3.1.0 67 . For the four identified penton proteins and 3 DJR capsid proteins we additionally performed pentameric or trimeric structural predictions using AlphaFold v.3.0.1, respectively. All predicted proteins from all isolate genomes and MAGs that were classified as Aliimimivirinae in GVDB 30 were subjected to ortholog identification using OrthoFinder 68 v.2.5.4. For the different strains of ChlorVs, CroV and Chlorella Virus XW01, all-vs-all proteins searches were performed using MMseqs2 69 v.14.7e284. Based on these searches, genes in ChlorV genomes with at least 70% sequence identity were manually grouped together. Modifications For ChlorV-2,3 and 4, basecalling of modified bases using dorado v.0.7.0 was performed using the context-free models [email protected]_4mC_5mC@v3 for 4mC and 5mC methylations and [email protected]_6mA@v3 for 6mA methylations. For ChlorV-1, the rerio 70 models [email protected]_6mA@v2 and [email protected]_4mC_5mC@v1 were used. The basecalled reads were mapped to the respective genome using minimap2 71 v.2.24-r1122 and summarized per-base using modkit 72 pileup v.0.4.2. Modkit motif was used to find short sequence motifs that were enriched in methylations. For each of the four DNA methyltransferases putatively linked to a methylation motif, we performed additionally AlphaFold 3 structural predictions including DNA Sequences either including the motif or not, and including the protein both as a monomer or as a monodimer. 16 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint The genomes were additionally scanned for restriction-modification enzymes and systems using DefenseFinder 73 v.2.0.0 and diamond v.2.0.15.153 searches against the Restriction Enzyme Database 43 . Data and code availability All sequence data, including raw nanopore, PacBio and Illumina reads, assemblies and host rRNA genes (OZ264022-OZ264049) have been deposited in the European Nucleotide Archive under project PRJEB90372. Further data such as multiple sequence alignments, phylogenetic trees, AlphaFold 3 models and other relevant data have been deposited in Edmond (https://doi.org/10.17617/3.CC4JL7). Custom code and scripts are available at https://github.com/maxemil/chlorv-genomes.

We would like to thank Chris Roome for IT-support.

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Supplementary Figures: Figure S1: Location of the open-ocean collection point Station ALOHA approximately 100 km north of O‘ahu. 24 812 813 814 815 816 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure S2: Phylogenetic tree of chlorarachniophyte host strains and references. 18S and 28S rRNA gene sequence alignments were concatenated and used to reconstruct a phylogenetic tree under the TIM3+F+R3 model (selected by automatic model selection) in IQ-TREE with 1000 ultrafast bootstraps. 25 817 818 819 820 821 822 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure S3: Flow cytometric detection of ChlorV-1 particles. Viral particles are clearly detectable after glutaraldehyde fixation and staining with SYBR Gold on an Attune NxT™ flow cytometer equipped with a small particle SSC filter (488/10). The viral population is the circular cloud of dots with the yellow/red center; diffuse populations of dots at the top represent bacteria that are present in the cultures. Figure S4: ANI and pairwise alignment coverage of ChlorVs and isolated virus genomes in Aliimimivirinae . 26 823 824 825 826 827 828 829 830 831 832 833 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure S5: Aliimimivirinae gene sharing network and genome sizes. All predicted proteins of isolate and cultivation-independently acquired genomes within the subfamily Aliimimivirinae were subjected to orthogroup prediction. The orthogroups were then used to cluster the genomes. Genomes in the cluster Aliimimivirinae I are labelled blue, while members of Aliimimivirinae II are labelled orange. The genome oPacV_662 was not confidently placed in either of these groups and thus labelled green. 27 834 835 836 837 838 839 840 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure S6: Promoter motifs in ChlorV-1. Two significant motifs were found, likely representing an early (AAAAATTGA) and a late (TCTA) promoter, similar to the related Cafeteria roenbergensis virus. The position relative to the transcription start site (TSS) differed between these two motifs, with AAAAATTGA showing a peak around -34 and TCTA around -14. 28 841 842 843 844 845 846 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure S7: Modification frequency in ChlorVs for N4-methylcytosine (4mC), C5-methylcytosine (5mC) and N6-methyladenine (6mA). The ‘fraction modified’ is the ratio of reads corroborating a specific modification per site and the total coverage. For ChlorV-2,3 and 4 slightly different models ([email protected]_4mC_5mC@v3 for 4mC and 5mC methylations and [email protected]_6mA@v3 for 6mA) were used to basecall the data than for ChlorV-1 (rerio models 29 847 848 849 850 851 852 853 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint [email protected]_6mA@v2 and [email protected]_4mC_5mC@v1) due to delays between sequencing runs. Depth of coverage for ChlorV-1 was 2328, while ChlorV-2 had an average depth of 434, ChlorV-3 983 and ChlorV-4 81. Figure S8: AlphaFold 3 prediction of a dimer of ChlorV-1..374 with the DNA sequence ATTAACAT CAATTGTATG AAAATT. Protein domains are colored as in Figure 4. pTM=0.85, ipTM=0.8. 30 854 855 856 857 858 859 860 861 862 863 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure S9: Genome position plot of ChlorV-1 virion proteins. Genes encoding proteins that were detected in purified virions by mass spectrometry are shown as circles in ascending order of their respective gene number. The 10 most abundant virion proteins are marked in red and labeled with their gene number. Figure S10: Trimeric structure predictions of the putative ChlorV-1 DJR capsid proteins 153, 160, and 178 with AlphaFold 3. All proteins are experimentally verified virion components. 31 864 865 866 867 868 869 870 871 872 873 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint Figure S11: Pentameric structure predictions of the putative ChlorV-1 penton proteins 087, 095, 265 and 266 with AlphaFold 3. ChlorV-1..087 and ChlorV-1..266 are experimentally verified virion proteins. Supplements: Table S1 Isolation times and location of hosts and viruses. Table S2 Sequence-based annotation for all predicted proteins from all four ChlorV strains Table S3 ChlorV gene clusters and sequence/structure-based selected annotations. Table S4 Summary of ipTM and pTM values for all structural prediction of dimeric or monomeric methyltransferases and different DNA sequences. Table S5 Experimentally detected proteins using mass spectrometry. Table S6 Sequencing read accessions and basecalling models. Table S7 Chlorarachniophyte hosts 18S and 28S gene accessions. Supplementary File 1: Single gene trees for giant virus phylogenetic markers. Supplementary File 2: Protein structure predictions using AlphaFold 3 and structure-based annotation. Datashare: Trees and alignments, AlphaFold 3 models, tabular methylation calls, defense finder result files. 32 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.688996doi: bioRxiv preprint