Complete genome sequence of a novel totivirus isolated from the leaves of Myrica rubra.

Chinese bayberry is a fruit that is appreciated for its taste. A novel totivirus associated with rolling, disfiguring, chlorotic and vein-clearing symptoms on the leaf apices of Chinese bayberry was identified by transcriptome sequencing and reverse transcription PCR (RT-PCR). The complete genome of the virus was determined to be 4959 nucleotides long, and it contains two open reading frames (ORFs). Its genomic organization is similar to that of previously reported totiviruses. ORF1 encodes a putative coat protein (CP) of 765 aa, and ORF2 encodes an RNA-dependent RNA polymerase (RdRp) of 815 aa. These two putative proteins share 55.1% and 62.6%, amino acid sequence identity, respectively, with the corresponding proteins of Panax notoginseng virus A, respectively. According to the demarcation criteria for totivirus species established by the International Committee on Taxonomy of Viruses (ICTV), the new virus should be considered a member of a new species in the genus totivirus, family Orthototiviridae, which we have tentatively named ''Myrica rubra-associated totivirus'' (MRaTV).

A Simple Multiplex Reverse Transcription-PCR Method for the Diagnosis of L-A and M Totiviruses in Saccharomyces cerevisiae.

Killer yeasts and their toxins have many potential applications in environmental, medical, and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait are usually performed phenotypically based on their toxin production and immunity. In this study, we describe a simple and specific reverse transcription (RT) multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated with the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primer addition to the RT reaction. Using a mixture of specific primers at the PCR step, this multiplex RT-PCR protocol provided an accurate diagnosis of both L-A and M totivirus in all its known variants, L-A-1/M1, L-A-2/M2, L-A-28/M28, and L-A-lus/Mlus, found in infected killer yeasts. Using this method, the expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified but, importantly, asymptomatic L-A-2/M2 infected cells were found in addition to unexpected L-A-lus/M2 totiviral associations. IMPORTANCE The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, the development of simple methods for their detection significantly facilitates their study. The simplified multiplex RT-PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depended on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provided valuable insights into L-A/M viral associations and their coadaptational events in nature.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Molecular characterization of a novel totivirus infecting the basal fungus Conidiobolus heterosporus.

Mycoviruses are widely distributed in fungi, but only a few mycoviruses have been reported in basal fungi to date. Here, we characterized a novel totivirus isolated from the basal fungus Conidiobolus heterosporus, and we designated this virus as "Conidiobolus heterosporus totivirus 1" (ChTV1). The complete genome of ChTV1 contains two discontinuous open reading frames (ORFs), ORF1 and ORF2, encoding a putative coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRP), respectively. Phylogenetic analysis based on RdRP sequences showed that ChTV1 clustered with members of the genus Totivirus. The RdRP of ChTV1 has 51% sequence identity to that of Trichoderma koningiopsis totivirus 1 (TkTV1), which is the highest among mycoviruses. However, TkTV1 formed a distinct cluster with Wuhan insect virus 27, with 63% RdRP sequence identity, although Wuhan insect virus 27 has not been described, and its host represents a different kingdom. Therefore, we propose that ChTV1 is a new member of the genus Totivirus, family Totiviridae.

Identification and molecular characterization of tea-oil camellia-associated totivirus 1.

A novel dsRNA virus was identified by high-throughput sequencing from tea oil trees in China. Its complete genome of 4714 bp contains two open reading frames (ORFs). ORF1 encodes a putative coat protein (CP) of 702 amino acids (aa), and ORF2 codes for an RNA-dependent RNA polymerase (RdRp) of 855 aa. The virus shares the highest aa sequence identity of 45.21% in RdRp with taro-associated totivirus L (MN_119621), a member of the genus Totivirus in the family Totiviridae. Phylogenetic analysis of the aa sequences of the RdRp places the new virus in a group with other totiviruses, suggesting that this virus, which is provisionally named "tea-oil camellia-associated totivirus 1", should be considered a member of the genus Totivirus.

A novel totivirus isolated from the phytopathogenic fungus Rhodosporidiobolus odoratus strain GZ2017.

To our knowledge, there have been no reports of mycoviruses infecting Rhodosporidiobolus odoratus. Here, we describe the sequence of a novel mycovirus isolated from R. odoratus, which was designated "Rhodosporidiobolus odoratus RNA virus 1" (RoRV1). Sequence analysis revealed that RoRV1 has two discontinuous open reading frames (ORFs), ORF1 and ORF2, potentially encoding a hypothetical protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on RdRp sequences clearly placed RoRV1 in the genus Totivirus, family Totiviridae. The fungus also contains two additional, smaller dsRNAs, which might represent RoRV1 satellite RNAs.

Identification and genetic analysis of a totivirus isolated from the Culex tritaeniorhynchus in northern China.

Totiviridae, a viral family of double-stranded RNA (dsRNA) viruses, contain a single dsRNA genome 4.6-7.0 kb in length. Totiviridae were initially only known to infect fungi and other eukaryotes as well as plants, but an increase in totiviruses has been detected in insects, mosquitoes, and bats. Here, we describe the isolation and characterization of a strain belonging to the family Totiviridae isolated from Culex tritaeniorhynchus in Kenli, China, in 2016. We isolated a totivirus from field-collected mosquitoes in China by cell culture in Aedes albopictus C6/36 cells, identified the virus by morphological observation and complete genome sequencing, and characterized it by phylogenetic analysis. Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 35-40 nm. The genome was 7612 bp in length, including two open reading frames (ORFs). ORF1 (5058 nt) encodes the capsid protein, while ORF2 (2216 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). Nucleotide and amino acid homology analysis of isolate showed higher levels of sequence identity with isolate CTV_NJ2 (China, 2010) with 94.87% nucleic acid identity and 97.32% amino acid identity. The isolate was designated C. tritaeniorhynchus totivirus KL (CTV-KL). This is the first identification of a totivirus in a C. tritaeniorhynchus in northern China. Analysis of the virus's morphology, characteristic and genome organization will further enrich our understanding of the molecular and biological characteristics of dsRNA Totiviridae viruses.

Genome characterization of the newly identified maize-associated totivirus Anhui.

Maize-associated totivirus Anhui (MATV-Ah) is a novel totivirus with a 5536-nt genome and two large ORFs that encode a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRP). The two ORFs share amino acid identities of 32 and 56% when compared to other plant-associated totiviruses, respectively. Based on genome sequence similarity and phylogenetic analysis, MATV-Ah is proposed to be a member of the family Totiviridae genus Totivirus.

Two novel totiviruses in the white-backed planthopper, Sogatella furcifera.

There is little information about commensal viruses in the white-backed planthopper, Sogatella furcifera, although it is an important agricultural insect. Here, two novel double-stranded RNA viruses related to the viruses in the family Totiviridae were identified using next-generation sequencing and tentatively named Sogatella furcifera totivirus 1 and 2 (SfTV1 and SfTV2). Their complete genomes consist of 6310 and 6303 nt, respectively, showing typical genomic features with viruses in the family Totiviridae. Identity, phylogenetic and conserved sequence analyses showed that SfTV1, SfTV2 and three other insect viruses may form a proposed novel genus of the family Totiviridae. Vertical transmission of the two viruses was highly efficient, and they were detected in all insect tissues and developmental stages, with the highest titres in the adult and in the haemolymph and reproductive organs. To our knowledge, this is the first report of viruses in the family Totiviridae found in a hemipteran insect.

Novel and divergent viruses associated with Australian orchid-fungus symbioses.

Terrestrial orchids represent a symbiotic union between plants and mycorrhizal fungi. This study describes the occurrence and nature of viruses associated with one population of wild Pterostylis sanguinea orchids, including their fungal symbionts, over two consecutive years. A generic sequencing approach, which combined dsRNA-enrichment from plant and mycelial tissues, random amplification and high throughput shotgun sequencing was used to identify novel viruses. The majority of the virus-like sequences represent partial genomes, and their identification is based solely on de novo assembly of sequencing data. In orchid leaf tissues we found three isolates of a novel totivirus and an unclassified virus; both resemble fungus-infecting viruses. Two isolates of Ceratobasidium sp that were isolated from orchid underground stems contained at least 20 viruses, 16 of which were previously described as alphapartitiviruses and betapartitiviruses. A novel hypovirus and a mitovirus were genetically distant from existing members of the genera and did not readily fit into recognised subgroups.

Discovery of new orbiviruses and totivirus from Anopheles mosquitoes in Eastern Australia.

Three new viruses classifiable within the Totivirus and Orbivirus genera were detected from Anopheles mosquito species collected in Eastern Australia. The viruses could not be isolated in C6/36 mosquito cell cultures but were shown to replicate in their mosquito hosts by small RNA analysis. The viruses grouped phylogenetically with other viruses recently detected in insects. These discoveries contribute to a better understanding of commensal viruses in Australian mosquitoes and the evolution of these viruses.

Complete genome sequence of a variant of maize-associated totivirus from Ecuador.

The complete genomic sequence of a variant of the recently reported maize-associated totivirus (MATV) from China was obtained from commercial maize in Ecuador. The genome of MATV-Ec (Ecuador) (4,998 bp) is considerably longer than that of MATV-Ch (China) (3,956 bp), the main difference due to a ≈ 1-kb-long capsid-protein-encoding fragment that is completely absent from the Chinese genome. Sequence alignments between MATV-Ec and MATV-Ch showed an overall identity of 82% at the nucleotide level, whereas at the amino acid level, the viruses exhibited 95% and 94% identity for the putative capsid protein and the RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the viral RdRp domain indicated that MATV-Ec and MATV-Ch share a common ancestor with other plant-associated totiviruses, with Panax notoginseng virus A as the closest relative. MATV-Ec was detected in 46% (n = 80) of maize plants tested in this study, but not in endophytic fungi isolated from plants positive for the virus.

Molecular detection of a novel totivirus from golden shiner (Notemigonus crysoleucas) baitfish in the USA.

During regulatory and routine surveillance sampling of apparently healthy baitfish from the state of Minnesota, a novel totivirus (tentatively named "golden shiner totivirus", GSTV) was detected in a homogenate of kidney and spleen of golden shiner (Notemigonus crysoleucas). The nearly complete genome is 7788 nt long with a complete 5' untranslated region (UTR) of 135 nt (1-135 nt position), complete open reading frames (ORFs) and a partial 3' UTR of 54 nt (7734-7788). The sequence is comprised of two ORFs (ORF1 and ORF2). The larger ORF1 encodes a 1659-aa polypeptide in frame +1 from nt position 136 to 5115 (4980 nt) with a start codon at position 136-138 and a stop codon at position 5113-5115. The ORF1 is 54 aa longer than the 1605-aa ORF1-encoded protein of a reference strain of infectious myonecrosis virus (IMNV), ID-EJ-12-1(AIC34743.1). The predicted ORF1 and ORF2 fusion protein sequence was NFQDGG. Hence, an overlapping region of 99 nt was observed, which is shorter than the 172-nt and 199-nt overlapping regions in Armigeres subalbatus totivirus (AsTV) and IMNV, respectively. GSTV formed a separate lineage based on phylogenetic analysis of ORF1-encoded major capsid protein (MCP) and ORF2-encoded RNA-dependent RNA polymerase (RdRp) sequences. Based on ORF1 MCP sequence analysis, GSTV was most closely related to IMNV, with maximum aa sequence identity of 26.42-27.86 %, followed by 26.59, 22.94 and 21.75 % for Drosophila totivirus (DTV), AsTV and Omono River virus (OMRV), respectively. Similar to ORF1, the ORF2 (RdRp) of GSTV formed a separate clade with maximum identity of 38.10 % and 38.50 % to IMNV and DTV, respectively. The virus identified here differs enough from its closest relative that it may represent a new genus in the family Totiviridae. The disease-causing potential and management impact of this novel virus is unknown at this time.

The complete genome sequence of a novel maize-associated totivirus.

Deep sequencing of small RNA (sRNA) populations in maize plants from southwest China resulted in the identification of a previously unknown dsRNA virus with a sequence and genome organization resembling that of a totivirus. The complete viral genome is 3,956 nucleotides in length and contains two open reading frames (ORFs) with the potential to produce a ORF1-ORF2 fusion protein through a -1 ribosomal frameshift translation mechanism. ORF1 encodes the putative capsid protein (CP), whereas the predicted product of ORF2 contains motifs typical of an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis using the amino acid sequences of putative RdRp fusion proteins showed that the new virus was grouped in a clade together with the totiviruses, suggesting that it is a new member of the genus Totivirus of the family Totiviridae. The virus is tentatively named "maize-associated totivirus (MATV)". Our findings demonstrate that it is feasible to identify totiviruses by deep sequencing of small RNAs.

Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi.

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a -1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.

Characterization of virus-like particles and identification of capsid proteins in Xanthophyllomyces dendrorhous.

Two dsRNAs of estimated lengths of 5 (L1) and 3.7 (L2) kpb are commonly found in strains of the basidiomycetous yeast Xanthophyllomyces dendrorhous, and the presence of virus-like particles (VLPs) have been described in some strains. Recently, two putative totiviruses (XdV-L1A and XdV-L1B) were identified from L1 dsRNA and one (XdV-L2) from L2 dsRNA in the strain UCD 67-385. In some strains, there are smaller dsRNAs (0.9-1.4 kb) that probable are satellite elements. In this work, the VLPs from several strains of X. dendrorhous, which differ in their dsRNAs content, were separated by sucrose gradient and characterized in relation to the dsRNAs and proteins that compose them. It was found that all types of dsRNAs were encapsidated into VLPs, supporting the hypothesis that the smaller dsRNAs are satellite molecules. A main protein of approx. 76 or 37 kDa composed the virions that only have the L1-dsRNA or L2-dsRNA, respectively. In the strain UCD 67-385, these both proteins were identified as viral capsid protein (CP), allow to confirm the gag predicted ORFs in XdV-L1A, XdV-L1B, and XdV-L2, with CPs of 76.6, 76.2, and 38.8 kDa, respectively. Analysis of predicted structures of CPs of XdV-L1A and XdV-L1B, showed high similitudes with the CPs of ScV-L-A and other totiviruses.

Sequence determination of a quadripartite dsRNA virus isolated from Aspergillus foetidus.

Virus infection of Aspergillus foetidus was documented over 40 years ago and was one of the first mycovirus infections described in a filamentous fungus. The virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. Here, we report the complete nucleotide sequence of the AfV-F genome isolated from virions purified from the prototype isolate of the fungus. The AfV-F double-stranded (ds) RNA genome is tetra-segmented, and the plus strands of each of the four segments, but not the minus strands, are polyadenylated. The organisation and sequences of the four AfV-F dsRNAs are similar to those described for Alternaria alternata virus 1, which we propose is a member of an emerging mycovirus genus ("Alternavirus") and family ("Alternaviridae"), which also includes AfV-F.

The complete nucleotide sequence of a totivirus from Aspergillus foetidus.

Virus infection of Aspergillus foetidus was first described in the 1970s, and the purified virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. AfV-S consists of a mixture of two viruses, the larger of which, called AfV-S1, is a dsRNA-containing member of the genus Victorivirus in the family Totiviridae, and its complete nucleotide sequence is described here.

Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous.

BACKGROUND: Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation in viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNAs are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. RESULTS: The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totiviridae family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. CONCLUSIONS: Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.