Large Open Reading Frame Research Articles

A new potyvirus isolated from Pennisetum alopecuroides with the potential to infect cereal crops

Pennisetum plants (Pennisetum alopecuroides L.), displaying a dwarfing phenotype along with delayed flowering and mosaic symptom on leaves, were found in Beijing, China. Flexuous filamentous particles with a size of approximate 15 × 850 nm were observed in symptomatic leaves via transmission electron microscopy. Deep sequencing of small RNAs (sRNA) from symptomatic leaves and analysis of sRNA populations were then conducted to determine the genome sequence of the viral agent in diseased plant tissues. It showed that the viral agent had one positive-sense and single-stranded RNA genome, which consisted of 9717 nucleotides (nts) excluding poly(A) tail. The complete viral genome contained a large open reading frame, encoding a polyprotein of 3131 amino acids (aa). Sequence comparison and phylogenetic analysis demonstrated that the viral agent belonged to the genus Potyvirus in the family Potyviridae. In the cladogram it was most closely related to johnsongrass mosaic virus, sharing 72% nt and 65% aa sequence identity. This viral agent was provisionally named pennisetum alopecuroides mosaic virus (PalMV). Subsequently, it was confirmed that PalMV is the causal agent of this new disease in P. alopecuroides by Koch’s postulates and reverse transcription-polymerase chain reaction analysis. Moreover, maize, millet, wheat, sorghum and rice plants were experimentally infected by PalMV via rub inoculation. Consequently, we proposed that PalMV could be a potentially dangerous virus threating a wide range of cereal crops.

A novel mitovirus isolated from the phytopathogenic fungus Botryosphaeria dothidea.

In this study, we isolated and determined the complete genome sequence of a novel mitovirus, "Botryosphaeria dothidea mitovirus 2" (BdMV2), from the phytopathogenic fungus Botryosphaeria dothidea isolate DT-5. BdMV2 has a genome 2,482 nt in length with an A+U content of 67%. The genome of BdMV2 contains a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 717 amino acids (aa) with a molecular mass of 81.86 kDa. A BLASTp comparison of the RdRp sequence showed the highest identity (66.67%) with that of Alternaria arborescens mitovirus 1 (AbMV1). Sequence comparisons and phylogenetic analysis revealed that BdMV2 is a new member of the genus Mitovirus of the family Mitoviridae.

The complete genome sequence of a new mitovirus from the phytopathogenic fungus Colletotrichum higginsianum.

In this study, a novel mitovirus designed "Colletotrichum higginsianum mitovirus 1" (ChMV1) was isolated from the phytopathogenic fungus Colletotrichum higginsianum. The genome of this mitovirus is 2,893 nt in length with an A + U content of 61% and contains a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). A BLASTp analysis revealed that the RdRp domain of ChMV1 had 30.25% to 61.72% sequence identity to those of members of the genus Mitovirus and showed the highest degree of similarity (61.72% identity) to Botrytis cinerea mitovirus 3 (BcMV3). Phylogenetic analysis further indicated that ChMV1 is a member in the genus Mitovirus of the family Mitoviridae. To the best of our knowledge, this is the first report of a mitovirus in C. higginsianum.

A novel previously undescribed fusarivirus from the phytopathogenic fungus Setosphaeria turcica.

A putative mycovirus belonging to the proposed family "Fusariviridae" was discovered in Setosphaeria turcica by sequencing a double-stranded RNA extracted from this phytopathogenic fungus. The virus was tentatively named "Setosphaeria turcica fusarivirus 1" (StFV1). StFV1 has a genome comprising 6685 nucleotides. The genome contains three open reading frames (ORF). The largest ORF, ORF1, is preceded by an untranslated region (UTR) of 16 nucleotides and separated from ORF2 by an intergenic region of 63 nucleotides. The smallest ORF, ORF3, overlaps ORF2 by 16 nucleotides and is followed by a 3'-UTR of 82 nucleotides. The protein encoded by ORF1 is 71.8%, 67.4% and 68.1% identical to the RNA-dependent RNA polymerases (RdRps) of Pleospora typhicola fusarivirus 1 (PtFV1), Plasmopara viticola lesion-associated fusarivirus 1 (PvlaFV1), and Plasmopara viticola lesion-associated fusarivirus 3 (PvlaFV3), respectively, but has less than 47% amino acid sequence identity to the RdRps of other fusariviruses. To our knowledge, this is the first fusarivirus discovered in S. turcica and the first virus to be identified in this fungus using conventional cloning methods.

Complete genome sequence of pleioblastus mosaic virus, a distinct member of the genus Potyvirus.

Pleioblastus mosaic virus (PleMV) is a tentative member of the genus Potyvirus in the family Potyviridae and was discovered in bamboo with mosaic symptoms in Tokyo, Japan. Since no information on the genome sequence of PleMV has been reported, its taxonomic position has long been uncertain. Here, we report the first complete genome sequences of two distinct PleMV isolates. Excluding the 3'-terminal poly(A) tail, their genomic RNA sequences consist of 9,634 and 9,643 nucleotides (nt); both contain a large open reading frame (ORF) encoding a polyprotein and a small ORF termed PIPO. The large ORFs of the two isolates share 79.2% and 87.6% sequence identity at the nucleotide (nt) and amino acid (aa) level, respectively, and were found to have the highest nt and aa sequence identity (69.0% and 69.9%) to the potyvirus johnsongrass mosaic virus (JGMV). Phylogenetic analysis showed that PleMV is most closely related to JGMV but forms its own clade. These results suggest that PleMV is a distinct member of the genus Potyvirus.

Cryphonectria nitschkei chrysovirus 1 with unique molecular features and a very narrow host range

Cryphonectria nitschkei chrysovirus 1 (CnCV1), was described earlier from an ascomycetous fungus, Cryphonectria nitschkei strain OB5/11, collected in Japan; its partial sequence was reported a decade ago. Complete sequencing of the four genomic dsRNA segments revealed molecular features similar to but distinct from previously reported members of the family Chrysoviridae. Unique features include the presence of a mini-cistron preceding the major large open reading frame in each genomic segment. Common features include the presence of CAA repeats in the 5′-untranslated regions and conserved terminal sequences. CnCV1-OB5/11 could be laterally transferred to C. nitschkei and its relatives C. radicalis and C. naterciae via coculturing, virion transfection and protoplast fusion, but not to fungal species other than the three species mentioned above, even within the genus Cryphonectria, suggesting a very narrow host range. Phenotypic comparison of a few sets of CnCV1-infected and -free isogenic strains showed symptomless infection in new hosts.

Molecular characterization of a novel mitovirus from the plant‑pathogenic fungus Botryosphaeria dothidea.

Here, a novel mycovirus, Botryosphaeria dothidea mitovirus 1 (BdMV1), was isolated from a phytopathogenic fungus, Botryosphaeria dothidea, and its molecular characteristics were determined. BdMV1 has a genome of 2,667 nt that contains a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 727 amino acids with a molecular mass of 81.64 kDa. BLASTp analysis revealed that the RdRp domain of BdMV1 has 39.59% and 39.18% sequence identity to Plasmopara viticola associated mitovirus 43 and Setosphaeria turcica mitovirus 1, respectively. Phylogenetic analysis further suggested that BdMV1 is a new member of the genus Mitovirus within the family Mitoviridae. To the best of our knowledge, this is the first report of a mitovirus in B. dothidea.

Co-infection of a novel fusagravirus and a partitivirus in a Korean isolate of Rosellinia necatrix KACC40168.

We report here the presence of dsRNA mycoviruses in a Korean isolate of Rosellinia necatrix. A multiple band pattern of double-stranded RNA (dsRNA) from R. necatrix suggested mixed mycovirus infection. Next-generation sequencing analysis of purified dsRNAs indicated the presence of two dsRNA mycoviruses related to the members of families "Fusagraviridae" (proposed) and Partitiviridae. The first dsRNA virus revealed that the complete genome sequence was 8868bp in size and contained two large open reading frames (ORFs 1 and 2), overlapped by 22bp containing a canonical (- 1) slippery heptanucelotide sequence of UUUAAAC. The deduced amino acid sequence of ORF1 and ORF2 showed highest similarity to the hypothetical protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 3 (RnFGV3). Phylogenetic analysis showed that this dsRNA virus clustered with RnFGV3 and other fusagraviruses. Gene organization, sequence similarity, and phylogenetic analysis indicate that this virus seems to belong to a novel species of "Fusagraviridae", which we have named Rosellinia necatrix fusagravirus 4. The second virus has two dsRNA segments with sizes of 1907bp and 1918bp, each of which encoded a single ORF showing highest similarity to the RdRp and capsid protein of known members of Partitiviridae. Evaluation of genome structure, sequence similarity, and phylogeny indicate this to be a new member of the genus Alphapartitivirus in the family Partitiviridae, hereafter designated as Rosellinia necatrix partitivirus 26. This is the first report of the presence of a fusagravirus in an Asian R. necatrix isolate and of its mixed infection with a partitivirus.

Molecular characterization of the complete genome sequence of human Parechovirus 1 in Pakistan

Human parechoviruses (HPeVs) are highly common pathogens in children under 2 years of age. Of the 19 distinct HPeV genotypes identified worldwide, HPeV1 is still the most prevalent type associated with respiratory and gastrointestinal symptoms in infants and young children. Pakistan's previous studies have focused only on the detection and partial sequencing of HPeV genotypes. In the present study, we have obtained the complete genomes of 2 HPeV1 strains (PAK419 and PAK663) from children using NGS method on Illumina Hiseq Platform. These samples were collected from children suffering from acute gastroenteritis in Rawalpindi, Pakistan during 2016. The near complete genome sequences obtained for two HPeV1 strains (PAK419 and PAK663) consist of total 6877 nucleotides with a single, large open reading frame (ORF) encoding a polyprotein gene. Phylogenetic analysis showed that both HPeV1 strains exhibited maximum amino acid similarity (97 %) to HPeV1 strains from The Nederlands (2007-863, GQ183034) and clustered closely with this and with other HPeV1 strains isolated from other countries in the world (Ethiopia, Taiwan, Russia and Brazil). A motif of arginine-glycine-aspartic acid (RGD) in the VP1 (Outer capsid protein) C-terminus region that is suggested to help virus entry into the host cell also identified in PAK419 and PAK663. SimPlot analysis revealed that intergenotypic recombination events may have take place in the non-structural region between both HPeV1 strains (PAK419, PAK663), two major strains of HPeV1 (GQ183034 and MG873157) and four minor strains of HPeV4 (AM235750), HPeV7 (EU556224), HPeV15 (MN265386) and HPeV18 (KT879915). The full genome of HPeV1 strains characterized in the current study will provide complete information on these newly isolated strains for further preventive or treatment measures.

Genome analysis and phylogenetic characterization of two deformed wing virus strains from Apis cerana in Vietnam.

BackgroundDeformed wing virus (DWV) is a virulent virus that causes honeybee disease. DWV can exist as a latent infection in honeybees, outbreak into epidemics, and cause serious damage to beekeeping cross the world, including Vietnam.MethodsThe two DWV strains circulating in Vietnamese honeybee, Apis cerana, were first isolated from adult honeybees in North Vietnam (DWV-NVN) and South Vietnam (DWV-SVN). Their complete nucleotide sequences were determined, aligned, and compared with other DWV strains.ResultsThe two Vietnamese DWV strains comprised 10,113 bp and contained a large single open reading frame (ORF) of 2,893 amino acids, initiating at nucleotide 1,130 and terminating at nucleotide 9,812. Multiple nucleotide sequence alignment between these two DWV-VN strains and DWV strains in A. mellifera was performed. The DWV-VN strains showed a low genetic identity (from 91.4% to 92.0%) with almost of these strains, but lower identities (89.2% and 89.4%) with UK2 and (89.6%) with the China2 strain. Low identities (91.7% and 91.9%) were also observed between the China3 strain (in A. cerana) and the DWV-VN strains, respectively. The deduced amino acid sequence alignment showed high genetic similarities (97.0%–97.9%) when the USA1, Chile, Italy1, France, UK1, UK2, Japan, Korea2, China1, China2 and China3 strains were compared to the DWV-VN strains. This ratio was 96.7% and 96.8% when the Korea1 strain was compared to the DWV-SVN and DWV-NVN strains, respectively. Numerous amino acid substitutions were identified in the L, VP3, and RdRp sequences. Notably, we observed six substitutions positioned at amino acids 27 (E > I), 98 (S > T), 120 (A > V), 153 (M > T), 170 (D > F), and 174 (Y > F) in the L protein, two amino acid changes at positions 980 (S > A) and 1032 (E > T) in VP3, and one amino acid change at position 2627 (R > C) unique to the DWV-VN strains. Phylogenetic analysis based on complete genome sequences, RdRp sequences and Simplot analysis indicated that there was a significant difference between DWV-VN strains in A. cerana and DWV strains in A. mellifera. The results suggested that the genetic variations of the DWV-VN strains in A. cerana help them to adapt geographical conditions and may lead to change the viral pathogenicity of DWV-VN strains.

Costus stripe mosaic virus, a tentative new member of the genus Potyvirus.

Morphological, biological, serological, and molecular tests underpin the description of costus stripe mosaic virus (CoSMV) as a new member of the genus Potyvirus, family Potyviridae. Found affecting the native ornamental Costus spiralis in Brazil, the pathogen showed a severely restricted natural and experimental host range. Excluding the poly(A) tail, the CoSMV genome contains a large open reading frame (ORF) of 9,446 nucleotides that encodes a polyprotein with 3,046 amino acids, which is potentially cleaved into ten products, and a small ORF (77 amino acids) knows as PIPO. Genome analysis demonstrated the highest CoSMV nucleotide sequence identity to onion yellow dwarf virus (51.79%). No evidence of recombination was detected in the CoSMV genome, and phylogenetic analysis revealed its basal position in a group formed by members of the genus Potyvirus, along with Cyrtanthus elatus virus A (Vallota speciosa virus) and canna yellow streak virus. CoSMV was not transmitted by aphids of the species Aphis solanella, Myzus persicae or Uroleucon sonchi, which could be due to mutations in the HC-Pro motifs required for aphid transmission. A divergence in the P1 protein cleavage site was found when compared to other members of the family Potyviridae. Based on its unique biological and molecular characteristics and the current species demarcation criteria, we propose CoSMV to be a new tentative member of the genus Potyvirus.

A novel mitovirus detected in Diaporthe rudis, a fungus associated with Phomopsis dieback on grapevines.

The genome sequence of a mitovirus found in an isolate of Diaporthe rudis, one of the causal agents of Phomopsis dieback on grapevines, was determined by two high-throughput sequencing approaches, small RNA and total RNA sequencing. The genome of this mitovirus is 2,455 nt in length and includes a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). A BLASTx comparison of the full-length genome sequence showed the highest similarity (54.15%) with that of Colletotrichum falcatum mitovirus 1 (CfMV1). Our results reveal a new member of the genus Mitovirus first detected in D. rudis (Fr.) Nitschke, with the proposed name "Diaporthe rudis mitovirus 1" (DrMV1).

Complete genome sequence of a novel potyvirus isolated from Polygonatum kingianum.

The complete genome sequence of a putative novel potyvirus, tentatively named "Polygonatum kingianum virus 1" (PKgV1), infecting Polygonatum kingianum in China was determined (GenBank accession no. MK427056). PKgV1 has a genome organization that is typical of potyviruses, with a single large open reading frame (nt 123-9236) that encodes a 3037-aa polyprotein that is predicted to be cleaved into 10 mature proteins by virus-encoded proteases. Nine cleavage sites and several conserved motifs were identified in PKgV1 by comparative sequence analysis. Pairwise comparisons revealed that the PKgV1 polyprotein shares 52.0-56.2% nucleotide and 49.2-52.8% amino acid sequence identity with members of the genus Potyvirus. Phylogenetic analysis indicated that PKgV1 clustered with members of the genus Potyvirus and that it is closely related to but distinct from lettuce mosaic virus (LMV, accession no. KJ161186). These results suggest that Polygonatum kingianum virus 1 (PKgV1) is a new member of the genus Potyvirus of the family Potyviridae.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

Integrative functional genomics decodes herpes simplex virus 1

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.

Genomic characterization of cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm.

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30nm in diameter and averaging 120nm in length. The viral genome is 9227bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus).

The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.

A Single ssRNA Segment Encoding RdRp Is Sufficient for Replication, Infection, and Transmission of Ourmia-Like Virus in Fungi.

Recently, an increasing number of ourmia-like viruses have been found in fungi; however, the features of these viruses remain unknown. Here, we report a novel ourmia-like virus isolated from Sclerotinia sclerotiorum. This virus, named S. sclerotiorum ourmia-like virus 4 (SsOLV4), has a genome 2,982 nt in length with a G-pentamer (GGGGG) at the 5′-terminus and a C-pentamer (CCCCC) at the 3′-terminus. The SsOLV4 genome has only one large putative open reading frame (ORF) predicted with both standard codes and mitochondrial codes and encodes an RNA-dependent RNA polymerase (RdRp). SsOLV4 is closely phylogenetically related to Pyricularia oryzae ourmia-like virus 1, with 42% identity between the RdRp amino acid sequences. We constructed full-length cDNA of SsOLV4 and synthesized RNA in vitro using the T7 RNA polymerase. The synthesized RNA could transfect S. sclerotiorum protoplasts efficiently. We further found that viral RNA could infect mycelia when mixed with PEG buffer. Our study suggests that a novel genus in family Botourmiaviridae should be established for SsOLV4 and other related viruses and demonstrates that one single-stranded RNA segment encoding RdRp is sufficient for ourmia-like viruses in fungi.

Whole-genome sequence analysis of paris virus 1: a novel member of the genus Potyvirus infecting Paris polyphylla var. yunnanensis.

The complete genome sequence of a novel potyvirus, tentatively named "paris virus 1" (ParV1, GenBank accession no. MN549985), infecting Paris polyphylla var. yunnanensis was determined in this study. A single large open reading frame (nt 96-9818) encoding a 3240-aa polyprotein that is predicted to be cleaved into 10 mature proteins was detected in the ParV1 genome. Comparative analysis of the ParV1 genome sequence with those of other potyviruses identified nine cleavage sites and conserved motifs that are typical features of potyviruses. Pairwise sequence comparisons showed that the ParV1 polyprotein shares 49.6-65.1% nucleotide and 47.1-68.9% amino acid sequence identity with viruses of the genus Potyvirus. BLAST analysis revealed that ParV1 shares 65.1% nucleotide and 68.9% amino acid sequence identity with Thunberg fritillary mosaic virus (TFMV, accession no. CAI59123), its closest known relative. These results suggest that paris virus 1 (ParV1) is a new member of the genus Potyvirus.

M6A-dependent biogenesis of circular RNAs in male germ cells.

The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m6A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m6A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m6A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m6A-modified start codons in junction sequences.