# An Update on Eukaryotic Viruses Revived from Ancient Permafrost

Jean-Marie Alempic, Audrey Lartigue, Artemiy E Goncharov, Guido Grosse, Jens Strauss, Alexey N Tikhonov, Alexander N Fedorov, Olivier Poirot, Matthieu Legendre, Sébastien Santini, et al.

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Jean-Marie Alempic, Audrey Lartigue, Artemiy E Goncharov, Guido Grosse, Jens Strauss, et al. An Update on Eukaryotic Viruses Revived from Ancient Permafrost. Viruses, 2023, 15 (2), pp.564. 10.3390/xxxxx.10-04207147

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Article

# An update on eukaryotic viruses revived from ancient permafrost

Jean-Marie Alempic¹,², Audrey Lartigue¹,³, Artemiy E Goncharov², Guido Grosse³,⁴, Jens Strauss³, Alexey N. Tikhonov⁵, Alexander N. Fedorov⁶, Olivier Poirot¹, Matthieu Legendre¹, Sébastien Santini¹, Chantal Abergel¹, and Jean-Michel Claverie¹,\*

**Citation:** Alempic, J.-M.; Lartigue, A.; Goncharov, A.E.; Grosse, G.; Strauss, J.; Tikhonov, A.N.; Fedorov, A.N.; Poirot, O.; Legendre, M.; Santini, S.; et al. An update on eukaryotic viruses revived from ancient permafrost. *Viruses* **2022**, *14*, x. https://doi.org/10.3390/xxxxx

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**Copyright:** © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

¹ IGS, Information Génomique & Structurale (UMR7256), Institut de Microbiologie de la Méditerranée (FR 3489), Institut Microbiologie, Bioénergies et Biotechnologie, and Institut Origines, CNRS, Aix Marseille University, Marseille, 13288, France

² Department of Molecular Microbiology, Institute of Experimental Medicine, Saint Petersburg, Russia, Department of Epidemiology, Parasitology and Disinfectology, Northwestern State Medical Mechnikov University, Saint Petersburg, 195067, Russia

³ Permafrost Research Section, Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, 14473, Potsdam, Germany

⁴ Institute of Geosciences, University of Potsdam, 14478 Potsdam, Germany.

⁵ Laboratory of theriology, Zoological Institute of Russian Academy of Science, St. Petersburg, 199034, Russia.

⁶ Melnikov Permafrost Institute, 677010 Yakutsk, Russia

\# These authors contributed equally.

\* Correspondence: Jean-Michel.Claverie@univ-amu.fr; Tel.: +33 413946777

**Abstract:** One quarter of the Northern hemisphere is underlain by permanently frozen ground, referred to as permafrost. Due to climate warming, irreversibly thawing permafrost is releasing organic matter frozen for up to a million years, most of which decomposes into carbon dioxide and methane, further enhancing the greenhouse effect. Part of this organic matter also consists of revived cellular microbes (prokaryotes, unicellular eukaryotes) as well as viruses that remained dormant since prehistorical times. While the literature abounds on descriptions of the rich and diverse prokaryotic microbiomes found in permafrost, no additional report about "live" viruses have been published since the two original studies describing pithovirus (in 2014) and mollivirus (in 2015). This wrongly suggests that such occurrences are rare and that "zombie viruses" are not a public health threat. To restore an appreciation closer to reality, we report the preliminary characterizations of 13 new viruses isolated from 7 different ancient Siberian permafrost samples, 1 from the Lena river and 1 from Kamchatka cryosol. As expected from the host specificity imposed by our protocol, these viruses belong to 5 different clades infecting *Acanthamoeba* spp. but not previously revived from permafrost: pandoravirus, cedratvirus, megavirus, and mancpmanvirus, in addition to a new pithovirus strain.

**Keywords:** Permafrost; Acanthamoeba; Giant Virus; Pleistocene; Siberia; Kamchatka

## 1. Introduction

Ongoing international modeling and monitoring studies keep confirming that the continuous release of greenhouse gas (mostly CO₂) due to human activities since the industrial revolution is causing significant climate change through global warming. It is now widely acknowledged that an average temperature increase of 1.5°C relative to 1850–1900 would be exceeded during the 21st century, under all realistic circumstances [1] even though the adequacy of present climate models to predict regional changes remains in debate [2]. For instance, climate warming is particularly noticeable in the Arctic where average temperatures appear to increase twice as fast as in temperate regions [3]. One of the most visible consequences is the global thawing of permafrost at increasing depths [4,
5] and the rapid erosion of permafrost bluffs [6], a phenomenon most visible in Siberia where deep continuous permafrost underlays most of the North Eastern territories. 47 48

The thawing of permafrost has significant microbiological consequences. First, above freezing temperatures, the return of liquid water triggers the metabolic reactivation of numerous soil microorganisms (bacteria, archaea, protists, fungi)[7-11], exposing the organic material previously trapped in permafrost to decomposition, releasing additional CO2 and methane further contributing greenhouse gas to the atmosphere [5, 12, 13]. Yet, a more immediate public health concern is the physical release of live bacteria (or archaea) that have remained in cryptobiosis trapped in deep permafrost, isolated from the Earth's surface for up to 2 million years [7, 14] (although a more consensual limit would be half a million years [15]). On a shorter time scale, the periodical return of anthrax epidemics devastating reindeer populations has been linked to the deeper thawing of the permafrost active layer at the soil surface during exceptionally hot summers, allowing century-old *Bacillus anthracis* spores from old animals burial grounds or carcasses to resurface [16-18] 49 50 51 52 53 54 55 56 57 58 59 60

One could imagine that very deep permafrost layers (i.e. million-year-old), such as those extracted by open-pit mining, could release totally unknown pathogens [19]. Finally, the abrupt thawing vertically operating along the whole wall of permafrost bluffs (consisting of specific ice-rich deposits called "yedoma") such as seen in the Kolyma lowland or around the Yukon river Alaska, causes the simultaneous release of ancient microorganisms from frozen soils dating from the whole Holocene to the late Pleistocene (i.e. up to 120.000 years ago) [20]. Many culture-based and culture-independent studies (i.e. barcoding and/or metagenomics) have documented the presence of a large diversity of bacteria in ancient permafrost [7-9, 14, 21-25], a significant proportion of which are thought to be alive, although estimates vary greatly with the depth (age) and soil properties [14, 26, 27]. These bacterial populations include relatives of common contemporary pathogens (Acinetobacter, Bacillus anthracis, Brucella, Campylobacter, Clostridia, Mycoplasma, various Enterobacteria, Mycobacteria, Streptococci, Staphylococci, Rickettsia) [8, 9, 21, 26, 28]. Fortunately, we can reasonably hope that an epidemic caused by a revived prehistoric pathogenic bacterium could be quickly controlled by the modern antibiotics at our disposal, as they target cellular structures (e.g. ribosomes) and metabolic pathways (transcription, translation or cell wall synthesis) conserved during the evolution of all bacterial phyla [29], even though bacteria carrying antibiotic-resistance genes appear to be surprisingly prevalent in permafrost [23, 28, 30]. 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79

The situation would be much more disastrous in the case of plant, animal, or human diseases caused by the revival of an ancient unknown virus. As unfortunately well documented by recent (and ongoing) pandemics [31, 32], each new virus, even related to known families, almost always requires the development of highly specific medical responses, such as new antivirals or vaccines. There is no equivalent to "broad spectrum antibiotics" against viruses, because of the lack of universally conserved druggable processes across the different viral families [33, 34]. It is therefore legitimate to ponder the risk of ancient viral particles remaining infectious and getting back into circulation by the thawing of ancient permafrost layers. Focusing on eukaryote-infecting viruses should also be a priority, as bacteriophages are no direct threat to plants, animals, or humans, even though they might shape the microbial ecology of thawing permafrost [35]. 80 81 82 83 84 85 86 87 88 89 90

Our review of the literature shows that very few studies have been published on this subject. To our knowledge, the first one was the isolation of Influenza RNA from one frozen biopsy of the lung of a victim buried in permafrost since 1918 [36] from which the complete coding sequence of the hemagglutinin gene was obtained. Another one was the detection of smallpox virus DNA in a 300-year-old Siberian mummy buried in permafrost [37]. Probably for safety/regulatory reasons, there was not follow up studies attempting to "revive" these viruses (fortunately). The first isolation of two fully infectious eukaryotic viruses from 30,000-y old permafrost was thus performed in our laboratory and published in 2014 and 2015 [38, 39]. A decisive advantage of our approach was to choose *Acan-* 91 92 93 94 95 96 97 98 99
*thamoeba* spp. as a host, to act as a specific bait to potentially infectious viruses, thus eliminating any risk for crops, animals or humans. However, no other isolation of a permafrost virus has been published since, which might suggest that these were lucky shots and that the abundance of viruses remaining infectious in permafrost is very low. This in fact is wrong, as numerous other *Acanthamoeba*-infecting viruses have been isolated in our laboratory, but not yet published pending their complete genome assembly, annotation, or detailed analysis. In the present article we provide an update on thirteen of them, most of which remain at a preliminary stage of characterization. These isolates will be available for collaborative studies upon formal request through a material transfer agreement. The ease with which these new viruses were isolated suggests that infectious particles of viruses specific to many other untested eukaryotic hosts (protozoans or animals) remain probably abundant in ancient permafrost.

## 2. Materials and Methods

### 2.1. *Permafrost sampling*

The various on-site sampling protocols have been previously described in [28, 40] for samples #3 and #5 (collected in the spring 2015), in [41] for sample #4, in [42] for sample #6, and [43, 44] for samples #7-9 (see Table 1). Liquid sample #2 and #4 were collected in pre-sterilized 50 ml Falcon tube in august 2019, as well as sample #1 consisting of surface soil without vegetation from the Shapina river bank collected on 07/15/2017 and since maintained frozen at -20 C in the laboratory.

### 2.2. *Sample preparation for culturing*

About 1g of sample is resuspended in 40 mM Tris pH 7.5, from 2-10% V/V depending on its nature (liquid, mud, solid soil) and vortexed at room temperature. After decanting for 10 minutes, the supernatant is taken up, then centrifugated at 10,000 g for one hour. The pellet is then resuspended in 40 mM Tris pH 7.5 with a cocktail of antibiotics (Ampicillin 100µg/mL, Chloramphenicol 34µg/mL, Kanamycin 20µg/mL). This preparation is then deposited one drop at a time onto two 15 cm-diameter Petri dishes (Sarsted 82.1184.500) one previously seeded with *Acanthamoeba castellanii* at 500 cells/cm², the other with *A. castellanii* cells previously adapted to Fongizone (1.25%).

### 2.3. *Detection of virus infection*

Morphological changes (rounding up, non-adherent cells, encystment, ...) of the *A. castellanii* cells might eventually become detectable after 72 h, but might be due to a variety of irrelevant causes (overconfluency, presence of a toxin, fungal proliferation, ...). Under a light microscope, the areas exhibiting the most visible changes are spotted using a p1000 pipetman. This 1 mL volume is then centrifugated (13,000 g for 30 minutes), the pellet resuspended in 100 µL and scrutinized under a light microscope. This subsample is also used to seed further T25 cell culture flasks of fresh *A. castellanii* cells.

### 2.4. *Preliminary identification of infecting viruses*

Potential viral infections are suggested by intracellular changes (presence of cytoplasmic viral factories, nuclear deformation, lysis), or by the direct visualization of giant virus particles. Using a set of in-house-designed family-specific primers (Table 2), a PCR test is performed using the Terra PCR Direct Polymerase Mix (Takara Bio Europe SAS, Saint-Germain-en-Laye, France). Amplicons are then sequenced (Eurofins Genomics, Ebersberg, Germany) to confirm the finding of new isolates.

### 2.5. *Further characterization of new virus isolates*

Positive subcultures are then reseeded and passaged in T25 then T75 cell culture flasks (Nunc™ EasYFlasks™, Thermofisher scientific, Waltham, MA USA) until the den-
sity/quantity of viral particles allows their further characterization by Electron Microscopy (Figure 3). New viral isolates of particular interest are then eventually cloned and their whole genome sequenced.

## 2.6. Viral genome sequencing

Virus cloning, virus particles purification using a cesium chloride gradient, and DNA extraction from approximately 5 x 10⁹ purified particles (using the Purelink Genomic extraction mini kit, ThermoFisher) have been previously described [45]. Sequence data was generated from the Illumina HiSeq X platform provided by Novogene Europe (Cambridge, UK). Genome data assembly was performed in-house as previously described [45]. The draft genome sequences listed in Table 3-6, were only minimally annotated using automated BlastP similarity searches, pending further human-supervised validation and analyses, and final submission to the EMBL/Genbank database.

## 2.7. Design of virus-specific PCR primers

Clusters of protein-coding genes common to all known members of a viral family or clade were identified using Orthofinder [46]. The protein sequence alignments of these clusters were converted into nucleotide alignments using Pal2nal [47], and sorted using the “most unrelated pair criteria”. These alignments were thus visually inspected to select the variable regions flanked by strictly conserved sequences suitable as PCR primers. The primers and their genes of origin are listed in Table 2.

<table><caption>**Table 1.** Samples and virus description.</caption><thead><tr><th>Sample #</th><th>GPS coordinates</th><th>Description</th><th>Isolated virus</th></tr></thead><tbody><tr><td>1</td><td>N 55°06'54''<br>E 159°57'48''</td><td>Surface soil, Shapina river bank, Kamchatka Modern</td><td>Cedratvirus kamchatka (strain P5)</td></tr><tr><td>2</td><td>N 62°06'23'92''<br>E 129°48'35''</td><td>Lena river, Yakustk Modern</td><td>Cedratvirus lena (strain DY0)<br>Pandoravirus lena (strain DY0)</td></tr><tr><td>3</td><td>N 61° 45' 39.1''<br>E 130° 28' 28.78''</td><td>Talik, -6.5 m below a lake, Yukechi Alas [40]<br>Isolation: >53 y BP</td><td>Pandoravirus talik (strain Y4)</td></tr><tr><td>4</td><td>N 68°38'21.1''<br>E 159°3'20.67''</td><td>Melting ice wedge Duvanny yar [20, 41]<br>Mixed ages</td><td>Cedratvirus duvanny (strain DY1)<br>Pandoravirus duvanny (strain DY1)</td></tr><tr><td>5</td><td>N 61° 45' 39.1''<br>E 130° 28' 28.78''</td><td>-16 m below a lake, Yukechi Alas [40]<br>Isolation: >48,500 y BP</td><td>Pandoravirus yedoma (strain Y2)</td></tr><tr><td>6</td><td>N 74°13'00''<br>E 141°03'48''</td><td>Woolly mammoth stomach content, Maly Lyakhovsky Island [42]<br>Isolation: >28,600 y BP</td><td>Pandoravirus mammoth (strain Mm38)</td></tr><tr><td>7</td><td>N 70°43'25''<br>E 135°25'47''</td><td>Soil with mammoth wool RHS paleolithic site, Yana river left bank [43, 44]<br>Isolation: >27,000 BP</td><td>Megavirus mammoth (strain Yana14)<br>Pithovirus mammoth (strain Yana14)<br>Pandoravirus mammoth (strain Yana14)</td></tr><tr><td>8*</td><td>N 70°43'25''<br>E 135°25'47''</td><td>Fossil wolf (Canis lupus) intestinal content, RHS paleolithic site [43, 44]<br>Isolation: >27,000 y BP</td><td>Pandoravirus lupus (strain Tums1)</td></tr><tr><td>9*</td><td>N 70°43'25''<br>E 135°25'47''</td><td>Fossil wolf (Canis lupus) intestinal content, RHS paleolithic site [43, 44]<br>Isolation: >27,000 y BP</td><td>Pacmanvirus lupus (strain Tums2)</td></tr></tbody></table>
* Same location, but different frozen remains.

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<table><caption>**Table 2.** PCR primers used to identify the newly isolated viruses.</caption><thead><tr><th>Virus family or subgroups</th><th>Primer sequences (forward & reverse)</th><th>Prototype gene #</th></tr></thead><tbody><tr><td>Pandoraviridae</td><td>F: TCGTGGATCGACATTGGCGTGCAGTT<br>R: CTGGTAGGTGACGGCAAAGTT</td><td>P. salinus (NC_022098)<br>CDS_1260<br>Putative oxidoreductase</td></tr><tr><td>cedratviruses</td><td>F: AAACCTAGGTTGCTAACTGTAGATCCTTG<br>R: GGAACCAGCGTTACCGAGTGCATCTTC</td><td>Cedratvirus A11 (NC_032108)<br>BQ3484_149<br>Hypothetical protein</td></tr><tr><td>Pithoviridae</td><td>F: GTGGTCCAAAACTGGAAGAACTA<br>R: GCGTCAAGCTCAACATCAAGTTC</td><td>P. sibericum (NC_023423)<br>pv_393<br>DNA/RNA helicase</td></tr><tr><td>Mimiviridae<br>(A, B, C lineages)</td><td>F: TGGAATAATGGTGATGGTATTGATGT<br>R: ACTGGTACCTAATCCTTTGTAATATTT</td><td>M. chilensis (NC_016072)<br>mg403<br>Topoisomerase 2</td></tr><tr><td>pacmanviruses</td><td>F: GTCTCAATGGGCCACTTGAGCTG<br>R: CCCGCTCTTGACCTCTGGGTTCC</td><td>Pacmanvirus A23 (LT706986)<br>PACV_217<br>Major Capsid Protein</td></tr></tbody></table>

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<figure><img src="image_4.png" /><figcaption>**Figure 1.** Morphological features guiding the early identification of newly isolated viruses (Negative staining, Transmission Electron Microscopy). (A) The large ovoid particle (1,000 nm in length) of Pandoraviruses with its characteristic apex ostiole (white arrowhead). (B) A mixture of Pandoravirus particles and Megavirus icosahedral particles exhibiting a “stargate” (white starfish-like structure crowning a vertex, white arrowhead). (C) The elongated particle of a Cedratvirus (1,500 nm in length) exhibits two apex cork-like structures (white arrowheads). (D) The elongated particle of a Pithovirus (1,900 nm in length) exhibits a single apex cork-like structure (white arrowhead). (E) The large (770 nm in diameter) “hairy” icosahedral particle of a Megavirus, with its prominent “stargate” (white arrowhead). (F) The smaller icosahedral particle (200 nm in diameter) typical of Asfarviruses/Pacmanviruses.</figcaption></figure>

**3. Results**

**3.1. *Pandoraviruses***

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A majority (4/7) of the viruses that we isolated from ancient permafrost layers (sample #5-9, Table 1) belong to the proposed family Pandoraviridae (not yet endorsed by ICTV). One more was isolated from the Lena river (in August 2019, sample #2, Table 1) in Yakutsk, the capital city of the Sakha (Yakutia) Republic, built on permafrost and known as the coldest city on earth. Pandoraviruses remain the most enigmatic of giant viruses infecting Acanthamoeba spp. with the largest known viral genomes (a linear DNA molecule up to 2.6 Mb) [48, 49], a puzzling bias in their nucleotidic 4-mer composition [50], the largest proportion of protein-coding genes of unknown origins [49, 51], and the apparent capacity to create such genes de novo from non-coding DNA segments at a relatively high frequency [49, 51]. This study is the first to document their presence in ancient soils, in contrast with their scarcity in metagenomic data [52]. Already visible under the light microscope during the early phase of the isolation process, Pandoraviruses are easy to identify by electron microscopy thanks to their typical ovoid particles (1 µm in length and 0.5 µm in diameter) enclosed in a 70-nm thick electron-dense tegument, interrupted by an ostiole-like apex (Figure 1.A). The replication cycle of Pandoraviruses in A. castellanii cells lasts from 10 to 15 hours and is initiated by the internalization of individual particles via phagocytic vacuoles. Eight to 10 hours after infection, the Acanthamoeba cells become rounded, lose their adherence, and new particles appear at the periphery of the region formerly occupied by the nucleus. The replicative cycle ends when the cells lyse releasing about a hundred particles each.

<table><caption>**Table 3.** PCR identification of previous and new pandoravirus isolates.</caption><thead><tr><td>Virus</td><td>Accession #</td><td>Base pairs (contigs)</td><td>Amplicon identity</td></tr></thead><tbody><tr><td>P. salinus (prototype)</td><td>NC_022098</td><td>2,473,870</td><td>100% (1203/1203)</td></tr><tr><td>Pandoravirus_celtis</td><td>MK174290</td><td>2,028,440</td><td>93% (1128/1203)</td></tr><tr><td>Pandoravirus_quercus</td><td>NC_037667</td><td>2,077,288</td><td>93% (1125/1203)</td></tr><tr><td>Pandoravirus_inopinatum</td><td>NC_026440</td><td>2,243,109</td><td>93% (1133/1203)</td></tr><tr><td>Pandoravirus_dulcis</td><td>NC_021858</td><td>1,908,524</td><td>92% (1122/1203)</td></tr><tr><td>Pandoravirus_neocaledonia</td><td>NC_037666</td><td>2,003,191</td><td>86% (1045/1203)</td></tr><tr><td>Pandoravirus_macleodensis</td><td>NC_037665</td><td>1,838,258</td><td>86% (1039/1203)</td></tr><tr><td>Pandoravirus lena (strain DY0)</td><td>-</td><td>2,030,260 (6)</td><td>93% (1131/1203)</td></tr><tr><td>Pandoravirus duvanny (strain DY1)</td><td>-</td><td>unassembled</td><td>92% (1114/1203)</td></tr><tr><td>Pandoravirus talik (strain Y4)</td><td>-</td><td>1,817,546 (1)</td><td>92% (1114/1203)</td></tr><tr><td>Pandoravirus mammoth (strain Yana14)</td><td>-</td><td>not yet sequenced</td><td>91% (1104/1203)</td></tr><tr><td>Pandoravirus_yedoma (strain Y2)</td><td>-</td><td>not yet sequenced</td><td>91% (1095/1203)</td></tr><tr><td>Pandoravirus lupus (strain Tums1)</td><td>-</td><td>not yet sequenced</td><td>91% (1092/1203)</td></tr><tr><td>Pandoravirus mammoth (strain Mm38)</td><td>-</td><td>1,776,082 (2)</td><td>86% (1040/1203)</td></tr></tbody></table>

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<figure><img src="image_5.png" /><figcaption>**Figure 2.** Neighbor-joining tree of the available pandoravirus isolates. The tree was built from 2052 gap-free sites in the multiple alignment of 17 RNA polymerases (RPB1) protein sequences using the MAFFT online facility from the Osaka University server [53]. The inclusion of the permafrost isolates confirms the ancient branching of the Pandoraviridae in two separate clades. Accession numbers are indicated following the isolate name when available.</figcaption></figure>

## 3.2. *Cedratviruses* and *pithoviruses*

Three of the newly isolated viruses belong to the recently proposed "cedratviruses" [54, 55], a new clade (a new genus or a new subfamily) within the Pithoviridae family [56]. One was cultivated from the same Lena river water sample previously cited (Sample #2, Table 1), one from surface cryosol in Kamchatka collected during the summer (Sample #1, Table 1), and one from mud flowing into the Kolyma river at Duvanny yar, resulting from the thawing of permafrost layers of mixed ages (Sample #4, Table 1). One additional member of the Pithoviridae was isolated from a 27.000-y old permafrost sample containing a large amount of mammoth wool (Sample #7, Table 1). It is worth recalling that the prototype of this family was previously isolated from an ancient permafrost layer of more than 30,000-y BP [56]. Other members of this family are the most abundant in a recent metagenomic study of various Siberian permafrost samples focusing on eukaryotic viruses [52].

Even though cedratviruses and pithoviruses produce large ovoid particles like those of Pandoraviruses, they are easily distinguishable thanks to several morphological features. Their particles are usually more elongated (up to 2µm), have a much thinner wall,

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and exhibit unique characteristic cork-like structures at their extremity (often two on each side for cedravirus particles) [54, 55](Figure 1.C, 1.D). 226

Cedratvirus/Pithoviruses are very different from pandoraviruses, with much smaller circular DNA genomes of only 460 to 600 kbp, although a non-isolated member appear to reach 1.6 Mb [52]. After entering Acanthamoeba cells by phagocytosis, the particles are stripped from their corks, allowing the viral genome to be delivered through the apical opening into the cytoplasm after membrane fusion. This is then followed by an eclipse phase and the formation of a cytoplasmic electron-lucent viral factory, where a complex morphogenesis occurs. After ~12 h of infection, mature viral particles are released by cell lysis. In contrast with Pandoravirus infections, the cell nucleus maintains its shape throughout the entire Pithovirus replication cycle, a trademark of fully cytoplasmic viruses carrying a functional transcription apparatus in their particles [57]. 227
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<table><caption>**Table 4.** PCR Identification of previous and newly isolated Cedratvirus and Pithovirus species. 238</caption><thead><tr><th>Virus</th><th>Accession #</th><th>Base pairs</th><th>Amplicon identity</th></tr></thead><tbody><tr><td>Cedratvirus A11 (prototype)</td><td>NC_032108</td><td>589,068</td><td>100% (1239/1239)</td></tr><tr><td>Cedratvirus lausannensis</td><td>LT907979</td><td>575,161</td><td>94% (1176/1239)</td></tr><tr><td>Cedratvirus kamchatka</td><td>MN873693</td><td>466,767</td><td>88% (1091/1239)</td></tr><tr><td>Cedratvirus lena (strain DY0)</td><td>-</td><td>465,544</td><td>87% (1090/1239)</td></tr><tr><td>Cedratvirus duvanny (strain DY1)</td><td>-</td><td>472,117</td><td>87% (1087/1239)</td></tr><tr><td>Pithovirus sibericum (P1084-T)</td><td>NC_023423</td><td>610,033</td><td>100% (593/593)</td></tr><tr><td>Pithovirus mammoth (strain Yana14)</td><td>-</td><td>610,309</td><td>97% (581/593)</td></tr></tbody></table>

### 3.3. *Megavirus mammoth* 239

Megavirus mammoth (strain Yana14) is the first Mimiviridae family member ever rescued from ancient permafrost. It was isolated from the highly productive sample (dated > 27.000-y BP) exhibiting fossil mammoth wool (sample #7, Table 1) together with two other viruses: Pithovirus mammoth and Pandoravirus mammoth. The prototype of the Mimiviridae family, Mimivirus, was isolated in 2003 [58]. Its landmark discovery revealed the existence of giant viruses with particles visible by light microscopy and genetic size (>1 Mb) exceeding that of many parasitic bacteria [59]. Initially thought to be exclusively associated to amoebal hosts, the Mimiviridae family has diversified enormously, and now includes many divergent members infecting a variety of microalgae [60-62] and heterotrophic protozoans [63, 64]. Several large-scale metagenomic studies have suggested that the Mimiviridae family (now comprising several subgroups) constitutes the largest part of the eukaryotic virome in both freshwater [65] and marine aquatic environments [66], with hundreds of reported Metagenomic Assembled Genomes (MAG) [67]. A recent metagenomic study revealed the presence of Mimiviridae-related DNA in several ancient permafrost layers [52]. The Mimiviridae particles exhibit the icosahedral shape encountered in many smaller viruses, except that their diameter is around 0.5 µm, to which an external layer of dense fibrils (up to 125 nm thick) is added, making them easily visible by light microscopy. In electron microscopy, the internal nucleoid appears as a dark electron-dense sphere, surrounded by a lipidic membrane. Strikingly, one vertex exhibits a well-defined starfish-like structure, called the "stargate", the opening of which delivers the nucleoid inside the cytoplasm of the infected cells after membrane fusion [68]. These features (icosahedral symmetry, large size, fibrils, and stargate) are unique to Mimiviridae members, making their identification straightforward (Figure 1.E). The replication cycle of the first megavirus genus member was fully described previously [69]. As other giant viruses infecting Acanthamoeba spp., their particles penetrate by phagocytosis. Six hours p.i. infected cells start rounding and losing adherence. New particles are then produced in very large cytoplasmic viral factories, leaving the cell nucleus intact. 240
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They are then released in large quantities (burst size ≈ 500) through cell lysis after 12-17 hours (depending on the virus and host strain)[69]. Those parameters can be strongly affected by the presence of the ubiquitous virophages, a small satellite virus, parasitising the various Mimiviridae family members [70, 71].

<table><caption>**Table 5.** Identification of Megavirus mammoth among previously isolated Megavirinae members.</caption><thead><tr><td>Virus</td><td>Accession #</td><td>Base pairs</td><td>Amplicon identity</td></tr></thead><tbody><tr><td>Megavirus chilensis</td><td>NC_016072</td><td>1,259,197</td><td>100% (1497/1497)</td></tr><tr><td>Megavirus vitis</td><td>MG807319</td><td>1,242,360</td><td>99% (1493/1497)</td></tr><tr><td>Megavirus mammoth (strain Yana14)</td><td>-</td><td>1,260,651</td><td>99% (1493/1497)</td></tr><tr><td>Megavirus powai lake</td><td>KU877344</td><td>1,208,707</td><td>93% (1396/1497)</td></tr><tr><td>Megavirus baoshan</td><td>MH046811</td><td>1,224,839</td><td>92% (1379/1497)</td></tr><tr><td>Moumouvirus</td><td>NC_020104</td><td>1,021,348</td><td>83% (1248/1497)</td></tr><tr><td>Moumouvirus australiensis</td><td>MG807320</td><td>1,098,002</td><td>82% (1244/1497)</td></tr><tr><td>Mimivirus</td><td>NC_014649</td><td>1,181,549</td><td>77% (1158/1497)</td></tr></tbody></table>

The PCR results indicate that Megavirus mammoth is a member of the clade C Megamimivirinae (i.e. the Megavirus genus) (Table 5). In addition, it encodes all the trademark proteins of the genus: the MutS-like DNA mismatch DNA repair enzyme (ORF 570, 99% identical residues), the glutamine-dependent asparagine synthetase (ORF 434, 98% identical residues), and the 5 amino-acyl tRNA ligase: Ileu AARS (ORF 383, 99% ID), Asp AARS (ORF 771, 100% ID), Met AARS (ORF 798, 99% ID), Arg AARS (ORF 834, 99% ID), Cys AARS (ORF 837, 98% ID), Trp AARS (ORF 876, 96% ID), and Tyr AARS (ORF 944, 97% ID).

### 3.4. *Pacmanvirus lupus*

*Pacmanviruses* are recently discovered *Acanthamoeba*-infecting viruses distantly related to the African swine fever virus, until then the only known members of the *Asfarviridae* family that infects pigs [72]. We now report the isolation of a third member of this newly defined group from the frozen intestinal remains of a Siberian wolf (*Canis lupus*) preserved in a permafrost layer dated >27,000-y BP. At variance with the other truly giant viruses (i.e. associated with unusually large particles), their icosahedral particles are about 220 nm in diameter (Figure 1.F), hence not individually discernable under the light microscope (Nomarski optic). However, DAPI (4', 6-diamidino-2-phenylindole) fluorescence staining can be used to reveal cytoplasmic viral DNA replication in *Acanthamoeba* cultures suspected to undergo viral infection. In culture of *A. castellanii*, *Pacmanvirus lupus* replicated slowly, producing a low yield of particles, without causing massive cell lysis.

*Pacmanvirus lupus* genome consists of a double-stranded DNA linear molecule of 407,705 bp, comparable in size to that of the previously studied members of this group (Table 6). However, out of its 506 predicted protein-coding genes, only 241 (47.6%) exhibit homologs in the two previously sequenced paccmanvirus genomes, and 221 (43.7%) are ORFans. Thus, if *P. lupus* appears definitely closer to paccmanviruses than to any other known viruses, its evolutionary distance is larger than usually observed within a subfamily or a genus. This large global discrepancy in gene content, is reflected in the low level of similarity observed between various core genes of *P. lupus* and their homologs in other paccmanviruses and closest relatives (Table 7). The asfarviruses appear even more distant with half the number of genes and half the genome size, calling into question the initial classification of paccmanviruses as truly related to family *Asfarviridae* (Table 7) [72]. Yet, in a phylogenetic tree built on these sequences, *P. lupus* appears unambiguously clustered with the other *Paccmanviruses*, justifying its given name (Figure 3).

<caption>**Table 6.** Identification of *Paccmanvirus lupus* (strain Tums2) among previously isolated paccmanviruses.</caption>
<table><caption>**Table 7.** Closest virus relatives of *Pacmanvirus lupus*.</caption><thead><tr><th>Virus</th><th>Accession #</th><th>Base pairs</th><th>Amplicon identity</th></tr></thead><tbody><tr><td><i>Pacmanvirus A23</i></td><td>NC_034383</td><td>395,405</td><td>100% (470/470)</td></tr><tr><td><i>Pacmanvirus S19</i></td><td>MZ440852</td><td>418,588</td><td>93% (439/470)</td></tr><tr><td><i>Pacmanvirus lupus</i> (strain Tums2)</td><td>-</td><td>407,705</td><td>&lt;85% (399/468)<br/>+ two large insertions</td></tr></tbody></table>

<table><thead><tr><th>Pacmanvirus lupus<br/>Predicted protein</th><th>Pacmanvirus A23<br/>NC_034383<br/>%ID (aa)</th><th>Pacmanvirus S19<br/>MZ440852<br/>%ID (aa)</th><th>Faustovirus<br/>KJ614390<br/>%ID (aa)</th><th>Kaumoebavirus<br/>NC_034249<br/>%ID (aa)</th><th>Asfarviruses<br/>NC_044958<br/>%ID (aa)</th></tr></thead><tbody><tr><td>RNA polymerase<br/>(RPB1) ORF 302</td><td>79%<br/>(1124/1415)</td><td>80%<br/>(1130/1415)</td><td>49%<br/>(707/1434)</td><td>42%<br/>(598/1429)</td><td>41%<br/>(596/1457)</td></tr><tr><td>RNA polymerase<br/>(RPB2) ORF 33</td><td>85%<br/>(1093/1289)</td><td>85%<br/>(1104/1301)</td><td>55%<br/>(681/1241)</td><td>44%<br/>(528/1211)</td><td>43%<br/>(526/1228)</td></tr><tr><td>DNA polymerase<br/>(PolB) ORF 265</td><td>65%<br/>(1036/1591)</td><td>65%<br/>(1032/1591)</td><td>37%<br/>(513/1385)</td><td>27.5%<br/>(344/1250)</td><td>33%<br/>(382/1163)</td></tr><tr><td colspan="6">Genome size</td></tr><tr><td>407,705 bp</td><td>395,405 bp</td><td>418,588 bp</td><td>457-491 kb</td><td>351-363 kb</td><td>172-191 kb</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Neighbor-joining tree of the closest *Pacmanvirus lupus* relatives (using RPB1 homologs, Table 7). The closest *Mimiviridae* RPB1 sequences are used as an outgroup. The alignment is based on 1300 gap-free sites. Although *P. lupus* is well clustered with other *pacmanviruses*, this clade (together with *faustovirus*) clearly appears as a sister group rather than *bona fide* members within the *Asfarviridae* (ASFV) family. The tree was built from 1300 gap-free sites in the multiple alignment of 9 RNA polymerases (RPB1) protein sequences using the MAFFT online facility from the Osaka University server [53]. Accession numbers are indicated following the isolate name when available.</figcaption></figure>

## 4. Discussion

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Following initial reports published more than 5 years ago [38, 39], this study confirms the capacity of large DNA viruses infecting *Acanthamoeba* to remain infectious after more than 48,500 years spent in deep permafrost. Our results extend our previous findings to 3 additional virus families or groups: 4 new members of the *Pandoraviridae*, one member of the *Mimiviridae*, and one mancanvirus (Table 1). One additional pithovirus was also revived from a particularly productive sample dated 27,000-y BP (sample#7, Table 1) exhibiting mammoth wool. Given these viruses' diversity both in their particle structure and replication mode, one can reasonably infer that many other eukaryotic viruses infecting a variety of hosts much beyond *Acanthamoeba spp.* may also remain infectious in similar conditions. Genomic traces of such viruses are detected in a recent large-scale metagenomic study of ancient permafrost [52] as well as in Arctic lake sediments [73]. They include well documented human and vertebrate pathogens such as poxviruses, herpesviruses, and asfarviruses, although in lower proportions than protozoan infecting viruses.

In our recent metagenomic study [52], Pandoraviruses are notably absent while they constitute the large majority of the viruses revived from permafrost and cryosols. Such a discrepancy might originate from the fact that the extraction of genomic DNA from their sturdy particles requires a much harsher treatment than for most other viruses. Their abundance in environmental viromes might thus be much larger than the small fraction they contribute to the DNA pool. Such DNA extraction bias may apply to many other microbes, and is a serious limitation to the validity of metagenomic approaches for quantitative population studies.

The types of viruses revived in our study are indeed the results of even stronger biases. First, the only viruses we can expect to detect are those infecting species of *Acanthamoeba*. Second, because we rely on "sick" amoeba to point out potentially virus-replicating cultures, we strongly limit ourselves to the detection of lytic viruses. Third, we favor the identification of "giant" viruses, given the important role given to light microscopy in the early detection of viral replication. It is thus likely that many small, non-lytic viruses do escape our scrutiny, as well as those infecting many other protozoa that can survive in ancient permafrost [10].

However, we believe that the use of *Acanthamoeba* cells as a virus bait is nevertheless a good choice for several reasons. First, *Acanthamoeba* spp. are free-living amoebae that are ubiquitous in natural environments, such as soils and fresh, brackish, and marine waters, but are also in dust particles, pools, water taps, sink drains, flowerpots, aquariums, sewage, as well as medical settings hydrotherapy baths, dental irrigation equipment, humidifiers, cooling systems, ventilators, and intensive care units [74]. The detection of their virus may thus provide a useful test for the presence of any other live viruses in a given setting. Second, if many *Acanthamoeba* species can be conveniently propagated in axenic culture conditions, they remain "self-cleaning" thanks to phagocytosis, and are capable of tolerating heavy contamination by bacteria (that they eat) as well as high doses of antibiotics and antifungals. The third, but not the least, advantage is that of biological security. When we use *Acanthamoeba spp.* cultures to investigate the presence of infectious unknown viruses in prehistorical permafrost (in particular from paleontological sites, such as RHS [43, 44]), we are using its billion years of evolutionary distance with human and other mammals as the best possible protection against an accidental infection of laboratory workers or the spread of a dreadful virus once infecting Pleistocene mammals to their contemporary relatives. The biohazard associated with reviving prehistorical amoeba-infecting viruses is thus totally negligible, compared to the search for "paleoviruses" directly from permafrost-preserved remains of mammoths, woolly rhinoceros, or prehistoric horses, as it is now pursued in the Vector laboratory (Novosibirsk, Russia) [75], fortunately a BSL4 facility. Without the need of embarking on such a risky project, we believe our results with *Acanthamoeba*-infecting viruses can be extrapolated to many other DNA viruses capable of infecting humans or animals. It is thus likely that ancient permafrost (eventually much older than 50,000 years, our limit solely dictated by the validity range of radiocarbon dating) will release these unknown viruses upon thawing. How long these
viruses could remain infectious once exposed to outdoor conditions (UV light, oxygen, heat), and how likely they will be to encounter and infect a suitable host in the interval, is yet impossible to estimate. But the risk is bound to increase in the context of global warming when permafrost thawing will keep accelerating, and more people will be populating the Arctic in the wake of industrial ventures. 372
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**Author Contributions:** Conceptualization and funding, C.A. and J.-M.C.; resources (samples), A.E.G., G.G, J.S., A.N.T.; virus isolation and characterization: J.-M.A., A.L.; bioinformatic analysis, O.P., M.L., S.S., J.-M.C.; writing, J.-M.C.; All authors have read and agreed to the published version of the manuscript. 377
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**Funding:** This work was supported by the Agence Nationale de la Recherche grant (ANR-10-INBS-09-08) to J-MC and the CNRS Projet de Recherche Conjoint (PRC) grant (PRC1484-2018) to CA, as well as recurrent CNRS funding to the IGS laboratory. GG and JS were funded by a European Research Council starting grant (PETA-CARB, #338335) and the Helmholtz Association of German Research Centres (HGF) Impulse and Networking Fund (ERC-0013). 381
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**Data Availability Statement:** The newly isolated viruses described here are available for collaborative studies upon formal request through a material transfer agreement. 386
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**Acknowledgments:** We thank our volunteer collaborator, Alexander Morawitz, for collecting the Kamchatka soil samples and Dr. Lyubov Shmakova (Soil Cryology Lab, Pushchino, Russia) for her generous gift of Siberian permafrost samples from which, unfortunately, no viruses were rescued in the present study. We acknowledge the computing support of the PACA BioInfo platform. We thank M. Ulrich (DFG project #UL426/1-1) and P. Konstantinov for helping with fieldwork at the Yukechi site, as well as the Alfred Wegener Institute and Melnikov Permafrost Institute logistics for field support and sample acquisition. 388
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**Conflicts of Interest:** The authors declare no conflict of interest. 395

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