2 pattern in archaeal virus communities - biorxiv · 1/16/2020 · whereas bicaudaviruses appear...

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New virus isolates from Italian hydrothermal environments underscore the biogeographic 1

pattern in archaeal virus communities 2

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Diana P. Baquero1,2, Patrizia Contursi3, Monica Piochi4, Simonetta Bartolucci3, Ying Liu1, 6

Virginija Cvirkaite-Krupovic1, David Prangishvili1,* and Mart Krupovic1,* 7

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1 Archaeal Virology Unit, Department of Microbiology, Institut Pasteur, 75015 Paris, France 10

2 Sorbonne Universités, UPMC Univ, 75005 Paris, France 11

3 University of Naples Federico II, Department of Biology, Naples, Italy 12

4 Istituto Nazionale di Geofisica e Vulcanologia, Naples, Osservatorio Vesuviano, Italy 13

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* Correspondence to: [email protected] and [email protected] 15

Institut Pasteur, Department of Microbiology, 16

75015 Paris, France 17

Tel: 33 (0)1 40 61 37 22 18

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Running title 21

Biogeographic pattern in archaeal virus communities 22

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Competing Interests 25

The authors declare that they have no competing interests. 26

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.CC-BY-NC-ND 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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ABSTRACT 32

Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, 33

showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we 34

investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, 35

Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, 36

droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed 37

to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and 38

characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus 39

Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family 40

Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the 41

order Sulfolobales, namely, Saccharolobus, Acidianus and Metallosphaera. Notably, 42

Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. 43

Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear 44

biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting 45

geographical structuring of virus communities in extreme geothermal environments. Furthermore, 46

we propose a revised classification of the Rudiviridae family, with establishment of five new 47

genera. Collectively, our results further show that high-temperature continental hydrothermal 48

systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses. 49



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INTRODUCTION 50

One of the most remarkable features of hyperthermophilic archaea is the diversity and uniqueness 51

of their viruses. Most of these viruses infect members of the phylum Crenarchaeota and are 52

evolutionarily unrelated to viruses infecting bacteria, eukaryotes or even archaea thriving at 53

moderate temperate [1-4]. Thus far, unique to hyperthermophilic archaea are rod-shaped viruses 54

of the families Rudiviridae and Clavaviridae; filamentous enveloped viruses of the families 55

Lipothrixviridae and Tristromaviridae; as well as spherical (Globuloviridae), ellipsoid 56

(Ovaliviridae), droplet-shaped (Guttaviridae), coil-shaped (Spiraviridae) and bottle-shaped 57

(Ampullaviridae) viruses [1,5]. Hyperthermophilic archaea are also infected by two types of 58

spindle-shaped viruses, belonging to the families Fuselloviridae and Bicaudaviridae [6,7]. 59

Whereas bicaudaviruses appear to be restricted to hyperthermophiles, viruses distantly related to 60

fuselloviruses are also known to infect hyperhalophilic archaea [8], marine hyperthermophilic 61

archaea [9,10] and marine ammonia-oxidizing archaea from the phylum Thaumarchaeota [11]. 62

Finally, hyperthermophilic archaea are also infected by three groups of viruses with icosahedral 63

virions, Turriviridae [12], Portogloboviridae [13] and two closely related, unclassified viruses 64

infecting Metallosphaera species [14]. Portoglobovirus SPV1 is structurally and genomically 65

unrelated to other known viruses [13,15], whereas viruses structurally similar to turriviruses are 66

widespread in all three domains of life [1,16,17]. Structural studies on filamentous and spindle-67

shaped crenarchaeal viruses have illuminated the molecular details of virion organization and 68

further underscored the lack of relationship to viruses of bacteria and eukaryotes [18-24]. 69

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The uniqueness of hyperthermophilic archaeal viruses extends to their genomes, with ∼75% of the 71

genes lacking detectable homologues in sequence databases [25]. All characterized archaeal 72

viruses have DNA genomes, which can be single-stranded (ss) or double-stranded (ds), linear or 73

circular. Comparative genomic and bipartite network analyses have shown that viruses of 74

hyperthermophilic archaea share only few genes with the rest of the virosphere [26]. Furthermore, 75

most of the genes in viruses from different families are family-specific, albeit a handful of genes 76

encoding transcription regulators, glycosyltransferases and anti-CRISPR proteins display broader 77

distribution [27]. These observations, in combination with the structural studies, led to the 78

suggestion that crenarchaeal viruses have originated on multiple independent occasions and 79

constitute a unique part of the virosphere [1]. 80



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Although the infection cycles of crenarchaeal viruses has been studied for just a handful of 82

representative viruses, the available data has already provided valuable insight into the virus-host 83

interaction strategies in archaea. For instance, members of the Fuselloviridae, Guttaviridae, 84

Turriviridae and Bicaudaviridae are temperate and their genomes can be site-specifically 85

integrated into the host chromosome by means of a virus-encoded integrase [28-33]. In the case of 86

SSV1, the most extensively characterized member of the Fuselloviridae family, the virus 87

integrates its genome into the host chromosome at an arginyl-tRNA gene upon cell infection. After 88

exposure to UV light, the excision and replication of the SSV1 viral genomes is induced, leading 89

to virion release [34,35]. Two different egress strategies have been elucidated. The enveloped 90

virions of fusellovirus SSV1 are assembled at the host cell membrane and are released from the 91

cell by a budding mechanism similar to that of some eukaryotic enveloped viruses [36]. By 92

contrast, lytic crenarchaeal viruses belonging to three unrelated families, Rudiviridae, Turriviridae 93

and Ovaliviridae, employ a unique release mechanism based on the formation of pyramidal 94

protrusions on the host cell surface, leading to perforation of the cell envelope and release of 95

intracellularly assembled mature virions [5,37,38]. 96

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Single-cell sequencing combined with environmental metagenomics of hydrothermal microbial 98

community from Yellowstone National Park [39] led to the estimation that >60% of cells contain 99

at least one virus type and a majority of these cells contain two or more virus types [40]. However, 100

despite their diversity, distinctiveness and abundance, the number of isolated species of viruses 101

infecting hyperthermophilic archaea remains low compared to the known eukaryotic or bacterial 102

viruses [2]. Indeed, it has been estimated that only about 0.01-0.1% of viruses present in 103

geothermal acidic environments have been isolated [41]. Similarly, using a combination of viral 104

assemblage sequencing and network analysis, it has been estimated that out of 110 identified virus 105

groups, less than 10% represent known archaeal viruses, suggesting that the vast majority of virus 106

clusters represent unknown viruses, likely infecting archaeal hosts [42]. Furthermore, the evolution 107

and structuring of virus communities in terrestrial hydrothermal settings remains poorly 108

understood. Here, to improve understanding on these issues, we explored the diversity of archaeal 109

viruses at the active solfataric field of the Campi Flegrei volcano [43,44] in Pozzuoli, Italy, namely 110

the Pisciarelli hydrothermal area. We report on the isolation and characterization of five new 111



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archaeal viruses belonging to three different families and infecting hosts from five different 112

crenarchaeal genera. 113



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MATERIALS AND METHODS 114

Enrichment cultures 115

Nine samples were collected from hot springs, mud pools and hydrothermally altered terrains at 116

the solfataric field of the Campi Flegrei volcano in Pozzuoli, Italy — the study area known as 117

Pisciarelli — with temperatures ranging from 81 to 96°C and pH between 1 and 7 (Supplementary 118

Information, Table S1). Each sample was inoculated into medium favoring the growth of 119

Sulfolobus/Saccharolobus (basal salt solution supplemented with 0.2% tryptone, 0.1% yeast 120

extract, 0.1% sucrose, pH adjusted to 3.5), Acidianus (basal salt solution supplemented with 0.2% 121

tryptone, 0.1% yeast extract, sulfur, pH adjusted to 3.5), and Pyrobaculum (0.1% yeast extract, 1% 122

tryptone, 0.1-0.3% Na2S2O3 x 5 H2O, pH adjusted to 7) species [45,46]. The enrichment cultures 123

were incubated for 10 days at 75°C, except for Pyrobaculum cultures, which were grown at 90°C 124

for 15 days without shaking. 125

126

VLP concentration and purification 127

VLPs were collected after removal of cells from the enrichment culture (7,000 rpm, 20 min, Sorvall 128

1500 rotor) and concentrated by ultracentrifugation (40,000 rpm, 2h, 10°C, Beckman SW41 rotor). 129

The particles were resuspended in buffer A: 20 mM KH2PO4, 250 mM NaCl, 2.14 mM MgCl2, 130

0.43 mM Ca(NO3)2, <0.001% trace elements of Sulfolobales medium, pH 6 [47] and visualized 131

under the transmission electron microscope (TEM) FEI Tecnai Biotwin as described below. VLPs 132

were further purified by centrifugation in a CsCl buoyant density gradient (0.45 g ml−1) with a 133

Beckman SW41 rotor at 39,000 rpm for 20 h at 10°C. All opalescent bands were collected with a 134

needle and a syringe, dialyzed against buffer A and analyzed by TEM for the presence of VLPs. 135

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Transmission electron microscopy and VLP analysis 137

For negative-stain TEM, 5 µl of the samples were applied to carbon‐coated copper grids, 138

negatively stained with 2% uranyl acetate and imaged with the transmission electron microscope 139

FEI Tecnai Biotwin. The width and length of the negatively stained virus particles were determined 140

using ImageJ (n=80) [48]. 141

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Isolation of virus-host pairs 143



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Strains of cells were colony purified by plating dilutions of the Sulfolobus/Saccharolobus 144

enrichment cultures onto Phytagel (0.7% [wt/vol]) plates incubated for 7 days at 75 °C. Brownish 145

colonies were toothpicked, inoculated into 1 mL of growth medium and incubated at 75°C for 3 146

days. To isolate the sensitive hosts of the observed VLPs, 5-µl aliquots of concentrated VLP 147

preparations were spotted onto Phytagel (0.3% [wt/vol]) plates containing lawns of each isolate, 148

as described previously [46]. Inhibition zones were cut out from the phytagel and inoculated into 149

20 mL of exponentially growing cultures of the corresponding isolates. After incubation at 75°C 150

for 3 days, the VLPs were concentrated by ultracentrifugation (40,000 rpm, 2h, 10°C, Beckman 151

SW41 rotor), resuspended in buffer A and observed by TEM. Pure virus strains were obtained by 152

two rounds of single-plaque purification, as described previously [34]. 153

154

For Pyrobaculum enrichment cultures, a different approach was carried out since it was not 155

possible to obtain single strain isolates. Growing liquid cultures of P. arsenaticum 2GA [45], P. 156

arsenaticum PZ6 (DSM 13514) [49], P. calidifontis VA1 (DSM 21063) [50] and P. 157

oguniense TE7 (DSM 13380) [51] were mixed with concentrated VLPs and incubated for 15 days 158

at 90°C. Increase in the number of extracellular viral particles was verified by TEM. In order to 159

obtain cultures producing just one type of viral particles, eight ten-fold serial dilutions (10 ml) of 160

infected cells were established, incubated at 90°C for 22 days and observed by TEM. Because a 161

plaque assay could not be established for VLPs infecting Pyrobaculum, we relied on TEM 162

observations for assessment of virus-host interactions. 163

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Host range 165

The following laboratory collection strains were used to test the ability to replicate the viruses 166

MRV1, ARV3 and SSRV1: A. convivator [31], A. hospitalis [52], Saccharolobus solfataricus P1 167

(GenBank accession no. NZ_LT549890) and S. solfataricus P2 [53], Sulfolobus islandicus strains 168

REN2H1 [46], HVE10/4 [54], LAL14/1 [55], REY15A [54], ΔC1C2 [56], and Sulfolobus 169

acidocaldarius strain DSM639 [57]. CsCl gradient-purified virus particles were added directly to 170

the growing cultures of Acidianus species and virus propagation was evaluated by TEM. For 171

Sulfolobus and Saccharolobus species, spot tests were performed and the replication of the virus 172

was evidenced by the presence of an inhibition zone on the lawn of the tested strains. 173

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The host ranges of PFV2 and PSV2 were examined by adding purified virions to the growing 175

cultures of P. arsenaticum PZ6 (DSM 13514) [49], P. calidifontis VA1 (DSM 21063) and P. 176

oguniense TE7 (DSM 13380) [51]. The virus propagation was monitored by TEM since plaque 177

assays could not be established. 178

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16S rDNA sequence determination 180

DNA was extracted from exponentially growing cell cultures using the Wizard Genomic DNA 181

Purification Kit (Promega). The 16S rRNA genes were amplified by PCR using 519F (5'-182

CAGCMGCCGCGGTAA) and 1041R (5'-GGCCATGCACCWCCTCTC) primers [58]. The 183

identity of the isolated strains was determined by blastn search of the corresponding 16S rRNA 184

gene sequences against the non-redundant nucleotide sequence database at NCBI. 185

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Viral DNA extraction, sequencing and analysis of viral genomes 187

Viral DNA was extracted from purified virus particles as described previously [45]. Sequencing 188

libraries were prepared from 100 ng of DNA with the TruSeq DNA PCR-Free library Prep Kit 189

from Illumina and sequenced on Illumina MiSeq platform with 150-bp paired-end read lengths 190

(Institut Pasteur, France). Raw sequence reads were processed with Trimmomatic v.0.3.6 [59] and 191

assembled using SPAdes 3.11.1 [60] with default parameters. Open reading fragments (ORF) were 192

predicted by GeneMark.hmm [61] and RAST v2.0 [62]. Each predicted ORF was manually 193

inspected for the presence of putative ribosome-binding sites upstream of the start codon. The in 194

silico-translated protein sequences were analyzed by BLASTP [63] against the non-redundant 195

protein database at the National Center for Biotechnology Information with an upper threshold E-196

value of 1e-03. Searches for distant homologs were performed using HHpred [64] against PFAM 197

(Database of Protein Families), PDB (Protein Data Bank) and CDD (Conserved Domains 198

Database) databases. Transmembrane domains were predicted using TMHMM [65]. The 199

sequences of the viral genomes have been deposited in GenBank, with accession numbers listed 200

in Table 2. 201

202

Phylogenomic analysis 203

All pairwise comparisons of the nucleotide sequences of rudivirus genomes were conducted using 204

the Genome-BLAST Distance Phylogeny (GBDP) method implemented in VICTOR, under 205



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settings recommended for prokaryotic viruses [66]. The resulting intergenomic distances derived 206

from pairwise matches (local alignments) were used to infer a balanced minimum evolution tree 207

with branch support via FASTME including SPR postprocessing for D6 formula, which 208

corresponds to the sum of all identities found in high-scoring segment pairs divided by total 209

genome length. Branch support was inferred from 100 pseudo-bootstrap replicates each. The tree 210

was rooted with members of the family Lipothrixviridae. 211

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Genome sequences of 19 rudiviruses were further compared using the Gegenees, a tool for 213

fragmented alignment of multiple genomes [67]. The comparison was done in the BLASTN mode, 214

with the settings 200/100. The cutoff threshold for non-conserved material was 40%. 215

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RESULTS 217

Diversity of virus-like particles in enrichments cultures 218

Nine environmental samples (I1-I9) were collected in hot springs, mud pools and hydrothermally 219

altered terrains of the solfataric fields of the Campi Flegrei volcano in Pozzuoli, Italy, with 220

temperatures ranging from 81 to 96°C and pH values between 1 and 7 (Supplementary 221

Information, Table S1). The enrichment cultures were obtained by inoculating the samples into 222

different media favoring the growth of hyperthermophilic members of the genera 223

Sulfolobus/Saccharolobus, Acidianus and Pyrobaculum [45,46]. Virus-like particles (VLPs) were 224

collected from cell-free culture supernatants and visualized under transmission electron 225

microscope (TEM) as described in Materials and Methods. 226

227

A variety of VLPs were detected in the Sulfolobus/Saccharolobus enrichment cultures of samples 228

I3 and I9 and the Pyrobaculum enrichment culture of sample I4 (Figure 1). Based on virion 229

morphologies, the VLPs detected in the two samples inoculated in Sulfolobus/Saccharolobus 230

medium could be assigned to five archaeal virus families: Fuselloviridae (Figure 1A), 231

Bicaudaviridae (Figure 1B), Ampullaviridae (Figure 1C), Rudiviridae (Figure 1D) 232

and Lipothrixviridae (Figure 1E). VLPs propagated in Pyrobaculum medium resembled members 233

of the families Globuloviridae (Figure 1F), Tristromaviridae (Figure 1G) and Guttaviridae (Figure 234

1H). We next set out to establish pure cultures of these different viruses and to isolate their 235

respective hosts. 236

237

Isolation of virus-host pairs 238

In order to isolate VLP-propagating strains, 215 single strain isolates were colony purified from 239

the two enrichment cultures established in the Sulfolobus medium. Concentrated VLPs were first 240

tested against the isolates by spot test. In case of cell growth inhibition, a liquid culture of the 241

isolate was established and infected with the halo zone observed in the spot test. The production 242

of the viral particles was subsequently verified by TEM. As a result, three strains replicating VLPs 243

were identified. Comparison of their 16S rRNA gene sequences showed that the three strains 244

belong to three different genera of the order Sulfolobales, namely, Saccharolobus (until recently 245

known as Sulfolobus), Acidianus and Metallosphaera. The 16S rRNA genes of these strains, 246

POZ9, POZ149 and POZ202, respectively, displayed 100%, 99% and 99% identity to the 247



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corresponding genes of Acidianus brierleyi DSM 1651 (NZ_CP029289), Saccharolobus 248

solfataricus Ron 12/III (X90483) and Metallosphaera sedula SARC-M1 (CP012176). Rod-shaped 249

particles of different sizes were propagated successfully in the three isolated strains (Figure 2A-250

C) and, following the nomenclature used for other rudiviruses, were named Metallosphaera rod-251

shaped virus 1 (MRV1), Acidianus rod-shaped virus 3 (ARV3) and Saccharolobus solfataricus 252

rod-shaped virus 1 (SSRV1), respectively. 253

254

Negatively stained MRV1, ARV3 and SSRV1 virions are rod-shaped particles measuring 630 ± 255

20 × 25 ± 2 nm, 670 ± 40 × 23 ± 4 nm and 750 ± 30 × 24 ± 3 nm, respectively (Figure 2A-C). 256

Similar to members of the Rudiviridae family, the three viral particles have terminal fibers located 257

at each end of the virion, which have been shown to play a role in host recognition in the case of 258

Sulfolobus islandicus rod-shaped virus 2 (SIRV2) [68]. Interestingly, ARV3 virions appear to be 259

more flexible than MRV1 and SSRV1 particles, resembling Acidianus rod-shaped virus 1 (ARV1) 260

[69]. 261

262

A different approach was chosen to identify the hosts of the viral particles detected in the 263

Pyrobaculum medium. Exponentially growing liquid cultures of Pyrobaculum strains were mixed 264

with concentrated VLPs and incubated for 15 days at 90°C (see Materials and Methods). The 265

replication of the particles was monitored by TEM. P. arsenaticum 2GA propagated filamentous 266

and spherical particles, named Pyrobaculum filamentous virus 2 (PFV2; Figure 2D) and 267

Pyrobaculum spherical virus 2 (PSV2; Figure 2E), respectively. Because P. arsenaticum 2GA 268

could not be grown as a lawn on solid medium, dilutions of infected cells were made in order to 269

establish cultures infected with just one type of viral particles. 270

271

Negatively stained virions of PFV2 are filamentous and flexible particles of about 450 ± 20 × 34 272

± 4 nm in size with terminal filaments of up to 130 nm in length attached to one or both ends of 273

the virions (Figure 2D), similar to what has been reported for PFV1 [45]. PSV2 virions are 274

spherical particles of around 90 ± 20 nm of diameter, with a variable number of bulging protrusions 275

on their surface (Figure 2E), resembling Pyrobaculum spherical virus (PSV) particles [70]. 276



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Unfortunately, the hosts for other VLPs shown in Figure 1 could not be isolated, either due to 277

unfavorable growth conditions in the laboratory or due to virus-mediated extinction of the 278

corresponding host cell populations. 279

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Host Range 281

The host ranges of MRV1, ARV3 and SSRV1 were investigated using strains of the 282

genera Acidianus, Sulfolobus and Saccharolobus (Table 1). Only ARV3 could be propagated on 283

A. hospitalis, whereas none of the other tested strains served as a host for MRV1 and SSRV1, even 284

though S. solfataricus P1 and P2 were also isolated from the solfataric field in Pozzuoli [53]. The 285

host ranges of PFV2 and PSV2 were studied using strains of the genus Pyrobaculum (Table 1). 286

None of the tested strains supported propagation of PSV2. By contrast, apparent increase in the 287

number of PFV2 particles was observed by TEM in the presence of P. oguniense TE7, albeit not 288

to the same extent as with P. arsenaticum 2GA, indicating that P. oguniense TE7 is not optimal 289

for the virus propagation. 290

291

Genome organization 292

Genomes of the five viruses were isolated from the purified virions and treated with DNase I, type 293

II restriction endonucleases (REases) and RNase A. None of the viral genomes were sensitive to 294

RNase A, but could be digested by DNase I and REases, indicating that all viral genomes consist 295

of dsDNA molecules. The genomes were sequenced on Illumina MiSeq platform, with the 296

assembled contigs corresponding to complete or near-complete virus genomes. The general 297

properties of the virus genomes are summarized in Table 2. 298

299

New species in the Globuloviridae family 300

The linear dsDNA genome of PSV2 is 18,212 bp in length and has a GC content of 45%, which is 301

similar to that of other Pyrobaculum-infecting viruses (45-48%) [45,70]. The coding region of the 302

PSV2 genome is flanked by perfect 55 bp-long terminal inverted repeats (TIR), confirming the 303

linear topology and (near) completeness of the genome. It contains 32 predicted open reading 304

frames (ORFs), all located on the same strand. Thirteen PSV ORFs contain at least one predicted 305

membrane-spanning region. Notably, two of them (ORFs 3 and 9) have nine predicted 306

transmembrane domains. 307



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308

Globuloviruses stand out as some of the most mysterious among archaeal viruses, with 98% of 309

their proteins showing no similarity to sequences in public databases [25]. In part, this is due to 310

the fact that the family is represented by only two members, PSV and TTSV1, infecting 311

Pyrobaculum [70] and Thermoproteus tenax [71], respectively, which precludes generation of 312

sensitive sequence profiles for remote homology searches. Previous structural genomics initiatives 313

were also not very illuminating [72]. As a result, the vast majority of the globulovirus proteins lack 314

functional annotation. Homologs of PSV2 proteins were identified exclusively in members of the 315

Globuloviridae (Figure 3), corroborating the initial affiliation of PSV2 into the family 316

Globuloviridae based on the morphological features of the virion. Nineteen PSV2 ORFs, including 317

those encoding the three major structural proteins (VP1-VP3), have closest homologs in PSV [70] 318

with amino acid sequence identities ranging between 28% and 65% (Figure 3); 13 of these ORFs 319

are also shared with TTSV1. The remaining 13 PSV2 ORFs yielded no significant matches to 320

sequences in public databases. The three genomes display no appreciable similarity at the 321

nucleotide sequence level, indicating considerable sequence diversity within the Globuloviridae 322

family. Notably, among five PSV proteins for which high-resolution structures are available [72], 323

only one protein with a unique fold, PSV gp11 (ORF239), is conserved in PSV2. 324

325

Sensitive profile-profile comparisons using HHpred allowed functional annotation of only four 326

PSV2 proteins. The PSV2 ORF2 encodes a protein with an AAA+ ATPase domain, which is most 327

closely related to those found in ClpB-like chaperones and heat shock proteins (HHpred 328

probability of 99.6%; Supplementary Information, Table S2). ORF3 encodes one of the two 329

proteins with nine putative transmembrane domains and is predicted to function as a membrane 330

transporter, most closely matching bacterial and archaeal cation exchangers (HHpred probability 331

of 95.5%). Interestingly, the product of ORF4 is predicted to be a circadian clock protein KaiB, 332

albeit with a lower probability (HHpred probability of 93%). Finally, ORF32 shares homology 333

with a putative transcriptional regulator with the winged helix-turn-helix domain (wHTH) of 334

Saccharolobus solfataricus (HHpred probability of 94.4%). In addition, ORF11 encodes a 335

functionally uncharacterized DUF1286-family protein conserved in archaea and several 336

Saccharolobus-infecting viruses (Supplementary Information, Table S2). 337

338



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New member in the Tristromaviridae family 339

The linear genome of the isolated PFV2 carries 17,602 bp and contains 39 ORFs, all except one 340

located on the same strand. The coding region is flanked by 59 bp-long TIRs. The GC content 341

(45.3%) of the genome is similar to that of PSV2 and other Pyrobaculum-infecting viruses [45,70], 342

but is considerably lower than in Pyrobaculum arsenaticum PZ6 (58.3%), and Pyrobaculum 343

ogunienseTE7 (55.1%). Eleven of the PFV2 ORFs were predicted to encode proteins with one or 344

more membrane-spanning domains (Supplementary Information, Table S2). 345

346

The PFV2 genome is 98.9% identical over 70% of its length to that of PFV1, the type species of 347

the Tristromaviridae family [45]. PFV1 and PFV2 were isolated ~3 years apart, from the same 348

solfataric field in Pozzuoli [45], suggesting that the population of tristromaviruses is relatively 349

stable over time. Accordingly, 36 of the 39 PFV2 ORFs are nearly identical (average amino acid 350

identity of 96.5%) to those of PFV1. Two events account for the differences between PFV1 and 351

PFV2: (i) a deletion spanning most of the PFV1 gene 27 (including codons 72-495) as well as the 352

downstream genes 28-30, and (ii) insertion of a four-gene block between PFV1 genes 36 and 37 353

in PFV2 (Figure 4). PFV1 gene 27 encodes a minor virion protein, whereas genes 29 and 30 encode 354

putative small, lectin-like carbohydrate-binding proteins (Rensen et al., 2016). The absence of the 355

corresponding genes in PFV2 genome suggests that they are dispensable for the PFV1/PFV2 356

infection cycle. Notably, a homolog of the PFV1 glycoside hydrolase gene 28 is reinserted into 357

PFV2 genome as part of the four-gene block (PFV2 ORF34). However, the two genes do not 358

appear to be orthologous in PFV1 and PFV2 genomes, because they share much lower sequence 359

similarity (50% versus average 96.5% identity) compared to other orthologous genes. By contrast, 360

PFV2 ORF35 has no counterpart in PFV1 but is homologous to the glycosyltransferase gene of 361

Thermoproteus tenax virus 1 (TTV1) [73], the only other known member of the Tristromaviridae 362

family [74](Figure 4). Thus, comparison of the closely related tristromavirus sequences revealed 363

active genome remodeling in this virus group, involving both deletions and horizontal acquisition 364

of new genes. 365

366

New rod-shaped viruses 367

The linear genomes of the isolated rudiviruses have a length ranging from 20,269 to 26,079 bp and 368

a GC content varying between 32.02% and 34.12%. The MRV1 genome contains 80 bp-long TIR, 369



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suggesting that the genome is coding-complete (i.e., contains all protein-coding genes). Although 370

no TIRs could be identified for ARV3 and SSRV1, comparison with the genomes of other 371

rudiviruses (Figure 5) suggests that the two genomes are also nearly complete. 372

373

The genomes of MRV1, ARV3 and SSRV1 contain 27, 33 and 37 ORFs, respectively, and display 374

high degree of gene synteny (Figure 5). Comparison of the three genomes showed that they share 375

26 putative proteins, with amino acid sequence identities ranging between 35.1% and 93.9%. 376

Among the previously reported rudiviruses, the three newly isolated viruses share the highest 377

similarity with ARV2 (ANI of ~78%), which was metagenomically sequenced from samples 378

collected in the same Pozzuoli area [75]; this group of viruses shares 20 genes. 379

380

Rudiviruses represent one of the most extensively studied families of archaeal viruses with many 381

of the viral proteins being functionally and structurally characterized [76]. Most of the MRV1, 382

ARV3 and SSRV1 ORFs have orthologs in at least some members of the Rudiviridae. Genes 383

shared with other rudiviruses include those for the major and minor capsid proteins, several 384

transcription factors with ribbon-helix-helix motifs, three glycosyltransferases, GCN5-family 385

acetyltransferase, Holliday junction resolvase, SAM-dependent methyltransferase and a gene 386

cassette encoding the ssDNA-binding protein, ssDNA annealing ATPase and Cas4-like ssDNA 387

exonuclease (Figure 5). The conservation of these core proteins in the expanding collection of 388

rudiviruses underscores their critical role in viral reproduction. Conspicuously missing from the 389

MRV1 and ARV3 are homologs of the SIRV2 P98 protein responsible for the formation of 390

pyramidal structures for virion egress and anti-CRISPR (Acr) proteins encoded by diverse 391

crenarchaeal viruses [77,78]. Notably, SSRV1 carries a gene for the recently characterized AcrIII-392

1, which blocks antiviral response of type III CRISPR-Cas systems by cleaving the cyclic 393

oligoadenylate second messenger [79]. Given that CRISPR-Cas systems are highly prevalent in 394

hyperthermophilic archaea[80], the lack of identifiable anti-CRISPR genes in MRV1 and ARV3 395

is somewhat unexpected, suggesting that the two viruses encode novel Acr proteins. Similarly, 396

lack of the genes encoding recognizable P98-like pyramid proteins in MRV1 and ARV3 (as well 397

as in ARV1 and ARV2) suggests that these viruses have evolved a different solution for virion 398

release. 399

400



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Besides the core genes, rudiviruses are known to carry a rich complement of variable genes, which 401

typically occupy the termini of linear genomes and are shared with viruses isolated from the same 402

geographical location [81]. For instance, MRV1, ARV3 and SSRV1 carry several genes, which 403

are exclusive to Italian rudiviruses. These include a divergent glycoside hydrolase, putative metal-404

dependent deubiquitinase, alpha-helical DNA-binding protein, transcription initiation factor and 405

several short hypothetical proteins, which are likely candidates for Acrs (Supplementary 406

Information, Table S2). Notably, some of these hypothetical proteins are shared with other 407

crenarchaeal viruses (families Lipothrixviridae and Bicaudaviridae) isolated from the same 408

location. 409

410

A biogeographic pattern in the Rudiviridae family 411

To gain insight into the global architecture of the rudivirus populations and the factors that govern 412

it, we performed phylogenomic analysis of all available rudivirus genomes using the Genome-413

BLAST Distance Phylogeny method implemented in VICTOR [66]. Our results unequivocally 414

show that the 19 sequenced rudiviruses fall into six clades corresponding to the geographical 415

origins of the virus isolation (Figure 6), suggesting local adaptation of the corresponding viruses. 416

Thus, on the global scale, horizontal spread of rudivirus virions between geographically remote 417

continental hydrothermal systems appears to be restricted. 418

419

Remarkably, despite forming a monophyletic group, all rudiviruses originating from Italy infect 420

relatively distant hosts, belonging to three different genera of the order Sulfolobales. Such pattern 421

was highly unexpected, because closely related hyperthermophilic archaeal viruses typically infect 422

hosts from the same genus. Thus, we hypothesized that such pattern of host specificities might 423

signify host switching events in the history of the Italian rudivirus assemblage. CRISPR arrays, 424

which contain spacer sequences derived from mobile genetic elements, keep memory of past 425

infections and are commonly used as indicators for matching the uncultivated viruses to their 426

potential hosts [82-84]. Thus, to investigate which hosts were exposed to Italian rudiviruses, we 427

searched the CRISPRdb database [85] for the presence of spacers matching the corresponding viral 428

genomes. Spacer matches were found for all five virus genomes, albeit with different levels of 429

identity. Matches with 100% identity were obtained for ARV2, ARV3, SSRV1 and MRV1 430

(Supplementary Information, Table S3). Spacers matching ARV2 and ARV3 were found in 431



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Saccharolobus solfataricus P1, whereas ARV2 was also targeted by a CRISPR spacer (100% 432

identity) from Metallosphaera sedula DSM 5348. Unexpectedly, MRV1, which infects 433

Metallosphaera species, was matched by spacers from different strains of S. solfataricus. 434

Conversely, SSRV1 infecting S. solfataricus was targeted by multiple spacers from CRISPR arrays 435

of M. sedula DSM 5348 (Supplementary Information, Table S3). These results are consistent with 436

the possibility that in the recent history of Italian rudiviruses, host switching, even across the genus 437

boundary, has been relatively common. 438

439

Revised classification of rudiviruses 440

Of the 19 rudiviruses for which (near) complete genome sequences are available, only three 441

(SIRV1, SIRV2 and ARV1) are officially classified. All three viruses are included in the same 442

genus, Rudivirus. Here, we propose a taxonomic framework for classification of all cultivated and 443

uncultured rudiviruses for which genome sequences are available. As mentioned above, 444

phylogenomic analysis revealed six different clades coinciding with the origin of virus isolation 445

(Figure 6), highlighting considerable diversity of the natural rudivirus population, which has 446

stratified into several assemblages, warranting their classification into distinct, genus-level 447

taxonomic units. To this end, we compared the genome sequences using the Gegenees tool [67], 448

which fragments the genomes and calculates symmetrical identity scores for each pairwise 449

comparison based on BLASTn hits and a genome length. The analysis revealed seven clusters of 450

related genomes, which were generally consistent with those obtained in the phylogenomic 451

analysis (Table S4). Notably, due to considerable sequence divergence, ARV1 falls into a separate 452

cluster from other Italian rudiviruses. Consistently, in the phylogenomic tree, ARV1 forms a sister 453

group to other Italian rudiviruses. Furthermore, among the five Italian rudiviruses, the genome of 454

ARV1 is most divergent, displaying multiple gene and genomic segment inversions compared to 455

the other viruses (Figure 5). Viruses from the seven clades also differ considerably in terms of the 456

variable gene contents. For instance, viruses SIRV1 and SIRV2 isolated in Iceland share 11 genes 457

that are absent from the USA SIRVs [81]. Thus, to acknowledge the differences between the 458

known rudiviruses, we propose to classify them into seven genera: “Icerudivirus” (former 459

Rudivirus,to include SIRV1-SIRV3), “Mexirudivirus” (SMRV1), “Azorudivirus” (SRV), 460

“Itarudivirus” (ARV1), “Hoswirudivirus” (ARV2, ARV3, MRV1, SSRV1; hoswi-, for host 461

switching), “Japarudivirus” (SBRV1) and “Usarudivirus” (SIRV4-SIRV11). 462



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463

DISCUSSION 464

Virus discovery in the “age of metagenomics” is increasingly performed by culture-independent 465

methods [86], whereby viral genomes are sequenced directly from the environment. This is a 466

powerful approach which has already yielded thousands of viral genomes, providing 467

unprecedented insights into virus diversity, environmental distribution and evolution [82,87-90]. 468

The limitation of viral metagenomics, however, is that the host species for the sequenced viruses 469

typically remain unknown, even if occasionally predicted. Furthermore, truly novel viruses, which 470

lack similarity to known reference virus genomes, often escape identification and remain in the so-471

called “viral dark matter” [91-94]. Finally, although many features of viruses can be deduced from 472

the genome sequence alone, different molecular aspects of virus-host interactions remain obscure. 473

Thus, to get a full(er) picture of virus biology and to further expand the knowledge on virus 474

diversity, classical approaches of virus isolation and characterization remain highly valuable. Here, 475

we reported the results of our exploration of the diversity of hyperthermophilic archaeal viruses at 476

the solfataric field in Pisciarelli, Pozzuoli. 477

478

Previous sampling of archaeal viruses in the thermal springs in Pisciarelli led to the isolation of 479

viruses from the families Ampullaviridae, Bicaudaviridae, Lipothrixviridae, Rudiviridae and 480

Tristromaviridae [31,45,69,75,95], whereas those of the families Fuselloviridae, Globuloviridae 481

and Guttaviridae, which we observed in the initial enrichment cultures, have not been previously 482

reported from the sampled Pisciarelli sites. Nevertheless, similar virion morphologies have been 483

observed in high-temperature continental hydrothermal systems from other geographical locations 484

across the globe [29,32,70,96,97], pointing to global distribution of most groups of 485

hyperthermophilic archaeal viruses. However, how these virus communities are structured and 486

whether geographically remote hydrothermal ecosystems undergo virus immigration is not fully 487

understood. 488

489

A previous study has revealed a biogeographic pattern among Sulfolobus islandicus-infecting 490

rudiviruses isolated from hot springs in Iceland and United States [81]. That is, viruses from the 491

same geographical location were more closely related to each other than they were to viruses from 492

other locations and the larger the distance the more divergent the virus genomes were. Similar 493



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observation has been made for the Sulfolobus-infecting spindle-shaped viruses of the family 494

Fuselloviridae [97-99]. By contrast, a study focusing on three relatively closely spaced hot springs 495

in Yellowstone National Park concluded that horizontal virus movement, rather than mutation, is 496

the dominant factor controlling the viral community structure [100]. 497

498

Phylogenomic and comparative genomic analysis of the rudiviruses reported herein and those 499

sequenced previously revealed a strong biogeographic pattern. All rudiviruses formed clades 500

corresponding to the geographical origins of their isolation. This observation strongly suggests 501

that diversification and evolution of rudiviruses is influenced by special confinement within 502

discrete high-temperature continental hydrothermal systems, with little horizontal migration of 503

viral particles over large distances. Consistently, analyses of the CRISPR spacers carried by 504

hyperthermophilic archaea predominantly target local viruses, further indicating geographically 505

defined co-evolution of viruses and their hosts [97,101]. This is in stark contrast with the global 506

architecture of virus communities associated with hyperhalophilic archaea, where genomic 507

similarity between viruses does not correspond to geographical distance [102,103]. Indeed, it has 508

been suggested that hypersaline environments worldwide function like a single habitat, with 509

extensive exchange of microbes and their viruses between remote hypersaline sources [102]. It has 510

been shown that small salt crystals harboring halophilic archaea are carried on bird feathers, 511

suggesting that bird migration is a driving force of haloarchaeal spread across the globe [104]. 512

Obviously, similar route of dissemination is hardly imaginable for microbial communities thriving 513

in acidic high-temperature hydrothermal settings, offering an explanation for the different patterns 514

observed for hyperthermophilic and hyperhalophilic archaeal viruses. Notably, however, it has 515

been suggested that reversible silicification of virus particles, which is conceivable in hot spring 516

environments, might promote long-distance host-independent virus dispersal [105]. 517

518

We also show, for the first time, that relatively closely related rudiviruses infect phylogenetically 519

distant hosts, belonging to three different genera. This observation is inconsistent with stable 520

coevolution of rudiviruses with their hosts, but rather points to host switch events in the recent 521

history of Italian rudiviruses. At least in the case of rudivirises, relatively few genetic changes 522

appear to be necessary for gaining the ability to infect a new host. In tailed bacteriophages, host 523

range switches are typically associated with mutations in genes encoding the tail fiber proteins 524



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responsible for host recognition [106]. Several molecular mechanisms underlying mutability of 525

the tail fiber genes have been described, including genetic drift, diversity generating retroelements 526

and phase variation cassettes. The latter two systems have been demonstrated to function also in 527

viruses infecting anaerobic methane-oxidizing (ANME) and hyperhalophilic archaea, respectively 528

[107,108]. Both mechanisms depend on specific enzymes, namely, reverse-transcriptase and 529

invertase. However, neither of the two systems is present in rudiviruses, suggesting that genetic 530

drift is the most likely mechanism responsible for generating diversity in the host recognition 531

module of rudiviruses. 532

533

Analysis of CRISPR spacers from hyperthermophilic archaea confirmed that viruses closely 534

related to those isolated in this study were infecting highly different hosts in the recent past. Indeed, 535

viruses infecting Saccharolobus and Metallosphaera species were targeted by spacers from 536

CRISPR arrays of Metallosphaera and Saccharolobus, respectively. This observation further 537

reinforces a relatively recent host switch event. Notably, this phenomenon appears to be also 538

applicable to rudivirus from other geographical locations. In particular, SBRV1, a rudivirus from 539

a Japanese hot spring [109], was found to be targeted by 521 unique CRISPR spacers associated 540

with all four principal CRISPR repeat sequences present in Sulfolobales [101], suggesting either 541

very broad host range or frequent host switches. Furthermore, we have recently shown that 542

CRISPR targeting is an important factor driving the genome evolution of hyperthermophilic 543

archaeal viruses [101]. Given the presence of multiple CRISPR spacers matching rudivirus 544

genomes, we hypothesize that necessity to switch hosts might be, to a large extent, driven by 545

CRISPR targeting. Notably, matching of CRISPR spacers to protospacers in viral genomes is one 546

of the most widely used approaches of host identification for viruses discovered by metagenomics, 547

with the estimated host genus prediction accuracy of 70–90% [83,84,89]. Our results suggest that 548

in the case of rudiviruses, spacer matching might not provide accurate predictions beyond the rank 549

of family (i.e., Sulfolobaceae). 550

551

Collectively, we show that terrestrial hydrothermal systems harbor a highly diverse virome 552

represented by multiple families with unique virus morphologies not described in other 553

environments. Genomes of the newly isolated viruses, especially those infecting Pyrobaculum 554

species, remain a rich source of unknown genes, which could be involved in novel mechanisms of 555



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virus-host interactions. Furthermore, our results suggest that global rudivirus communities display 556

biogeographic pattern and diversify into distinct lineages confined to discrete geographical 557

locations. This diversification appears to involve relatively frequent host switching, potentially 558

evoked by host CRISPR-Cas immunity systems. Future studies should focus on characterization 559

of molecular mechanisms allowing rudiviruses to avoid CRISPR targeting by infecting new hosts. 560

561

562

ACKNOWLEDGEMENTS 563

This work was supported by l’Agence Nationale de la Recherche (France) project ENVIRA (to 564

M.K.) and the European Union’s Horizon 2020 research and innovation program under grant 565

agreement 685778, project VIRUS X (to D.P.). D.P.B. is part of the Pasteur – Paris University 566

(PPU) International PhD Program. This project has received funding from the European Union's 567

Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant 568

agreement No 665807. We are also grateful to the Ultrastructural BioImaging (UTechS UBI) unit 569

of Institut Pasteur for access to electron microscopes and Marc Monot from the Biomics Platform 570

of Institut Pasteur for helpful discussions on genome assembly. 571

572

COMPETING INTERESTS 573

The authors declare that they have no conflict of interest. 574

575



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108. Klein R, Rossler N, Iro M, Scholz H and Witte A. Haloarchaeal myovirus phiCh1 harbours a phase 850 variation system for the production of protein variants with distinct cell surface adhesion 851 specificities. Mol Microbiol. 2012; 83:137-50. 852

109. Liu Y, Brandt D, Ishino S, Ishino Y, Koonin EV, Kalinowski J, Krupovic M and Prangishvili D. New 853 archaeal viruses discovered by metagenomic analysis of viral communities in enrichment 854 cultures. Environ Microbiol. 2019; 21:2002-2014. 855

856

857



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FIGURES AND LEGENDS 858

859

Figure 1. Electron micrographs of the VLPs observed in enrichment cultures: (A) fuselloviruses 860

(tailless lemon-shaped virions), (B) bicaudaviruses (large, tailed lemon-shaped virions), (C) 861

ampullaviruses (bottle-shaped virions), (D) rudiviruses (rod-shaped virions), (E) lipothrixviruses 862

(filamentous virions), (F) globuloviruses (spherical enveloped virions), (G) tristromaviruses 863

(filamentous enveloped virions) and (H) guttaviruses (droplet-shaped virions). Samples were 864

negatively stained with 2% (wt/vol) uranyl acetate. Bars. 200 nm. 865

866



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867

Figure 2. Electron micrographs of the five isolated viruses. A. Metallosphaera rod-shaped virus 868

1. B. Acidianus rod-shaped virus 3. C. Saccharolobus solfataricus rod-shaped virus 1.D. 869

Pyrobaculum filamentous virus 2. E. Pyrobaculum spherical virus 2. Samples were negatively 870

stained with 2% (wt/vol) uranyl acetate. Scale bars: 500 nm; in insets: 100 nm. 871

872

873

874

875

876

877



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878

Figure 3. Genome alignment of the three members of the Globuloviridae family. The open reading 879

frames (ORFs) are represented by arrows that indicate the direction of transcription. The terminal 880

inverted repeats (TIRs) are denoted by black bars at the ends of the genomes. Genes encoding the 881

major structural proteins are shown in dark grey. The functional annotations of the predicted ORFs 882

are depicted above/below the corresponding ORF. Homologous genes are connected by shading 883

in grayscale based on the level of amino acid sequence identity. VP, virion protein; wHTH, winged 884

helix-turn-helix domain. 885

886

887

888

889

Figure 4. Genome comparison of the three members of the Tristromaviridae family. The open 890

reading frames (ORFs) are represented by arrows that indicate the direction of transcription. The 891

terminal inverted repeats (TIRs) are denoted by black bars at the ends of the genomes. Genes 892

encoding the major structural proteins are shown in dark grey, whereas the four-gene block 893

discussed in the text is shown in black. The functional annotations of the predicted ORFs are 894

depicted above/below the corresponding ORFs. Homologous genes are connected by shading in 895

grayscale based on the level of amino acid sequence identity. The dotted line represents the 896

incompleteness of the TTV1 genome. GHase, glycoside hydrolase; GTase, glycosyltransferase; 897

TP/VP, virion protein. 898



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899

900

Figure 5. Genome alignment of the Italian rudiviruses. ARV3, MRV1 and SSRV1 are newly 901

isolated members of the Rudiviridae family, whereas ARV1 and ARV2 were reported previously 902

[69,75]. The open reading frames (ORFs) are represented by arrows that indicate the direction of 903

transcription. The terminal inverted repeats (TIRs) are denoted by black bars at the ends of the 904

genomes. The functional annotations of the predicted ORFs are depicted above/below the 905

corresponding ORFs. Homologous genes are connected by shading in grayscale based on the 906

amino acid sequence identity. ATase, acetyltransferase; GHase, glycoside hydrolase; GTase, 907

glycosyltransferase; HJR, Holliday junction resolvase; MCP, major capsid protein; MTase, 908

methyltransferase; SSB, ssDNA binding protein; TFB, Transcription factor B; ThyX, thymidylate 909

synthase; wHTH, winged helix-turn-helix domain. 910

911



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912

Figure 6. Inferred phylogenomic tree of all known members of the Rudiviridae family based on 913

whole genome VICTOR [66] analysis at the amino acid level. The tree is rooted with 914

lipothrixviruses, and the branch length is scaled in terms of the Genome BLAST Distance 915

Phylogeny (GBDP) distance formula D6. Only branch support values >70% are shown. For each 916

genome, the abbreviated virus name, GenBank accession number and host organism (when 917

known) are indicated. Question marks denote that the host is not known. The tree is divided into 918

colored blocks according to the geographical origin of the compared viruses. 919

920



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Table 1. Host range of the archaeal viruses isolated in this study. 921

Newly-isolated archaeal virus

Host strain MRV1 ARV3 SSRV1 PSV2 PFV2

Metallosphaera sedula POZ202 H - - - -

Acidianus brierleyi POZ9 - H - - -

Acidianus convivator - - - - -

Acidianus hospitalis - + - - -

Saccharolobus solfataricus P1 - - - - -

Saccharolobus solfataricus P2 - - - - -

Saccharolobus solfataricus POZ149 - - H - -

Sulfolobus islandicus LAL14/1 - - - - -

Sulfolobus islandicus REN2H1 - - - - -

Sulfolobus islandicus HVE10/4 - - - - -

Sulfolobus islandicus REY15A - - - - -

Sulfolobus islandicus ΔC1C2 - - - - -

Sufolobus acidocaldarius DSM639 - - - - -

Pyrobaculum arsenaticum PZ6 (DSM 13514) - - - - -

Pyrobaculum arsenaticum 2GA - - - H H

Pyrobaculum calidifontis VA1 (DSM 21063) - - - - -

Pyrobaculum oguniense TE7 (DSM 13380) - - - - +

H, isolation host; +, supports virus replication. 922

923

Table 2. Genomic features of the hyperthermophilic archaeal viruses isolated in this study. 924

Virus Host Size (bp) TIR Topology GC% ORFs Accession #

MRV1 Metallosphaera sedula POZ202 20269 + linear 34.12 27 MN876843

ARV3 Acidianus brierleyi POZ9 23666 - linear 32.02 33 MN876842

SSRV1 Saccharolobus solfataricus POZ149 26097 - linear 32.31 37 MN876841

PSV2 Pyrobaculum arsenaticum 2GA 18212 + linear 45.01 32 MN876845

PFV2 Pyrobaculum arsenaticum 2GA 17602 + linear 45.32 39 MN876844

TIR, terminal inverted repeats; ORFs, open reading frames. 925

926



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2 pattern in archaeal virus communities - biorxiv · 1/16/2020 · whereas bicaudaviruses appear...

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