Identification of a novel waikavirus infecting Pittosporum tobira in China.

A novel waikavirus, tentatively named "Pittosporum tobira waikavirus" (PtWV), was identified in Pittosporum tobira plants exhibiting mosaic and ringspot symptoms on foliage in Yunnan, China. The full-length genomic sequence was determined by high-throughput sequencing and rapid amplification of cDNA ends. The genome of PtWV is 12,709 nt in length and has a large open reading frame (ORF) of 11,010 nt, encoding a polyprotein, and a small ORF that encodes a 13.2-kDa bellflower vein chlorosis virus (BVCV)-like protein. Phylogenetic analysis and sequence alignment revealed that PtWV is closely related to actinidia yellowing virus 1 (AcYV1), which shares the highest amino acid (aa) sequence similarity (50.1% identity) in the Pro-RdRp region. To the best of our knowledge, this is the first report of a novel waikavirus in P. tobira.

Characterization of a novel psyllid-transmitted waikavirus in carrots.

Carrots collected from the Western Negev region in Israel during the winter of 2019 showed disease symptoms of chlorosis, leaf curling, a loss of apical dominance, and multiple lateral roots that were not associated with known pathogens of the carrot yellows disease. Symptomatic carrots were studied for a possible involvement of plant viruses in disease manifestations using high throughput sequencing analyses. The results revealed the presence of a waikavirus, sharing a ∼70% nucleotide sequence identity with Waikavirus genus members. Virions purified from waikavirus-positive carrots were visualized by transmission electron microscopy, showing icosahedral particle diameter of ∼28 nm. The genome sequence was validated by overlapping amplicons by designed 12 primer sets. A complete genome sequence was achieved by rapid amplification of cDNA ends (RACE) for sequencing the 5' end, and RT-PCR with oligo dT for sequencing the 3' end. The genome encodes a single large ORF, characteristic of waikaviruses. Aligning the waikavirus-deduced amino-acid sequence with other waikavirus species at the Pro-Pol region, a conserved sequence between the putative proteinase and the RNA-dependent RNA polymerase, showed a ∼40% identity, indicating the identification of a new waikavirus species. The amino-acid sequence of the three coat proteins and cleavage sites were experimentally determined by liquid chromatography-mass spectrometry. A phylogenetic analysis based on the Pro-Pol region revealed that the new waikavirus clusters with persimmon waikavirus and actinidia yellowing virus 1. The new waikavirus genome was localized in the phloem of waikavirus-infected carrots. The virus was transmitted to carrot and coriander plants by the psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae).

Complete nucleotide sequence of hackberry virus A, a tentative member of the genus Waikavirus.

The complete genomic sequence of a waikavirus from Chinese hackberry in Zhejiang province, China, named "hackberry virus A" (HVA), was determined using high-throughput sequencing (HTS) combined with reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The bicistronic genomic RNA of HVA was found to consist of 12,691 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and to encode a large polyprotein of 3783 amino acids (aa) and an additional 10.3-kDa protein. The aa sequences of the Pro-Pol and the CP regions of this virus share 39.8-44.2% and 25.5-36.4% identity, respectively, with currently known waikaviruses. These values are significantly below the current species demarcation threshold (< 75% and < 80% aa identity for the CP and Pro-Pol region, respectively) for the family Secoviridae, indicating that HVA represents a new species in the genus Waikavirus. This is the first report of a virus infecting Chinese hackberry.

Broadening the host range and genetic diversity of waikaviruses.

Waikaviruses are monopartite, positive sense, single-stranded RNA viruses that cause economically important plant diseases. Despite their importance, waikaviruses are poorly understood and only ten members are currently recognized. The present study on Sequence Read Archive (SRA)-based data-driven virus discovery (DDVD) identified 22 putative new waikaviruses, nearly doubling the number of known waikaviruses, in SRA libraries of diverse plant species, from ferns to trees. Besides, a highly divergent secoviral sequence with distinct genome features was identified in a wheat transcriptome. Other significant findings of the study include identification of a new waikavirus in a library derived from diseased water chestnut sample wherein a caulimovirus was reported, prediction of coiled-coils in hypothetical protein region of waikaviral polyprotein alignment and phylogenetic clustering of tree-infecting waikaviruses. The study not only reiterates the importance of DDVD in unveiling hitherto hidden viral sequences in plant SRA libraries but also deepens our understanding of waikaviral diversity.

Critical residues for proteolysis activity of maize chlorotic dwarf virus (MCDV) 3C-like protease and comparison of activity of orthologous waikavirus proteases.

Maize chlorotic dwarf virus (MCDV) encodes a 3C-like protease that cleaves the N-terminal polyprotein (R78) as previously demonstrated. Here, we examined amino acid residues required for catalytic activity of the protease, including those in the predicted catalytic triad, amino acid residues H2667, D2704, and C2798, as well as H2817 hypothesized to be important in substrate binding. These and other residues were targeted for mutagenesis and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that abolished catalytic activity. Mutations that altered the predicted catalytic triad residues and H2817 disrupted MCDV-S protease activity, as did mutagenesis of a conserved tyrosine residue, Y2774. The protease activity and R78 cleavage of orthologs from divergent MCDV isolates MCDV-Tn and MCDV-M1, and other waikavirus species including rice tungro spherical virus (RTSV) and bellflower vein chlorosis virus (BVCV) were also examined.

Complete nucleotide sequence of sweetbriar rose curly-top associated virus, a tentative member of the genus Waikavirus.

A novel virus, tentatively named "sweetbriar rose curly-top associated virus" (SRCTaV), was identified in sweetbriar rose (Rosa rubiginosa) using high-throughput sequencing. The complete genome sequence of SRCTaV was determined and characterized. Phylogenetic analysis revealed that SRCTaV is closely related to members of the genus Waikavirus.

Complete genome sequence of camellia virus A, a tentative new member of the genus Waikavirus.

A new positive-strand RNA virus genome was discovered in Camellia japonica plants. The complete genome of the virus is 12,570 nt in size, excluding the poly(A) tail, and contains one large open reading frame (ORF1) and two small open reading frames (ORF2, ORF3). ORF1 and ORF2 are homologous to sequences of waikaviruses, while ORF3 has no relatives in the databases. ORF1 encodes a putative polyprotein precursor that is putatively processed into eight smaller proteins, as in typical waikaviruses. Comprehensive analysis, including BLAST searches, genome organization and pairwise sequence comparisons, and phylogeny reconstructions, invariably placed the virus with the waikaviruses. Furthermore, due to lower amino acid sequence identity to known waikaviruses than the threshold species demarcation cutoff, this virus may represent a new species in the genus Waikavirus, family Secoviridae, and we have tentatively named it "camellia virus A" (CamVA). Finally, a field survey was conducted to assess the occurrence of CamVA in camellias and its associated symptoms.

A novel Waikavirus (the family Secoviridae) genome sequence identified in rapeseed (Brassica napus).

The genome sequence of a novel species of the genus Waikavirus (the family Secoviridae), which we named Brassica napus RNA virus 1 (BnRV1), was identified in a rapeseed (Brassica napus) transcriptome dataset. The BnRV1 genome was 12,293 nucleotides long followed by a poly(A) tail. Two open reading frames (ORFs), called ORF1 and ORFX, were predicted. The larger ORF, ORF1, encodes a polyprotein of 3,471 amino acids and the smaller ORF, ORFX, overlaps ORF1 and encodes an 87 aa long protein of unknown function. The BnRV1 ORF1 polyprotein was predicted to undergo proteolytic processing to yield seven mature proteins, including an RNA-dependent RNA polymerase and three distinct coat proteins. The ORF1 and ORFX proteins share sequence similarities with the respective proteins of viruses in the genus Waikavirus, including the bellflower vein chlorosis virus, rice tungro spherical virus, and maize chlorotic dwarf virus. A phylogenetic tree inferred from a conserved segment of the polyproteins of several Secoviridae viruses confirmed that BnRV1 is a novel species of the genus Waikavirus. The BnRV1 genome sequence identified in this study may be useful for the study of waikavirus biology and waikavirus-derived diseases. Keywords: Brassica napus RNA virus 1; Waikavirus; Secoviridae; rapeseed.

Assessment of resistance to rice tungro disease in popular rice varieties in India by introgression of a transgene against Rice tungro bacilliform virus.

Rice crops in South and Southeast Asian countries suffer critical yield losses due to rice tungro disease caused by joint infection with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Previously, for generating RNA interference-based transgenic resistance against tungro viruses, RTBV ORF IV was used as a transgene to develop RTBV resistance in a popular high-yielding scented rice variety. The transgene from this line was then introgressed into five popular high-yielding but tungro-susceptible rice varieties by marker-assisted backcross breeding with a view to combine the resistant trait with the agronomic traits. The present work includes a resistance assay of the BC3F5 lines of these varieties under glasshouse conditions. Out of a total of 28 lines tested, each consisting of 12 individual plants, eight lines showed significant amelioration in height reduction and 100- to 1000-fold reduction in RTBV titers. The RNAi-mediated resistance was clearly manifested by the presence of virus-derived small RNA (vsRNA) specific for RTBV ORF IV in the transgenic backcrossed lines.

Small RNA-based interactions between rice and the viruses which cause the tungro disease.

Rice tungro disease is caused by a complex of two viruses, Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). To examine the RNAi-based defence response in rice during tungro disease, we characterized the virus-derived small RNAs and miRNAs by Deep Sequencing. We found that, while 21 nt/22 nt (nucleotide) siRNAs are predominantly produced in a continuous, overlapping and asymmetrical manner from RTBV, siRNA accumulation from RTSV were negligible. Additionally, 54 previously known miRNAs from rice, predicted to be regulating genes involved in plant defence, hormone signaling and developmental pathways were differentially expressed in the infected samples, compared to the healthy ones. This is the first study of sRNA profile of tungro virus complex from infected rice plants. The biased response of the host antiviral machinery against the two viruses and the differentially-expressed miRNAs are novel observations, which entail further studies.

Complete nucleotide sequence and genome organization of red clover associated virus 1 (RCaV1), a putative member of the genus Waikavirus (family Secoviridae, order Picornavirales).

Using high-throughput sequencing, a novel waikavirus was identified in a mixed virus infection of red clover (Trifolium pratense L.). Its complete genomic sequence was determined and characterized. The virus, tentatively named red clover associated virus 1 (RCaV1), is phylogenetically related to members of the genus Waikavirus (family Secoviridae, order Picornavirales).

Simultaneous resistance against the two viruses causing rice tungro disease using RNA interference.

Rice tungro is the most important viral disease affecting rice in South and Southeast Asia, caused by two viruses rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Transgenic resistance using RNA-interference (RNAi) has been reported individually against RTBV and RTSV earlier. Here we report the development of transgenic rice plants expressing RNAi against both RTBV and RTSV simultaneously. A DNA construct carrying 300 bp of RTBV DNA and 300 bp of RTSV cDNA were cloned as the two arms in hairpin orientation in a binary plasmid background to generate RNAi against both viruses simultaneously. Transgenic rice plants were raised using the above construct and their resistance against RTBV and RTSV was quantified at the T1 plants. Levels of both the viral nucleic acids showed a fall of 100- to 500-fold in the above plants, compared with the non-transgenic controls, coupled with the amelioration of stunting. The transgenic plants also retained higher chlorophyll levels than the control non-transgenic plants after infection with RTBV and RTSV. Small RNA analysis of virus inoculated transgenic plants indicated the presence of 21 nt and 22 nt siRNAs specific to RTBV and RTSV. The evidence points towards an active RNAi mechanism leading to resistance against the tungro viruses in the plants analysed.

Development of an Indirect ELISA and Dot-Blot Assay for Serological Detection of Rice Tungro Disease.

Rice tungro disease (RTD) is one of the most destructive diseases of rice in South and Southeast Asia. RTD is routinely detected based on visual observation of the plant. However, it is not always easy to identify the disease in the field as it is often confused with other diseases or physiological disorders. Here we report the development of two serological based assays for ease of detection of RTD. In this study we had developed and optimized an indirect ELISA and dot-blot assay for detection of RTD. The efficiency of both assays was evaluated by comparing the specificity and sensitivity of the assays to PCR assay using established primer sets. The indirect ELISA showed 97.5% and 96.6%, while the dot-blot assay showed 97.5% and 86.4% sensitivity and specificity, respectively, when compared to established PCR method. The high sensitivity and specificity of the two assays merit the use of both assays as alternative methods to diagnose RTD. Furthermore, the dot-blot assay is a simple, robust, and rapid diagnostic assay that is suitable for field test for it does not require any specialized equipment. This is a great advantage for diagnosing RTD in paddy fields, especially in the rural areas.

Identification of a maize chlorotic dwarf virus silencing suppressor protein.

Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389kDa proteolytically processed polyprotein. We show that the N-terminal 78kDa polyprotein (R78) of MCDV acts as a suppressor of RNA silencing in a well-established assay system. We further demonstrate that R78 is cleaved by the viral 3C-like protease into 51 and 27kDa proteins (p51 and p27), and that p51 is responsible for silencing suppressor activity. Silencing suppressor activity of R78 is conserved in three divergent MCDV strains (MCDV-Severe, MCDV-M1, and MCDV-Tennessee), as well as the waikavirus Bellflower vein chlorosis virus, but was not detected for orthologous protein of Rice tungro spherical virus (RTSV-A) or the similarly-positioned protein from the sequivirus Parsnip yellow fleck virus (PYFV). This is the first identification of a virus suppressor of RNA silencing encoded by a waikavirus.

Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.

Evaluation of virus resistance and agronomic performance of rice cultivar ASD 16 after transfer of transgene against Rice tungro bacilliform virus by backcross breeding.

Severe losses of rice yield in south and southeast Asia are caused by Rice tungro disease (RTD) induced by mixed infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to develop transgene-based resistance against RTBV, one of its genes, ORF IV, was used to generate transgenic resistance based on RNA-interference in the easily transformed rice variety Pusa Basmati-1, and the transgene was subsequently introgressed to rice variety ASD 16, a variety popular in southern India, using transgene marker-assisted selection. Here, we report the evaluation of BC3F4 and BC3F5 generation rice plants for resistance to RTBV as well as for agronomic traits under glasshouse conditions. The BC3F4 and BC3F5 generation rice plants tested showed variable levels of resistance, which was manifested by an average of twofold amelioration in height reduction, 1.5-fold decrease in the reduction in chlorophyll content, and 100- to 10,000-fold reduction in the titers of RTBV, but no reduction of RTSV titers, in three backcrossed lines when compared with the ASD 16 parent. Agronomic traits of some of the backcrossed lines recorded substantial improvements when compared with the ASD 16 parental line after inoculation by RTBV and RTSV. This work represents an important step in transferring RTD resistance to a susceptible popular rice variety, hence enhancing its yield in areas threatened by the disease.

Complete genome sequence of bellflower vein chlorosis virus, a novel putative member of the genus Waikavirus.

The complete genome sequence of a new virus isolated from a bellflower (Campanula takesimana) plant was determined. The genome of this virus is composed of monopartite single-stranded RNA of 11,649 nucleotides in length. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 42% (with 99% coverage) to the polyprotein of the isolate Orissa of rice tungro spherical virus (RTSV; genus Waikavirus). Phylogenetic analysis strongly supports that the identified virus is a member of a new species of the genus Waikavirus. The name bellflower vein chlorosis virus (BVCV) is proposed for this new virus.

Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus.

Rice tungro spherical virus (RTSV), also known as Rice waika virus, does not cause visible symptoms in infected rice plants. However, the virus plays a critical role in spreading Rice tungro bacilliform virus (RTBV), which is the major cause of severe symptoms of rice tungro disease. Recent studies showed that RNA interference (RNAi) can be used to develop virus-resistance transgenic rice plants. In this report, we presented simple procedures and protocols needed for the creation of transgenic rice plants capable of producing small interfering RNA specific against RTSV sequences. Notably, our study showed that 60 out of 64 individual hygromycin-resistant lines (putative transgenic lines) obtained through transformation carried transgenes designed for producing hairpin double-stranded RNA. Northern blot analyses revealed the presence of small interfering RNA of 21- to 24-mer in 46 out of 56 confirmed transgenic lines. Taken together, our study indicated that transgenic rice plants carrying an inverted repeat of 500-bp fragments encoding various proteins of RTSV can produce small interfering RNA from the hairpin RNA transcribed from that transgene. In light of recent studies with other viruses, it is possible that some of these transgenic rice lines might be resistant to RTSV.

Viruses in maize and Johnsongrass in southern Ohio.

The two major U.S. maize viruses, Maize dwarf mosaic virus (MDMV) and Maize chlorotic dwarf virus (MCDV), emerged in southern Ohio and surrounding regions in the 1960s and caused significant losses. Planting resistant varieties and changing cultural practices has dramatically reduced virus impact in subsequent decades. Current information on the distribution, diversity, and impact of known and potential U.S. maize disease-causing viruses is lacking. To assess the current reservoir of viruses present at the sites of past disease emergence, we used a combination of serological testing and next-generation RNA sequencing approaches. Here, we report enzyme-linked immunosorbent assay and RNA-Seq data from samples collected over 2 years to assess the presence of viruses in cultivated maize and an important weedy reservoir, Johnsongrass (Sorghum halepense). Results revealed a persistent reservoir of MDMV and two strains of MCDV in Ohio Johnsongrass. We identified sequences of several other grass-infecting viruses and confirmed the presence of Wheat mosaic virus in Ohio maize. Together, these results provide important data for managing virus disease in field corn and sweet corn maize crops, and identifying potential future virus threats.

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.