Single ORF Research Articles

New Iflavirus Species Characterized from Mosquitoes Captured in the Sao Paulo Zoological Facilities.

Metagenomic studies of mosquito viromes demonstrated a more diverse composition than just an exclusive composition of pathogenic arboviruses transmitted to humans. In our study, the virome of 866 female mosquitoes collected throughout 2020 at the São Paulo Zoo, located in the city of São Paulo/SP-Brazil, was obtained. Specifically, in this paper, we describe a new virus found by viral RNA extraction and next-generation MiSeq sequencing of a group of 23 specimens of Anopheles (Nys.) strodei. The complete genome with a length of 9709 nucleotides was characterized by a positive orientation and a single strand, with a single large ORF, which encodes a polyprotein of 2987 amino acids. The phylogenetic analysis showed an association with the viral family Iflaviridae and the Riboviria realm. We carried out comparisons with translated sequences of the capsid regions of other iflavirus, and the identities in relation to our sequence were below the minimum limit of 90%, indicating that possibly it is a new species of iflavirus. Our findings contribute to expanding knowledge of virome composition among mosquito species in Brazil and globally. Moreover, we provide a viral genome reference specific to this geographic region and Culicidae family of mosquitoes. This resource facilitates future in silico recognition and assembly of viral genomes within metagenomic datasets.

Complete genome sequence of a novel partitivirus with a dsRNA3 segment, isolated from Fusarium commune strain CP-SX-3 causing strawberry root rot.

A novel partitivirus, Fusarium commune partitivirus 1 (FcoPV1), was identified in Fusarium commune strain CP-SX-3 isolated from diseased roots of strawberry with symptoms of root rot. The complete genome of FcoPV1 comprises three double-stranded RNAs (dsRNAs): dsRNA1 (1,825 nt), dsRNA2 (1,592 nt), and dsRNA3 (1,421 nt). dsRNA1 contains a single open reading frame (ORF1) encoding an RNA-dependent RNA polymerase (RdRp), and dsRNA2 contains a single ORF (ORF2) encoding a coat protein (CP). dsRNA3 is a possible satellite RNA that does not appear to encode a known protein. BLASTp analysis revealed that RdRp (86.59%) and CP (74.13%) encoded by the two ORFs (ORF1 and ORF2) had the highest sequence similarity to their counterparts in Fusarium equiseti partitivirus 1 (FePV1). Phylogenetic analysis based on the complete amino acid sequence of RdRp suggested that FcoPV1 should be considered a member of a new species in the proposed genus "Zetapartitivirus" within the family Partitiviridae. To the best of our knowledge, this is the first report of a zetapartitivirus infecting phytopathogenic F. commune.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

Complete genome sequence of a new mitovirus associated with walking iris (Trimezia northiana).

The complete genome sequence of a new member of the family Mitoviridae was obtained from walking iris (Trimezia northiana (Schneev.) Ravenna by high-throughput sequencing. This is the first putative mitovirus identified in a monocotyledonous plant. The new mitovirus was tentatively named "walking iris virus 1" (WIV1). The complete genome of WIV1 is 2,858 nt in length with a single ORF encoding a viral replicase (RdRp). The highest level of amino acid sequence identity was 45% to Beta vulgaris mitovirus 1. In the viral replicase, a conserved protein domain for mitovirus RNA-dependent RNA polymerase and six highly conserved motifs were detected, consistent with other members of the family Mitoviridae. Phylogenetic inferences placed WIV1 among members of the genus Duamitovirus (family Mitoviridae) in a monophyletic clade with other plant mitoviruses. Sequence comparison and phylogenetic analysis support the classification of WIV1 as a new member of the genus Duamitovirus (family Mitoviridae).

Complete genome sequence of a novel virus isolated from the phytopathogenic fungus Ceratobasidium sp. AG-A strain SHX-YJLC-1.

A novel mycovirus, Ceratobasidium bipartite virus 1 (CBV1), was identified in Ceratobasidium sp. AG-A strain SHX-YJLC-1 isolated from diseased potato stems. The complete genome of CBV1 consists of two double-stranded RNA (dsRNA) segments: dsRNA1 (2311 bp) and dsRNA2 (1761 bp). dsRNA1 contains a single open reading frame (ORF1) encoding an RNA-dependent RNA polymerase (RdRp), while dsRNA2 contains a single ORF (ORF2) encoding a hypothetical protein (HP) with unknown function. BLASTp analysis revealed that RdRp (75.04%) and HP (61.86%) encoded by the two ORFs have the highest sequence similarity to their counterparts in Rhizoctonia solani dsRNA virus 11 (RsRV11). The genome organization and phylogenetic analysis indicated that the closest relatives to CBV1 are members of the proposed family "Bipartitiviridae". Based on the collective results, CBV1 is inferred to be a new member of the proposed family "Bipartitiviridae". This is the first report on the complete genome sequence of the novel bipartitivirus CBV1, which infects Ceratobasidium sp. AG-A strain SHX-YJLC-1.

Characterization of a novel psyllid-transmitted waikavirus in carrots

Carrots collected from the Western Negev region in Israel during the winter of 2019 showed disease symptoms of chlorosis, leaf curling, a loss of apical dominance, and multiple lateral roots that were not associated with known pathogens of the carrot yellows disease. Symptomatic carrots were studied for a possible involvement of plant viruses in disease manifestations using high throughput sequencing analyses. The results revealed the presence of a waikavirus, sharing a ∼70% nucleotide sequence identity with Waikavirus genus members. Virions purified from waikavirus-positive carrots were visualized by transmission electron microscopy, showing icosahedral particle diameter of ∼28 nm. The genome sequence was validated by overlapping amplicons by designed 12 primer sets. A complete genome sequence was achieved by rapid amplification of cDNA ends (RACE) for sequencing the 5′ end, and RT-PCR with oligo dT for sequencing the 3′ end. The genome encodes a single large ORF, characteristic of waikaviruses. Aligning the waikavirus-deduced amino-acid sequence with other waikavirus species at the Pro-Pol region, a conserved sequence between the putative proteinase and the RNA-dependent RNA polymerase, showed a ∼40% identity, indicating the identification of a new waikavirus species. The amino-acid sequence of the three coat proteins and cleavage sites were experimentally determined by liquid chromatography-mass spectrometry. A phylogenetic analysis based on the Pro-Pol region revealed that the new waikavirus clusters with persimmon waikavirus and actinidia yellowing virus 1. The new waikavirus genome was localized in the phloem of waikavirus-infected carrots. The virus was transmitted to carrot and coriander plants by the psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae).

A Temperate Sinorhizobium Phage, AP-16-3, Closely Related to Phage 16-3: Mosaic Genome and Prophage Analysis.

Soil Sinorhizobium phage AP-16-3, a strain phylogenetically close to Rhizobium phage 16-3, was isolated in a mountainous region of Dagestan, belonging to the origin of cultivated plants in the Caucasus, according to Vavilov N.I. The genome of phage AP-16-3 is 61 kbp in size and contains 62 ORFs, of which 42 ORFs have homologues in the genome of Rhizobium phage 16-3, which was studied in the 1960s-1980s. A search for Rhizobium phage 16-3-related sequences was performed in the genomes of modern strains of root nodule bacteria belonging to different species, genera, and families. A total of 43 prophages of interest were identified out of 437 prophages found in the genomes of 42 strains, of which 31 belonged to Sinorhizobium meliloti species. However, almost all of the mentioned prophages contained single ORFs, and only two prophages contained 51 and 39 ORFs homologous to phages related to 16-3. These prophages were detected in S. meliloti NV1.1.1 and Rh. leguminosarum OyaliB strains belonging to different genera; however, the similarity level of these two prophages did not exceed 14.7%. Analysis of the orphan genes in these prophages showed that they encoded predominantly virion structural elements, but also enzymes and an extensive group of hypothetical proteins belonging to the L, S, and E regions of viral genes of phage 16-3. The data obtained indicate that temperate phages related to 16-3 had high infectivity against nodule bacteria and participated in intragenomic recombination events involving other phages, and in horizontal gene transfer between rhizobia of different genera. According to the data obtained, it is assumed that the repetitive lysogenic cycle of temperate bacteriophages promotes the dissolution of the phage genetic material in the host bacterial genome, and radical updating of phage and host bacterial genomes takes place.

Apis mellifera Solinvivirus-1, a Novel Honey Bee Virus That Remained Undetected for over a Decade, Is Widespread in the USA.

A metagenomic analysis of the virome of honey bees (Apis mellifera) from an apiary with high rates of unexplained colony losses identified a novel RNA virus. The virus, which was named Apis mellifera solinvivirus 1 (AmSV1), contains a 10.6 kb positive-strand genomic RNA with a single ORF coding for a polyprotein with the protease, helicase, and RNA-dependent RNA polymerase domains, as well as a single jelly-roll structural protein domain, showing highest similarity with viruses in the family Solinviviridae. The injection of honey bee pupae with AmSV1 preparation showed an increase in virus titer and the accumulation of the negative-strand of AmSV1 RNA 3 days after injection, indicating the replication of AmSV1. In the infected worker bees, AmSV1 was present in heads, thoraxes, and abdomens, indicating that this virus causes systemic infection. An analysis of the geographic and historic distribution of AmSV1, using over 900 apiary samples collected across the United States, showed AmSV1 presence since at least 2010. In the year 2021, AmSV1 was detected in 10.45% of apiaries (95%CI: 8.41-12.79%), mostly sampled in June and July in Northwestern and Northeastern United States. The diagnostic methods and information on the AmSV1 distribution will be used to investigate the connection of AmSV1 to honey bee colony losses.

A Novel Mitovirus PsMV2 Facilitates the Virulence of Wheat Stripe Rust Fungus.

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), seriously affects wheat production. Here, we report the complete genome sequence and biological characterization of a new mitovirus from P. striiformis strain GS-1, which was designated as "Puccinia striiformis mitovirus 2" (PsMV2). Genome sequence analysis showed that PsMV2 is 2658 nt in length with an AU-rich of 52.3% and comprises a single ORF of 2348 nt encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that PsMV2 is a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 multiplied highly during Pst infection and it suppresses programmed cell death (PCD) triggered by Bax. Silencing of PsMV2 in Pst by barley stripe mosaic virus (BSMV)-mediated Host Induced Gene Silencing (HIGS) reduced fungal growth and decreased pathogenicity of Pst. These results indicate PsMV2 promotes host pathogenicity in Pst. Interestingly, PsMV2 was detected among a wide range of field isolates of Pst and may have coevolved with Pst in earlier times. Taken together, our results characterized a novel mitovirus PsMV2 in wheat stripe rust fungus, which promotes the virulence of its fungal host and wide distribution in Pst which may offer new strategies for disease control.

Complete genome sequence of a novel alternavirus infecting the fungus Ilyonectria crassa

A novel dsRNA mycovirus named Ilyonectria crassa alternavirus 1 (IcAV1) was found in Ilyonectria crassa isolate NW-FVA 1829. The fungus was isolated from an ash (Fraxinus excelsior L.) necrotic trunk disc infected with Hymenoscyphus fraxineus [(T. Kowalski) Baral, Queloz, Hosoya] causing ash dieback. The complete genome of IcAV1 is composed of three segments, each containing a single ORF on the positive-sense RNA. The extreme 5’ UTRs of dsRNA 1 (3604 bp), dsRNA 2 (2547 bp), and dsRNA 3 (2518 bp) share a conserved hexadecamer sequence (5’-GGCTGTGTGTTTAGTT-3’) and are capped. The 3’ UTRs are polyadenylated. In silico analysis showed that the viral RdRP is encoded on dsRNA 1 and the capsid-protein subunits are encoded on dsRNA 3. Maximum-likelihood analysis of the aa sequence of the viral RdRP showed that IcAV1 clusters with alternaviruses from Fusarium spp., while the type member of the proposed family "Alternaviridae", Alternaria alternata virus 1 (AaV1), formed a clade together with Stemphylium lycopersici mycovirus (SlV). The function of the protein encoded on segment 2 is unknown. Based on its genome organization and its phylogenetic position, IcAV1 is suggested to be a new member of the proposed family "Alternaviridae". This is the first report of a mycovirus infecting I. crassa.

A novel picorna-like virus identified in the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil (CBW; Anthonomus grandis; Coleoptera: Curculionidae) is considered the major insect pest of cotton, causing considerable losses in yield and fiber quality. An increase in the boll weevil population due to increasingly inefficient chemical control measures is of great concernamong cotton producers. The absence of conventional or transgenic cultivars with minimal resistance to CBW has stimulated the search for new molecular and biological tools for efficient control of this insect pest. In this study, we used a metagenomic approach based on RNA deep sequencing to investigate the presence of viruses and coding viral RNA in apparently healthy native adult CBW insects collected from cotton crops in Mato Grosso state, Brazil. Using an Illumina HiSeq 2000 paired-end platform, 138,798 virus-related reads were obtained, and a consensus sequence of a putative new virus, 10,632 nucleotides in length, was assembled. The sequences of the 5' and 3' untranslated regions (UTRs) were determined by rapid amplification of cDNA ends (RACE), followed by Nanopore sequencing. The complete genome sequence included a 5'-UTR (1,158 nucleotides), a 3'-UTR (561 nucleotides), and a single ORF of 8,913 nucleotides encoding a large polyprotein. Sequence analysis of the putative polyprotein showed several regions with high sequence similarity to structural and non-structural proteins of viruses of the family Iflaviridae. Pairwise alignments of polyprotein amino acid sequences showed the highest sequence identity (32.13%) to a partial polyprotein sequence of a putative iflavirus (QKN89051.1) found in samples from wild zoo birds in China. Phylogenetic analysis based on full polyprotein sequences of different iflaviruses indicated that this new picorna-like virus is most closely related to iflaviruses found in lepidopteran insects, and it was therefore tentatively named "Anthonomus grandis iflavirus 1" (AgIV-1). This is, to our knowledge, the first complete viral genome sequence found in CBW, and it could provide a basis for further studies about the infectivity and transmission of this virus and its possible association with symptoms or acute disease. AgIV-1 could potentially be used to develop biological or molecular tools, such as a viral vector to carry interfering RNA molecules for CBW control.

A novel narnavirus is widespread in Saccharomyces cerevisiae and impacts multiple host phenotypes.

RNA viruses are a widespread, biologically diverse group that includes the narnaviridiae, a family of unencapsidated RNA viruses containing a single ORF that encodes an RNA-dependent RNA polymerase. In the yeast Saccharomyces cerevisiae, the 20S and 23S RNA viruses are well-studied members of the narnaviridiae, which are present at low intracellular copy numbers, unless induced by stress or unfavorable growth conditions (Wickneret al. 2013), and are not known to affect host fitness. In this study, we describe a new Saccharomyces cerevisiae narnavirus that we designate as N1199. We show that N1199 is uniquely present as a double-stranded RNA at a high level relative to other known members of this family in one strain background, YJM1199, and is present as a single-stranded RNA at lower levels in 98 of the remaining 100-genomes strains. Further, we see a strong association between the presence of high level N1199 and host phenotype defects, including greatly reduced sporulation efficiency and growth on multiple carbon sources. Finally, we describe associations between N1199 abundance and host phenotype defects, including autophagy.

Efficient production of protein complexes in mammalian cells using a poxvirus vector.

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

First report of a chrysovirus infecting a member of the fungal genus Ilyonectria

The fungus Ilyonectria pseudodestructans belongs to the family Nectriaceae and was found to be part of the endophytic microbiome of apple trees (Malus x domestica, Borkh.) with apple replant disease (ARD). After dsRNA extraction, a mycoviral infection became evident. Here, we report the identification of a new virus, tentatively named “Ilyonectria pseudodestructans chrysovirus 1” (IpCV1), as the first member of the proposed new species “Alphachrysovirus ilyonectriae” within the genus Alphachrysovirus. This is the first report of a chrysovirus infecting a member of the fungal genus Ilyonectria. IpCV1 has a tripartite dsRNA genome with a total length of 8944 bp. The segments are 3439 bp, 2850 bp, and 2655 bp in length, and each dsRNA carries a single ORF. The encoded viral proteins are a 125.92-kDa RNA-dependent RNA polymerase, a 100.75-kDa coat protein, and one protein of unknown function with a predicted molecular mass of 93.04 kDa. The 5´ and 3´ UTRs are comparatively short and are 79 to 91 bp and 62 to 148 bp in length, respectively. This study provides the basis for further investigations of the impact of IpCV1 on its host and the etiology of ARD.

Three new mycoviruses identified in the apple replant disease (ARD)-associated fungus Rugonectria rugulosa

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease.

Complete genome sequence of a new quadrivirus infecting a member of the genus Thelonectria

A novel dsRNA virus named “Thelonectria quadrivirus 1” (TQV1) was found in a member of the genus Thelonectria (Ascomycota), isolated from a root associated with stem collar necrosis of Fraxinus excelsior L. The complete genome of TQV1 is composed of four segments, each containing a single ORF on the positive sense RNA. The sequence of the 5´ (5´-(C/T)ACGAAAAA-3´) and 3´termini (5´AT(T/G)AGCAATG(T/C)GC(G/A)CG-3’) of dsRNA 1 (4876 bp), dsRNA 2 (4312 bp), dsRNA 3 (4158 bp), and dsRNA 4 (3933 bp) are conserved. Based on its genome organization and phylogenetic position, TQV1 is suggested to be a new member of the family Quadriviridae. This is the first report of a mycovirus infecting a member of the genus Thelonectria.

Characterization of a Novel Mitovirus Infecting Melanconiella theae Isolated From Tea Plants.

A dsRNA segment was identified in the fungus Melanconiella theae isolated from tea plants. The complete dsRNA sequence, determined by random cloning together with RACE protocol, is 2,461 bp in length with an AU-rich content (62.37%) and comprises a single ORF of 2,265-nucleotides encoding an RNA-dependent RNA-polymerase (RdRp, 754 amino acids in size). The terminus sequences can fold into predicted stable stem-loop structures. A BLASTX and phylogenetic analysis revealed the dsRNA genome shows similarities with the RdRp sequences of mitoviruses, with the highest identity of 48% with those of grapevine-associated mitovirus 20 and Colletotrichum fructicola mitovirus 1. Our results reveal a novel member, tentatively named Melanconiella theae mitovirus 1 (MtMV1), belongs to the family Mitoviridae. MtMV1 is capsidless as examined by transmission electron microscope, efficiently transmitted through conidia as 100 conidium-generated colonies were analyzed, and easily eliminated by hyphal tipping method combined with green-leaf tea powder. MtMV1 has a genomic sequence obviously divergent from those of most members in the family Mitoviridae and some unique characteristics unreported in known members. This is the first report of a mycovirus infecting Melanconiella fungi to date.

The complete genome sequence of a novel chrysovirus from the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana, an entomopathogenic fungus, is used for arthropod pest control worldwide. Here, we report the discovery and characterization of a novel double-stranded RNA (dsRNA) mycovirus, Beauveria bassiana chrysovirus 2 (BbCV-2), isolated from a Chinese B. bassiana strain. The genome sequence of the virus was determined by metagenomic sequencing, RT-PCR, and RACE cloning and was found to consist of four dsRNA segments that are 3441bp, 2779bp, 2925bp, and 2688bp long, respectively. Each dsRNA segment contains a single ORF. The ORF of dsRNA1 encodes a 1114-amino-acid (aa) protein (123.4kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif, the sequence of which showed the highest identity of only 16.13% to that of Beauveria bassiana chrysovirus-1 (BbCV-1). The ORF of dsRNA2 encodes an 805-aa coat protein (CP) (84.7kDa). The ORFs of dsRNAs 3 and 4 encodes proteins of undetermined function. The virus is a new member of the family Chrysoviridae from B. bassiana.

An Ancient Clade of Penelope-Like Retroelements with Permuted Domains Is Present in the Green Lineage and Protists, and Dominates Many Invertebrate Genomes.

Penelope-like elements (PLEs) are an enigmatic clade of retrotransposons whose reverse transcriptases (RTs) share a most recent common ancestor with telomerase RTs. The single ORF of canonical endonuclease (EN)+ PLEs encodes RT and a C-terminal GIY–YIG EN that enables intrachromosomal integration, whereas EN− PLEs lack EN and are generally restricted to chromosome termini. EN+ PLEs have only been found in animals, except for one case of horizontal transfer to conifers, whereas EN− PLEs occur in several kingdoms. Here, we report a new, deep-branching PLE clade with a permuted domain order, whereby an N-terminal GIY–YIG EN is linked to a C-terminal RT by a short domain with a characteristic CxC motif. These N-terminal EN+ PLEs share a structural organization, including pseudo-LTRs and complex tandem/inverted insertions, with canonical EN+ PLEs from Penelope/Poseidon, Neptune, and Nematis clades, and show insertion bias for microsatellites, but lack canonical hammerhead ribozyme motifs. However, their phylogenetic distribution is much broader. The Naiads, found in numerous invertebrate phyla, can reach tens of thousands of copies per genome. In spiders and clams, Naiads independently evolved to encode selenoproteins containing multiple selenocysteines. Chlamys, which lack the CCHH motif universal to PLE ENs, occur in green algae, spike mosses (targeting ribosomal DNA), and slime molds. Unlike canonical PLEs, RTs of N-terminal EN+ PLEs contain the insertion-in-fingers domain (IFD), strengthening the link between PLEs and telomerases. Additionally, we describe Hydra, a novel metazoan C-terminal EN+ clade. Overall, we conclude that PLE diversity, taxonomic distribution, and abundance are comparable with non-LTR and LTR-retrotransposons.

Molecular characterization of a novel partitivirus and a fusarivirus coinfecting the fungus Nigrospora sphaerica.

In this report, we describe the molecular characterization of two novel mycoviruses coinfecting the plant pathogenic fungus Nigrospora sphaerica, which were designated "Nigrospora sphaerica fusarivirus 1" (NsFV1) and "Nigrospora sphaerica partitivirus 1" (NsPV1). NsFV1 has an undivided genome measuring 6,147 nt, excluding the polyA tail, and was predicted to contain two nonoverlapping open reading frames (ORF1 and 2). The larger ORF1 encodes a polyprotein containing a conserved RNA-dependent RNA polymerase (RdRp) and a helicase domain that has functions related to RNA replication, and the smaller ORF2 encodes a putative protein with an unknown function. NsPV1 consists of two genome segments, which measure 1,796 bp and 1,455 bp in length. Each of the two dsRNAs has a single ORF, and they are predicted to encode proteins with homology to viral RdRps and coat proteins of members of the family Partitiviridae. Phylogenetic analysis indicated that NsFV1 is a member of the recently proposed family "Fusariviridae", while NsPV1 was determined to belong to the genus Gammapartitivirus in the family Partitiviridae. To the best of our knowledge, this report is the first to describe mycoviruses infecting N. sphaerica.