Adoption of the 2A Ribosomal Skip Principle to Track Assembled Virions of Pepper Mild Mottle Virus in Nicotiana benthamiana.

The coat protein (CP) is an important structural protein that plays many functional roles during the viral cycle. In this study, the CP of pepper mild mottle virus (PMMoV) was genetically fused to GFP using the foot-and-mouth disease virus peptide 2A linker peptide and the construct (PMMoV-GFP2A) was shown to be infectious. The systemic spread of the virus was monitored by its fluorescence in infected plants. Electron microscopy and immunocolloidal gold labelling confirmed that PMMoV-GFP2A forms rod-shaped particles on which GFP is displayed. Studies of tissue ultrastructure and virion self-assembly confirmed that PMMoV-GFP2A could be used to monitor the real-time dynamic changes of CP location during virus infection. Aggregations of GFP-tagged virions appeared as fluorescent plaques in confocal laser microscopy. Altogether, PMMoV-GFP2A is a useful tool for studying the spatial and temporal changes of PMMoV CP during viral infection.

Complete genome sequence of Paris polyphylla chlorotic mottle virus infecting Paris polyphylla var. yunnanensis in southwest China.

A novel virus infecting a Paris polyphylla var. yunnanensis plant, tentatively named "Paris polyphylla chlorotic mottle virus" (PpCMV), was discovered in the city of Lijiang, Yunnan Province, China. Its genome consists of 6384 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and contains two open reading frames: ORF1 and ORF2. ORF1 is 6150 nt in length, encoding a large 2050-aa polyprotein with at least two conserved regions encoding a replication-associated protein and a coat protein, the latter of which is located at the 3' end of ORF1. ORF2, consisting of 1185 nt, is located within ORF1 but has a different reading frame. It encodes a 394-aa-long putative movement protein. Phylogenetic analysis based on amino acid sequences revealed that the newly discovered virus exhibited the closest relationship to Hobart betaflexivirus 1 and rhodiola betaflexivirus 1, both of which belong to the genus Capillovirus, sharing 48.8% and 36.5% amino acid sequence identity, respectively, in the structural protein. This is the first report of the complete genome sequence of PpCMV in China.

Complete genome sequence of iris potyvirus B infecting Lilium lancifolium in China.

The complete genome sequence of a virus from lily (Lilium lancifolium Thunb.) growing in Huoshan County, Anhui Province, China, was determined. The whole genome consists of 9558 nucleotides, excluding the poly(A) tail, and encodes a 3061-amino-acid polyprotein (GenBank number ON365558) typical of potyviruses. This is the first complete genome sequence of iris potyvirus B (IPB), for which only a partial sequence from Iris domestica was reported previously. Comparative analysis of this genome sequence with those of closely related potyviruses identified nine cleavage sites and the conserved motifs typical of potyviruses. The complete polyprotein ORF shares 73.6% nucleotide and 81.6% amino acid sequence identity with that of iris potyvirus A (IPA, GenBank number MH898493). Phylogenetic analysis showed that IPB is related to IPA and clusters in a group with lily yellow mosaic virus (LYMV). This is the first report of IPB infecting lily plants.

Tumor Microenvironment Responsive Pepper Mild Mottle Virus-Based Nanotubes for Targeted Delivery and Controlled Release of Paclitaxel.

Plant virus nanoparticles (PVNPs) have been widely used for drug delivery, antibody development and medical imaging because of their good biodegradation and biocompatibility. Particles of pepper mild mottle virus (PMMoV) are elongated and may be useful as drug carriers because their shape favours long circulation, preferential distribution and increased cellular uptake. Moreover, its effective degradation in an acidic microenvironment enables a pH-responsive release of the encapsulated drug. In this study, genetic engineering techniques were used to form rod-shaped structures of nanoparticles (PMMoV) and folated-modified PMMoV nanotubes were prepared by polyethylene glycol (PEG) to provide targeted delivery of paclitaxel (PTX). FA@PMMoV@PTX nanotubes were designed to selectively target tumor cells and to release the encapsulated PTX in response to pH. Efficient cell uptake of FA@PMMoV@PTX nanotubes was observed when incubated with tumor cells, and FA@PMMoV@PTX nanotubes had superior cytotoxicity to free PTX, as reflected by cell survival and apoptosis. This system is a strong candidate for use in developing improved strategies for targeted treatment of tumors.

eIF4A, a target of siRNA derived from rice stripe virus, negatively regulates antiviral autophagy by interacting with ATG5 in Nicotiana benthamiana.

Autophagy is induced by viral infection and has antiviral functions in plants, but the underlying mechanism is poorly understood. We previously identified a viral small interfering RNA (vsiRNA) derived from rice stripe virus (RSV) RNA4 that contributes to the leaf-twisting and stunting symptoms caused by this virus by targeting the host eukaryotic translation initiation factor 4A (eIF4A) mRNA for silencing. In addition, autophagy plays antiviral roles by degrading RSV p3 protein, a suppressor of RNA silencing. Here, we demonstrate that eIF4A acts as a negative regulator of autophagy in Nicotiana benthamiana. Silencing of NbeIF4A activated autophagy and inhibited RSV infection by facilitating autophagic degradation of p3. Further analysis showed that NbeIF4A interacts with NbATG5 and interferes with its interaction with ATG12. Overexpression of NbeIF4A suppressed NbATG5-activated autophagy. Moreover, expression of vsiRNA-4A, which targets NbeIF4A mRNA for cleavage, induced autophagy by silencing NbeIF4A. Finally, we demonstrate that eIF4A from rice, the natural host of RSV, also interacts with OsATG5 and suppresses OsATG5-activated autophagy, pointing to the conserved function of eIF4A as a negative regulator of antiviral autophagy. Taken together, these results reveal that eIF4A negatively regulates antiviral autophagy by interacting with ATG5 and that its mRNA is recognized by a virus-derived siRNA, resulting in its silencing, which induces autophagy against viral infection.

Biological and Genetic Characterization of Pod Pepper Vein Yellows Virus-Associated RNA From Capsicum frutescens in Wenshan, China.

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with some viruses of the genus Polerovirus. Pod pepper vein yellows virus (PoPeVYV) was recently reported as a new recombinant polerovirus causing interveinal yellowing, stunting, and leaf rolling in Capsicum frutescens plants at Wenshan city, Yunnan province, China. The complete genome sequence of its associated RNA has now been determined by next-generation sequencing and reverse transcription (RT) polymerase chain reaction (PCR). PoPeVYV-associated RNA (PoPeVYVaRNA) (GenBank Accession No. MW323470) has 2970 nucleotides and is closely related to other group II tlaRNAs, particularly tobacco bushy top disease-associated RNA (TBTDaRNA, GenBank Accession No. EF529625). In infection experiments on Nicotiana benthamiana and C. frutescens plants, synergism between PoPeVYVaRNA and PoPeVYV was demonstrated, leading to severe interveinal yellowing of leaves and stunting of plants. The results provide further information on the genetic and biological properties of the various agents associated with pepper vein yellows disease (PeVYD).

Construction of an infectious full-length cDNA clone of potato aucuba mosaic virus.

Potato aucuba mosaic virus (PAMV), a positive single-strand RNA virus, has one of the longest genomes of the viruses in the genus Potexvirus. In 2019, potato samples with mottle and crinkling symptoms from Huzhou, Zhejiang province, China, were identified to be infected with PAMV, potato virus X (PVX), and potato virus Y (PVY) by transcriptome sequencing. To study the effects of single infection by PAMV, the full-length sequence of PAMV from Huzhou (MT193476) was determined and an infectious full-length cDNA clone was constructed. This cDNA clone was infectious by agro-infiltration, leading to systemic symptoms in Nicotiana benthamiana, tomato, pepper, and potato.

Identification of viral particles in the apoplast of Nicotiana benthamiana leaves infected by potato virus X.

The apoplast is the extracellular space for signalling, nutrient transport, and plant-microbe interactions, but little is known about how plant viruses use the foliar apoplast. Proteomic analysis of the apoplasts isolated from potato virus X (PVX)-infected Nicotiana benthamiana plants showed that the coat protein (CP) is the dominant viral component. The presence of the CP in the apoplast was confirmed by western blot, viral nucleic acid was detected by reverse transcription-PCR and northern blot, and viral particles were observed by transmission electron microscopy (TEM). The apoplast from infected leaves was infectious if rubbed onto healthy leaves but not when infiltrated into them. The exosomes were separated from the apoplast fluid by high-speed centrifugation and TEM showed that PVX particles were not associated with the exosomes. These results suggest that PVX virions are released to the N. benthamiana apoplast in a one-way manner and do not share the bidirectional transport of exosomes.

Pod pepper vein yellows virus, a new recombinant polerovirus infecting Capsicum frutescens in Yunnan province, China.

Pepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5' half of its genome (encoding P0 to P4), PoPeVYV is most similar (93.1% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3'-part encoding P5, where it is most similar (91.7% nt identity) to tobacco vein distorting virus (TVDV, EF529624) suggesting a recombinant origin. Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt, with PeVYV-3 as the major parent but with the region 4126-5192 nt derived from TVDV as the minor parent. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.

Two distinct begomoviruses associated with an alphasatellite coinfecting Emilia sonchifolia in Thailand.

Emilia sonchifolia is a traditionally used medicinal plant that is widespread in tropical and subtropical regions of the world. Yellow vein symptoms were observed in E. sonchifolia plants in fields in the county of Koh Samui, Surat Thani Province, Thailand, in August 2015. Two distinct begomoviruses, designated TH4872-6 and TH4872-9, and an associated alphasatellite were obtained from an E. sonchifolia leaf sample (TH4872). Sequence analysis showed that the full-length sequence of TH4872-6 was most closely related to that of ageratum yellow vein China virus (AYVCNV), with 85.7% identity, suggesting that it is a novel begomovirus, while the TH4872-9 sequence closely resembled cotton leaf curl Multan virus (CLCuMuV) with 99.1% identity. The alphasatellite sequence showed the highest nucleotide sequence identity (92.8%) to an isolate of tobacco curly shoot alphasatellite (TbCSA) originating from China. Recombination analysis revealed that the isolate TH4872-6 is a potential recombinant begomovirus, derived from ageratum yellow vein virus (AYVV) and tobacco leaf curl Thailand virus (TbLCTHV). This study represents the first report of begomoviruses identified in E. sonchifolia in Thailand.

Anti-TMV activity and functional mechanisms of two sesquiterpenoids isolated from Tithonia diversifolia.

Unlike chemical pesticides, antiviral plants are biodegradable, replenishable and safe. In this study, 14 sesquiterpene compounds from Tithonia diversifolia were tested for their activities against Tobacco mosaic virus (TMV) using the half-leaf method. Tagitinin C (Ses-2) and 1ß-methoxydiversifolin-3-0-methyl ether (Ses-5) were found to have in vivo curative activities of 62.86% and 60.27% respectively, at concentrations of 100µg/mL, respectively. In contrast, the in vivo curative inhibition rate of control agent ningnanmycin was 52.48%. Indirect enzyme-linked immunosorbent assay (ID-ELISA) also verified Ses-2 and Ses-5 had higher inhibition activities than the control agent ningnanmycin. Additionally, qRT-PCR showed that both Ses-2 and Ses-5 can partly inhibit the expression of CP and RdRp, two genes that play key roles in TMV infection. When TMV started to systemically spread, Ses-2 inhibited CP expression while Ses-5 inhibited RdRp expression. These results suggest that the two bio-agents have anti-TMV activities and may be used as bio-pesticides to control the plant virus.

Full-length M and L RNA sequences of tospovirus isolate 2009-GZT, which causes necrotic ringspot on tomato in China.

The virus isolate 2009-GZT, collected from tomato in Guizhou province of China, was identified as a new member of the genus Tospovirus based on its S RNA sequence. Because its provisional name, "tomato necrotic spot virus" (TNSV), was identical to an already existing member of the genus Ilarvirus, 2009-GZT was renamed "tomato necrotic spot-associated virus" (TNSaV). In this study, the full-length sequences of the genomic M and L RNAs of TNSaV were determined and analyzed. The M RNA has 4,773 nucleotides (nt), encoding the NSm protein of 309 aa (34.4 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1123 aa (128 kDa) in the viral complementary (vc) strand. The NSm and Gn/Gc of TNSaV share the highest aa sequence identity (86.2 % and 86.9 %, respectively) with those of tomato zonate spot virus. The L RNA contains 8,908 nt and codes for the putative RNA-dependent RNA polymerase (RdRp) of 2885 aa (332 kDa) in the vc strand. The RdRp of TNSaV shares the highest aa sequence identity (85.2 %) with that of calla lily chlorotic spot virus (CCSV). Serological assays showed that TNSaV cross-reacts with rabbit antisera against watermelon silver mottle virus (WSMoV) NP and CCSV NP, indicating that TNSaV is a member of the WSMoV serogroup.

Identification of a new tospovirus causing necrotic ringspot on tomato in China.

BACKGROUND: Emerging tospoviruses cause significant yield losses and quality reduction in vegetables, ornamentals, and legumes throughout the world. So far, eight tospoviruses were reported in China. Tomato fruits displaying necrotic and concentric ringspot symptoms were found in Guizhou province of southwest China. FINDING: ELISA experiments showed that crude saps of the diseased tomato fruit samples reacted with antiserum against Tomato zonate spot virus (TZSV). Electron microscopy detected presence of quasi-spherical, enveloped particles of 80-100 nm in such saps. The putative virus isolate was designated 2009-GZT. Mechanical back-inoculation showed that 2009-GZT could infect systemically some solanaceous crop and non-crop plants including Capiscum annuum, Datura stramonium, Nicotiana benthamiana, N. rustica, N. tabacum and Solanum lycopersicum. The 3012 nt full-length sequence of 2009-GZT S RNA shared 68.2% nt identity with that of Calla lily chlorotic spot virus (CCSV), the highest among all compared viruses. This RNA was predicted to encode a non-structural protein (NSs) (459 aa, 51.7 kDa) and a nucleocapsid protein (N) (278 aa, 30.3 kDa). The N protein shared 85.8% amino acid identity with that of CCSV. The NSs protein shared 82.7% amino acid identity with that of Tomato zonate spot virus(TZSV). CONCLUSION: Our results indicate that the isolate 2009-GZT is a new species of Tospovirus, which is named Tomato necrotic spot virus (TNSV). This finding suggests that a detailed survey in China is warranted to further understand the occurrence and distribution of tospoviruses.

Weak affinity ligands selection using quartz crystal microbalance biosensor: multi-hydroxyl amine ligands for protein separation.

Multi-hydroxyl amines including tris(hydroxymethyl)aminomethane (Tris), serinol and ethanolamine were selected as weak affinity ligands using a rapid screening by quartz crystal microbalance (QCM) biosensor. Based on the specific recognition between the ligands and two proteins, lysozyme (LZM) and cytochrome c (Cyt c), a weak affinity chromatography method was developed for specific separation of the two proteins. The frontal analysis results showed that the apparent dissociation constants (K(D)) of ligand-protein complexes were all in the order of weak affinity (10(-4) M). By weak affinity columns modified with the three multi-hydroxyl amines individually, LZM and Cyt c were baseline separated as retarded peaks from non-specific protein and each other in a single cycle of loading and eluting. Moreover, the Tris-modified column typically showed the satisfactory repeatability and stability as a new type of weak affinity columns. The present strategy composed of QCM selecting and affinity chromatography separating was promising to extend the variety of weak affinity ligands and develop inexpensive specific affinity methods for separation and purification of multiple proteins on one single column.

Characterization of tomato zonate spot virus, a new tospovirus in China.

An isolate of a new tospovirus species, causing concentric zoned ringspots on fruits and necrotic lesions on leaves of infected plants, was characterised based on particle morphology, host range and serological properties. The complete nucleotide sequences of large (L), medium (M), and small (S) RNAs of this virus were found to contain 8919, 4945, and 3279 nts respectively. The L RNA encoded the RNA-dependent RNA polymerase (RdRp) (2885 aa, 332.7 kDa). The M RNA encoded a non-structural (NSm) protein (309 aa, 34.4 kDa) and a viral glycoprotein precursor (Gn/Gc) (1122 aa, 127.4 kDa). The S RNA encoded a non-structural protein (NSs) (459 aa, 51.9 kDa) and the nucleocapsid (N) protein (278 aa, 30.6 kDa). This N protein shared amino acid identities of 80.9% with those of calla lily chlorotic spot virus. Our results suggest that the virus studied here belongs to a new tospovirus species, for which the name tomato zonate spot virus is proposed.