Single Open Reading Frame Research Articles

Identification of a putative novel cholivirus in the transcriptome of Gymnema sylvestre R. Br

Gymnema sylvestre is a tropical climber species that is widely used in traditional medicine since ages. In the present study, the transcriptome datasets of G. sylvestre available in public domain were screened for the presence of novel plant viral sequences and a putative novel virus tentatively named as Gymnema sylvestre virus 1 (GysV1) was identified. Coding-complete genome segments of GysV1 that are 6.35kb (RNA1) and 3.98kb (RNA2) long possessed a single large open reading frame coding for a polyprotein. BLASTp, sequence identity and phylogenetic analyses revealed the relatedness of GysV1 to the members of the subgenus Cholivirus (genus Sadwavirus; family Secoviridae; order Picornavirales). Based on the species demarcation criteria of the family Secoviridae, GysV1 can be regarded as a new cholivirus member.

Molecular characterization of horse nettle virus A, a new member of subgroup B of the genus Nepovirus.

A new positive-strand RNA virus was discovered in a horse nettle plant, using high-throughput sequencing (HTS), and its complete genome, consisting of RNA1 and RNA2, which are 7522 and 4710 nucleotides in length, respectively, was characterized. Each genome segment contains a single open reading frame flanked by 5' and 3' untranslated regions (UTRs), followed by a poly(A) tail at the 3' end. The encoded proteins have the highest amino acid sequence identity (55% and 45%) to the polyprotein encoded by RNA1 of tomato black ring virus (TBRV) and RNA2 of potato virus B (PVB), respectively. Its genome organization and phylogenetic relationship to other nepoviruses suggested that this virus is a novel member of subgroup B, and recombination analysis revealed its evolutionary history within the subgroup. These results suggest the new virus, provisionally named "horse nettle virus A", represents a new species within the genus Nepovirus.

Identification and complete genome sequencing of a novel betapartitivirus naturally infecting the mycorrhizal desert truffle Terfezia claveryi.

Viruses that naturally infect fungal species and capable of establishing mycorrhizae are largely unknown. In this study, we identified and characterized a new partitivirus inhabiting the ascomycete, mycorrhizal desert truffle species Terfezia claveryi, and named it "Terfezia claveryi partitivirus 1" (TcPV1). The entire genome of TcPV1, sequenced by both high throughput sequencing of the total dsRNA extracts and by Sanger sequencing of the RLM-RACE PCR products comprised two dsRNA segments of 2404bp and 2374bp, respectively. Both dsRNA genome segments harbored a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. The BLASTp search of the RdRp and CP sequences revealed the highest sequence identities (41.92% and 24.13% identity, respectively) to those of Bipolaris maydis partitivirus 2 and Plasmopara viticola lesion associated partitivirus 5. Molecular phylogenetic analyses of the RdRp sequence showed that TcPV1 fall within a clade composed entirely of members of the genus Betapartitivirus, belonging to the family Partitiviridae. In light of this molecular evidence, TcPV1 is a new member of the genus Betapartitivirus. This is the first report of a new partitivirus hosted by the ascomycete, mycorrhizal fungus T. claveryi.

Genome sequence and characterization of a novel fabavirus infecting Cirsium setidens (gondre) in South Korea.

The complete nucleotide sequence of a novel gondre (Cirsium setidens)-infecting virus, provisionally named "cirsium virus A" (CiVA), was determined by high-throughput and Sanger sequencing, revealing a genome organization typical of fabaviruses. RNA1 and RNA2 are 5,828 and 3,478 nucleotides long, excluding the 3'-terminal poly(A) tails, each containing a single open reading frame. The highest sequence identity values for the CiVA coat protein and proteinase-polymerase, compared with known fabavirus sequences, were 59.09% and 69.68%, respectively, falling below the current thresholds for Fabavirus species demarcation. Our findings support classifying CiVA as a novel putative member of the genus Fabavirus, subfamily Comovirinae, family Secoviridae.

Complete genome sequence of a novel mycovirus from Pleurotus citrinopileatus

The complete genome sequence of a novel single-stranded [+ ssRNA] positive-sense (+) RNA mycovirus, designated as "Pleurotus citrinopileatus ourmiavirus 1" (PcOV1), isolated from Pleurotus citrinopileatus strain CCMJ2141, was determined. The complete genome of PcOV1 is composed of 2,535 nucleotides. It contains a single open reading frame (ORF), which encodes a protein of 657 amino acids (aa) containing conserved domains of an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on RdRp sequences revealed that PcOV1 is a new member of the genus Ourmiavirus in the family Botourmiaviridae. This is the first virus from P. citrinopileatus to be characterized.

Complete genome sequence of polygonatum mosaic-associated virus 1, a novel member of the genus Potyvirus in China

The complete genome sequence of a putative novel potyvirus, tentatively named "polygonatum mosaic-associated virus 1" (PMaV1), was sequenced from naturally infected Polygonatum cyrtonema Hua in China. PMaV1 has a typical genome organization of potyviruses with a single large open reading frame (nt 119-9448) that encodes a 3109-aa polyprotein that is predicted to be cleaved into 10 mature proteins by virus-encoded proteases. Pairwise comparisons revealed that PMaV1 shares 71.50% complete genome sequence identity with Polygonatum kingianum virus 4 and 80.00% amino acid sequence identity with Polygonatum kingianum virus 3 of the genus Potyvirus. Phylogenetic analysis indicated that PMaV1 clustered with other potyviruses and that it was most closely related to Polygonatum kingianum virus 3 and Polygonatum kingianum virus 4. These results suggest that PMaV1 is a new member of the genus Potyvirus of the family Potyviridae (Nucleotide sequence data reported are available in the GenBank databases under the accession number OP380926).

Molecular and biological characteristics of a novel chrysovirus infecting the fungus phytopathogenic Setosphaeria turcica f.sp. sorghi

A new double-stranded RNA (dsRNA) virus has been identified in the filamentous fungus Setosphaeria turcica f.sp. sorghi, whose genome consists of four segments (dsRNA1–4). Each dsRNA carries single open reading frame (ORF) flanked by 5′ and 3′ untranslated regions (UTRs) containing strictly conserved termini. The putative protein encoded by dsRNA1 showed 80.50% identity to the RNA-dependent RNA polymerase (RdRp) of the most closely related virus, Alternaria alternata chrysovirus 1 (AaCV1), belonging to the Chrysoviridae. dsRNA2 encodes the putative coat protein, while dsRNA3 and dsRNA4 respectively encode the hypothetical proteins of unknown functions. Phylogenetic analysis based on the RdRp protein indicated the virus clustered with members of the genus Betachrysovirus in the family Chrysoviridae. Based on the dsRNA profile, amino acid sequence comparisons, and phylogenetic analyses, the mycovirus is thought to be a new member of the family Chrysoviridae and designated as Setosphaeria turcica chrysovirus 1 (StCV1). Moreover, obvious differences were observed in the colony, mycelial and spore morphology between StCV1-infected and virus-cured strains of S. turcica f.sp. sorghi. StCV1 infection strongly reduced colony growth rate, spore production ability and virulence on host fungus. To our knowledge, this is the first report about mycovirus infecting S. turcica f.sp. sorghi and also the first chrysovirus infecting S. turcica.

The essential role of the quasi-long terminal repeat sequence for replication and gene expression of an endogenous pararetrovirus, petunia vein clearing virus.

Petunia vein clearing virus (PVCV) is a type member of the genus Petuvirus within the Caulimoviridae family and is defined as one viral unit consisting of a single open reading frame (ORF) encoding a viral polyprotein and one quasi-long terminal repeat (QTR) sequence. Since some full-length PVCV sequences are found in the petunia genome and a vector for horizontal transmission of PVCV has not been identified yet, PVCV is referred to as an endogenous pararetrovirus. Molecular mechanisms of replication, gene expression and horizontal transmission of endogenous pararetroviruses in plants are elusive. In this study, agroinfiltration experiments using various PVCV infectious clones indicated that the replication (episomal DNA synthesis) and gene expression of PVCV were efficient when the QTR sequences are present on both sides of the ORF. Whereas replacement of the QTR with another promoter and/or terminator is possible for gene expression, it is essential for QTR sequences to be on both sides for viral replication. Although horizontal transmission of PVCV by grafting and biolistic inoculation was previously reported, agroinfiltration is a useful and convenient method for studying its replication and gene expression.

A novel polymycovirus infecting the entomopathogenic fungus Metarhizium brunneum.

In this study, at least four distinct double-stranded RNA viruses, including two partitiviruses, one bipartite virus, and a novel polymycovirus, were detected in the entomopathogenic fungus Metarhizium brunneum strain RCEF0736. We describe the characterization of one of these viruses, a novel polymycovirus, which we have named "Metarhizium brunneum polymycovirus 1" (MbPmV1). The genome of MbPmV1 has four dsRNA segments, ranging from 1153 to 2421 bp in length. dsRNA1, 2, and 3 of MbPmV1 each contain a single large open reading frame (ORF), while dsRNA4 has two ORFs. BLASTp analysis indicated that dsRNA1, 2, 3, and 4 of MbPmV1 have a high degree of similarity to the putative RdRp (59.45%), serine protease (44.22%), putative methyltransferase (48.76%), and proline-alanine-serine-rich protein (PASrp) (52.80%), respectively, of Phaeoacremonium minimum tetramycovirus 1 (PmTmV1). MbPmV1 was grouped in a cluster with members of the genus Polymycovirus and was most closely related to PmTmV1 in the phylogenetic tree. Thus, we propose that MbPmV1 represents a novel species of the genus Polymycovirus, family Polymycoviridae. To our knowledge, this is the first report of a polymycovirus in a member of the genus Metarhizium.

Identification, characterization and prevalence in southern China of a new iflavirus in the leafhopper Recilia dorsalis (Hemiptera: Cicadellidae)

The leafhopper Recilia dorsalis (Hemiptera: Cicadellidae) is not only a significant pest in agriculture but also an important vector involved in transmitting numerous pathogens that are known to cause economic losses by affecting rice crops. Here, a new iflavirus was discovered in the leafhopper R. dorsalis by employing a transcriptomic approach. The complete viral genome was determined to be 10,711 nucleotides (nt) in length and contains a single open reading frame (ORF) encoding a putative polyprotein comprised of 3,161 amino acids (aa), which is flanked by 5′ and 3′ untranslated regions. The full viral genome nt and the deduced polyprotein aa sequence showed the highest similarity (71.6% and 77.8%, respectively) with Langfang leafhopper iflavirus. Phylogenetic analysis based on the RdRp domain indicated that the isolated virus, which we have tentatively named Recilia dorsalis iflavirus 2 (RdIV2), is clustered with the members of the family Iflaviridae. Moreover, the results of our surveys indicate that RdIV2 predominates in southwestern Guangdong and southeastern Guangxi, China, and was absent in the other three species of leafhoppers; Nephotettix cincticeps, N. virescens and N. nigropictus. Notably, R. dorsalis was found to be co-infected with RdIV2 and rice stripe mosaic virus (RSMV; a well-known rice-infecting virus vectored by R. dorsalis) in rice fields, although the co-infection rate is low.

Comparison of Iflavirus Diversity in Agricultural and Forest Pest Lepidoptera

Iflaviridae is a novel and important family of viruses in the Picornavirales order that infects invertebrates. Iflaviruses are non-enveloped, linear, positive single-stranded RNA viruses. The importance of iflaviruses in agriculture and forestry is twofold because these viruses, both cause economic loss by making insects such as honey bees and silkworms ill and prevent economic loss by making agricultural and forest pest insects ill. Therefore, iflaviruses contain highly interesting isolates that infect both beneficial and harmful insects in agriculture. Its genome structure, like most RNA viruses, has a very small genome size (8.8-10 kb), with a single open reading frame in the entire genome. The genomes of 14 infective iflaviruses that infect agricultural and forest pest insects from different countries have been analyzed so far. In this study, the similar relationship between these 14 viruses, whose complete genomes are available, was analyzed according to the complete sequence of the polyprotein. The results showed that the virus isolates obtained from forest pest insects were closely related to each other. Similarly, it was revealed that the iflaviruses obtained from insects that damage agricultural products are more similar to each other. In addition, the results from this study support previous studies on adding a new genus to the Iflaviridae family, which has only one genus.

Molecular characterization and transcriptomic analysis of a novel polymycovirus in the fungus Talaromyces amestolkiae.

Talaromyces amestolkiae is an important fungal species owing to its ubiquity in soils, plants, air, and food. In this study, we identified a novel six-segmented polymycovirus, Talaromyces amestolkiae polymycovirus 1 (TaPmV-1). Each of the double-stranded (ds) RNA segments of TaPmV-1 contained a single open reading frame, and the proteins encoded by dsRNA1, dsRNA2, dsRNA3, and dsRNA 5 shared significant amino acid identities of 56, 40, 47, and 43%, respectively, with the corresponding proteins of Aspergillus fumigatus polymycovirus-1(AfuPmV-1). DsRNA1, dsRNA3, and dsRNA5 of TaPmV-1 encoded an RNA-dependent RNA polymerase (RdRp), a viral methyltransferase, and a PAS-rich protein, respectively. The functions of the proteins encoded by dsRNA2, dsRNA4, and dsRNA6 have not been elucidated. Comparison of the virus-infected strain LSH3 with virus-cured strain LSHVF revealed that infection with TaPmV-l may reduce the production of red pigments and induce the clustering of fungal sclerotia. Furthermore, transcriptomic analyses demonstrated that infection with TaPmV-l downregulated the expression of transcripts related to metabolism, and may correlate with the reduced production of red pigments and clustering of sclerotia in T. amestolkiae. These results of this study provide novel insights into the mechanism of fungal gene regulation by polymycovirus infections at the transcriptome level, and this study is the first to report a novel polymycovirus of T. amestolkiae.

Molecular characterization of a novel endornavirus isolated from Ophiostoma bicolor associated with bark beetles.

Ophiostoma bicolor is a pathogenic fungus associated with bark beetles that can cause serious damage to host plants. In this study, a novel fungal virus, "Ophiostoma bicolor endornavirus 1" (ObEV1), was obtained from O. bicolor, and its complete genome sequence was determined. ObEV1 has a single-stranded positive-sense (+ ss) RNA genome of 10,119 nucleotides. Sequence annotation and comparison showed that the viral genome has a single large open reading frame (ORF) encoding a polyprotein of 362.48 kDa. The polyprotein contains seven conserved domains: RNA-dependent RNA polymerase (RdRp), viral RNA helicase 1 (VHel1), viral methyltransferase (VMet), DEAD-like helicase (DEXDc), gliding-GltJ (G1), large tegument protein UL36 (PHA), and YlqF-related-GTPase (Y). Sequence comparisons and phylogenetic analysis showed that ObEV1 is a novel mycovirus belonging to the genus Betaendornavirus of the family Endornaviridae. This is the first report of a mycovirus in the ophiostomatoid fungus O. bicolor.

Unravelling the characterization of hypoxia‐inducible factor‐1α ( HIF‐1α ) and antioxidant enzymes in clam Cyclina sinensis in response to hypoxia

Aquatic hypoxia has negative or detrimental effects on clams. To cope with hypoxia stress, a complex series of physiological and biochemical strategies can be employed in clams. Hypoxia-inducible factor-1α (HIF-1α) and antioxidant enzymes are commonly found in aquatic animals as an adaptation against hypoxia. In the present study, we cloned the HIF-1α sequence in clam Cyclina sinensis, and it consists of 2654 base pairs (bp) carrying a single open-reading frame that encompasses 2235 bp of the coding region. The predicted C. sinensis HIF-1α protein (744 amino acids) has the conserved HLH-PAS domain, which contained one N-terminal basic helix–loop–helix (bHLH) domain and two Per-ARNT-Sim A/B (PAS) domains, followed by PAS-associated C-terminal (PAC) domain. HIF-1α is expressed predominantly in gills. Exposed to hypoxia, the transcript level of HIF-1α was significantly up-regulated in response to hypoxia, and then, the transcript level declined continuously with the time elapsed. The antioxidant enzymes (SOD, CAT and GSH-PX) in gills were increased initially and then decreased after acute hypoxia exposure. Our study indicates that the HIF-1α gene and antioxidant enzymes may play an important role in cope with acute hypoxia exposure.

A new flavi-like virus identified in populations of wild carrots

We report the discovery of a new flavi-like virus identified in wild carrots (Daucus carota subsp. carota), using a double-stranded (ds)RNA high-throughput sequencing (HTS) approach. The new virus, tentatively named "carrot flavi-like virus 1" (CtFLV-1), has a large genome of 21.8kb that harbours a single open reading frame encoding a 7,078-aa polyprotein with conserved RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. The new virus is phylogenetically related to recently described flavi-like viruses from arthropods, but its closest relative is a plant-associated virus, gentian Kobu-sho-associated virus (GKSaV). A pairwise comparison showed that these two viruses share 38.4% amino acid (aa) sequence identity in their polyproteins and 73% and 47.8% aa sequence identity in their conserved RdRp and Hel domains, respectively. Based on their similar genome organization and phylogenetic relationship, GKSaV and CtFLV-1 could form the basis for a new genus of plant-associated viruses, possibly within the family Flaviviridae, for which the name "Koshovirus" is proposed.

Complete Genomic RNA Sequence of Tuberose Mild Mosaic Virus and Tuberose Mild Mottle Virus Acquired by High-Throughput Sequencing.

Tuberose (Polianthes tuberosa) is an ornamental flowering crop of the Amaryllidaceae family. Tuberose mild mosaic virus (TuMMV) and tuberose mild mottle virus (TuMMoV), members of the genus Potyvirus, are ubiquitously distributed in most tuberose growing countries worldwide with low biological incidence. Here, we report the first coding-complete genomic RNA of TuMMV and TuMMoV obtained through high-throughput sequencing (HTS) and further, the presence of both the viruses were confirmed using virus-specific primers in RT-PCR assays. Excluding the poly (A) tail, the coding-complete genomic RNA of TuMMV and TuMMoV was 9485 and 9462 nucleotides (nts) in length, respectively, and contained a single large open reading frame (ORF). Polyprotein encoded by both the viral genomes contained nine putative cleavage sites. BLASTn analysis of TuMMV and TuMMoV genomes showed 72.40–76.80% and 67.95–77% nucleotide sequence similarities, respectively, with the existing potyviral sequences. Phylogenetic analysis based on genome sequences showed that TuMMV and TuMMoV clustered in a distinct clade to other potyviruses. Further studies are required to understand the mechanism of symptom development, distribution, genetic variability, and their possible threat to tuberose production in India.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Biological activity and genome composition of a Tunisian isolate of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV-Tun2)

BackgroundThe baculovirus Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) is an entomopathogenic virus utilized as a biological control agent of the Egyptian cotton leaf worm, Spodoptera littoralis. Several studies have focused on the identification of different SpliNPV isolates from a biological and molecular point of view, but few of them conducted in-depth analyses of the genomic composition of these isolates.ResultsIdentification of a novel isolate of SpliNPV, termed Tun2, which was purified from infected S. littoralis larvae from Tunisia was reported. This isolate was propagated in vivo and its median lethal concentration (LC50) was determined to be 1.5 × 104 occlusion bodies (OBs)/ml for third instar S. littoralis larvae at 7 days of post-infection. OB production in late fourth instar larvae was estimated to be at least 2.7 × 109 OBs/g larval weight. The completely sequenced genome of SpliNPV-Tun2 was 137,099 bp in length and contained 132 open reading frames (ORF). It showed a 98.2% nucleotide identity to the Egyptian isolate SpliMNPV-AN1956, with some striking differences; between both genomes, insertion and deletion mutations were noticed in 9 baculovirus core genes, and also in the highly conserved polyhedrin gene. The homologs of ORF 106 and ORF 107 of SpliNPV-AN1956 appeared to be fused to a single ORF 106 in SpliNPV-Tun2, similar to the homologous ORF 110 in SpltNPV-G2.ConclusionSpliNPV-Tun2 is proposed as a new variant of SpliNPV and a potential candidate for further evaluation as a biocontrol agent for S. littoralis and probably other Spodoptera species.

Molecular characterization of a novel botourmiavirus with inverted complementary termini from the rice blast fungus Magnaporthe oryzae isolate HF04.

A novel positive-sense single-stranded RNA mycovirus, designated as "Magnaporthe oryzae botourmiavirus 10" (MoBV10), was identified in the rice blast fungus Magnaporthe oryzae isolate HF04. MoBV10 has a single genomic RNA segment consisting of 2,448 nucleotides, which contains a single open reading frame encoding an RNA-dependent RNA polymerase. Genome comparison and phylogenetic analysis indicated that MoBV10 is a new member of the genus Betascleroulivirus in the family Botourmiaviridae. The 5'- and 3'-terminal sequences of the genomic RNA of MoBV10 have inverted complementarity and potentially form a panhandle structure, which is very rare in RNA viruses.

Functional characterization of piggyBac-like elements from Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

PiggyBac is a transposable DNA element originally discovered in the cabbage looper moth (Trichoplusia ni). The T. ni piggyBac transposon can introduce exogenous fragments into a genome, constructing a transgenic organism. Nevertheless, the comprehensive analysis of endogenous piggyBac-like elements (PLEs) is important before using piggyBac, because they may influence the genetic stability of transgenic lines. Herein, we conducted a genome-wide analysis of PLEs in the brown planthopper (BPH) Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), and identified a total of 28 PLE sequences. All N. lugens piggyBac-like elements (NlPLEs) were present as multiple copies in the genome of BPH. Among the identified NlPLEs, NlPLE25 had the highest copy number and it was distributed on five chromosomes. The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals, as well as a single open reading frame transposase encoding 546 amino acids. Furthermore, NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision. A cross-recognition between the NlPLE25 transposon and the piggyBac transposon was also revealed in this study. These findings provide useful information for the construction of transgenic insect lines.