Polysaccharide Peptide Treatment Eliminates Strawberry Viruses and Promotes Strawberry Plant Growth and Rooting in Tissue Culture Media.

Based on our previous finding that polysaccharide peptide (PSP) has substantial antiviral activity, we cultured strawberry plants infected with strawberry mild yellow edge virus (SMYEV) or strawberry vein banding virus (SVBV) in Murashige and Skoog (MS) media supplemented with PSP to test its ability to eliminate these viruses. PSP not only improved the elimination of SMYEV and SVBV but also promoted the growth and rooting of strawberry plants in tissue culture. On the 45th day, the average height of the 'Ningyu' strawberry plants in the 1-mg/ml PSP treatment group was 1.91 cm, whereas that of the plants in the control group was 1.51 cm. After the same time point, the number of new leaves on the tissue culture media supplemented with 1 mg/ml and 500 µg/ml of PSP and without PSP were 4.92, 4.41, and 3.53, respectively. PSP also promoted strawberry rooting and significantly increased both the length and number of roots. In addition, after treatment with the 1-mg/ml PSP treatment in tissue culture for 45 days followed by meristem-shoot-tip culture, the elimination rates of SMYEV and SVBV in regenerated 'Ningyu' strawberry plants ranged from 60 to 100%. This study investigated the use of the antiviral agent PSP for virus elimination. PSP has a low production cost and thus has great application potential for virus elimination in crop plants.

Probing the Nucleic Acid Flexibility to Disarm the Viral Counter-Defense Machinery: Design and Characterization of Potent p19 Inhibitors.

Plant viruses are highly destructive and significant contributors to several global pandemics and epidemics in plants. A viral disease outbreak in plants can cause a scarcity of food supply and is a severe concern to humanity. The siRNA (small interfering RNA)-mediated RNA-induced silencing complex (RISC) formation is a primary defense mechanism in plants against viruses, where the RISC binds and degrades viral mRNAs. As a counter-defense, many viruses encode RNA-silencing suppressor proteins (e.g., the p19 protein from the Tombusviridae family) for viral proliferation in plants. The functional form of p19 (homodimer) binds to plant siRNA with high affinities, thereby interrupting the RISC formation and thus preventing the viral mRNA silencing in plants. By altering the RISC formation, the p19 protein helps the virus invasion in the plant and ultimately stunts host growth. In this study, we designed several modified siRNA-based molecules for p19 inhibition. The viral p19 protein is known to interact predominantly through H-bonds with 2'-OH and phosphates of the plant siRNA. We utilized this information and in silico-designed flexible substituents of siRNA, where we removed the C2'-C3' bond in each nucleotide unit. We performed all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicates each) for control/modified siRNA─p19 complexes (8 in total) followed by energetic estimations. Strikingly, in a few modified complexes, the siRNA not only retained the double-helical structural integrity but also displayed remarkably enhanced p19 binding compared to the control siRNA; hence, we consider it important to perform biological and chemical in vitro and in vivo studies on proposed flexible nucleic acids as p19 inhibitors for crop protection.

Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

Field application of nanoliposomes delivered quercetin by inhibiting specific hsp70 gene expression against plant virus disease.

BACKGROUND: The annual economic loss caused by plant viruses exceeds 10 billion dollars due to the lack of ideal control measures. Quercetin is a flavonol compound that exerts a control effect on plant virus diseases, but its poor solubility and stability limit the control efficiency. Fortunately, the development of nanopesticides has led to new ideas. RESULTS: In this study, 117 nm quercetin nanoliposomes with excellent stability were prepared from biomaterials, and few surfactants and stabilizers were added to optimize the formula. Nbhsp70er-1 and Nbhsp70c-A were found to be the target genes of quercetin, through abiotic and biotic stress, and the nanoliposomes improved the inhibitory effect at the gene and protein levels by 33.6 and 42%, respectively. Finally, the results of field experiment showed that the control efficiency was 38% higher than that of the conventional quercetin formulation and higher than those of other antiviral agents. CONCLUSION: This research innovatively reports the combination of biological antiviral agents and nanotechnology to control plant virus diseases, and it significantly improved the control efficiency and reduced the use of traditional chemical pesticides.

Recent Advances in Natural Products with Antiviral Activities.

Virus is a type of noncellular organism, which is simple in structure, small in size and contains only one kind of nucleic acid (RNA or DNA). It must be parasitized in living cells and proliferates by replication. Viruses can infect plants or animals, which leads to many epidemic diseases, such as the current pandemic COVID-19. Viral infectious diseases have brought serious threats to the health of people around the world. Natural products are chemical substances that are usually produced by living organisms and have biological or pharmacological activity. Many of these natural products show antiviral activity. Based on the increasing importance of antiviral research, this paper focuses on the discovery and development of antiviral natural products since 2010.

Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid.

While the potato spindle tuber viroid (PSTVd) variant, PSTVd-Dahlia (PSTVd-D or PSTVd-Dwt) induces very mild symptoms in tomato cultivar 'Rutgers', PSTVd-Intermediate (PSTVd-I or PSTVd-Iwt) induces severe symptoms. These two variants differ by nine nucleotides, of which six mutations are located in the terminal left (TL) to the pathogenicity (P) domains. To evaluate the importance of mutations located in the TL to the P domains, ten types of point mutants were created by swapping the nucleotides between the two viroid variants. Bioassay in tomato plants demonstrated that two mutants created on PSTVd-Iwt at positions 42 and 64 resulted in symptom attenuation. Phenotypic and RT-qPCR analysis revealed that mutation at position 42 of PSTVd-Iwt significantly reduced disease severity and accumulation of the viroid, whereas mutation at position 64 showed a significant reduction in stunting when compared to the PSTVd-Iwt infected plant. RT-qPCR analysis on pathogenesis-related protein 1b1 and chalcone synthase genes showed a direct correlation with symptom severity whereas the expansin genes were down-regulated irrespective of the symptom severity. These results indicate that the nucleotides at positions 42 and 64 are in concert with the ones at positions 43, 310, and 311/312, which determines the slower and stable accumulation of PSTVd-D without eliciting excessive host defense responses thus contributing in the attenuation of disease symptom.

Plant Virus Genome Is Shaped by Specific Dinucleotide Restrictions That Influence Viral Infection.

The presence of CpG and UpA dinucleotides is restricted in the genomes of animal RNA viruses to avoid specific host defenses. We wondered whether a similar phenomenon exists in nonanimal RNA viruses. Here, we show that these two dinucleotides, especially UpA, are underrepresented in the family Potyviridae, the most important group of plant RNA viruses. Using plum pox virus (PPV; Potyviridae family) as a model, we show that an increase in UpA frequency strongly diminishes virus accumulation. Remarkably, unlike previous observations in animal viruses, PPV variants harboring CpG-rich fragments display just faint (or no) attenuation. The anticorrelation between UpA frequency and viral fitness additionally demonstrates the relevance of this particular dinucleotide: UpA-high mutants are attenuated in a dose-dependent manner, whereas a UpA-low variant displays better fitness than its parental control. Using high-throughput sequencing, we also show that UpA-rich PPV variants are genetically stable, without apparent changes in sequence that revert and/or compensate for the dinucleotide modification despite its attenuation. In addition, we also demonstrate here that the PPV restriction of UpA-rich variants works independently of the classical RNA silencing pathway. Finally, we show that the anticorrelation between UpA frequency and RNA accumulation applies to mRNA-like fragments produced by the host RNA polymerase II. Together, our results inform us about a dinucleotide-based system in plant cells that controls diverse RNAs, including RNA viruses.IMPORTANCE Dinucleotides (combinations of two consecutive nucleotides) are not randomly present in RNA viruses; in fact, the presence of CpG and UpA is significantly repressed in their genomes. Although the meaning of this phenomenon remains obscure, recent studies with animal-infecting viruses have revealed that their low CpG/UpA frequency prevents virus restriction via a host antiviral system that recognizes, and promotes the degradation of, CpG/UpA-rich RNAs. Whether similar systems act in organisms from other life kingdoms has been unknown. To fill this gap in our knowledge, we built several synthetic variants of a plant RNA virus with deoptimized dinucleotide frequencies and analyzed their viral fitness and genome adaptation. In brief, our results inform us for the first time about an effective dinucleotide-based system that acts in plants against viruses. Remarkably, this viral restriction in plants is reminiscent of, but not identical to, the equivalent antiviral response in animals.

Synthesis of Four Optical Isomers of Antiviral Agent NK0209 and Determination of Their Configurations and Activities against a Plant Virus.

Previously, we reported for the first time that harmala alkaloids harmine and tetrahydroharmine exhibit activity against plant viruses, and we developed an analogue, designated NK0209, that efficiently prevents and controls plant virus diseases. Here, to investigate the influence of the spatial configuration of NK0209 on its antiviral activities, we synthesized its four optical isomers, determined their configurations, and evaluated their activities against tobacco mosaic virus. All four isomers were significantly more active than ningnanmycin, which is one of the most successful commercial antiviral agents, with in vivo inactivation, cure, and protection rates of 57.3 ± 1.9, 54.2 ± 3.3, and 55.0 ± 4.1% at 500 µg/mL. Furthermore, analysis of structure-activity relationships demonstrated for the first time that the spatial conformation of NK0209 is an important determinant of its antiviral activity, and our results provide information about the possible optimum configuration for interaction of this molecule with its target protein.

Rice black-streaked dwarf virus-encoded P5-1 regulates the ubiquitination activity of SCF E3 ligases and inhibits jasmonate signaling to benefit its infection in rice.

SCF (Skp1/Cullin1/F-box) complexes are key regulators of many cellular processes. Viruses encode specific factors to interfere with or hijack these complexes and ensure their infection in plants. The molecular mechanisms controlling this interference/hijack are currently largely unknown. Here, we present evidence of a novel strategy used by Rice black-streaked dwarf virus (RBSDV) to regulate ubiquitination in rice (Oryza sativa) by interfering in the activity of OsCSN5A. We also show that RBSDV P5-1 specifically affects CSN-mediated deRUBylation of OsCUL1, compromising the integrity of the SCFCOI1 complex. We demonstrate that the expressions of jasmonate (JA) biosynthesis-associated genes are not inhibited, whereas the expressions of JA-responsive genes are down-regulated in transgenic P5-1 plants. More importantly, application of JA to P5-1 transgenic plants did not reduce their susceptibility to RBSDV infection. Our results suggest that P5-1 inhibits the ubiquitination activity of SCF E3 ligases through an interaction with OsCSN5A, and hinders the RUBylation/deRUBylation of CUL1, leading to an inhibition of the JA response pathway and an enhancement of RBSDV infection in rice.

Opuntin B, the antiviral protein isolated from prickly pear (Opuntia ficus-indica (L.) Miller) cladode exhibits ribonuclease activity.

An antiviral protein, designated Opuntin B, was purified from Prickly Pear (Opuntia ficus-indica (L.) Miller) Cladode by heat treatment of the extract, protein precipitation by ammonium sulfate treatment followed by ion-exchange chromatography. Assessment of enzymatic activity of the purified protein showed that it degrades total plant genomic RNA, while causing electrophoretic mobility shifting of Cucumber mosaic virus (CMV) RNAs. However, heat-denatured viral RNA became sensitive to degradation upon treatment with antiviral protein. Opuntin B had no DNase activity on native and heat-denatured apricot genomic DNA, and on PCR-amplified coat protein gene of CMV. Using CMV as prey protein and Opuntin B as bait protein, no interaction was found between the antiviral protein and viral coat protein in far western dot blot analysis.

Synthesis of Anthranilic Diamide Derivatives Containing Moieties of Trifluoromethylpyridine and Hydrazone as Potential Anti-Viral Agents for Plants.

A series of novel anthranilic diamide derivatives (5a-5ab) containing moieties of trifluoromethylpyridine and hydrazone was designed and synthesized. The synthesized compounds were evaluated in vivo for their activities against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Most of the synthesized compounds displayed good to excellent antiviral activities. The compounds 5i, 5k, 5s, 5w, 5x, and 5z had the curative activity over 65% against TMV at the concentration of 500 µg/mL, which were significantly higher than those of ningnanmycin (55.0%) and ribavirin (37.9%). Notably, the curative activity of compound 5i was up to 79.5%, with the EC50 value of 75.9 µg/mL, whereas the EC50 value of ningnanmycin was 362.4 µg/mL. The pot experiments also further demonstrated the significantly curative effect of 5i. Meanwhile, compounds 5h, 5p and 5x displayed more protective activities on TMV than that of ningnanmycin. Moreover, compounds 5a, 5e, 5f, and 5i showed inactivation activity similar to ningnanmycin at 500 µg/mL, and the EC50 value of 5e (41.5 µg/mL) was lower than ningnanmycin (50.0 µg/mL). The findings of transmission electron microscopic (TEM) indicated that the synthesized compounds exhibited strong and significant binding affinity to TMV coat protein (CP) and could obstruct the self-assembly and increment of TMV particles. Microscale thermophoresis (MST) studies on TMV-CP and CMV CP revealed that some of the active compounds, particularly 5i, exhibited a strong binding capability to TMV-CP or CMV-CP. This study revealed that anthranilic diamide derivatives containing moieties of trifluoromethylpyridine and hydrazone could be used as novel antiviral agents for controlling the plant viruses.

Induction of in planta resistance by flagellin (Flg) and elongation factor-TU (EF-Tu) of Bacillus amyloliquefaciens (VB7) against groundnut bud necrosis virus in tomato.

Role of plant growth promoting rhizobacteria (PGPR) in growth promotion and induction of resistance against various plant pathogens have been extensively studied. However, MAMP (Microbe Associated Molecular Pattern) triggered immunity (MTI) against plant viruses are not well exploited. The present study enlightens the role of two MAMP genes including, flagellin (Flg) and elongation factor (EF-Tu) in the induction of plant defense against GBNV infecting tomato. Secondary metabolites of Bacillus amyloliquefaciens (VB7), effectively suppressed GBNV symptom expression up to 84% compared to untreated control in cowpea, the indicator host plant. Agrobacterium tumefaciens EHA105 clones expressing the MAMP genes were drenched in the root zone to assess the induction of resistance against GBNV in tomato. Treatment with A. tumefaciens EHA105 clones containing flagellin (Ag- Ba.Flg) and elongation factor-TU (Ag-Ba.EF-Tu) genes as soil drench and foliar spray, reduced virus titre,0.369 OD and 0.379 OD respectively as compared to control 1.249 OD. The disease severity was reduced up to 15% in Ag-Ba.Flg treated plants compared to 88.25% in inoculated control. Further there was an increased expression of defense associated genes including, MAPKK1, WRKY33BB, NPR1 and PR1.The present investigation clearly indicated the efficiency of MAMP genes in triggering defense mechanism in tomato against GBNV.

Pharmacological analysis of transmission activation of two aphid-vectored plant viruses, turnip mosaic virus and cauliflower mosaic virus.

Turnip mosaic virus (TuMV, family Potyviridae) and cauliflower mosaic virus (CaMV, family Caulimoviridae) are transmitted by aphid vectors. They are the only viruses shown so far to undergo transmission activation (TA) immediately preceding plant-to-plant propagation. TA is a recently described phenomenon where viruses respond to the presence of vectors on the host by rapidly and transiently forming transmissible complexes that are efficiently acquired and transmitted. Very little is known about the mechanisms of TA and on whether such mechanisms are alike or distinct in different viral species. We use here a pharmacological approach to initiate the comparison of TA of TuMV and CaMV. Our results show that both viruses rely on calcium signaling and reactive oxygen species (ROS) for TA. However, whereas application of the thiol-reactive compound N-ethylmaleimide (NEM) inhibited, as previously shown, TuMV transmission it did not alter CaMV transmission. On the other hand, sodium azide, which boosts CaMV transmission, strongly inhibited TuMV transmission. Finally, wounding stress inhibited CaMV transmission and increased TuMV transmission. Taken together, the results suggest that transmission activation of TuMV and CaMV depends on initial calcium and ROS signaling that are generated during the plant's immediate responses to aphid manifestation. Interestingly, downstream events in TA of each virus appear to diverge, as shown by the differential effects of NEM, azide and wounding on TuMV and CaMV transmission, suggesting that these two viruses have evolved analogous TA mechanisms.

Plant defense against virus diseases; growth hormones in highlights.

Phytohormones are critical in various aspects of plant biology such as growth regulations and defense strategies against pathogens. Plant-virus interactions retard plant growth through rapid alterations in phytohormones and their signaling pathways. Recent research findings show evidence of how viruses impact upon modulation of various phytohormones affecting plant growth regulations. The opinion is getting stronger that virus-mediated phytohormone disruption and alteration weaken plant defense strategies through enhanced replication and systemic spread of viral particles. These hormones regulate plant-virus interactions in various ways that may involve antagonism and cross talk to modulate small RNA (sRNA) systems. The article aims to highlight the recent research findings elaborating the impact of viruses upon manipulation of phytohormones and virus biology.

High-throughput small RNA sequencing for evaluation of grapevine sanitation efficacy.

This study describes the application of high-throughput sequencing of small RNA analysis of the efficacy of using Ribavirin to eliminate Grapevine leafroll-associated virus 1, Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus from Vitis vinifera cv. Riesling. The original plant used for sanitation by Ribavirin treatment was one naturally infected with all the viruses mentioned above as confirmed by RT-PCR. A tissue cultures of the plant were established and plantlets obtained were sanitized using Ribavirin. Three years after sanitation, a small RNA sequencing method for virus detection, targeting 21, 22 and 24 nt-long viral small RNAs (vsRNAs), was used to analyze both the mother plant and the sanitized plants. The results showed that the mother plant was infected by the three mentioned viruses and additionally by two viroids - Hop stunt viroid and Grapevine yellow speckle viroid 1. After Ribavirin treatment, the plants contained only the two viroids, with the complete elimination of all the viruses previously present.

Natural Products for Drug Discovery: Discovery of Gramines as Novel Agents against a Plant Virus.

Plant viral diseases seriously affect crop yield and quality. The natural product gramine (1) and its simple structural analogues 2-35 were synthesized from indoles, amines, and aldehydes in one step. The antiviral effects of these alkaloids were evaluated systematically. Most of these compounds were found to have higher antiviral effects than commercial ribavirin for the first time. Especially compounds 22, 30, and 31 exhibited significantly higher effects than ningnanmycin, thereby emerging as novel antiviral leads for further optimization. The preliminary implementation indicated that these compounds likely inhibit the assembly of tobacco mosaic virus (TMV) by cross-linking TMV capsid protein. Gramine analogues were also found to have broad-spectrum fungicidal effects. Although gramine has been reported to have influence on germination and development of Erysiphe graminis, these compounds displayed no fungicidal effects against Blumeria graminis f. sp. tritici on wheat in our test. Some of these compounds also exhibited certain insecticidal activities.

Discovery of Pimprinine Alkaloids as Novel Agents against a Plant Virus.

Plant viral diseases cause tremendous decreases in crop yield and quality. Natural products have always been a valuable source for lead discovery in medicinal and agricultural chemistry. A series of pimprinine alkaloids and their derivatives were prepared and identified by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). The antiviral activities of these alkaloids against tobacco mosaic virus (TMV) were systematically investigated for the first time. Most of the compounds exhibited higher antiviral activities than ribavirin. Compounds 5l, 9h, and 10h, which had similar or higher antiviral activities than ningnanmycin (perhaps the most widely used antiviral agent at present), emerged as new antiviral pilot compounds. This systematic structure-activity-relationship research lays the foundation for simplifying the structure of these alkaloids. The ring-open products, acylhydrazones 9a-9u, were also found to possess good antiviral activities. Moreover, all the synthesized compounds displayed broad-spectrum fungicidal activities. This study provides important information for the research and development of pimprinine alkaloids as novel antiviral agents.

Design, Synthesis, Antiviral Bioactivity, and Defense Mechanisms of Novel Dithioacetal Derivatives Bearing a Strobilurin Moiety.

A series of dithioacetal derivatives bearing a strobilurin moiety were designed and synthesized on the basis of our previous work. The antiviral activities of these compounds against Potato virus Y (PVY), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV) were systematically evaluated. Bioassay results indicated that C14 elicited excellent curative and protective activities against PVY, CMV, and TMV. The former had 50% effective concentrations (EC50) of 125.3, 108.9, and 181.7 µg/mL, respectively, and the latter had 148.4, 113.2, and 214.6 µg/mL, respectively, which were significantly superior to those of lead compound 6f (297.6, 259.6, and 582.4 µg/mL and 281.5, 244.3, and 546.3 µg/mL, respectively), Ningnanmycin (440.5, 549.1, and 373.8 µg/mL and 425.3, 513.3, and 242.7 µg/mL, respectively), Chitosan oligosaccharide (553.4, 582.8, and 513.8 µg/mL and 547.3, 570.6, and 507.9 µg/mL, respectively), and Ribavirin (677.4, 690.3, and 686.5 µg/mL and 652.7, 665.4, and 653.4 µg/mL, respectively). Moreover, defensive enzyme activities and RT-qPCR analysis demonstrated that the antiviral activity was associated with the changes of SOD, CAT, and POD activities in tobacco, which was proved by the related proteins of abscisic acid signaling pathway. This work provided a basis for further design, structural modification, and development of dithioacetal derivatives as new antiviral agents.

Elevated CO2 increases R gene-dependent resistance of Medicago truncatula against the pea aphid by up-regulating a heat shock gene.

Resistance against pathogens and herbivorous insects in many plant results from the expression of resistance (R) genes. Few reports, however, have considered the effects of elevated CO2 on R gene-based resistance in plants. The current study determined the responses of two near isogenic Medicago truncatula genotypes (Jester has an R gene and A17 does not) to the pea aphid and elevated CO2 in open-top chambers in the field. Aphid abundance, mean relative growth rate and feeding efficiency were increased by elevated CO2 on A17 plants but were reduced on Jester plants. According to proteomic and gene expression data, elevated CO2 enhanced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) but decreased the effector-triggered immunity (ETI) in aphid-infested A17 plants. For aphid-infested Jester plants, by contrast, elevated CO2 enhanced the ETI-related heat shock protein (HSP) 90 and its co-chaperones, the jasmonic acid (JA) signaling pathway, and ubiquitin-mediated proteolysis. In a loss-of-function experiment, silencing of the HSP90 gene in Jester plants impaired the JA signaling pathway and ubiquitin-mediated proteolysis against the aphid under ambient CO2 , and negated the increased resistance against the aphid under elevated CO2 . Our results suggest that increases in expression of HSP90 are responsible for the enhanced resistance against the aphid under elevated CO2 .

Roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant RNA viruses in Nicotiana benthamiana.

Reactive oxygen species (ROS), including superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2- production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2- can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2- scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2- and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.