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Established in 2016, the family Pleolipoviridae comprises globally distributed archaeal viruses that produce pleomorphic particles. Pseudo-spherical enveloped virions of pleolipoviruses are membrane vesicles carrying a nucleic acid cargo. The cargo can be either a single-stranded or double-stranded DNA molecule, making this group the first family introduced in the 10 th Report on Virus Taxonomy including both single-stranded and double-stranded DNA viruses. The length of the genomes is approximately 7-17 kilobase pairs, or kilonucleotides in the case of single-stranded molecules. The genomes are circular single-stranded DNA, circular double-stranded DNA, or linear double-stranded DNA molecules. Currently, eight virus species and seven proposed species are classified in three genera: Alphapleolipovirus (five species), Betapleolipovirus (nine species), and Gammapleolipovirus (one species). Here, we summarize the updated taxonomy of the family Pleolipoviridae to reflect recent advances in this field, with the focus on seven newly proposed species in the genus Betapleolipovirus: Betapleolipovirus HHPV3, HHPV4, HRPV9, HRPV10, HRPV11, HRPV12, and SNJ2.

Competing Interests: The authors have declared that no competing interests exist.

Metagenomics is a helpful tool for the analysis of unculturable organisms and viruses. Viruses that target bacteria and archaea play important roles in the microbial diversity of various ecosystems. Here we show that Methanosarcina virus MV (MetMV), the second Methanosarcina sp. virus with a completely determined genome, is characteristic of hydrocarbon pollution in environmental (soil and water) samples. It was highly abundant in Hungarian hydrocarbon polluted samples and its genome was also present in the NCBI SRA database containing reads from hydrocarbon polluted samples collected in Canada, indicating the stability of its niche and the marker feature of this virus. MetMV, as the only currently identified marker virus for pollution in environmental samples, could contribute to the understanding of the complicated network of prokaryotes and their viruses driving the decomposition of environmental pollutants., Competing Interests: J. Molnár, Á. L. Kovács, I. K. Nagy and T. Kovács are employees of Enviroinvest Corp., B. Magyar is employee of Biocentrum Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The Nanoarchaeota are small cells with reduced genomes that are found attached to and dependent on a second archaeal cell for their growth and replication. Initially found in marine hydrothermal environments and subsequently in terrestrial geothermal hot springs, the Nanoarchaeota species that have been described are obligate ectobionts, each with a different host species. However, no viruses had been described that infect the Nanoarchaeota. Here, we identify a virus infecting Nanoarchaeota by the use of a combination of viral metagenomic and bioinformatic approaches. This virus, tentatively named Nanoarchaeota Virus 1 (NAV1), consists of a 35.6-kb circular DNA genome coding for 52 proteins. We further demonstrate that this virus is broadly distributed among Yellowstone National Park hot springs. NAV1 is one of the first examples of a virus infecting a single-celled organism that is itself an ectobiont of another single-celled organism. IMPORTANCE Here, we present evidence of the first virus found to infect Nanoarchaeota, a symbiotic archaean found in acidic hot springs of Yellowstone National Park, USA. Using culture-independent techniques, we provide the genome sequence and identify the archaeal host species of a novel virus, NAV1. NAV1 is the first example of a virus infecting an archaeal species that is itself an obligate symbiont and dependent on a second host organism for growth and cellular replication. On the basis of annotation of the NAV1 genome, we propose that this virus is the founding member of a new viral family, further demonstrating the remarkable genetic diversity of archaeal viruses., (Copyright © 2020 American Society for Microbiology.)

CRISPR-Cas immunity is at the forefront of antivirus defense in bacteria and archaea and specifically targets viruses carrying protospacers matching the spacers catalogued in the CRISPR arrays. Here, we perform deep sequencing of the CRISPRome-all spacers contained in a microbiome-associated with hyperthermophilic archaea of the order Sulfolobales recovered directly from an environmental sample and from enrichment cultures established in the laboratory. The 25 million CRISPR spacers sequenced from a single sampling site dwarf the diversity of spacers from all available Sulfolobales isolates and display complex temporal dynamics. Comparison of closely related virus strains shows that CRISPR targeting drives virus genome evolution. Furthermore, we show that some archaeal viruses carry mini-CRISPR arrays with 1-2 spacers and preceded by leader sequences but devoid of cas genes. Closely related viruses present in the same population carry spacers against each other. Targeting by these virus-borne spacers represents a distinct mechanism of heterotypic superinfection exclusion and appears to promote archaeal virus speciation.

The diversity of archaeal viruses is severely undersampled compared with that of viruses infecting bacteria and eukaryotes, limiting our understanding on their evolution and environmental impacts. Here, we describe the isolation and characterization of four new viruses infecting halophilic archaea from the saline Lake Retba, located close to Dakar on the coast of Senegal. Three of the viruses, HRPV10, HRPV11 and HRPV12, have enveloped pleomorphic virions and should belong to the family Pleolipoviridae, whereas the forth virus, HFTV1, has an icosahedral capsid and a long non-contractile tail, typical of bacterial and archaeal members of the order Caudovirales. Comparative genomic and phylogenomic analyses place HRPV10, HRPV11 and HRPV12 into the genus Betapleolipovirus, whereas HFTV1 appears to be most closely related to the unclassified Halorubrum virus HRTV-4. Differently from HRTV-4, HFTV1 encodes host-derived minichromosome maintenance helicase and PCNA homologues, which are likely to orchestrate its genome replication. HFTV1, the first archaeal virus isolated on a Haloferax strain, could also infect Halorubrum sp., albeit with an eightfold lower efficiency, whereas pleolipoviruses nearly exclusively infected autochthonous Halorubrum strains. Mapping of the metagenomic sequences from this environment to the genomes of isolated haloarchaeal viruses showed that these known viruses are underrepresented in the available viromes., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Marine Group I (MGI) Thaumarchaeota are some of the most abundant microorganisms in the deep ocean and responsible for much of the ammonia oxidation occurring in this environment. In this work, we present 35 sequences assembled from metagenomic samples of the first uncultivated Caudovirales viruses associated with Thaumarchaeota, which we designated marthavirus. Most of the sequences were obtained from cellular metagenomes confirming that they represent an important tool to study environmental viral communities due to cells retrieved while undergoing viral lysis. Metagenomic recruitment showed that this viral population is formed by very divergent entities with high intrapopulation homogeneity. However, metatranscriptomic analyses revealed the same differential expression profile with the capsid as major transcript, indicative of viruses during the lytic cycle. The cobalamine biosynthesis gene cobS, an auxiliary metabolic gene, was also highly expressed during the infection. These analyses expand our understanding of the global diversity of archaeal viruses., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Viruses are ubiquitous in the biosphere and greatly affect the hosts they infect. It is generally accepted that members of every microbial taxon are susceptible to at least one virus, and a plethora of bacterial viruses are known. In contrast, knowledge of the archaeal virosphere is still limited. Here, a novel lytic archaeal virus is described, designated "Drs3", as well as its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its virus were isolated from the anaerobic digester of an experimental biogas plant in Germany. The tailed virus has an icosahedral head with a diameter of approximately 60 nm and a long non-contractile tail of approximately 230 nm. These structural observations suggest that the new isolate belongs to the family Siphoviridae, but it could not be assigned to an existing genus. Lysis of the host Khl10 was observed 40-44 h after infection. Lysis of the type strain Methanobacterium formicicum DSMZ 1535 was not observed in the presence of Drs3, pointing towards resistance in the type strain or a rather narrow host range of this newly isolated archaeal virus. The complete 37-kb linear dsDNA genome of Drs3 contains 39 open reading frames, only 12 of which show similarity to genes with predicted functions.

Background: The genomes of halophilic archaea (haloarchaea) often comprise multiple replicons. Genomic variation in haloarchaea has been linked to viral infection pressure and, in the case of Antarctic communities, can be caused by intergenera gene exchange. To expand understanding of genome variation and biogeography of Antarctic haloarchaea, here we assessed genomic variation between two strains of Halorubrum lacusprofundi that were isolated from Antarctic hypersaline lakes from different regions (Vestfold Hills and Rauer Islands). To assess variation in haloarchaeal populations, including the presence of genomic islands, metagenomes from six hypersaline Antarctic lakes were characterised., Results: The sequence of the largest replicon of each Hrr. lacusprofundi strain (primary replicon) was highly conserved, while each of the strains' two smaller replicons (secondary replicons) were highly variable. Intergenera gene exchange was identified, including the sharing of a type I-B CRISPR system. Evaluation of infectivity of an Antarctic halovirus provided experimental evidence for the differential susceptibility of the strains, bolstering inferences that strain variation is important for modulating interactions with viruses. A relationship was found between genomic structuring and the location of variation within replicons and genomic islands, demonstrating that the way in which haloarchaea accommodate genomic variability relates to replicon structuring. Metagenome read and contig mapping and clustering and scaling analyses demonstrated biogeographical patterning of variation consistent with environment and distance effects. The metagenome data also demonstrated that specific haloarchaeal species dominated the hypersaline systems indicating they are endemic to Antarctica., Conclusion: The study describes how genomic variation manifests in Antarctic-lake haloarchaeal communities and provides the basis for future assessments of Antarctic regional and global biogeography of haloarchaea.

Viruses of archaea represent one of the most enigmatic parts of the virosphere. Most of the characterized archaeal viruses infect extremophilic hosts and display remarkable diversity of virion morphotypes, many of which have never been observed among viruses of bacteria or eukaryotes. The uniqueness of the virion morphologies is matched by the distinctiveness of the genomes of these viruses, with ∼75% of genes encoding unique proteins, refractory to functional annotation based on sequence analyses. In this review, we summarize the state-of-the-art knowledge on various aspects of archaeal virus genomics. First, we outline how structural and functional genomics efforts provided valuable insights into the functions of viral proteins and revealed intricate details of the archaeal virus-host interactions. We then highlight recent metagenomics studies, which provided a glimpse at the diversity of uncultivated viruses associated with the ubiquitous archaea in the oceans, including Thaumarchaeota, Marine Group II Euryarchaeota, and others. These findings, combined with the recent discovery that archaeal viruses mediate a rapid turnover of thaumarchaea in the deep sea ecosystems, illuminate the prominent role of these viruses in the biosphere. Finally, we discuss the origins and evolution of archaeal viruses and emphasize the evolutionary relationships between viruses and non-viral mobile genetic elements. Further exploration of the archaeal virus diversity as well as functional studies on diverse virus-host systems are bound to uncover novel, unexpected facets of the archaeal virome., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the past decade, molecular surveys of viral diversity have revealed that viruses are the most diverse and abundant biological entities on Earth. In culture, however, most viral isolates that infect microbes are represented by a few variants isolated on type strains, limiting our ability to study how natural variation affects virus-host interactions in the laboratory. We screened a set of 137 hot spring samples for viruses that infect a geographically diverse panel of the hyperthemophilic crenarchaeon Sulfolobus islandicus. We isolated and characterized eight SIRVs ( Sulfolobus islandicus rod-shaped viruses) from two different regions within Yellowstone National Park (USA). Comparative genomics revealed that all SIRV sequenced isolates share 30 core genes that represent 50-60% of the genome. The core genome phylogeny, as well as the distribution of variable genes (shared by some but not all SIRVs) and the signatures of host-virus interactions recorded on the CRISPR (clustered regularly interspaced short palindromic repeats) repeat-spacer arrays of S. islandicus hosts, identify different SIRV lineages, each associated with a different geographic location. Moreover, our studies reveal that SIRV core genes do not appear to be under diversifying selection and thus we predict that the abundant and diverse variable genes govern the coevolutionary arms race between SIRVs and their hosts., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Hypersaline environments that are subject to salinity changes are particularly rich in viruses. Here we report a newly isolated archaeal halovirus, Haloarcula hispanica pleomorphic virus 3 (HHPV3). Its reproduction significantly retards host growth and decreases cell viability without causing lysis. HHPV3 particles require a minimum of 3M NaCl for stability and maintain high infectivity even in saturated salt. Notably, virions are irreversibly inactivated at ~1.5M NaCl in neutral pH, but tolerate this salinity at alkaline pH. The HHPV3 virion is a pleomorphic membrane vesicle containing two major protein species and lipids acquired nonselectively from the host membrane. The circular double-stranded DNA genome contains a conserved gene block characteristic of pleolipoviruses. We propose that HHPV3 is a member of the Betapleolipovirus genus (family Pleolipoviridae). Our findings add insights into the diversity observed among the pleolipoviruses found in hypersaline environments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length., Importance: Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life, Archaea This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea, Bacteria, and Eukarya). Traditionally, viral metagenomic studies provide information about viral gene content but rarely provide knowledge about virion morphology and/or cellular host identity. Here we describe a new virus, Acidianus tailed spindle virus (ATSV), initially identified by bioinformatic analysis of viral metagenomic data sets from a high-temperature (80°C) acidic (pH 2) hot spring located in Yellowstone National Park, followed by more detailed characterization using only environmental samples without dependency on culturing. Characterization included the identification of the large tailed spindle virion morphology, determination of the complete 70.8-kb circular double-stranded DNA (dsDNA) viral genome content, and identification of its cellular host. Annotation of the ATSV genome revealed a potential three-domain gene product containing an N-terminal leucine-rich repeat domain, followed by a likely posttranslation regulatory region consisting of high serine and threonine content, and a C-terminal ESCRT-III domain, suggesting interplay with the host ESCRT system. The host of ATSV, which is most closely related to Acidianus hospitalis, was determined by a combination of analysis of cellular clustered regularly interspaced short palindromic repeat (CRISPR)/Cas loci and dual viral and cellular fluorescence in situ hybridization (viral FISH) analysis of environmental samples and confirmed by culture-based infection studies. This work provides an expanded pathway for the discovery, isolation, and characterization of new viruses using culture-independent approaches and provides a platform for predicting and confirming virus hosts., Importance: Virus discovery and characterization have been traditionally accomplished by using culture-based methods. While a valuable approach, it is limited by the availability of culturable hosts. In this research, we report a virus-centered approach to virus discovery and characterization, linking viral metagenomic sequences to a virus particle, its sequenced genome, and its host directly in environmental samples, without using culture-dependent methods. This approach provides a pathway for the discovery, isolation, and characterization of new viruses. While this study used an acidic hot spring environment to characterize a new archaeal virus, Acidianus tailed spindle virus (ATSV), the approach can be generally applied to any environment to expand knowledge of virus diversity in all three domains of life., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Viral communities of two different salt pans located in the Namib Desert, Hosabes and Eisfeld, were investigated using a combination of multiple displacement amplification of metaviromic DNA and deep sequencing, and provided comprehensive sequence data on both ssDNA and dsDNA viral community structures. Read and contig annotations through online pipelines showed that the salt pans harbored largely unknown viral communities. Through network analysis, we were able to assign a large portion of the unknown reads to a diverse group of ssDNA viruses. Contigs belonging to the subfamily Gokushovirinae were common in both environmental datasets. Analysis of haloarchaeal virus contigs revealed the presence of three contigs distantly related with His1, indicating a possible new lineage of salterproviruses in the Hosabes playa. Based on viral richness and read mapping analyses, the salt pan metaviromes were novel and most closely related to each other while showing a low degree of overlap with other environmental viromes.

Viruses infecting archaea show a variety of virion morphotypes, and they are currently classified into more than ten viral families or corresponding groups. A pleomorphic virus morphotype is very common among haloarchaeal viruses, and to date, several such viruses have been isolated. Here, we propose the classification of eight such viruses and formation of a new family, Pleolipoviridae (from the Greek pleo for more or many and lipos for lipid), containing three genera, Alpha-, Beta-, and Gammapleolipovirus. The proposal is currently under review by the International Committee on Taxonomy of Viruses (ICTV). The members of the proposed family Pleolipoviridae infect halophilic archaea and are nonlytic. They share structural and genomic features and differ from any other classified virus. The virion of pleolipoviruses is composed of a pleomorphic membrane vesicle enclosing the genome. All pleolipoviruses have two major structural protein species, internal membrane and spike proteins. Although the genomes of the pleolipoviruses are single- or double-stranded, linear or circular DNA molecules, they share the same genome organization and gene synteny and show significant similarity at the amino acid level. The canonical features common to all members of the proposed family Pleolipoviridae show that they are closely related and thus form a new viral family.

Proviral regions have been identified in the genomes of many haloarchaea, but only a few archaeal halophilic temperate viruses have been studied. Here, we report a new virus, SNJ2, originating from archaeal strain Natrinema sp. J7-1. We demonstrate that this temperate virus coexists with SNJ1 virus and is dependent on SNJ1 for efficient production. Here, we show that SNJ1 is an icosahedral membrane-containing virus, whereas SNJ2 is a pleomorphic one. Instead of producing progeny virions and forming plaques, SNJ2 integrates into the host tRNA(Met) gene. The virion contains a discontinuous, circular, double-stranded DNA genome of 16 992 bp, in which both nicks and single-stranded regions are present preceded by a 'GCCCA' motif. Among 25 putative SNJ2 open reading frames (ORFs), five of them form a cluster of conserved ORFs homologous to archaeal pleolipoviruses isolated from hypersaline environments. Two structural protein encoding genes in the conserved cluster were verified in SNJ2. Furthermore, SNJ2-like proviruses containing the conserved gene cluster were identified in the chromosomes of archaea belonging to 10 different genera. Comparison of SNJ2 and these proviruses suggests that they employ a similar integration strategy into a tRNA gene., (© 2015 John Wiley & Sons Ltd.)

Background: Viruses are the most abundant and genetically diverse biological entities on earth, yet the repertoire of viral proteins remains poorly explored. As the number of sequenced virus genomes grows into the thousands, and the number of viral proteins into the hundreds of thousands, we report a systematic computational analysis of the point of first-contact between viruses and their hosts, namely viral transmembrane (TM) proteins., Results: The complement of α-helical TM proteins in double-stranded DNA viruses infecting bacteria and archaea reveals large-scale trends that differ from those of their hosts. Viruses typically encode a substantially lower fraction of TM proteins than archaea or bacteria, with the notable exception of viruses with virions containing a lipid component such as a lipid envelope, internal lipid core, or inner membrane vesicle. Compared to bacteriophages, archaeal viruses are substantially enriched in membrane proteins. However, this feature is not always stable throughout the evolution of a viral lineage; for example, TM proteins are not part of the common heritage shared between Lipothrixviridae and Rudiviridae. In contrast to bacteria and archaea, viruses almost completely lack proteins with complicated membrane topologies composed of more than 4 TM segments, with the few detected exceptions being obvious cases of relatively recent horizontal transfer from the host., Conclusions: The dramatic differences between the membrane proteomes of cells and viruses stem from the fact that viruses do not depend on essential membranes for energy transformation, ion homeostasis, nutrient transport and signaling.

Understanding of viral assemblage structure in natural environments remains a daunting task. Total viral assemblage sequencing (for example, viral metagenomics) provides a tractable approach. However, even with the availability of next-generation sequencing technology it is usually only possible to obtain a fragmented view of viral assemblages in natural ecosystems. In this study, we applied a network-based approach in combination with viral metagenomics to investigate viral assemblage structure in the high temperature, acidic hot springs of Yellowstone National Park, USA. Our results show that this approach can identify distinct viral groups and provide insights into the viral assemblage structure. We identified 110 viral groups in the hot springs environment, with each viral group likely representing a viral family at the sub-family taxonomic level. Most of these viral groups are previously unknown DNA viruses likely infecting archaeal hosts. Overall, this study demonstrates the utility of combining viral assemblage sequencing approaches with network analysis to gain insights into viral assemblage structure in natural ecosystems.

Hypersaline environments around the world are dominated by archaea and their viruses. To date, very little is known about these viruses and their interaction with the host strains when compared to bacterial and eukaryotic viruses. We performed the first culture-dependent temporal screening of haloarchaeal viruses and their hosts in the saltern of Samut Sakhon, Thailand, during two subsequent years (2009, 2010). Altogether we obtained 36 haloarchaeal virus isolates and 36 archaeal strains, significantly increasing the number of known archaeal virus isolates. Interestingly, the morphological distribution of our temporal isolates (head-tailed, pleomorphic, and icosahedral membrane-containing viruses) was similar to the outcome of our previous spatial survey supporting the observations of a global resemblance of halophilic microorganisms and their viruses. Myoviruses represented the most abundant virus morphotype with strikingly broad host ranges. The other viral morphotypes (siphoviruses, as well as pleomorphic and icosahedral internal membrane-containing viruses) were more host-specific. We also identified a group of Halorubrum strains highly susceptible to numerous different viruses (up to 26). This high virus sensitivity, the abundance of broad host range viruses, and the maintenance of infectivity over a period of one year suggest constant interplay of halophilic microorganisms and their viruses within an extreme environment.

Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2 with an environmental virus mixture isolated from Yellowstone National Park (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012). Experimental studies of isolated genetic elements from this mixture revealed that SMV1 (S ulfolobus Monocauda Virus 1), a tailed spindle-shaped virus, can induce spacer acquisition in CRISPR loci of Sulfolobus species from a second coinfecting conjugative plasmid or virus (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012; Erdmann et al. Mol Microbiol 91:900-917, 2014). Here we describe, firstly, the isolation of archaeal virus mixtures from terrestrial hot springs and the techniques used both to infect laboratory strains with these virus mixtures and to obtain purified virus particles. Secondly, we present the experimental conditions required for activating SMV1-induced spacer acquisition in two different Sulfolobus species.

Isolated archaeal viruses comprise only a few percent of all known prokaryotic viruses. Thus, the study of viruses infecting archaea is still in its early stages. Here we summarize the most recent discoveries of archaeal viruses utilizing a virion-centered view. We describe the known archaeal virion morphotypes and compare them to the bacterial counterparts, if such exist. Viruses infecting archaea are morphologically diverse and present some unique morphotypes. Although limited in isolate number, archaeal viruses reveal new insights into the viral world, such as deep evolutionary relationships between viruses that infect hosts from all three domains of life., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years.

Plaque assay is the method traditionally used to isolate and purify lytic viruses, to determine the viral titer and host range. Whereas most bacterioviruses are either temperate or lytic, the majority of known archeoviruses are not lytic (i.e. they are temperate or chronic). In view of the widespread occurrence of such viruses in extreme environments, we designed an original method, called the inverted spot test, to determine the host range and infectivity of viruses isolated from anaerobic hyperthermophilic and sulfur-reducing microorganisms. Here, we used this approach to prove for the first time the infectivity of Pyrococcus abyssi virus 1 (PAV1) and to confirm the host range of Thermococcus prieurii virus 1 (TPV1), the only two viruses isolated so far from any of the described marine hyperthermophilic archaea (Euryarchaeota phylum, Thermococcales order).

Extremophilic archaea, both hyperthermophiles and halophiles, dominate in habitats where rather harsh conditions are encountered. Like all other organisms, archaeal cells are susceptible to viral infections, and to date, about 100 archaeal viruses have been described. Among them, there are extraordinary virion morphologies as well as the common head-tailed viruses. Although approximately half of the isolated archaeal viruses belong to the latter group, no three-dimensional virion structures of these head-tailed viruses are available. Thus, rigorous comparisons with bacteriophages are not yet warranted. In the present study, we determined the genome sequences of two of such viruses of halophiles and solved their capsid structures by cryo-electron microscopy and three-dimensional image reconstruction. We show that these viruses are inactivated, yet remain intact, at low salinity and that their infectivity is regained when high salinity is restored. This enabled us to determine their three-dimensional capsid structures at low salinity to a ∼10-Å resolution. The genetic and structural data showed that both viruses belong to the same T-number class, but one of them has enlarged its capsid to accommodate a larger genome than typically associated with a T=7 capsid by inserting an additional protein into the capsid lattice.

This review presents a personal account of research on archaeal viruses and describes many new viral species and families, demonstrating that viruses of Archaea constitute a distinctive part of the virosphere and display morphotypes that are not associated with the other two domains of life, Bacteria and Eukarya. I focus primarily on viruses that infect hyperthermophilic members of the phylum Crenarchaeota. These viruses' distinctiveness extends from their morphotypes to their genome sequences and the structures of the proteins they encode. Moreover, the mechanisms underlying the interactions of these viruses with their hosts also have unique features. Studies of archaeal viruses provide new perspectives concerning the nature, diversity, and evolution of virus-host interactions. Considering these studies, I associate the distinctions between bacterial and archaeal viruses with the fundamental differences in the envelope compositions of their host cells.

Halovirus PH1 infects Haloarcula hispanica and was isolated from an Australian salt lake. The burst size in single-step growth conditions was 50-100 PFU/cell, but cell density did not decrease until well after the rise (4-6 hr p.i.), indicating that the virus could exit without cell lysis. Virions were round, 51 nm in diameter, displayed a layered capsid structure, and were sensitive to chloroform and lowered salt concentration. The genome is linear dsDNA, 28,064 bp in length, with 337 bp terminal repeats and terminal proteins, and could transfect haloarchaeal species belonging to five different genera. The genome is predicted to carry 49 ORFs, including those for structural proteins, several of which were identified by mass spectroscopy. The close similarity of PH1 to SH1 (74% nucleotide identity) allowed a detailed description and analysis of the differences (divergent regions) between the two genomes, including the detection of repeat-mediated deletions. The relationship of SH1-like and pleolipoviruses to previously described genomic loci of virus and plasmid-related elements (ViPREs) of haloarchaea revealed an extensive level of recombination between the known haloviruses. PH1 is a member of the same virus group as SH1 and HHIV-2, and we propose the name halosphaerovirus to accommodate these viruses.

Known viruses build their particles using a restricted number of redundant structural solutions. Here, we describe the Aeropyrum coil-shaped virus (ACV), of the hyperthermophilic archaeon Aeropyrum pernix, with a virion architecture not previously observed in the viral world. The nonenveloped, hollow, cylindrical virion is formed from a coiling fiber, which consists of two intertwining halves of a single circular nucleoprotein. The virus ACV is also exceptional for its genomic properties. It is the only virus with a single-stranded (ss) DNA genome among the known hyperthermophilic archaeal viruses. Moreover, the size of its circular genome, 24,893 nt, is double that of the largest known ssDNA genome, suggesting an efficient solution for keeping ssDNA intact at 90-95 °C, the optimal temperature range of A. pernix growth. The genome content of ACV is in line with its unique morphology and confirms that ACV is not closely related to any known virus.

There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.

Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interactions. Reliable laboratory conditions to propagate STIV and available genetic tools allowed structural characterization of the virus and viral components that lead to the proposal of common capsid ancestry with PRD1 (bacteriophage), Adenovirus (eukaryotic virus) and PBCV (chlorellavirus). Microarray and proteomics approaches systematically analyzed viral replication and the corresponding host responses. Cellular cryo-electron tomography and thin-section EM studies uncovered the assembly and maturation pathway of STIV and revealed dramatic cellular ultra-structure changes upon infection. The viral-induced pyramid-like protrusions on cell surfaces represent a novel viral release mechanism and previously uncharacterized functions in viral replication., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hypersaline environments are dominated by archaea and bacteria and are almost entirely devoid of eukaryotic organisms. In addition, hypersaline environments contain considerable numbers of viruses. Currently, there is only a limited amount of information about these haloviruses. The ones described in detail mostly resemble head-tail bacteriophages, whereas observations based on direct microscopy of the hypersaline environmental samples highlight the abundance of non-tailed virus-like particles. Here we studied nine spatially distant hypersaline environments for the isolation of new halophilic archaea (61 isolates), halophilic bacteria (24 isolates) and their viruses (49 isolates) using a culture-dependent approach. The obtained virus isolates approximately double the number of currently described archaeal viruses. The new isolates could be divided into three tailed and two non-tailed virus morphotypes, suggesting that both types of viruses are widely distributed and characteristic for haloarchaeal viruses. We determined the sensitivity of the hosts against all isolated viruses. It appeared that the host ranges of numerous viruses extend to hosts in distant locations, supporting the idea that there is a global exchange of microbes and their viruses. It suggests that hypersaline environments worldwide function like a single habitat., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

We describe a novel virus, TPV1 (Thermococcus prieurii virus 1), which was discovered in a hyperthermophilic euryarchaeote isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2700 m at the East Pacific Rise. TPV1 is the first virus isolated and characterized from the hyperthermophilic euryarchaeal genus Thermococcus. TPV1 particles have a lemon-shaped morphology (140 nm × 80 nm) similar to the structures previously reported for Fuselloviruses and for the unclassified virus-like particle PAV1 (Pyrococcus abyssi virus 1). The infection with TPV1 does not cause host lysis and viral replication can be induced by UV irradiation. TPV1 contains a double-stranded circular DNA of 21.5 kb, which is also present in high copy number in a free form in the host cell. The TPV1 genome encompasses 28 predicted genes; the protein sequences encoded in 16 of these genes show no significant similarity to proteins in public databases. Proteins predicted to be involved in genome replication were identified as well as transcriptional regulators. TPV1 encodes also a predicted integrase of the tyrosine recombinase family. The only two genes that are homologous between TPV1 and PAV1 are TPV1-22 and TPV1-23, which encode proteins containing a concanavalin A-like lectin/glucanase domain that might be involved in virus-host recognition., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Since their discovery in the early 1980s, viruses that infect the third domain of life, the Archaea, have captivated our attention because of their virions' unusual morphologies and proteins, which lack homologues in extant databases. Moreover, the life cycles of these viruses have unusual features, as revealed by the recent discovery of a novel virus egress mechanism that involves the formation of specific pyramidal structures on the host cell surface. The available data elucidate the particular nature of the archaeal virosphere and shed light on questions concerning the origin and evolution of viruses and cells. In this review, we summarize the current knowledge of archeoviruses, their interaction with hosts and plasmids and their role in the evolution of life., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Hypersaline environments harbour the highest number of viruses reported for aquatic environments. In crystallizer ponds from solar salterns, haloviruses coexist with extremely halophilic Archaea and Bacteria and present a high diversity although little is known about their activity. In this work, we analyzed the viral expression in one crystallizer using a metatranscriptomic approach in which clones from a metaviromic library were immobilized in a microarray and used as probes against total mRNA extracted from the hypersaline community. This approach has two advantages: (i) it overcomes the fact that there is no straightforward, unambiguous way to extract viral mRNA from bulk mRNAs and (ii) it makes the sequencing of all mRNAs unnecessary. Transcriptomic data indicated that the halovirus assemblage was highly active at the time of sampling and the viral groups with the highest expression levels were those related to high GC content haloarchaea and Salinibacter representatives, which are minor components in the environment. Moreover, the changes in the viral expression pattern and in the numbers of free viral particles were analyzed after submitting the samples to two stress conditions: ultraviolet-radiation and dilution. Results showed that Archaea were more sensitive than Bacteria to these stress conditions. The overexpression in the predicted archaeal virus fraction raised and the total numbers of free viruses increased. Furthermore, we identified some very closely related viral clones, displaying single-nucleotide polymorphisms, which were expressed only under certain conditions. These clones could be part of very closely related virus genomes for which we propose the term 'ecoviriotypes'.

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication., (Copyright © 2011. Published by Elsevier Inc.)

Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double-stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb), and derive apparently from archaeal viruses HAV1 and HAV2. Genomic DNA was obtained from samples enriched in filamentous and tadpole-shaped virus-like particles respectively. They yielded few significant matches in public sequence databases reinforcing, further, the wide diversity of archaeal viruses. Several variants of HAV1 exhibit major genomic alterations, presumed to arise from viral adaptation to different hosts. They include insertions up to 350 bp, deletions up to 1.5 kb, and genes with extensively altered sequences. Some result from recombination events occurring at low complexity direct repeats distributed along the genome. In addition, a 33.8 kb archaeal plasmid pHA1 was characterized, encoding a possible conjugative apparatus, as well as three cryptic plasmids of thermophilic bacterial origin, pHB1 of 2.1 kb and two closely related variants pHB2a and pHB2b, of 5.2 and 4.8 kb respectively. Strategies are considered for assembling genomes of smaller genetic elements from complex environmental samples, and for establishing possible host identities on the basis of sequence similarity to host CRISPR immune systems., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Hypersaline environments harbour the highest number of virus-like particles reported for planktonic systems. However, very little is known about the genomic diversity of these virus assemblages since most of the knowledge on halophages is based on the analysis of a few isolates infecting strains of hyperhalophilic Archaea that may not be representatives of the natural microbiota. Here, we report the characterization, through a metagenomic approach, of the viral assemblage inhabiting a crystallizer pond (CR30) from a multi-pond solar saltern in Santa Pola (SE Spain). A total of 1.35 Mbp were cloned that yielded a total of 620 kb sequenced viral DNA. The metavirome was highly diverse and different from virus communities of marine and other aquatic environments although it showed some similarities with metaviromes from high-salt ponds in solar salterns in San Diego (SW USA), indicating some common traits between high-salt viromes. A high degree of diversity was found in the halophages as revealed by the presence of 2479 polymorphic nucleotides. Dinucleotide frequency analysis of the CR30 metavirome showed a good correlation with GC content and enabled the establishment of different groups, and even the assignment of their putative hosts: the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Archaea often live in extreme, harsh environments such as acidic hot springs and hypersaline waters. To date, only two icosahedrally symmetric, membrane-containing archaeal viruses, SH1 and Sulfolobus turreted icosahedral virus (STIV), have been described in detail. We report the sequence and three-dimensional structure of a third such virus isolated from a hyperthermoacidophilic crenarchaeon, Sulfolobus strain G4ST-2. Characterization of this new isolate revealed it to be similar to STIV on the levels of genome and structural organization. The genome organization indicates that these two viruses have diverged from a common ancestor. Interestingly, the prominent surface turrets of the two viruses are strikingly different. By sequencing and mass spectrometry, we mapped several large insertions and deletions in the known structural proteins that could account for these differences and showed that both viruses can infect the same host. A combination of genomic and proteomic analyses revealed important new insights into the structural organization of these viruses and added to our limited knowledge of archaeal virus life cycles and host-cell interactions.

Archaeal organisms are generally known as diverse extremophiles, but they play a crucial role also in moderate environments. So far, only about 50 archaeal viruses have been described in some detail. Despite this, unusual viral morphotypes within this group have been reported. Interestingly, all isolated archaeal viruses have a double-stranded DNA (dsDNA) genome. To further characterize the diversity of archaeal viruses, we screened highly saline water samples for archaea and their viruses. Here, we describe a new haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1) that was isolated from a solar saltern and infects an indigenous host belonging to the genus Halorubrum. Infection does not cause cell lysis, but slightly retards growth of the host and results in high replication of the virus. The sequenced genome (7048 nucleotides) of HRPV-1 is single-stranded DNA (ssDNA), which makes HRPV-1 the first characterized archaeal virus that does not have a dsDNA genome. In spite of this, similarities to another archaeal virus were observed. Two major structural proteins were recognized in protein analyses, and by lipid analyses it was shown that the virion contains a membrane. Electron microscopy studies indicate that the enveloped virion is pleomorphic (approximately 44 x 55 nm). HRPV-1 virion may represent commonly used virion architecture, and it seems that structure-based virus lineages may be extended to non-icosahedral viruses.

The world of prokaryotic viruses, including the "traditional" bacteriophages and the viruses of Archaea, is currently in a period of renaissance, brought about largely by our new capabilities in (meta)genomics and by the isolation of diverse novel virus-host systems. In this review, we highlight some of the directions where we believe research on the prokaryotic virosphere will lead us in the near future.

Viruses are the largest reservoir of genetic material on the planet, yet little is known about the population dynamics of any virus within its natural environment. Over a 2-year period, we monitored the diversity of two archaeal viruses found in hot springs within Yellowstone National Park (YNP). Both temporal phylogeny and neutral biodiversity models reveal that virus diversity in these local environments is not being maintained by mutation but rather by high rates of immigration from a globally distributed metacommunity. These results indicate that geographically isolated hot springs are readily able to exchange viruses. The importance of virus movement is supported by the detection of virus particles in air samples collected over YNP hot springs and by their detection in metacommunity sequencing projects conducted in the Sargasso Sea. Rapid rates of virus movement are not expected to be unique to these archaeal viruses but rather a common feature among virus metacommunities. The finding that virus immigration rather than mutation can dominate community structure has significant implications for understanding virus circulation and the role that viruses play in ecology and evolution by providing a reservoir of mobile genetic material.

The Acidianus bottle-shaped virus, ABV, infects strains of the hyperthermophilic archaeal genus Acidianus and is morphologically distinct from all other known viruses. Its genome consists of linear double-stranded DNA, containing 23,814 bp with a G+C content of 35%, and it exhibits a 590-bp inverted terminal repeat. Of the 57 predicted ORFs, only three produced significant matches in public sequence databases with genes encoding a glycosyltransferase, a thymidylate kinase and a protein-primed DNA polymerase. Moreover, only one homologous gene is shared with other sequenced crenarchaeal viruses. The results confirm the unique nature of the ABV virus, and support its assignment to the newly proposed viral family the Ampullaviridae. Exceptionally, one region at the end of the linear genome of ABV is similar in both gene content and organization to corresponding regions in the genomes of the bacteriophage varphi29 and the human adenovirus. The region contains the genes for a putative protein-primed DNA polymerase, and a small putative RNA with a predicted secondary structure closely similar to that of the prohead RNA of bacteriophage varphi29. The apparent similarities in the putative mechanisms of DNA replication and packaging of ABV to those of bacterial and eukaryal viruses are most consistent with the concept of a primordial gene pool as a source of viral genes.

It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day-1 L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.

The discovery of archaeal viruses provides insights into the fundamental biochemistry and evolution of the Archaea. Recent studies have identified a wide diversity of archaeal viruses within the hot springs of Yellowstone National Park and other high-temperature environments worldwide. These viruses are often morphologically unique and code for genes with little similarity to other known genes in the biosphere, a characteristic that has complicated efforts to trace their evolutionary history. Comparative genomics combined with structural analysis indicate that spindle-shaped virus lineages might be unique to the Archaea, whereas other icosahedral viruses might share a common lineage with viruses of Bacteria and Eukarya. These studies provide insights into the evolutionary history of viruses in all three domains of life.

A novel virus, ATV, of the hyperthermophilic archaeal genus Acidianus has the unique property of undergoing a major morphological development outside of, and independently of, the host cell. Virions are extruded from host cells as lemon-shaped tail-less particles, after which they develop long tails at each pointed end, at temperatures close to that of the natural habitat, 85 degrees C. The extracellularly developed tails constitute tubes, which terminate in an anchor-like structure that is not observed in the tail-less particles. A thin filament is located within the tube, which exhibits a periodic structure. Tail development produces a one half reduction in the volume of the virion, concurrent with a slight expansion of the virion surface. The circular, double-stranded DNA genome contains 62,730 bp and is exceptional for a crenarchaeal virus in that it carries four putative transposable elements as well as genes, which previously have been associated only with archaeal self-transmissable plasmids. In total, it encodes 72 predicted proteins, including 11 structural proteins with molecular masses in the range of 12 to 90 kDa. Several of the larger proteins are rich in coiled coil and/or low complexity sequence domains, which are unusual for archaea. One protein, in particular P800, resembles an intermediate filament protein in its structural properties. It is modified in the two-tailed, but not in the tail-less, virion particles and it may contribute to viral tail development. Exceptionally for a crenarchaeal virus, infection with ATV results either in viral replication and subsequent cell lysis or in conversion of the infected cell to a lysogen. The lysogenic cycle involves integration of the viral genome into the host chromosome, probably facilitated by the virus-encoded integrase and this process can be interrupted by different stress factors.