Single Open Reading Frame Research Articles

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria tenuissima.

In this study, a novel positive-sense single-stranded RNA (+ ssRNA) mycovirus, Alternaria tenuissima mitovirus 1 (AtMV1), was identified in Alternaria tenuissima strain YQ-2-1, a phytopathogenic fungus causing leaf blight on muskmelon. The genome of AtMV1 is a single RNA molecule that is 3013 nt in length with an A + U content of 66.58% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a 313-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular mass of 35.48 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5' and 3' untranslated regions were predicted to fold into stem-loop and panhandle secondary structures. The results of a BLASTp search revealed that the amino acid (aa) sequence of RdRp of AtMV1 shared the highest sequence similarity (51.04% identity) with that of Sichuan mito-like virus 30, a member of the genus Duamitovirus within the family Mitoviridae. Phylogenetic analysis based on the aa sequence of the RdRp suggested that AtMV1 is a novel member of the genus Duamitovirus. To our knowledge, this is the first report of the complete genome sequence of a new mitovirus infecting A. tenuissima.

Improved influenza vaccine responses after expression of multiple viral glycoproteins from a single mRNA.

Influenza viruses cause substantial morbidity and mortality every year despite seasonal vaccination. mRNA-based vaccines have the potential to elicit more protective immune responses, but for maximal breadth and durability, it is desirable to deliver both the viral hemagglutinin and neuraminidase glycoproteins. Delivering multiple antigens individually, however, complicates manufacturingand increases cost, thus it would be beneficial to express both proteins from a single mRNA. Here, we develop an mRNA genetic configuration that allows the simultaneous expression of unmodified, full-length NA and HA proteins from a single open reading frame. We apply this approach to glycoproteins from contemporary influenza A and B viruses and, after vaccination, observe high levels of functional antibodies and protection from disease in female mouse and male ferret challenge models. This approach may further efforts to utilize mRNA technology to improve seasonal vaccine efficacy by efficiently delivering multiple viral antigens simultaneously and in their native state.

Identification and Characterization of Four Novel Viruses in Balclutha incisa.

Balclutha incisa (Cicadellidae: Deltocephalinae), a leafhopper prevalent in tropical and temperate regions, is notably abundant in grasses and rice. The virome of B. incisa was investigated using deep transcriptome sequencing, leading to the first identification of four viruses belonging to the families Aliusviridae, Iflaviridae, and Totiviridae in B. incisa. These viruses have been provisionally named B. incisa ollusvirus 1 (BiOV1), B. incisa ollusvirus 2 (BiOV2), B. incisa iflavirus 1 (BiIV1), and B. incisa totivirus 1 (BiTV1). The complete genome sequences of these viruses were obtained through rapid amplification of cDNA ends (RACE). BiOV1 has a linear genome of 15,125 nucleotides (nt), while BiOV2 possesses a circular genome of 14,853 nt. The BiIV1 genome, excluding the poly(A) tail, is 10,903 nt in length and encodes a single open reading frame (ORF) for a polyprotein consisting of 3194 amino acids (aa). The BiTV1 genome is 4357 nt long and contains two overlapping ORFs, with the viral RNA-dependent RNA polymerase (RdRp) translated via a -1 ribosomal frameshift. Phylogenetic and sequence identity analyses suggest that all these viruses are novel members of their respective families. This study significantly expands our understanding of the virome associated with B. incisa by reporting and characterizing these novel viruses.

Complete genome sequence of lima bean endornavirus 1: a putative new member of the genus Alphaendornavirus (family Endornaviridae).

In this study, we completely sequenced the genome of a new member of the genus Alphaendornavirus, family Endornaviridae, from lima bean (Phaseolus lunatus), for which we propose the name "lima bean endornavirus 1" (LbEV1). The complete genome of LbEV1 consists of 15,265 nucleotides, including a stretch of 12 cytosine residues at its 3' end, and contains a long single open reading frame (ORF) coding for a 4980-aa-long polyprotein. Analysis of the polyprotein sequence revealed the presence of four conserved functional domains (in order from the N- to C-terminus): viral helicase 1, peptidase _C97, glycosyltransferase_GTB-type, and viral RNA-dependent RNA polymerase (RdRP). The LbEV1 polyprotein showed the highest amino acid sequence similarity (63% identity and 98% coverage) to Phaseolus vulgaris endornavirus 3 (PvEV3) and also showed 42% identity (95% coverage) to Geranium carolinianum endornavirus. Phylogenetic analysis based on the viral RdRp domain showed that LbEV1 belongs to a subclade within the genus Alphaendornavirus that includes three other viruses infecting plants of the genus Phaseolus.

A novel ormycovirus isolated from the plant-pathogenic fungus Fusarium graminearum.

In this study, we identified a novel mycovirus, Fusarium graminearum ormycovirus 1 (FgOV1), from the pathogenic fungus Fusarium graminearum. The virus has two RNA segments, RNA1 and RNA2, with lengths of 2,591 and 1,801 nucleotides, respectively, excluding the polyA tail. Each segment contains a single open reading frame (ORF). The ORF in RNA1 encodes an RNA-dependent RNA polymerase, while the ORF in RNA2 encodes a hypothetical protein. Phylogenetic analysis showed that FgOV1 belongs to the gammaormycovirus clade, whose members are related to betaormycoviruses. To our knowledge, this is the first report of an ormycovirus in Fusarium graminearum.

Construction of Recombinant Marek's Disease Virus Co-Expressing VP1 and VP2 of Chicken Infectious Anemia Virus.

The chicken infectious anemia virus (CIAV) has been reported in major poultry-producing countries and poses a significant threat to the poultry industry worldwide. In this study, two Marek's disease virus (MDV) recombinants, rMDV-CIAV-1 and rMDV-CIAV-2, were generated by inserting the CIAV VP1 and VP2 genes into the MDV vaccine strain 814 at the US2 site using the fosmid-based rescue system. For rMDV-CIAV-1, an internal ribosome entry site was inserted between VP1 and VP2, so that both proteins were produced from a single open reading frame. In rMDV-CIAV-2, VP1 and VP2 were cloned into different open reading frames and inserted into the MDV genome. The recombinant viruses simultaneously expressed VP1 and VP2 in infected chicken embryo fibroblasts and exhibited growth kinetics similar to those of the parent MDV. The two recombinant viruses induced antibodies against CIAV in chickens. A single dose of the recombinant viruses provided strong protection against CIAV-induced anemia in chickens. These recombinant VP1- and VP2-expressing MDVs are potential vaccines against CIAV in chickens.

Detection and molecular characterization of a novel mitovirus associated with Passiflora edulis Sims.

Mitoviruses are cryptic capsidless viruses belonging to the family Mitoviridae that replicate and are maintained in the mitochondria of fungi. Complete mitovirus-like sequences were recently assembled from plant transcriptome data and plant leaf tissue samples. Passion fruit (Passiflora spp.) is an economically important crop for numerous tropical and subtropical countries worldwide, and many virus-induced diseases impact its production. From a large-scale genomic study targeting viruses infecting Passiflora spp. in Brazil, we detected a de novo-assembled contig with similarity to other plant-associated mitoviruses. The contig is ∼2.6kb long, with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRP). This contig has been named "passion fruit mitovirus-like 1" (PfMv1). An alignment of the predicted amino acid sequence of the RdRP of PfMv1 and those of other plant-associated mitoviruses revealed the presence of the six conserved motifs of mitovirus RdRPs. PfMv1 has 79% coverage and 50.14% identity to Humulus lupulus mitovirus 1. Phylogenetic analysis showed that PfMV1 clustered with other plant-associated mitoviruses in the genus Duamitovirus. Using RT-PCR, we detected a PfMv1-derived fragment, but no corresponding DNA was identified, thus excluding the possibility that this is an endogenized viral-like sequence. This is the first evidence of a replicating mitovirus associated with Passiflora edulis, and it should be classified as a member of a new species, for which we propose the name "Duamitovirus passiflorae".

Molecular characterization of a novel gammapartitivirus infecting the fungus Nigrospora oryzae.

Here, we identified a new mycovirus infecting the phytopathogenic fungus Nigrospora oryzae, which we have designated "Nigrospora oryzae partitivirus 2" (NoPV2). The genome of NoPV2 consists of two dsRNA segments (dsRNA 1 and dsRNA 2), measuring 1771 and 1440 bp in length, respectively. dsRNA 1 and dsRNA 2 each contain a single open reading frame (ORF) that encodes the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP), respectively. A BLASTp search showed that the RdRp of NoPV2 had significant sequence similarity to the RdRps of other partitiviruses, including Nigrospora sphaerica partitivirus 1 (75.61% identity) and Magnaporthe oryzae partitivirus 1 (67.53% identity). Phylogenetic analysis revealed that NoPV2 is a new member of the genus Gammapartitivirus in the family Partitiviridae. This study provides important information for understanding the diversity of mycoviruses in N. oryzae.

Screening and Characterization of a New Iflavirus Virus in the Fruit Tree Pest Pyrops candelaria.

Pyrops candelaria is one of the common pests of fruit trees, but the research on the pathogenic microorganisms it may carry is very limited. Therefore, it is essential to reveal the pathogenic microbes it carries and their potential hazards. This study found a new virus from the transcriptome of P. candelaria, which was first reported in P. candelaria and named PyCaV (Pyrops candelaria associated virus). RACE and bioinformatics assay revealed that the full length of PyCaV is 10,855 bp with the polyA tail, containing a single open-reading frame (ORF) encoding a polyprotein consisting of 3171 amino acid (aa). The virus has a typical iflavirus structure, including two rhv domains, an RNA helicase domain (HEL), a 3C cysteine protease domain (Pro), and an RNA-dependent RNA polymerase domain (RdRp). Further phylogenetic analysis revealed that this virus belongs to family Iflaviridae and sequence alignments analysis suggested PyCaV is a new member in an unassigned genus of family Iflaviridae. Further in-depth analysis of the virus infection showed that PyCaV is distributed throughout the whole P. candelaria, including its head, chest, and abdomen, but more PyCaV was identified in the chest. The distribution of PyCaV in different parts of P. candelaria was further explored, which showed that more PyCaV was detected in its piercing-sucking mouthparts and chest viscera. Statistical analysis showed that the PyCaV infection was affected by time and location.

Molecular characterization of a novel mitovirus from the plant-pathogenic fungus Nigrospora oryzae.

Here, we characterized a novel mitovirus from the fungus Nigrospora oryzae, which was named "Nigrospora oryzae mitovirus 3" (NoMV3). The NoMV3 genome is 2,492 nt in length with a G + C content of 33%, containing a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 775 amino acids with a molecular mass of 88.75 kDa. BLASTp analysis revealed that the RdRp of NoMV3 had 68.6%, 50.6%, and 48.6% sequence identity to those of Nigrospora oryzae mitovirus 2, Suillus luteus mitovirus 6, and Fusarium proliferatum mitovirus 3, respectively, which belong to the genus Unuamitovirus within the family Mitoviridae. Phylogenetic analysis based on amino acid sequences supported the classification of NoMV3 as a member of a new species in the genus Unuamitovirus within the family Mitoviridae.

Complete genome sequence of a novel mitovirus isolated from the phytopathogenic fungus Alternaria alternata causing apple leaf blotch.

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata.

Complete genome analysis of a novel hypovirus in the phytopathogenic fungus Monilinia fructicola.

Monilinia fructicola is one of the most devastating fungal diseases of rosaceous fruit crops, both in the field and postharvest, causing significant yield losses. Here, we report the discovery of a novel positive single-stranded RNA virus, Monilinia fructicola hypovirus 3 (MfHV3), in a strain (hf-1) of the phytopathogenic fungus Monilinia fructicola. The complete genome of MfHV3 is 9259 nucleotides (nt) in length and contains a single large open reading frame (ORF) from nt position 462 to 8411. This ORF encodes a polyprotein with three conserved domains, namely UDP-glycosyltransferase, RNA-dependent RNA polymerase (RdRp), and DEAD-like helicase. The MfHV3 polyprotein shares the highest similarity with Colletotrichum camelliae hypovirus 1. Phylogenetic analysis indicated that MfHV3 clustered with members of the genus Betahypovirus within the family Hypoviridae. Taken together, the results of genomic organization comparisons, amino acid sequence alignments, and phylogenetic analysis convincingly show that MfHV3 is a new member of the genus Betahypovirus, family Hypoviridae.

Complete genome sequence of a novel botourmiavirus infecting the fungus Phomopsis asparagi.

Here, we report a novel ourmia-like mycovirus, named "Phomopsis asparagi magoulivirus 1" (PaMV1), derived from the phytopathogenic fungus Phomopsis asparagi. The genome of PaMV1 consists of a positive-sense single-stranded RNA (+ ssRNA) that is 2,639 nucleotides in length, with a GC content of 57.13%. It contains a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) consisting of 686 amino acids with a molecular mass of 78.57 kDa. Phylogenetic analysis based on RdRp sequences revealed that PaMV1 grouped together with Diaporthe gulyae magoulivirus 1 (DgMV1) in a distinct clade. Sequence comparisons and phylogenetic analysis suggest that PaMV1 is a novel member of the genus Magoulivirus, family Botourmiaviridae.

Comparative analysis of multiple consensus genomes of the same strain of Marek's disease virus reveals intrastrain variation.

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloging divergent nucleotides between strains with different phenotypes. However, most comparative genomic studies of MDV rely on previously published consensus genomes despite the confirmed existence of MDV strains as mixed viral populations. To assess the reliability of interstrain genomic comparisons relying on published consensus genomes of MDV, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform three-way comparisons between multiple consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 236 positions involving 13 open reading frames (ORFs). By contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Notably, we were able to identify 3 single-nucleotide polymorphisms (SNPs) in the unique long region and 16 SNPs in the unique short (US) region of CVI988GenBank.BAC that were not present in either CVI988Pirbright.lab or CVI988USDA.PA.field. Recombination analyses of field strains previously described as natural recombinants of CVI988 yielded no evidence of crossover events in the US region when either CVI988Pirbright.lab or CVI988USDA.PA.field were used to represent CVI988 instead of CVI988GenBank.BAC. We were also able to confirm that both CVI988 and Md5 populations were mixed, exhibiting a total of 29 and 27 high-confidence minor variant positions, respectively. However, we did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988GenBank.BAC. Taken together, our findings suggest that continued reliance on the same published consensus genome of CVI988 may have led to an overestimation of genomic divergence between CVI988 and virulent strains and that multiple consensus genomes per strain may be necessary to ensure the accuracy of interstrain genomic comparisons.

Complete genome sequence and genetic characterization of a novel segmented RNA virus infecting Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is a significant agricultural pest capable of long-distance migration and transmission of viruses that cause severe disease in rice. In this study, we identified a novel segmented RNA virus in a BPH, and this virus exhibited a close relationship to members of a recently discovered virus lineage known as "quenyaviruses" within the viral kingdom Orthornavirae. This newly identified virus was named "Nilaparvata lugens quenyavirus 1" (NLQV1). NLQV1 consists of five positive-sense, single-stranded RNAs, with each segment containing a single open reading frame (ORF). The genomic characteristics and phylogenetic analysis support the classification of NLQV1 as a novel quenyavirus. Notably, all of the genome segments of NLRV contained the 5'-terminal sequence AUCUG. The characteristic virus-derived small interfering RNA (vsiRNA) profile of NLQV1 suggests that the antiviral RNAi pathway of the host BPH was activated in response to virus infection. These findings represent the first documented report of quenyaviruses in planthoppers, contributing to our understanding of quenyaviruses and expanding our knowledge of insect-specific viruses in planthoppers.

Complete genome sequence of a novel mitovirus identified in the phytopathogenic fungus Fusarium oxysporum f. sp. melonis strain T-SD3.

A novel mitovirus was identified in Fusarium oxysporum f. sp. melonis strain T-SD3 and designated as "Fusarium oxysporum mitovirus 3" (FoMV3). The virus was isolated from diseased muskmelon plants with the typical symptom of fusarium wilt. The complete genome of FoMV3 is 2269 nt in length with a predicted AU content of 61.40% and contains a single open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF was predicted to encode a polypeptide of 679 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain with a molecular mass of 77.39 kDa, which contains six conserved motifs with the highly conserved GDD tripeptide in motif IV. The 5'-untranslated region (UTR) and 3'-UTR of FoMV3 were predicted to fold into stem-loop structures. BLASTp analysis revealed that the RdRp of FoMV3 shared the highest aa sequence identity (83.85%) with that of Fusarium asiaticum mitovirus 5 (FaMV5, a member of the family Mitoviridae) infecting F. asiaticum, the causal agent of wheat fusarium head blight. Phylogenetic analysis further suggested that FoMV3 is a new member of the genus Unuamitovirus within the family Mitoviridae. This is the first report of a new mitovirus associated with F. oxysporum f. sp. melonis.

Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

Molecular and biological characterization of a novel partitivirus from Talaromyces pinophilus

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

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.

Bicistronic expression of nuclear transgenes in Chlamydomonas reinhardtii.

In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-α-bisabolene production of 7.95 mg L-1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome.