Complete genome sequence of Aconitum amalgavirus 1, a distinct member of the genus Amalgavirus.

A novel virus named Aconitum amalgavirus 1 (AcoAV-1) was identified in Chinese aconite (Aconitum carmichaelii) plants. The complete genome of AcoAV-1 is 3,370 nucleotides long, containing two partially overlapping open reading frames encoding a putative coat protein and a RNA-dependent RNA polymerase, respectively. Its fusion protein shares 34.9%-50.7% amino acid sequence identity with other amalgaviruses. Phylogenetic analysis showed that this virus formed a clade with blueberry latent virus and four other related viruses, suggesting that it belongs to the genus Amalgavirus in the family Amalgaviridae.

Genomic characterization of a new enamovirus infecting common bean.

A novel enamovirus was identified in common bean plants with disease symptoms. Its genome of 5,781 nucleotides (nt) contains five open reading frames. This virus and other members of the genus Enamovirus share 50.4-68.4% nucleotide sequence identity in the complete genome and 19.9-51.9% amino acid sequence identity in the P0 protein, 24.9-52.5% in P1, 33.4-62.9% in P1-P2, 30.6-81.1% in P3, and 32.3-74.2% in P3-P5. Phylogenetic analysis showed that the virus is most closely related to alfalfa enamovirus 1 and pea enation mosaic virus 1 in the genus Enamovirus of the family Solemoviridae. These results suggest that this virus, tentatively named "bean enamovirus 1", should be classified as a member of a new species in the genus Enamovirus.

First Report of Pea as a Natural Host of tomato mottle mosaic virus in China.

Tomato mottle mosaic virus, a new species in the genus Tobamovirus and family Virgaviridae, was first reported on tomato in Mexico in 2013 (Li et al. 2013). Subsequently, tomato mottle mosaic virus (ToMMV) was found infecting pepper and eggplant in China (Li et al. 2014; Chai et al. 2018). ToMMV was thought to pose a serious risk to solanaceous crops due to its potential to break resistance and numerous transmission ways (Li et al. 2020), meanwhile, some plant species in the families of Amaranthaceae, Brassicaceae, Cucurbitaceae, and Verbenaceae, were found as the hosts of ToMMV under experimental condition (Sui et al. 2017; Li et al. 2020). To clarify the occurrence of ToMMV in Yunnan province of China, 395 plant samples, including tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.) and pea (Pisum sativum L.), exhibiting viral-like symptoms were collected in major crop plantations in Yunnan province in 2020. Total nucleic acids were extracted from the diseased samples using a CTAB method (Li et al. 2008), and tested by reverse transcription polymerase chain reaction (RT-PCR) with ToMMV specific primers, ToMMVMPF (5'-ATGGCTCTAACTGTTAGTGGT-3') and ToMMVMPR (5'-TTAATACGAATCAGATCCCGCG-3'), which were designed based on the movement protein gene sequence of ToMMV YYMLJ isolate (KR824950). ToMMV was detected in 13 symptomatic samples (11 cherry tomato and 2 green pea plants) with a total detection rate of 3.29%. An 807-bp fragment was amplified from 2 out of 86 pea samples showing foliar chlorosis, mosaic, malformation and necrosis symptoms, and the amplicons were cloned and sequenced. Sequences obtained from the two pea samples were identical. Therefore, one sequence was deposited in the GenBank (accession no. MW561348). BLAST search result showed that the nucleotide sequence had the highest identity of 99.88% with the ToMMV TiLhaLJ isolate (KR824951). The ToMMV infection on the two pea samples was also verified by dot-enzyme immunoassay (Dot-ELISA) using ToMMV monoclonal antibody (kindly provided by Dr. Jianxiang Wu, Zhejiang University, China). To determine the pathogenicity of ToMMV on pea, ToMMV infectious cDNA clone was used to inoculate into 3-week-old healthy pea plants via an Agrobacterium-mediated method (Tu et al. 2021). Mottle or slight mosaic, chlorosis and malformation symptoms were observed on the upper leaves of the 8 out of 12 inoculated plants 8 days postinoculation, and ToMMV could be detected by RT-PCR from the 8 symptomatic plants but not from the asymptomatic and healthy control pea plants with the above described ToMMV specific primer pair. ToMMV has now only been detected in Mexico, China, Spain, USA, Israel and Australia on tomato, pepper and eggplant (Webster et al. 2014; Turina et al. 2016; Ambrós et al. 2017; Lovelock et al. 2020). To the best of our knowledge, this is the first report of natural infection of ToMMV in pea as well as the natural infection of ToMMV on plants outside of the family Solanaceae. The result also implies that ToMMV has a potential risk to more crops in the field. Since pea, pepper and tomato are economically important cash crops in China, proper virus management strategies for the cultivation of these crops should be adopted.

A Multiyear Survey and Identification of Pepper- and Tomato-Infecting Viruses in Yunnan Province, China.

During pepper and tomato production seasons in 2013-2017, large-scale virus disease surveys were conducted in different regions of Yunnan Province, China. A total of 1,267 pepper and tomato samples with various virus-like symptoms were collected and analyzed for virus infections through dot enzyme-linked immunosorbent assay (dot-ELISA), polymerase chain reaction (PCR), and reverse-transcription (RT)-PCR. The detection results showed that 19 different viruses were present in about 50.9% of the assayed samples, and among these viruses, seven viruses were found in both pepper and tomato samples. Mixed infections with two to three of the 15 identified mixed infection types were found in the pepper samples and 10 identified mixed infection types were found in the tomato samples. Among the infected samples, Tomato spotted wilt orthotospovirus (TSWV) was the most common virus, with a detection rate of about 20.0% followed by Pepper vein yellows virus (PeVYV, 13.0%). This survey revealed for the first time that pepper is a natural host of Tobacco vein distorting virus (TVDV) worldwide and tomato is a natural host of Potato leafroll virus (PLRV) in China. PeVYV, Tobacco mild green mosaic virus (TMGMV) and Wild tomato mosaic virus (WTMV) were first time found in pepper and Tomato mottle mosaic virus (ToMMV) and Chilli veinal mottle virus (ChiVMV) were first time found in tomato in Yunnan Province. Finally, the virus incidences were higher in Kunming, Yuxi, Chuxiong, and Honghe region than other regions.

The occurrence of Pea enation mosaic virus 1 and Pea enation mosaic virus 2 from disease-affected pea fields in China.

Pea (Pisum sativum L.) is an economically important legume crop that is commonly used as dry beans, fresh peas, pods and shoots (Guo et al. 2009). Pea enation mosaic is an important virus disease of pea caused by two viruses in an obligate symbiosis, pea enation mosaic virus 1 (PEMV-1, Enamovirus, Luteoviridae) and pea enation mosaic virus 2 (PEMV-2, Umbravirus, Tombusviridae) (Hema et al. 2014). In November 2019, foliar yellow mosaic and vein enations symptoms were observed from pea plants in five fields of Honghe autonomous prefecture, Yunnan province, China. Incidence of symptomatic plants ranged from 20 to 40% and was distributed in both small and large fields. Leaves with typical virus-like symptoms were collected from five symptomatic pea plants in two fields and used for total RNA extraction. The five extracts of equimolar quantities were pooled into a sample and subjected to High Throughput Sequencing (HTS) by Illumina HiSeq system. Analyses of raw RNA reads were performed using CLC Genomics Workbench 12 (Qiagen). A total of 60,009,746 RNA reads were obtained from the sample, and de novo assembly of the reads using the CLC Genomics generated 88,105 contigs. BLASTN searches revealed the presence of contigs with high similarities to PEMV-1, PEMV-2, Pea seed-borne mosaic virus, and Bean yellow mosaic virus. To confirm the presence of PEMV-1 and PEMV-2 in the samples, two virus-specific primer pairs were designed based on the contig sequences obtained by HTS in this study. Primer pairs PEMV-1F/PEMV-1R (5'-ATGCCGACTAGATCGAAATC-3'/5'-TCAGAGGGAGGCATTCATTA-3') that flank the cp gene of PEMV-1 and PEMV-2F/PEMV-2R (5'-ATGACGATAATCATTAATG-3'/5'-TCACCCGTAGTGAGAGGCA-3') that target the ORF3 region of PEMV-2 were used to amplify the two viruses in RT-PCR. DNA fragments of the expected sizes (PEMV-1, 570 bp; PEMV-2, 693 bp) were amplified from all five samples. The RT-PCR products were cloned and sequenced. Sequence analysis showed that the 570-bp amplicon (MT481989) shared the highest nucleotide sequence identity of 98.95% with PEMV-1 (Z48507), while the 693-bp fragment (MT481990) had the highest nucleotide sequence identity of 97.4% with PEMV-2 isolate JKI (MK948534). One gram of the symptomatic leaves from each of the five plants was homogenized with 5 mL of 0.01 M phosphate-buffered saline (PBS buffer), pH 7.0. Each of the resulted saps was used to inoculate onto five healthy pea seedlings. A total of 25 healthy pea seedlings were inoculated, and 16 inoculated plants developed yellowing and mottling at 10 days post inoculation (dpi); no symptoms were observed on control plants inoculated only with PBS buffer. The formation of the typical enation was observed along the veins of lower side of the symptomatic leaves of the inoculated plants at 30 dpi. PEMV-1 and PEMV-2 infection were confirmed by RT-PCR assays using the specific primer pairs described above. Although the presence of the pea enation mosaic virus complex was suspected in China based on symptomatology (Brunt et al. 1997), to our knowledge, this is the first molecular confirmation of PEMV-1 and PEMV-2 occurrence in China. The co-infection of PEMV-1 and PEMV-2 usually cause severe yield losses; therefore, integration of detection and control measures is important in pea production regions where the two viruses occurred.

Detection of tobamoviruses by RT-PCR using a novel pair of degenerate primers.

A generic RT-PCR assay was developed for the universal detection of viruses of the genus Tobamovirus using a novel pair of degenerate primers designed based on conserved regions on replicase genes of 32 tobamoviruses. The assay detected nine tobamoviruses, including six Solanaceae-infecting subgroup tobamoviruses of Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tomato mottle mosaic virus (ToMMV), Tobacco mottle green mosaic virus (TMGMV), Pepper mild mottle virus (PMMoV), Paprika mild mottle virus (PaMMV), one Orchidaceae-infecting tobamovirus of Odontoglossum ringspot virus (ORSV) and two Cucurbitaceae-infecting subgroup tobamoviruses of Cucumber green mottle mosaic virus (CGMMV) and Zucchini green mottle mosaic virus (ZGMMV), with high amplification efficiency, specificity and sensitivity. The assay was applied to detect tobamoviruses in pepper and tomato fields. Five tobamoviruses, PMMoV, TMV, ToMV, ToMMV and TMGMV, were detected from the pepper fields in single and mixed infections. Single infections of PMMoV, ToMV and ToMMV and mix-infection of ToMV + PMMoV were detected from the tomato fields. Among these viruses, PMMoV was first detected from tomato worldwide, while ToMMV was first detected from tomato plants in China. This generic assay is simple, cost-effective and has great potential to detect more tobamoviruses in the field.

A new putative deltapartitivirus recovered from Dianthus amurensis.

Two double stranded RNAs (dsRNA), likely representing the genome of a novel deltapartitivirus, provisionally named carnation cryptic virus 3 (CCV3), were recovered from Dianthus amurensis. The two dsRNAs were 1,573 (dsRNA1) and 1,561 (dsRNA2) bp in size, each containing a single open reading frame (ORF) encoding a 475- and 411-aa protein, respectively. The 475-aa protein contains a conserved RNA dependent RNA polymerase (RdRp) domain which shows significant homology to RdRps of established or putative partitiviruses, particularly those belonging to the genus Deltapartitivirus. However, it shares an amino acid identity of 75% with its closest relative, the RdRp of the deltapartitivirus beet cryptic virus 2 (BCV2), and is <62% identical to the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRps of selected partitiviruses, CCV3 clustered with BCV2 and formed a well-supported monophyletic clade with known or putative deltapartitiviruses.

The complete genome sequence, occurrence and host range of Tomato mottle mosaic virus Chinese isolate.

BACKGROUND: Tomato mottle mosaic virus (ToMMV) is a recently identified species in the genus Tobamovirus and was first reported from a greenhouse tomato sample collected in Mexico in 2013. In August 2013, ToMMV was detected on peppers (Capsicum spp.) in China. However, little is known about the molecular and biological characteristics of ToMMV. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and rapid identification of cDNA ends (RACE) were carried out to obtain the complete genomic sequences of ToMMV. Sap transmission was used to test the host range and pathogenicity of ToMMV. RESULTS: The full-length genomes of two ToMMV isolates infecting peppers in Yunnan Province and Tibet Autonomous Region of China were determined and analyzed. The complete genomic sequences of both ToMMV isolates consisted of 6399 nucleotides and contained four open reading frames (ORFs) encoding 126, 183, 30 and 18 kDa proteins from the 5' to 3' end, respectively. Overall similarities of the ToMMV genome sequence to those of the other tobamoviruses available in GenBank ranged from 49.6% to 84.3%. Phylogenetic analyses of the sequences of full-genome nucleotide and the amino acids of its four proteins confirmed that ToMMV was most closely related to Tomato mosaic virus (ToMV). According to the genetic structure, host of origin and phylogenetic relationships, the available 32 tobamoviruses could be divided into at least eight subgroups based on the host plant family they infect: Solanaceae-, Brassicaceae-, Cactaceae-, Apocynaceae-, Cucurbitaceae-, Malvaceae-, Leguminosae-, and Passifloraceae-infecting subgroups. The detection of ToMMV on some solanaceous, cucurbitaceous, brassicaceous and leguminous plants in Yunnan Province and other few parts of China revealed ToMMV only occurred on peppers so far. However, the host range test results showed ToMMV could infect most of the tested solanaceous and cruciferous plants, and had a high affinity for the solanaceous plants. CONCLUSIONS: The complete nucleotide sequences of two Chinese ToMMV isolates from naturally infected peppers were verified. The tobamoviruses were divided into at least eight subgroups, with ToMMV belonging to the subgroup that infected plants in the Solanaceae. In China, ToMMV only occurred on peppers in the fields till now. ToMMV could infect the plants in family Solanaceae and Cucurbitaceae by sap transmission.

Identification and molecular characterization of Panax notoginseng virus A, which may represent an undescribed novel species of the genus Totivirus, family Totiviridae.

A double-stranded RNA (dsRNA), PN-S, isolated from Panax notoginseng was sequenced and characterized. dsRNA PN-S was determined to be 5003 base pairs (bp) in length and to have a genome organization similar to that of totiviruses. The two deduced proteins encoded by dsRNA PN-S have the highest amino acid (aa) sequence identity to CP and RNA-dependent RNA polymerase (RdRp) of black raspberry virus F (BrVF), a tentative member of the genus Totivirus, family Totiviridae, at 39% and 54%, respectively. In a phylogenetic tree based on an alignment of the RdRp amino acid sequences of dsRNA PN-S and selected totiviruses, dsRNA PN-S formed a monophyletic clade with dsRNA viruses belonging to the genus Totivirus. These data suggest that dsRNA PN-S may represent the genome of a member of a novel species of the genus Totivirus, family Totiviridae, for which we propose the name Panax notoginseng virus A (PnVA).

Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR.

Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay was developed for simultaneous detection of the four causal agents of the disease. Four pairs of specific primers based on the conserved regions of each of the four disease agents were used in the one-tube RT-PCR. The RT-PCR products consisted of fragments of 1049 base pairs (bp) for TBTV, 792bp for TVDVaRNA, 598bp for Sat-TBTV and 357bp for TVDV, and their origins were confirmed by sequencing. Primer concentrations and cycling condition were optimized for the multiplex RT-PCR. The detection limit of the assay was up to 10(-4) dilution. The assay was evaluated using tobacco plants infected naturally with one to four target viruses, transmission vector of aphids and field samples collected from Yunnan, Hunan, and Guizhou province, China. The results show that the multiplex RT-PCR is reliable and sensitive as a simple, rapid and cost-effective method to detect these pathogens in tobacco and aphid. This assay will be useful for virus surveys when large numbers of samples are tested.