Diversity and Current Classification of dsRNA Bacteriophages.

Half a century has passed since the discovery of Pseudomonas phage phi6, the first enveloped dsRNA bacteriophage to be isolated. It remained the sole known dsRNA phage for a quarter of a century and the only recognised member of the Cystoviridae family until the year 2018. After the initial discovery of phi6, additional dsRNA phages have been isolated from globally distant locations and identified in metatranscriptomic datasets, suggesting that this virus type is more ubiquitous in nature than previously acknowledged. Most identified dsRNA phages infect Pseudomonas strains and utilise either pilus or lipopolysaccharide components of the host as the primary receptor. In addition to the receptor-mediated strictly lytic lifestyle, an alternative persistent infection strategy has been described for some dsRNA phages. To date, complete genome sequences of fourteen dsRNA phage isolates are available. Despite the high sequence diversity, similar sets of genes can typically be found in the genomes of dsRNA phages, suggesting shared evolutionary trajectories. This review provides a brief overview of the recognised members of the Cystoviridae virus family and related dsRNA phage isolates, outlines the current classification of dsRNA phages, and discusses their relationships with eukaryotic RNA viruses.

Influenza A virus reassortment is strain dependent.

RNA viruses can exchange genetic material during coinfection, an interaction that creates novel strains with implications for viral evolution and public health. Influenza A viral genetic exchange can occur when genome segments from distinct strains reassort in coinfected cells. Predicting potential genomic reassortment between influenza strains has been a long-standing goal. Experimental coinfection studies have shed light on factors that limit or promote reassortment. However, determining the reassortment potential between diverse Influenza A strains has remained elusive. To address this challenge, we developed a high throughput genotyping approach to quantify reassortment among a diverse panel of human influenza virus strains encompassing two pandemics (swine and avian origin), three specific epidemics, and both circulating human subtypes A/H1N1 and A/H3N2. We found that reassortment frequency (the proportion of reassortants generated) is an emergent property of specific pairs of strains where strain identity is a predictor of reassortment frequency. We detect little evidence that antigenic subtype drives reassortment as intersubtype (H1N1xH3N2) and intrasubtype reassortment frequencies were, on average, similar. Instead, our data suggest that certain strains bias the reassortment frequency up or down, independently of the coinfecting partner. We observe that viral productivity is also an emergent property of coinfections, but uncorrelated to reassortment frequency; thus viral productivity is a separate factor affecting the total number of reassortants produced. Assortment of individual segments among progeny and pairwise segment combinations within progeny generally favored homologous combinations. These outcomes were not related to strain similarity or shared subtype but reassortment frequency was closely correlated to the proportion of both unique genotypes and of progeny with heterologous pairwise segment combinations. We provide experimental evidence that viral genetic exchange is potentially an individual social trait subject to natural selection, which implies the propensity for reassortment is not evenly shared among strains. This study highlights the need for research incorporating diverse strains to discover the traits that shift the reassortment potential to realize the goal of predicting influenza virus evolution resulting from segment exchange.

Black box of phage-bacterium interactions: exploring alternative phage infection strategies.

The canonical lytic-lysogenic binary has been challenged in recent years, as more evidence has emerged on alternative bacteriophage infection strategies. These infection modes are little studied, and yet they appear to be more abundant and ubiquitous in nature than previously recognized, and can play a significant role in the ecology and evolution of their bacterial hosts. In this review, we discuss the extent, causes and consequences of alternative phage lifestyles, and clarify conceptual and terminological confusion to facilitate research progress. We propose distinct definitions for the terms 'pseudolysogeny' and 'productive or non-productive chronic infection', and distinguish them from the carrier state life cycle, which describes a population-level phenomenon. Our review also finds that phages may change their infection modes in response to environmental conditions or the physiological state of the host cell. We outline known molecular mechanisms underlying the alternative phage-host interactions, including specific genetic pathways and their considerable biotechnological potential. Moreover, we discuss potential implications of the alternative phage lifestyles for microbial biology and ecosystem functioning, as well as applied topics such as phage therapy.

ICTV Virus Taxonomy Profile: Finnlakeviridae.

Finnlakeviridae is a family of icosahedral, internal membrane-containing bacterial viruses with circular, single-stranded DNA genomes. The family includes the genus, Finnlakevirus, with the species, Flavobacterium virus FLiP. Flavobacterium phage FLiP was isolated with its Gram-negative host bacterium from a boreal freshwater habitat in Central Finland in 2010. It is the first described single-stranded DNA virus with an internal membrane and shares minimal sequence similarity with other known viruses. The virion organization (pseudo T=21 dextro) and major capsid protein fold (double-ß-barrel) resemble those of Pseudoalteromonas phage PM2 (family Corticoviridae), which has a double-stranded DNA genome. A similar major capsid protein fold is also found in other double-stranded DNA viruses in the kingdom Bamfordvirae. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Finnlakeviridae, which is available at ictv.global/report/finnlakeviridae.

Half a Century of Research on Membrane-Containing Bacteriophages: Bringing New Concepts to Modern Virology.

Half a century of research on membrane-containing phages has had a major impact on virology, providing new insights into virus diversity, evolution and ecological importance. The recent revolutionary technical advances in imaging, sequencing and lipid analysis have significantly boosted the depth and volume of knowledge on these viruses. This has resulted in new concepts of virus assembly, understanding of virion stability and dynamics, and the description of novel processes for viral genome packaging and membrane-driven genome delivery to the host. The detailed analyses of such processes have given novel insights into DNA transport across the protein-rich lipid bilayer and the transformation of spherical membrane structures into tubular nanotubes, resulting in the description of unexpectedly dynamic functions of the membrane structures. Membrane-containing phages have provided a framework for understanding virus evolution. The original observation on membrane-containing bacteriophage PRD1 and human pathogenic adenovirus has been fundamental in delineating the concept of "viral lineages", postulating that the fold of the major capsid protein can be used as an evolutionary fingerprint to trace long-distance evolutionary relationships that are unrecognizable from the primary sequences. This has brought the early evolutionary paths of certain eukaryotic, bacterial, and archaeal viruses together, and potentially enables the reorganization of the nearly immeasurable virus population (~1 × 1031) on Earth into a reasonably low number of groups representing different architectural principles. In addition, the research on membrane-containing phages can support the development of novel tools and strategies for human therapy and crop protection.

Recognition of six additional cystoviruses: Pseudomonas virus phi6 is no longer the sole species of the family Cystoviridae.

Cystoviridae is a family of bacterial viruses (bacteriophages) with a tri-segmented dsRNA genome. It includes a single genus Cystovirus, which has presently only one recognised virus species, Pseudomonas virus phi6. However, a large number of additional dsRNA phages have been isolated from various environmental samples, indicating that such viruses are more widespread and abundant than previously recognised. Six of the additional dsRNA phage isolates (Pseudomonas phages phi8, phi12, phi13, phi2954, phiNN and phiYY) have been fully sequenced. They all infect Pseudomonas species, primarily plant pathogenic Pseudomonas syringae strains. Due to the notable genetic and structural similarities with Pseudomonas phage phi6, we propose that these viruses should be included into the Cystovirus genus (and consequently into the Cystoviridae family). Here, we present an updated taxonomy of the family Cystoviridae and give a short overview of the properties of the type member phi6 as well as the putative new members of the family.

ICTV Virus Taxonomy Profile: Cystoviridae.

The family Cystoviridae includes enveloped viruses with a tri-segmented dsRNA genome and a double-layered protein capsid. The innermost protein shell is a polymerase complex responsible for genome packaging, replication and transcription. Cystoviruses infect Gram-negative bacteria, primarily plant-pathogenic Pseudomonas syringae strains. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Cystoviridae, which is available at http://www.ictv.global/report/cystoviridae.

Virus found in a boreal lake links ssDNA and dsDNA viruses.

Viruses have impacted the biosphere in numerous ways since the dawn of life. However, the evolution, genetic, structural, and taxonomic diversity of viruses remain poorly understood, in part because sparse sampling of the virosphere has concentrated mostly on exploring the abundance and diversity of dsDNA viruses. Furthermore, viral genomes are highly diverse, and using only the current sequence-based methods for classifying viruses and studying their phylogeny is complicated. Here we describe a virus, FLiP (Flavobacterium-infecting, lipid-containing phage), with a circular ssDNA genome and an internal lipid membrane enclosed in the icosahedral capsid. The 9,174-nt-long genome showed limited sequence similarity to other known viruses. The genetic data imply that this virus might use replication mechanisms similar to those found in other ssDNA replicons. However, the structure of the viral major capsid protein, elucidated at near-atomic resolution using cryo-electron microscopy, is strikingly similar to that observed in dsDNA viruses of the PRD1-adenovirus lineage, characterized by a major capsid protein bearing two ß-barrels. The strong similarity between FLiP and another member of the structural lineage, bacteriophage PM2, extends to the capsid organization (pseudo T = 21 dextro) despite the difference in the genetic material packaged and the lack of significant sequence similarity.

Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1.

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES.

New enveloped dsRNA phage from freshwater habitat.

Cystoviridae is a family of bacteriophages with a tri-segmented dsRNA genome enclosed in a tri-layered virion structure. Here, we present a new putative member of the Cystoviridae family, bacteriophage ϕNN. ϕNN was isolated from a Finnish lake in contrast to the previously identified cystoviruses, which originate from various legume samples collected in the USA. The nucleotide sequence of the virus reveals a strong genetic similarity (~80 % for the L-segments, ~55 % for the M-segments and ~84 % for the S-segments) to Pseudomonas phage ϕ6, the type member of the virus family. However, the relationship between ϕNN and other cystoviruses is more distant. In general, proteins located in the internal parts of the virion were more conserved than those exposed on the virion surface, a phenomenon previously reported among eukaryotic dsRNA viruses. Structural models of several putative ϕNN proteins propose that cystoviral structures are highly conserved.

Subcellular localization of bacteriophage PRD1 proteins in Escherichia coli.

Bacteria possess an intricate internal organization resembling that of the eukaryotes. The complexity is especially prominent at the bacterial cell poles, which are also known to be the preferable sites for some bacteriophages to infect. Bacteriophage PRD1 is a well-known model serving as an ideal system to study structures and functions of icosahedral internal membrane-containing viruses. Our aim was to analyze the localization and interactions of individual PRD1 proteins in its native host Escherichia coli. This was accomplished by constructing a vector library for production of fluorescent fusion proteins. Analysis of solubility and multimericity of the fusion proteins, as well as their localization in living cells by confocal microscopy, indicated that multimeric PRD1 proteins were prone to localize in the cell poles. Furthermore, PRD1 spike complex proteins P5 and P31, as fusion proteins, were shown to be functional in the virion assembly. In addition, they were shown to co-localize in the specific polar area of the cells, which might have a role in the multimerization and formation of viral protein complexes.

Bacteriophage P23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage.

It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.