Identification of a novel statovirus in a faecal sample from a calf with enteric disease.

A novel clade of RNA viruses was identified in the mammalian gastrointestinal tract by next-generation sequencing. Phylogenetically, these viruses are related to the genera Tombusviridae (plant viruses) and Flaviviridae, which includes mammalian, avian and insect hosts. Named in line with their characterization as stool-associated Tombus-like viruses, it is unclear if statoviruses infect mammals or are dietary in origin. Here, metagenomic sequencing of faecal material collected from a 10-week-old calf with enteric disease found that 20 % of the reads mapped to a de novo-assembled 4 kb contig with homology to statoviruses. Phylogenetic analysis of the statovirus genome found a clear evolutionary relationship with statovirus A, but, with only 47 % similarity, we propose that the statovirus sequence presents a novel species, statovirus F. A TaqMan PCR targeting statovirus F performed on faecal material found a cycle threshold of 11, suggesting a high titre of virus shed from the calf with enteric disease. A collection of 48 samples from bovine enteric disease diagnostic submissions were assayed by PCR to investigate statovirus F prevalence and 6 of 48 (12.5 %) were positive. An ELISA to detect antibodies to the coat protein found that antibodies to statovirus F were almost ubiquitous in bovine serum. Combined, the PCR and ELISA results suggest that statovirus F commonly infects cattle. Further research is needed to elucidate the aetiological significance of statovirus infection.

Characterization of Mollivirus kamchatka, the First Modern Representative of the Proposed Molliviridae Family of Giant Viruses.

Microbes trapped in permanently frozen paleosoils (permafrost) are the focus of increasing research in the context of global warming. Our previous investigations led to the discovery and reactivation of two Acanthamoeba-infecting giant viruses, Mollivirus sibericum and Pithovirus sibericum, from a 30,000-year old permafrost layer. While several modern pithovirus strains have since been isolated, no contemporary mollivirus relative was found. We now describe Mollivirus kamchatka, a close relative to M. sibericum, isolated from surface soil sampled on the bank of the Kronotsky River in Kamchatka, Russian Federation. This discovery confirms that molliviruses have not gone extinct and are at least present in a distant subarctic continental location. This modern isolate exhibits a nucleocytoplasmic replication cycle identical to that of M. sibericum Its spherical particle (0.6 µm in diameter) encloses a 648-kb GC-rich double-stranded DNA genome coding for 480 proteins, of which 61% are unique to these two molliviruses. The 461 homologous proteins are highly conserved (92% identical residues, on average), despite the presumed stasis of M. sibericum for the last 30,000 years. Selection pressure analyses show that most of these proteins contribute to virus fitness. The comparison of these first two molliviruses clarify their evolutionary relationship with the pandoraviruses, supporting their provisional classification in a distinct family, the Molliviridae, pending the eventual discovery of intermediary missing links better demonstrating their common ancestry.IMPORTANCE Virology has long been viewed through the prism of human, cattle, or plant diseases, leading to a largely incomplete picture of the viral world. The serendipitous discovery of the first giant virus visible under a light microscope (i.e., >0.3 µm in diameter), mimivirus, opened a new era of environmental virology, now incorporating protozoan-infecting viruses. Planet-wide isolation studies and metagenome analyses have shown the presence of giant viruses in most terrestrial and aquatic environments, including upper Pleistocene frozen soils. Those systematic surveys have led authors to propose several new distinct families, including the Mimiviridae, Marseilleviridae, Faustoviridae, Pandoraviridae, and Pithoviridae We now propose to introduce one additional family, the Molliviridae, following the description of M. kamchatka, the first modern relative of M. sibericum, previously isolated from 30,000-year-old arctic permafrost.

Changes to virus taxonomy and to the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2021).

This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2021. The entire ICTV was invited to vote on 290 taxonomic proposals approved by the ICTV Executive Committee at its meeting in October 2020, as well as on the proposed revision of the International Code of Virus Classification and Nomenclature (ICVCN). All proposals and the revision were ratified by an absolute majority of the ICTV members. Of note, ICTV mandated a uniform rule for virus species naming, which will follow the binomial 'genus-species' format with or without Latinized species epithets. The Study Groups are requested to convert all previously established species names to the new format. ICTV has also abolished the notion of a type species, i.e., a species chosen to serve as a name-bearing type of a virus genus. The remit of ICTV has been clarified through an official definition of 'virus' and several other types of mobile genetic elements. The ICVCN and ICTV Statutes have been amended to reflect these changes.

Genome characterization and diversity of trifolium virus 1: identification of a novel legume-infecting capulavirus.

A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.

Complete genome sequence of hollyhock vein yellowing virus, a novel monopartite begomovirus infecting hollyhock in Pakistan.

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

ICTV Virus Taxonomy Profile: Clavaviridae.

The family Clavaviridae includes viruses that replicate in hyperthermophilic archaea from the genus Aeropyrum. The non-enveloped rigid virions are rod-shaped, with dimensions of about 143×16 nm, and have terminal cap structures, one of which is pointed and carries short fibres, while the other is rounded. The virion displays helical symmetry and is constructed from a single major α-helical protein, which is heavily glycosylated, and several minor capsid proteins. The 5278 bp, circular, double-stranded DNA genome of Aeropyrum pernix bacilliform virus 1 is packed inside the virion as a left-handed superhelix. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Clavaviridae, which is available at www.ictv.global/report/clavaviridae.

A new Tanay virus isolated from mosquitoes in Guangxi, China.

In 2013, seven virus strains were isolated from Culex tritaeniorhynchus and C. quinquefasciatus collected in Guangxi Province, China. The viruses caused cytopathological effects (CPE) only in C6/36 cells, and not in mammalian cells, e.g. BHK21 and Vero cells. Comparative genomic analysis revealed that the genomes of the seven virus strains ranged from 9589 to 9595 nt with G+C contents of 37.51-37.59%, and nucleotide sequence similarity > 99.8%. The nucleotide sequence homology of the seven virus strains with a Tanay virus isolate was 79.8-80.0%, while the corresponding amino acid homology of ORF1, ORF2, and ORF3 was 79.8-80, 73.9-4.6, and 93.4-94.3%, respectively. In phylogenetic analysis, based on the RdRP amino acid sequence, the seven virus strains were located on the same evolutionary branch, yet distinct from Tanay virus isolated from the Philippines. These results indicated that the seven virus strains isolated from Guangxi represent a new Tanay virus, "Tanay-like virus", within the genus Sandewavirus of the newly proposed taxon Negevirus. This was the first isolation of Tanay virus in China.

Complete nucleotide sequences of dsRNA2 and dsRNA7 detected in the phytopathogenic fungus Sclerotium hydrophilum and their close phylogenetic relationship to a group of unclassified viruses.

Ten dsRNA segments were extracted from Sclerotium hydrophilum isolate (HZ11). The isolation of virus-like particles contained 10 dsRNA segments with the same number and migration as those extracted directly from the fungal mycelia. Two of these dsRNA segments, dsRNA2 and dsRNA7, were cloned and sequenced. They were 2121 and 1953 bp, respectively. The dsRNA2 encodes a RNA-dependent RNA polymerase. The dsRNA7 contains two open reading frames that encode putative proteins of unknown functions. Phylogenetic analysis of the putative proteins indicated that they are closely related to protein encoded by unclassified viruses, such as Cryphonectria parasitica bipartite mycovirus 1, Lactarius rufus RNA virus 1, Penicillium aurantiogriseum bipartite virus 1, and Curvularia thermal tolerance virus. The 5'- and 3'-untranslated regions of the two dsRNAs share significant sequence identity and contain conserved sequence stretches. It suggested that dsRNA2 and dsRNA7 have a common origin and a close phylogenetic relationship to a group of unclassified viruses.

A novel gemycircularvirus from experimental rats.

Recently, gemycircularviruses have been found in humans and various species of animals. Here, a novel gemycircularvirus named Ch-zjrat-01 from blood samples of experimental rats was characterized. The novel gemycircularvirus encodes two major proteins, including a capsid protein (Cap) and a replication-associated protein (Rep). Phylogenetic analysis based on the amino acid sequence of Rep indicated that Ch-zjrat-01 clusters with two gemycircularviruses discovered from bird (KF371635) and mosquito (HQ335086), sharing 48.7 and 49.4 % sequence identities with them, respectively.

Tanay virus, a new species of virus isolated from mosquitoes in the Philippines.

In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.

First isolation of a Marseillevirus in the Diptera Syrphidae Eristalis tenax.

OBJECTIVE: Giant viruses and amoebae are common in freshwater, where they can coexist with various insects. We screened insect larvae to detect giant viruses using a high-throughput method. METHODS: We analyzed 86 Eristalis tenax larvae obtained from stagnant water reservoirs in Tunisia. The larvae were decontaminated and then dissected to remove internal parts for coculture with Acanthamoeba polyphaga. Genome sequencing of isolated viruses was performed on a 454 Roche instrument, and comparative genomics were performed. RESULTS: One Marseillevirus, named Insectomime virus, was isolated. The genome assembly generated two scaffolds, which were 382,776 and 3,855 bp in length. Among the 477 identified predicted proteins, the best hit for 435 of the identified proteins was a Marseillevirus or Lausannevirus protein. Tunisvirus was the most closely related to Insectomime, with 446 orthologs. One Insectomime protein shared with Lausannevirus and Tunisvirus showed the highest similarity with a protein from an aphid. CONCLUSION: The isolation of a Marseillevirus from an insect expands the diversity of environments in which giant viruses have been isolated. The coexistence of larvae and giant viruses in stagnant water may explain the presence of the giant virus in the larva internal structures. This study illustrates the putative role of amoeba in lateral gene transfer not only between the organisms it phagocytoses, but also between organisms living in the same environment. © 2013 S. Karger AG, Basel.

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses.

Characterisation and Distribution of Karaka Okahu Purepure Virus-A Novel Emaravirus Likely to Be Endemic to New Zealand.

We report the first emaravirus on an endemic plant of Aotearoa New Zealand that is, to the best of our knowledge, the country's first endemic virus characterised associated with an indigenous plant. The new-to-science virus was identified in the endemic karaka tree (Corynocarpus laevigatus), and is associated with chlorotic leaf spots, and possible feeding sites of the monophagous endemic karaka gall mite. Of the five negative-sense RNA genomic segments that were fully sequenced, four (RNA 1-4) had similarity to other emaraviruses while RNA 5 had no similarity with other viral proteins. A detection assay developed to amplify any of the five RNAs in a single assay was used to determine the distribution of the virus. The virus is widespread in the Auckland area, particularly in mature trees at Okahu Bay, with only occasional reports elsewhere in the North Island. Phylogenetic analysis revealed that its closest relatives are pear chlorotic leaf spot-associated virus and chrysanthemum mosaic-associated virus, which form a unique clade within the genus Emaravirus. Based on the genome structure, we propose this virus to be part of the family Emaravirus, but with less than 50% amino acid similarity to the closest relatives in the most conserved RNA 1, it clearly is a novel species. In consultation with mana whenua (indigenous Maori authority over a territory and its associated treasures), we propose the name Karaka Okahu purepure virus in te reo Maori (the Maori language) to reflect the tree from which it was isolated (karaka), a place where the virus is prevalent (Okahu), and the spotted symptom (purepure, pronounced pooray pooray) that this endemic virus appears to cause.

Distribution and Pathogenicity of Two Cutthroat Trout Virus (CTV) Genotypes in Canada.

The sole member of the Piscihepevirus genus (family Hepeviridae) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye (Oncorhynchus nerka), pink (O. gorbuscha), and chinook salmon (O. tshawytscha) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon (Salmo salar) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.

Molecular Characterization of the First Alternavirus Identified in Fusarium oxysporum.

A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family "Alternaviridae". Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family "Alternaviridae" members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1-and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family "Alternaviridae". Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family "Alternaviridae", and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.

A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues.

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales.

Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.

Virus population structure in the ectomycorrhizal fungi Lactarius rufus and L. tabidus at two forest sites in Southern Finland.

Lactarius fungi belong to the Russulaceae family and have an important ecological role as ectomycorrhizal symbionts of coniferous and deciduous trees. Two Lactarius species, L. tabidus and L. rufus have been shown to harbor bisegmented dsRNA viruses belonging to an unclassified virus group including the mutualistic Curvularia thermal tolerance virus (CThTV). In this study, we characterized the first complete genome sequences of these viruses designated as Lactarius tabidus RNA virus 1 (LtRV1) and Lactarius rufus RNA virus 1 (LrRV1), both of which included two genome segments of 2241 and 2049 bp. We also analyzed spatial distribution and sequence diversity of the viruses in sixty host strains at two forest sites, and showed that the viruses are species-specific at sites where both host species co-occur. We also found that single virus isolates inhabited several different conspecific host strains, and were involved in persistent infections during up to eight years.

Comparative Genomics Unveils Regionalized Evolution of the Faustovirus Genomes.

Faustovirus is a recently discovered genus of large DNA virus infecting the amoeba Vermamoeba vermiformis, which is phylogenetically related to Asfarviridae. To better understand the diversity and evolution of this viral group, we sequenced six novel Faustovirus strains, mined published metagenomic datasets and performed a comparative genomic analysis. Genomic sequences revealed three consistent phylogenetic groups, within which genetic diversity was moderate. The comparison of the major capsid protein (MCP) genes unveiled between 13 and 18 type-I introns that likely evolved through a still-active birth and death process mediated by intron-encoded homing endonucleases that began before the Faustovirus radiation. Genome-wide alignments indicated that despite genomes retaining high levels of gene collinearity, the central region containing the MCP gene together with the extremities of the chromosomes evolved at a faster rate due to increased indel accumulation and local rearrangements. The fluctuation of the nucleotide composition along the Faustovirus (FV) genomes is mostly imprinted by the consistent nucleotide bias of coding sequences and provided no evidence for a single DNA replication origin like in circular bacterial genomes.

Isolation and characterization of a single-stranded RNA virus infecting the bloom-forming diatom Chaetoceros socialis.

Diatoms are very significant primary producers in the world's oceans. Various environmental factors affect the depletion of diatom populations. The importance of viruses as a potential mortality source has recently been recognized. We isolated and characterized a new diatom virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) causing the lysis of the bloom-forming species Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko. The virus infectious to C. socialis f. radians was isolated from water samples collected in Hiroshima Bay. Here we show the physiology, morphology, and genome characteristics of the virus clone. Virions were 22 nm in diameter and accumulated in the cytoplasm of the host cells. The latent period and the burst size were estimated to be <48 h and 66 infectious units per host cell, respectively. CsfrRNAV harbors a single-stranded RNA (ssRNA) genome and encodes at least three polypeptides of 32.0, 28.5, and 25.0 kDa. Sequencing analysis shows the length of the genome is 9,467 bases, excluding a poly(A) tail. The monophyly of CsfrRNAV and other diatom-infecting RNA viruses, Rhizosolenia setigera RNA virus and Chaetoceros tenuissimus RNA virus, was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domains. This suggested a new ssRNA virus family, Bacillariornaviridae. This discovery of CsfrRNAV may aid in further understanding the ecological dynamics of the C. socialis f. radians population in nature and the relationships between ssRNA diatom viruses and their hosts.