Large Open Reading Frame Research Articles

Whole genomic characterization of a Korean human parechovirus type 1 (HPeV1) identifies recombination events

A human parechovirus (HPeV) strain CAU10-NN was detected from a stool sample of a 2-year-old healthy female infant in South Korea using a metagenomic approach. The CAU10-NN virus was isolated using a cell culture system and its whole genome was analyzed. The RNA genome of the CAU10-NN strain consists of 7,348 nucleotides (nt), excluding a poly(A) tail. A large open reading frame of 6,540 nt that encodes a putative polyprotein precursor of 2,180 amino acids is flanked by a 5'-untranslated region (UTR) of 708 nt and 3'-UTR of 88 nt followed by a poly(A) tail. Phylogenetic analysis shows that the CAU10-NN strain belongs to HPeV1. SimPlot and Bootscan analyses reveal that the virus genome is composed of regions related to corresponding genomic regions of other HPeVs. Recombination analysis indicates that the CAU10-NN strain might be a product of more than one genomic recombination event that occurred among HPeV1, HPeV3, and HPeV4 strains.

Identification and genetic characterization of a novel picornavirus from chickens

A novel picornavirus from commercial broiler chickens (Gallus gallus domesticus) has been identified and genetically characterized. The viral genome consists of a single-stranded, positive-sense RNA genome of >9243 nt excluding the poly(A) tail and as such represents one of the largest picornavirus genomes reported to date. The virus genome is GC-rich with a G+C content of 54.5 %. The genomic organization is similar to other picornaviruses: 5' UTR-L-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3' UTR. The partially characterized 5' UTR of >373 nt appears to possess a type II internal ribosomal entry site (IRES), which is also found in members of the genera Aphthovirus and Cardiovirus. This IRES exhibits significant sequence similarity to turkey 'gallivirus A'. The 3' UTR of 278 nt contains the conserved 48 nt 'barbell-like' structure identified in 'passerivirus', 'gallivirus', Avihepatovirus and some Kobuvirus genus members. A predicted large open reading frame (ORF) of 8592 nt encodes a potential polyprotein precursor of 2864 amino acids. In addition, the virus contains a predicted large L protein of 462 amino acids. Pairwise sequence comparisons, along with phylogenetic analysis revealed the highest percentage identity to 'Passerivirus A' (formerly called turdivirus 1), forming a monophyletic group across the P1, P2 and P3 regions, with <40, <40 and <50 % amino acid identity respectively. Reduced identity was observed against 'gallivirus A' and members of the Kobuvirus genus. Quantitative PCR analysis estimated a range of 4×10(5) to 5×10(8) viral genome copies g(-1) in 22 (73 %) of 30 PCR-positive faeces. Based on sequence and phylogenetic analysis, we propose that this virus is the first member of a potential novel genus within the family Picornaviridae. Further studies are required to investigate the pathogenic potential of this virus within the avian host.

Complete genome sequence of a novel hypovirus infecting Phomopsis longicolla

The complete nucleotide sequence and genome organization of a hypovirus from the isolate ME711 of Phomopsis longicolla was determined and compared to sequences of members of the family Hypoviridae. The genome of the hypovirus, tentatively named Phomopsis longicolla hypovirus 1 (PlHV1-ME711), was determined to be 9760 nucleotides long, excluding the 3' poly (A) tail. The genome contains a single large open reading frame (ORF) encoding a polyprotein designated as P307. Its genomic organization is typical of members of the proposed genus Betahypovirus (Yaegashi et al. in Virus Res 165:143-50, 2012).

Cloning and characterization of the N-methyl-D-aspartate receptor subunit NR1 gene from chum salmon, Oncorhynchus keta (Walbaum, 1792)

Here, we report the information about molecular and expression characterization of NR1 gene in chum salmon for the first time. The complete NR1 subunit showed a large open-reading frame of 2844 bp in the total length of 3193 bp, and this cDNA contained a coding region encoding 948 amino acids and a stop codon. The organization of the NR1 subunit of chum salmon were similar of most other fishes, except C’ terminal. The expression of NR1 subunit was to show higher in the natal river near to the hatchery than near to the coast. We expect that the information reported herein may facilitate further investigations on the relationship between memory factors of natal rivers and homing mechanisms in Salmonidae.

Characterization of a novel dsRNA endornavirus in the plant pathogenic fungus Thielaviopsis basicola

Double-stranded (ds) RNA elements are commonly present in strains of the plant pathogenic fungus Thielaviopsis basicola which infects a wide range of plant species. To characterize a novel 12 kb dsRNA in strain NC1527 of this fungus, reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain an 11,602 bp cDNA sequence from overlapping cDNA clones. Northern blot analysis confirmed that 15 individual cDNA clones that covered the entire length of the cDNA sequence all hybridized to the 12 kb dsRNA. An open reading frame (ORF) search revealed that the 5′non-coding region of this sequence spans 27 nucleotides, followed by one large ORF of 11,575 bp nucleotides, which potentially encodes a large putative polyprotein of 3858 amino acid residues. Specialized Basic Local Alignment Search Tool (BLAST) searches of conserved domains indicated that the putative polyprotein contained a viral RNA helicase1 (Hel), glycosyl transferase (GT) and RNA-dependent RNA polymerase (RdRp) domain regions. BLASTp searches in the protein database using translated nucleotides showed that the cloned dsRNA had homology to endornaviruses, which are present in a few fungi as well as some plant species. The amino acid homologies ranged from 28% to 34% in the RdRp domain region, 23–32% in the helicase domain region and 29–30% in GT region. We designate this dsRNA element as a new endornavirus, Chalara elegans endornavirus 1 (CeEV1). Phylogenetic comparison of the sequence of RdRP with other endornavirus indicated that CeEV1 was relatively distant and may be an ancestral form.

Triple Trans-Splicing Adeno-Associated Virus Vectors Capable of Transferring the Coding Sequence for Full-Length Dystrophin Protein into Dystrophic Mice

Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11.1 kb). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7 kb). Here we developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying "in tandem" sequential exonic parts of the human DMD coding sequence enable the expression of the full-length protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences. This method of triple-AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11 kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.

A phylogeographical study of the cauliflower mosaic virus population in mid-Eurasia Iran using complete genome analysis

The full-length sequences of 34 Iranian cauliflower mosaic virus (CaMV) isolates were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic variability and patterns of genetic exchange in CaMV isolates from Iran. Based on the severity of symptoms and their ability to infect Brassica oleracea var. capitata, Iranian CaMV isolates were grouped into two distinct biotypes: latent/mild mottle (LI/MMo) and severe (S) infection. Recombination breakpoints were detected between the large intergenic region (LIR) and open reading frame (ORF) V (event 2); between ORF VII and ORF II (event 3), between ORF I and ORF III (event 4), and within ORF VI (event 1). Phylogenetic analysis indicated that Iranian CaMV isolates clustered into two subgroups belonging to group I (GI) that were distinct from North American and European isolates from group II (GII). Northeast Iranian isolates (subgroup B) and CaMV isolates from subgroup A closely corresponded to the S and LI/MMo biological groups, respectively. Genome-wide pairwise identity analysis of the CaMV isolates revealed three regions of pairwise identity representation: 92-94 % for GII and 94-96 % and 98-100 % for subgroups A and B. The within-population diversity was lower than the between-population diversity, suggesting the contribution of a founder effect on diversification of CaMV isolates. Amino acid sequences were conserved, with ω values ranging from 0.074 to 0.717 in different proteins. Thirteen amino acids in the deduced proteins of ORFs I, II, III, VI and VII were under positive selection (ω > 1), whereas purifying selection applied to the proteins encoded by ORFs IV and V. This study suggests that variation in the CaMV population can be explained by host-range differentiation and selection pressure. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of CaMV strains, providing valuable new information for understanding the diversity and evolution of caulimoviruses.

Tall oatgrass mosaic virus (TOgMV): a novel member of the genus Tritimovirus infecting Arrhenatherum elatius

A novel tritimovirus of the family Potyviridae was isolated from tall oatgrass, Arrhenatherum elatius, exhibiting mosaic symptoms. The virus, for which the name tall oatgrass mosaic virus (TOgMV) is coined, has a filamentous particle of 720 nm and is associated with pinwheel inclusion bodies characteristic of members of the family Potyviridae. The virus was mechanically transmitted to tall oatgrass seedlings, which subsequently exhibited mosaic symptoms. The experimental host range was limited to a few monocot species. The complete genome sequence of TOgMV was determined to be 9359 nucleotides, excluding the 3' polyadenylated tail. The viral RNA encodes one large putative open reading frame of 3029 amino acids with a genome organization typical of monopartite potyvirids. Pairwise comparison of putative mature proteins and proteinase cleavage sites indicated that TOgMV is most closely related to members of the genus Tritimovirus. Phylogenetic analysis of the complete polyprotein and CP sequences of representative members of the family Potyviridae indicate that TOgMV is a distinct tritimovirus naturally infecting tall oatgrass.

Complete Genome Sequences of Two Bovine Viral Diarrhea Viruses Isolated from Brain Tissues of Nonambulatory (Downer) Cattle

Here, we report the complete genome sequences of two bovine viral diarrhea viruses (BVDVs) (strains 11F011 and 12F004) isolated from brain tissues from nonambulatory (downer) cattle. The complete genomes of strains 11F011 and 12F004 contain 12,287 nucleotides (nt) with a single large open reading frame and 12,301 nt with a single large open reading frame, respectively. Phylogenetic analysis indicated that these strains belong to the BVDV-2a and -1b genotypes, respectively.

Genome Sequence of Bovine Viral Diarrhea Virus Strain 10JJ-SKR, Belonging to Genotype 1d

Here, we report the complete genome sequence of a bovine viral diarrhea virus (BVDV) belonging to genotype 1d, strain 10JJ-SKR, which was isolated from cattle. The complete genome is 12,267 nucleotides (nt) in length, with a single large open reading frame. This is the first report of a BVDV belonging to genotype 1d and will enable further study of the molecular and epidemiological characteristics of this virus.

The genome sequence and transmission of an iflavirus from the brown planthopper, Nilaparvata lugens

A previously unknown iflavirus has been identified in a laboratory colony of the brown planthopper, Nilaparvata lugens. The iflavirus-like sequence was first identified in contig sequences obtained from transcriptome sequencing (RNA-seq) of the brown planthopper. The complete viral genome was resequenced using the Sanger method. The positive-strand RNA genome was 10,937 nucleotides excluding the 3′ poly(A) tail, and contained a single large open reading frame encoding coat proteins in the 5′ region and replicases in the 3′ region. Conserved motifs for coat proteins, helicase, cysteine protease, and RNA-dependent RNA polymerase were identified in the deduced amino sequence, and the estimated molecular mass of the large polyprotein was 358.6kDa. RT-PCR detection of the viral genome indicated that viral shedding occurred through the honeydews of insects in the infected colony. To test transmission, the collected honeydews were used to feed insects in a non-viruliferous colony. After 7 days, the expected RT-PCR fragment was detected in the insects, indicating that the virus can be transmitted horizontally. This is the first iflavirus identified in planthoppers; thus, we propose the name of the virus as Nilaparvata lugens honeydew virus-1.

An Improved Method for oriT -Directed Cloning and Functionalization of Large Bacterial Genomic Regions

We have made significant improvements to a broad-host-range system for the cloning and manipulation of large bacterial genomic regions based on site-specific recombination between directly repeated oriT sites during conjugation. Using two suicide capture vectors carrying flanking homology regions, oriT sites are recombined on either side of the target region. Using a broad-host-range conjugation helper plasmid, the region between the oriT sites is conjugated into an Escherichia coli recipient strain, where it is circularized and maintained as a chimeric mini-F vector. The cloned target region is functionalized in multiple ways to accommodate downstream manipulation. The target region is flanked with Gateway attB sites for recombination into other vectors and by rare 18-bp I-SceI restriction sites for subcloning. The Tn7-functionalized target can also be inserted at a naturally occurring chromosomal attTn7 site(s) or maintained as a broad-host-range plasmid for complementation or heterologous expression studies. We have used the oriTn7 capture technique to clone and complement Burkholderia pseudomallei genomic regions up to 140 kb in size and have created isogenic Burkholderia strains with various combinations of genomic islands. We believe this system will greatly aid the cloning and genetic analysis of genomic islands, biosynthetic gene clusters, and large open reading frames.

New Volvovirus Isolates from Acheta domesticus (Japan) and Gryllus assimilis (United States)

A novel circular single-stranded DNA (ssDNA) virus, volvovirus, from the house cricket has been described recently. Here, we report the isolation of volvoviruses from Acheta domesticus in Japan and Gryllus assimilis in the United States. These Acheta domesticus volvovirus (AdVVV) isolates have genomes of 2,517 and 2,516 nucleotides (nt) and 4 large open reading frames (ORFs).

Complete genome sequence of the original Taiwanese isolate of sweet potato latent virus and its relationship to other potyviruses infecting sweet potato

The complete genome of sweet potato latent virus (SPLV) was determined to be 10081 nucleotides long excluding the 3' poly (A) tail. The genome contains a single large open reading frame encoding a polyprotein of 3247 amino acids. Its genomic organization is typical of potyviruses and contains motifs conserved in members of the genus Potyvirus. Pairwise comparisons show that SPLV shares identities of 50.0%-56.3% to other potyviruses at the genomic sequence level. Phylogenetic analysis shows that SPLV is closely related to four other sweet potato potyviruses in the sweet potato feathery mottle virus lineage, but it lacks the unique PISPO in the P1 region of those viruses. The genome analyses confirm that SPLV is a distinct sweet potato virus in the genus Potyvirus.

IRF-1 acts as a positive regulator in the transcription of grass carp (Ctenopharyngodon idella) IFN gene

Interferon regulatory factors (IRFs) are well-known to be crucial for modulating the innate immune responses to viral infections. In the present study, the IRF-1 gene of grass carp (Ctenopharyngodon idella) (termed CiIRF-1) was cloned and characterized. The complete genomic sequence of CiIRF-1 was 3150 bp in length and comprised 9 exons and 8 introns. The CiIRF-1 promoter sequence was 558 bp in length. The largest open reading frame (ORF) of the full CiIRF-1 cDNA sequence was 870 bp, and encoded a polypeptide of 289 amino acids. The putative CiIRF-1 was characterized by a conserved N-terminal DBD (113 aa), and included a signature of five conserved tryptophan residues. Phylogenetic relationship analysis revealed that CiIRF-1 was highly homologous to the counterparts of other teleosts and mammalians. CiIRF-1 was expressed at a low constitutive level but was significantly up-regulated following stimulation with either Poly I:C or recombinant grass carp (C. idella) IFN (rCiIFN) in all 6 tested tissues, especially in spleen and gill. The recombinant CiIRF-1 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. Three different fragments of promoter sequences from grass carp type I IFN (CiIFN) gene (GU139255) were amplified. These fragments included the proximal region (CiIFNP2), the distal region (CiIFNP6), and the full length of CiIFN promoter sequences (CiIFNP7). Gel mobility shift assays were employed to analyze the interaction between CiIRF-1 and CiIFN promoter sequences. The results revealed that CiIRF-1 could bind to CiIFN promoter with high affinity in vitro. Subsequently, the recombinant plasmid of pGL3-CiIFNPs and pcDNA3.1-CiIRF-1 were constructed and transiently co-transfected into C. idella kidney (CIK) cells. The impact of CiIRF-1 on CiIFN promoter sequences were measured by luciferase assays. These results demonstrated that CiIRF-1 acts as a positive regulator in the transcription of grass carp IFN gene.

Novel ssDNA viruses discovered in yellow-crowned parakeet (Cyanoramphus auriceps) nesting material

During routine monitoring of yellow-crowned parakeets in the Poulter Valley of the South Island of New Zealand, a dead parakeet chick was discovered in a nest. Known parrot-infecting viruses, such as beak and feather disease virus (BFDV), avian polyomavirus (APV), and parrot hepatitis B virus (PHBV), were not detected in the nesting material. However, we recovered two novel single-stranded DNA viruses (ssDNA), CynNCXV (2308nt) and CynNCKV (2087nt), which have genome architectures similar to those of circoviruses, characterised by circular genomes with two large bidirectional open reading frames (ORFs). Both contain a stem-loop element with a conserved nonanucleotide motif, known to be required for rolling-circle replication. The full genomes had no BLASTn similarity to known ssDNA viruses. However, in both genomes the larger ORFs have BLAST similarity to known replication-associated proteins (Reps). CynNCKV has 30% similarity to picobiliphyte nano-like virus (Picobiliphyte M5584-5) with 66-88% coverage (e-value of 5×10(-33)), whereas CynNCXV has 33% similarity to rodent stool-associated virus (RodSCV M-45) with 92-94% coverage (e-value of 5×10(-31)). Found within these ORFs were the rolling-circle replication motifs I, II, III and the helicase motifs Walker A and Walker B. Maximum-likelihood phylogenetic analysis of the Reps reveals that these are two novel ssDNA viruses. At this point, we are unable to attribute the death of the parakeet to these two new novel ssDNA viruses.

Complete Genome Sequence of Sacbrood Virus Strain SBM2, Isolated from the Honeybee Apis cerana in Vietnam

Here we report the complete genomic sequence of the SBM2 strain (VSBV-SBM2) of the sacbrood virus (SBV) that was isolated from the Asian honeybee (Apis cerana) in Northern Vietnam. The entire sequence excluding the 3′ poly(A) tail is 8,834 nucleotides in length and contains a single large open reading frame (ORF) of 8,580 nucleotides (position 178 to 8757), encoding 2,859 amino acids. VSBV-SBM2 shared 90 to 93% nucleotide identity and 95 to 96% amino acid homology to six complete genomes of SBV currently available in GenBank (two from China, three from Korea, and one from the United Kingdom). A hypervariable domain (amino acid [aa] position 712 to 729) and a conserved motif (position 2124 to 2143) in the precursor polypeptide of all seven SBVs are also described.

Identifying Novel Protein Complexes in Cancer Cells Using Epitope-Tagging of Endogenous Human Genes and Affinity-Purification Mass Spectrometry

Affinity-purification mass spectrometry (AP-MS) is the preeminent technique for identification of eukaryotic protein complexes in vivo. AP-MS workflows typically express epitope-tagged bait proteins, immunopurify, and then identify associated protein complexes using mass spectrometry. However, challenges of existing strategies include the construction of expression vectors for large open reading frames and the possibility that overexpression of bait proteins may result in expression of nonphysiological levels of the bait protein with concomitant perturbation of endogenous protein complexes. To address these issues, we use human cell lines with epitope-tagged endogenous genes as AP-MS substrates to develop a platform that we call "knock-in AP-MS", thereby avoiding the challenges of expression vector construction and ensuring that expression of tagged proteins is driven by endogenous regulatory mechanisms. Using three different bait genes (MRE11A, DNMT1 and APC), we show that cell lines expressing epitope-tagged endogenous genes make good substrates for sensitive and reproducible identification of protein interactions using AP-MS. In particular, we identify novel interactors of the important oncoprotein Adenomatous Polyposis Coli (APC), including an interaction with Flightless-1 homologue (FLII) that is enriched in nuclear fractions.

Identification and genetic characterization of new bovine viral diarrhea virus genotype 2 strains in pigs isolated in China

Classical swine fever (CSF)-like symptoms in pigs regarded as free from CSF has been reported previously. From sick pigs with CSF-like symptoms, and who had been inoculated with the hog cholera vaccine, samples were collected and subjected to RT-PCR using specific primers. Twelve bovine viral diarrhea virus 2 (BVDV-2) strains were screened and isolated. Homology comparison showed that the E2 genes of the twelve isolates were highly conserved. The genome of the one of the BVDV-2 isolates (named as SH-28) from the sick pigs, which showed a noncytopathic effect in MDBK cell cultures and strong reactivity with monoclonal antibody (MAb) Bz-53 raised against BVDV-2, was sequenced. The genome of SH-28 comprises 12,279 nucleotides and contains a large open reading frame beginning at nucleotide 386 and ending at nucleotide 12,073. Genomic comparison and phylogenetic analyzes showed that SH-28 fall into BVDV-2 subtype and was most similar to XJ-04 (nucleotide and amino acid homologies were 89.9-93.8% and 91.1-96.9%, respectively), but was genetically divergent from ZM-95 (pig BVDV-1).

Discovery of a novel circular single-stranded DNA virus from porcine faeces

A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (<30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.