Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design.

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105-106 times more sensitive than ELISA and 100-1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.

Complete genome sequence of a novel ophiovirus associated with chlorotic disease of pepper (Capsicum annuum L.) in Japan.

In July 2018, pepper plants (Capsicum annuum L.) with chlorotic leaves and fruits were observed in Kochi prefecture, Japan. High-throughput sequencing (HTS) identified the possible presence of an ophiovirus-like virus possessing three RNA segments in a chlorotic leaf. Using Sanger sequencing with primers designed based on the HTS results and a different source of RNA from the one used for HTS, the complete nucleotide sequences of three RNA segments encoding four proteins on their complementary strand were determined. The amino acid sequences of these four proteins showed similarity to those of the RNA-dependent RNA polymerase, RNA-silencing suppressor protein, movement protein, and coat protein, respectively, of ophioviruses, which are negative-sense single-stranded RNA viruses. However, the coat protein amino acid sequence of the virus found on pepper plants was no more than 61.9% identical to those of any known ophioviruses, which is lower than the species demarcation threshold of 85 %. Hence, we suggest that this virus, which we have named "pepper chlorosis associated virus" (PepCaV) should be considered a member of a new species in the genus Ophiovirus, for which we propose the name "Ophiovirus capsici". The results of phylogenetic analysis using coat protein amino acid sequences of PepCaV and other ophioviruses also supported this conclusion. PepCaV RNA was found to have conserved nucleotide sequences at both the 5' and 3' termini of the different RNA segments, and the conserved terminal nucleotide sequences were predicted to form a self-complementary double-stranded region, resulting in a panhandle structure in each of the genomic RNAs.

Complete genome sequence of vallota mosaic virus detected in a narcissus bulb imported from the United States to Japan.

Narcissus (Narcissus albidus) imported from the United States exhibited leaf chlorosis during post-entry quarantine. We employed next-generation sequencing (NGS) on symptomatic leaf samples and detected vallota mosaic virus (ValMV), belonging to the genus Potyvirus, family Potyviridae, as the viral agent. Sanger sequencing of PCR products and rapid amplification of cDNA ends based on NGS contigs revealed that ValMV is 9,451 nucleotides (nt) in length, excluding the poly(A) tail. Nucleotide and amino acid (aa) sequences of the coat protein region had over 98% identity to previously reported ValMV isolates. In each of the 10 regions encoding mature proteins, however, the sequence identity to other potyviruses was 49.5-71.9% nt and 18.3-78.9% aa, values that are below the species demarcation thresholds for the family Potyviridae. Phylogenetic analysis revealed that our ValMV isolate is most closely related to known ValMV isolates and is grouped with other potyviruses. Taken together, our results indicate that the newly isolated ValMV belongs to a distinct species in the genus Potyvirus. This study provides the first report of the complete ValMV genome sequence and the first record of this virus in narcissus.

Complete genome sequences of anemone mosaic virus and ranunculus mild mosaic virus isolated from anemone imported from the Netherlands into Japan.

Anemone mosaic virus (AnMV) and ranunculus mild mosaic virus (RanMMV) infect anemone plants, which exhibit characteristic mosaic patterns on their leaves. Employing next-generation sequencing of plant material imported from the Netherlands, the complete genome sequences of these two viruses were determined for the first time. AnMV and RanMMV have 9698 and 9537 nucleotides (nt), respectively, excluding the poly(A) tail. They share 80.0%/82.0% and 98.0%/97.0% nt/amino acid (aa) sequence identity, which is above the species demarcation value, in the previously reported AnMV and RanMMV coat protein sequences, but they share 69.0%/70.0% nt/aa sequence identity or less with other potyviruses in all 10 mature protein coding regions of the genome. Additionally, phylogenetic analysis confirmed the relationship of the AnMV and RanMMV genome sequences to previously reported partial sequences and placed them within the genus Potyvirus. These results show that these two viruses represent separate species within the genus Potyvirus.

Complete nucleotide sequence of chrysanthemum mosaic-associated virus, a novel emaravirus infecting chrysanthemum.

Here, we report the complete genome sequence of chrysanthemum mosaic-associated virus (ChMaV), a putative new member of the genus Emaravirus. The ChMaV genome comprises seven negative-sense RNA segments (RNAs 1, 2, 3a, 3b, 4, 5, and 6), and the amino acid sequences of its RNA-dependent RNA polymerase (RNA1), glycoprotein precursor (RNA2), nucleocapsid protein (RNA3), and movement protein (RNA4) showed the closest relationship to pear chlorotic leaf spot-associated virus. Phylogenetic analysis showed that it clusters with emaraviruses whose host plants originate from East Asia.

Novel degenerate primer sets for the detection and identification of emaraviruses reveal new chrysanthemum species.

Emaraviruses are a genus of plant viruses that have been newly described in the past decade. These viruses, some of which are transmitted by eriophyid mites, are important pathogens of cereals, fruits, and ornamental trees worldwide. This study used sequence data for emaraviruses to design new degenerate primer sets that identify an extensive range of known and unknown emaraviruses. Sequence alignment of the amino acid and nucleotide sequences of RNA-dependent RNA polymerases for 11 accessions among nine emaraviruses confirmed the presence of seven conserved motifs (Pre-A, F, A, B, C, D, and E). Subsequently, new degenerate primers were designed based on motifs F, A, and B, which were the most conserved among the seven motifs. Reverse transcription-polymerase chain reaction using these primers detected known emaraviruses more efficiently than previously known primers. These new primers enabled the identification of a partial nucleotide sequence of a putative novel emaravirus from chrysanthemum leaves showing mosaic or yellowish ringspot symptoms known to be associated with eriophyid mites, Paraphytoptus kikus. These sequences were specifically detected from the symptomatic leaves of a chrysanthemum, and the putative emaravirus was tentatively named chrysanthemum mosaic-associated virus.

First report of pear chlorotic leaf spot-associated virus on Japanese and European pears in Japan and its detection from an eriophyid mite.

In May 2018, three leaf samples were collected from Japanese pear trees cv. "Hosui" that exhibited typical chlorotic spot symptoms (Supplementary Figure S1) in a germplasm nursery in Tsukuba, Ibaraki. Total RNA was prepared using the rapid CTAB method (Gambino et al. 2008) for high-throughput sequencing, as described by Kubota et al. (2020). In brief, after removing ribosomal RNAs, a library was constructed by fragmenting RNA, synthesizing cDNA, and polymerase chain reaction (PCR) amplification. Sequencing was performed using NovaSeq 6000 sequencer (Illumina, San Diego, CA, U.S.A.) with paired-end 150 nt reads. De novo assembly was performed using CLC Genomics Workbench 11.0 Software (Qiagen, Hilden, Germany), with a minimum length of 500 bp. A total of 36,017 contigs derived from 33,565,182 reads were obtained and subjected to BLASTX search against the GenBank sequence database as of January 2019. Viruses commonly found in stone fruits, i.e., apple stem pitting virus, apple green crinkle-associated virus, apricot latent virus (foveaviruses), and apple stem grooving virus (a capillovirus), were detected. In addition, five contigs with amino acid sequence homologies to P1-P4 of known emaraviruses and the P7 of High Plains wheat mosaic virus (Tatineni et al. 2014) were detected and designated as PEV-Jp. The complete nucleotide (nt) sequences of the five segments of PEV-Jp were determined by Sanger sequencing of cloned reverse transcription (RT)-PCR amplification products using the primers shown in Supplementary Table S1; the 5'- and 3'-terminal sequences were RACE verified (Takara Bio, Shiga, Japan). In pairwise comparisons, the complete RNA1 to RNA5 of PEV-Jp (LC554756-760) shared 90.7% to 98.7% nt identities with those of PCLSaV-CG1 (MK602177-181), indicating that PEV-Jp is an isolate of PCLSaV. Using newly designed segment-specific primers (Supplementary Table S1), 12 symptomatic Japanese pear trees cv. "Kosui" sampled in 2020 from the same nursery tested positive for PCLSaV by RT-PCR while 12 symptomless trees were negative for the virus. Similar chlorotic spots, sometimes accompany necrotic spots, were observed on European pear (Pyrus communis) cv. "Le Lectier." (Fig. S1F) in Niigata in 2019; PCLSaV was detected by RT-PCR in leaf tissue samples from symptomatic trees (n = 3/3) but not in symptomless trees (n = 0/2). No vector for PCLSaV has been identified (Liu et al. 2020) but acaricide sprays in the early spring are effective for preventing occurrence of chlorotic spots in pear orchards (Nakai et al. 2018). Since the infestations of Eriophyes chibaensis Kadono, an eriophyid mite often observed on the Japanese pear (Fig. S1G to S1I) (Kadono, 1981), has been associated with occurrences of the chlorotic spots (Shimizu et al. 2019), samples of E. chibaensis individuals were collected from PCLSaV-positive Japanese pear cvs. "Akizuki" and "Kosui"and P. communis cv. "Le Lectier." for total nucleic acid isolations via phenol-chloroform extraction, followed by quantitative RT-PCR (Supplementary Table S1). The expected RNA1 and RNA5 specific 150 bp products were detected from mite samples collected from Akizuki (n = 6/12), Kosui (n = 13/18), and Le Lectier (n = 6/8). The results indicate that E. chibaensis can ingest PCLSaV and may be a potential vector for the virus, although additional experiments are needed to demonstrate its vector competency. To our knowledge, this is the first report of PCLSaV in Japan and the first report of its detection in E. chibaensis.

Perilla Mosaic Virus Is a Highly Divergent Emaravirus Transmitted by Shevtchenkella sp. (Acari: Eriophyidae).

Shiso (Perilla frutescens var. crispa) is widely grown as an important vegetable or herb crop in Japan. Beginning around the year 2000, occurrences of severe mosaic symptoms on shiso were documented and gradually spread across Kochi Prefecture, one of four major shiso production areas in Japan. Next generation sequencing and cloning indicated the presence of a previously unknown virus related to the members of the genus Emaravirus, for which we proposed the name Perilla mosaic virus (PerMV). The genome of PerMV consists of 10 RNA segments, each encoding a single protein in the negative-sense orientation. Of these proteins, P1, P2, P3a, P3b, P4, and P5 show amino acid sequence similarities with those of known emaraviruses, whereas no similarities were found in P6a, P6b, P6c, and P7. Characteristics of the RNA segments as well as phylogenetic analysis of P1 to P4 indicate that PerMV is a distinct and highly divergent emaravirus. Electron microscopy observations and protein analyses corresponded to presence of an emaravirus. Transmission experiments demonstrated that an eriophyid mite, Shevtchenkella sp. (family Eriophyidae), transmits PerMV with a minimum 30-min acquisition access period. Only plants belonging to the genus Perilla tested positive for PerMV, and the plant-virus-vector interactions were evaluated. The nucleotide sequences reported here are available in the DDBJ/ENA/GenBank databases under accession numbers LC496090 to LC496099.

Complete genome sequence of a divergent strain of potato virus P isolated from Solanum tuberosum in Russia.

Contigs with sequence similarity to potato virus P (PVP), which belongs to the genus Carlavirus, were identified by high-throughput sequencing analysis in potato tubers collected from a farmer's potato production field in Surazhevka, Artyom, Primorskiy Krai (Russia) in 2018. The complete genome sequence of this virus consisted of 8,394 nucleotides, excluding the poly(A) tail. This is the first report of PVP being detected outside South America. The isolate had high sequence similarity to PVP isolates from Argentina and Brazil, but low sequence similarity was observed in the genes encoding the RNA-dependent RNA polymerase (69% nucleotide sequence identity and 80% amino acid sequence identity) and coat protein (78% nucleotide sequence identity and 89% amino acid sequence identity). Phylogenetic analysis revealed that this PVP-like virus clustered with known PVP isolates but was distinct from them. Comparison of the sequences using the classification criteria of the ICTV indicated that this PVP-like virus is a strain of PVP.

Influence of the terminal left domain on horizontal and vertical transmissions of tomato planta macho viroid and potato spindle tuber viroid through pollen.

Viroids can be transmitted vertically and/or horizontally by pollen. Tomato planta macho viroid (TPMVd) has a high rate of horizontal transmission by pollen, whereas potato spindle tuber viroid (PSTVd) does not. To specify the domain(s) involved in horizontal transmission, four viroid chimeras were created by exchanging the terminal left (TL) and/or pathogenicity (P) domains between PSTVd and TPMVd. PSTVd-based chimeras containing TPMVd-TL and P, or TPMVd-TL alone, displayed a high rate of horizontal transmission. TPMVd-based chimeras containing PSTVd-TL and P lost infectivity, and those containing PSTVd-TL alone displayed a low rate of horizontal transmission. In addition, the vertical transmission rate was also higher in the mutants containing TPMVd-TL than in the others. These findings indicate that the sequences or structures in the TL and P (although the role is limited) domains are important not only for horizontal but also for vertical transmission by pollen.

Vertical and Horizontal Transmission of Pospiviroids.

Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens. Some viroids are vertically transmitted through both viroid-infected ovule and pollen. For example, potato spindle tuber viroid, a species that belongs to Pospiviroidae family, is delivered to the embryo through the ovule or pollen during the development of reproductive tissues before embryogenesis. In addition, some of Pospiviroidae are also horizontally transmitted by pollen. Tomato planta macho viroid in pollen infects to the ovary from pollen tube during pollen tube elongation and eventually causes systemic infection, resulting in the establishment of horizontal transmission. Furthermore, fertilization is not required to accomplish the horizontal transmission. In this review, we will overview the recent research progress in vertical and horizontal transmission of viroids, mainly by focusing on histopathological studies, and also discuss the impact of seed transmission on viroid dissemination and seed health.

Differences in dynamics of horizontal transmission of Tomato planta macho viroid and Potato spindle tuber viroid after pollination with viroid-infected pollen.

For viroids, pollen transmission is an important transmission pathway to progeny seeds and new hosts. In the current study, we found that Tomato planta macho viroid (TPMVd)-but not Potato spindle tuber viroid (PSTVd)-was horizontally transmitted by pollen from petunia plants. Using tissue-printing hybridization to track the changes in viroid distribution after pollination, we noted that TPMVd was present in petunia stigma, styles, and eventually ovaries, whereas PSTVd was detected in the stigma and upper style but not the ovary. These findings suggest that horizontal transmission of viroids depends on the infection of the lower style and ovary during the elongation of pollen tubes after pollination. Additionally, TPMVd was transmitted horizontally, leading to systematic infection, when we used TPMVd-infected petunia pollen to pollinate the flowers of healthy tomato plants. Fertilization typically does not occur after heterologous pollination and thus likely is not required to accomplish horizontal transmission of viroids.

Distribution of Tomato planta macho viroid in germinating pollen and transmitting tract.

Vertical and horizontal pollen transmission is important for efficient infection by viroids. Vertical pollen transmission of viroids is attributed to the infection by viroid in the embryo sac through infected pollen. To identify the viroid infection in pollen and pollen tubes elongating through the transmitting tract, we used in situ hybridization to histochemically analyze the distribution of Tomato planta macho viroid (TPMVd) in pollen grains, the stigma, and style of petunia plants. TPMVd was present in the generative nucleus and vegetative nucleus of mature infected pollen grains and germinating pollen grains. During pollen tube growth, TPMVd was present in the vegetative nucleus and two sperm nuclei, which were generated by division of the generative nucleus in the style transmitting tract. These findings indicated that viroid infection in sperm nuclei is responsible for vertical pollen transmission of viroids. TPMVd infection from TPMVd-infected pollen tubes to the transmitting tract was not observed. In addition, TPMVd signals were not confirmed in the stigma and transmitting tract of TPMVd-infected petunia plants, suggesting that viroids may not replicate in these tissues at the stage of mature style. Therefore, TPMVd may leak from the pollen tube somewhere in the ovary, except in the transmitting tract, during the horizontal transmission of TPMVd.

Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses.

The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as "DECS-C," is a powerful method for detecting novel plant viruses.

Matrix remodeling and cytological changes during spontaneous cartilage repair.

During the repair of articular cartilage, type I collagen (COL1)-based fibrous tissues change into a mixture of COL1 and type II collagen (COL2) and finally form hyaline cartilaginous tissues consisting of COL2. In order to elucidate the changes that occur in the matrix during cartilage repair and the roles of fibroblasts and chondrocytes in this process, we generated a minimal cartilage defect model that could be spontaneously repaired. Defects of 0.3 mm were created on the patellofemoral articular cartilage of rats using an Er:YAG laser and were observed histologically, ultrastructurally and histochemically. At week 2 after this operation, fibroblastic cells were found to be surrounded by COL1 throughout the area of the defect. These cells became acid phosphatase positive by week 4, both taking in and degrading collagen fibrils. Thereafter, the cells became rounded, with both COL1 and 2 evident in the matrix, and showed immunolocalized matrix metalloproteinase-1 or -9. In the region of the bone marrow, the cells became hypertrophic and were surrounded mainly by COL2 and proteoglycans. By the eighth week, the cartilaginous matrix was found to contain abundant COL2, in which collagen fibrils of various diameters were arranged irregularly. These morphological changes suggested that the fibroblastic cells both produce and resolve the matrix and undertake remodeling to become chondrocytes by converting from a COL1- into a COL2-dominant matrix. This process eventually forms new articular cartilage, but this is not completely identical to normal articular cartilage at the ultrastructural level.