Molecular characterization of a novel mycovirus isolated from Rhizoctonia solani AG-1 IA 9-11

The complete genome of the dsRNA virus isolated from Rhizoctonia solani AG-1 IA 9–11 (designated as Rhizoctonia solani dsRNA virus 11, RsRV11 ) were determined. The RsRV11 genome was 9,555 bp in length, contained three conserved domains, SMC, PRK and RT-like super family, and encoded two non-overlapping open reading frames (ORFs). ORF1 potentially coded for a 204.12 kDa predicted protein, which shared low but significant amino acid sequence identities with the putative protein encoded by Rhizoctonia solani RNA virus HN008 (RsRV-HN008) ORF1. ORF2 potentially coded for a 132.41 kDa protein which contained the conserved motifs of the RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that RsRV11 was clustered with RsRV-HN008 in a separate clade independent of other virus families. It implies that RsRV11, along with RsRV-HN008 possibly a new fungal virus taxa closed to the family Megabirnaviridae, and RsRV11 is a new member of mycoviruses.

Persistence of ambigrammatic narnaviruses requires translation of the reverse open reading frame

Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods and nematodes. Though initially described as simple single-gene non-segmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as “ambigrammatic” harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2 kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8 kb “Robin” RNA. We developed a system to launch CxNV1 in a naïve mosquito cell line, then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively-translating ribosomes on host mRNA or co-infecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission. IMPORTANCE Fundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome, relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses, and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.

An Umbra-related Virus Found in Vasconcellea X Heilbornii (Caricaceae)

The complete sequence of a new viral RNA from babaco (Vasconcellea x heilbornii) was determined. The genome consisted of 4,584 nucleotides organized in two non-overlapping open reading frames (ORFs 1 and 2), a 9-nt-long noncoding region (NCR) at the 5’ terminus and a 1,843 -nt-long NCR at the 3’ terminus. Sequence comparisons of ORF 2 revealed homology to the RNA-dependent-RNA-polymerase (RdRp) of several umbra- and umbra-related viruses. Phylogenetic analysis of the RdRp placed the new virus in a well-supported and cohesive clade that includes umbra-like viruses reported from papaya, citrus, opuntia, maize and sugarcane hosts. This clade shares a most recent ancestor with the umbraviruses but has different genomic features. The creation of a new genus, within the Tombusviridae, is proposed for the classification of these novel viruses.

The First Whole Genome Sequence and Characterisation of Avian Nephritis Virus Genotype 3

Avian nephritis virus (ANV) is classified in the Avastroviridae family with disease associations with nephritis, uneven flock growth and runting stunting syndrome (RSS) in chicken and turkey flocks, and other avian species. The whole genome of ANV genotype 3 (ANV-3) of 6959 nucleotides including the untranslated 5′ and 3′ regions and polyadenylated tail was detected in a metagenomic virome investigation of RSS-affected chicken broiler flocks. This report characterises the ANV-3 genome, identifying partially overlapping open reading frames (ORFs), ORF1a and ORF1b, and an opposing secondary pseudoknot prior to a ribosomal frameshift stemloop structure, with a separate ORF2, whilst observing conserved astrovirus motifs. Phylogenetic analysis of the Avastroviridae whole genome and ORF2 capsid polyprotein classified the first complete whole genome of ANV-3 within Avastroviridae genogroup 2.

Persistence of ambigrammatic narnaviruses requires translation of the reverse open reading frame

ABSTRACTNarnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods and nematodes. Though initially described as simple single-gene non-segmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as “ambigrammatic” harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2 kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8 kb “Robin” RNA. We developed a system to launch CxNV1 in a naïve mosquito cell line, then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively-translating ribosomes on host mRNA or co-infecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission.IMPORTANCEFundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome, relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses, and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.

Genome Sequences of Two Dual-Serotype-Specific Echovirus 20 Strains from Nigeria

Here, we describe nearly complete genome sequences (7,361 nucleotides [nt] and 6,893 nt) of two echovirus 20 (E20) isolates from Nigeria that were simultaneously typed as CVB and E20 (dual serotype) by neutralization assay. Both include two overlapping open reading frames (ORFs) of 67 and 2,183 amino acids that encoded a recently described gut infection-facilitating protein and the classic enterovirus proteins, respectively.

Genetic Analysis of Avian Gyrovirus 2 Variant-Related Gyrovirus Detected in Farmed King Ratsnake (Elaphe carinata): The First Report from China

Avian gyrovirus 2 (AGV2), which is similar to chicken infectious anemia virus, is a new member of the genus Gyrovirus. AGV2 has been detected not only in chicken but also in human tissues and feces. This study analyzed 91 samples (8 from liver tissue and 83 from fecal samples) collected from king ratsnakes (Elaphe carinata) from 7 separate farms in Hubei and Henan, China, for AGV2 DNA using PCR. The results demonstrated a low positive rate of AGV2 (6.59%, 6/91) in the snakes, and all the positive samples were collected from the same farm. The AGV2 strain HB2018S1 was sequenced, and its 2376 nt genome comprised three partially overlapping open reading frames: VP1, VP2, and VP3. Phylogenetic analysis revealed that the HB2018S1 and NX1506-1 strains from chickens in China belong to the same clade and that they have a nucleotide identity as high as 99.5%. Additionally, recombination analysis showed that HB2018S1 might originate from the recombination of viruses similar to those detected in chickens and a ferret. A total of 10 amino acid mutation sites (44(R/K), 74(T/A), 256 (C/R), 279(L/Q), and 373(V/A) in AGV2 VP1; 60(I/T), 125(T/I), 213(D/N), and 215(L/S) in AGV2 VP2; and 83(H/Y) in AGV2 VP3) different from those observed in most reference strains were found in the genome of HB2018S1, indicating that the differences may be related to a transboundary movement among hosts, which needs further elucidation. To the best of our knowledge, this study is the first report on an AGV2-infected poikilotherm, suggesting that cross-host transmission of viruses with circular single-stranded DNA genomes would be a public health concern.

A Victorivirus and Two Novel Mitoviruses Co-Infected the Plant Pathogen Nigrospora oryzae

Three dsRNAs, in sizes of approximately 2.5–5 kbp, were detected in the plant pathogenic fungus Nigrospora oryzae strain CS-7.5-4. Genomic analysis showed that the 5.0 kb dsRNA was a victorivirus named as Nigrospora oryzae victorivirus 2 (NoRV2). The genome of NoRV2 was 5166 bp in length containing two overlapping open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to the CPs of viruses belonging to the Totiviridae family. The stop codon of ORF1 and the start codon of ORF2 were overlapped by the tetranucleotide sequence AUGA. ORF2 was predicted to encode an RNA-dependent RNA polymerase (RdRp), which was highly similar to the RdRps of victoriviruses. Virus-like particle examination demonstrated that the genome of NoRV2 was solely encapsidated by viral particles with a diameter of approximately 35 nm. The other two dsRNAs that were less than 3.0 kb were predicted to be the genomes of two mitoviruses, named as Nigrospora oryzae mitovirus 1 (NoMV1) and Nigrospora oryzae mitovirus 2 (NoMV2). Both NoMV1 and NoMV2 were A-U rich and with lengths of 2865 and 2507 bp, respectively. Mitochondrial codon usage inferred that each of the two mitoviruses contains a major large ORF encoding a mitoviral RdRp. Horizontal transfer experiments showed that the NoMV1 and NoMV2 could be cotransmitted horizontally via hyphal contact to other virus-free N. oryzae strains and causes phenotypic change to the recipient, such as an increase in growth rate. This is the first report of mitoviruses in N. oryzae.

CCT: a coordinate conversion tool for hepatitis B virus

The hepatitis B virus (HBV) genome has ∼3 200 nucleotides, coding for seven proteins in four overlapping open reading frames (ORFs). Comparison of genomic coordinates between different samples and/or published literature requires manual conversion. An online tool is presented to convert nucleotide or amino acid positions between ORFs, regions and domains of the HBV genome. The user enters a position into an interactive web page, which then shows this position in all other applicable ORFs, regions or domains and plots it on a diagrammatic representation of the HBV genome. This tool assists researchers to convert coordinates, thereby facilitating comparisons between samples.

Classification, structure and molecular diagnostics of avian hepatitis E virus

Pathological syndromes caused by avian hepatitis E virus have been described as big liver and spleen disease; necrotic haemorrhagic hepatitis-splenomegaly syndrome; necrotic, haemorrhagic, hepatomegalic hepatitis; or hepatitis-liver haemorrhage syndrome. The aetiological factor of this syndrome belongs to the Hepeviridae family. Avian hepatitis E virus is a single-strand RNA virus whose genome consists of approx. 7,200 base pairs and contains a short non-coding 5’ terminus (27-35 nucleotides) followed by three partially overlapping open reading frames: ORF1, ORF3 and ORF2. Since the avian strains of hepatitis viruses do not replicate in cell cultures, molecular biology techniques are used in the diagnosis of infections. This article discusses in detail the structure and function of each ORF of avian hepatitis E virus, as well as methods for the identification of the genetic material of this pathogen.