Article

Antiviral activity of prickly pear ( Opuntia ficus-indica (L.) Miller) extract: Opuntin B, a second antiviral protein

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Abstract

We previously showed that aqueous extract of prickly pear (Opuntia ficus-indica) cladode has significant antiviral activity against Cucumber mosaic virus (CMV: family Bromoviridae). Antiviral activity of the extract was retained for a period of 9 days on treated faba bean leaves. This activity was considerably decreased at concentrations lower than 30 mg mL −1 , and by washing the treated leaves of faba bean plants 2 h after application of cactus extract, and was ineffective when applied 24 h after virus inoculation. No inhibitory effect was found against agro-inoculated strain of Beet curly top virus (BCTV: family Geminiviridae) on faba bean plants. Prickly pear extract reduced the efficiency of CMV transmission by Aphis gossypii on treated cucumber seedlings. An antiviral protein designated Opuntin B was isolated from the extract by heat treatment and differential centrifugation, and bulk protein precipitation by ammonium sulfate followed by cation and anion exchange chromatography. SDS-PAGE analysis in the presence of 2-ME revealed that Opuntin B is made of four protein components, in the molecular weight (Mr) range of 29-32 kDa, while in the absence of 2-ME, only two bands in the range of 26-28 kDa were visible. Native PAGE of the protein also showed 2 bands. Purified Opuntin B completely inhibited CMV on faba bean and zucchini plants at concentration of 40 μg mL −1 and had an IC 50 of 0.75 μg mL −1 for inhibition of the virus on faba bean leaves. The isolated antiviral protein exhibited inhibitory activity against Zucchini yellow mosaic virus (ZYMV: family Potyviridae) Squash mosaic virus (SqMV: family Secouviridae) and Tobacco mosaic virus, (TMV: family Virgaviridae), as well.

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... It exhibited ribonuclease activity against total RNA extract from CMV-infected plants and against CMV RNAs. A second antiviral protein, Opuntin B, was also isolated from prickly pear by the authors [12]. In contrast to Opuntin A, Opuntin B was adsorbed on CM-Sephadex C-50, was sensitive to 2-mercaptoethanol and was made of four protein components in the Mr range of 29-32 kDa. ...
... Prickly pear cladode extraction, extract clarification and protein precipitation were carried out as previously described [11]. Opuntin B was purified by heat treatment of the extract, bulk protein precipitation using ammonium sulfate followed by further protein purification using ion-exchange chromatography on CM-Sephadex C-50 (Sigma-Aldrich) and DEAE-cellulose columns, as previously described [12]. ...
... Overall, these results, viral RNA mobility shifting and no amplification of Opuntin B-treated viral RNAs indicate that Opuntin B is an antiviral protein that targets viral genomic RNA, not the virus particles. It exhibited CMV inhibition activity as pretreatment and post-treatment [12]. Therefore, it may enter plant cells along virus particles and affect viral RNAs after virus un-coating. ...
Article
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.
... A rich review has been carried out on the use of antiviral agents from natural sources against the tobacco mosaic virus (TMV), which is common among tobacco, cucumber, pepper, and ornamentals [51]. Some of these botanicals include Picrasma quassioides [136], Glycyrrhiza glabra L. [137], Arundina graminifolia [138], Tithonia diversifolia [139], Cephalotaxus sinensis [140], Actinidia chinensis [141], and prickly pear [142]. Similarly, Salix alba [48], Tanacetum vulgare [143], C. sinensis [140], prickly pear [144], and A. chinensis [141] showed an antiviral activity against cucumber mosaic virus (CMV) isolated from cucumber and watermelon. ...
... uja orientalis extract was effective against eggplant blister mottled virus (EBMV) isolated from eggplant [146], fig leaf mottle-associated virus-1 (FLMaV-1) was identified in fig trees [147], and watermelon mosaic potyvirus (WMV) and zucchini yellow mosaic virus (ZYMV) were common to watermelon [148,149]. Prickly pear has also exhibited a notable antiviral activity against zucchini yellow mosaic virus (ZYMV) and squash mosaic virus (SqMV) isolated from zucchini (Cucurbita pepo L.) [142]. ...
... ese include alkaloids, coumarins, flavonoids, phenolics, and terpenoids amongst others. Alkaloids: 1-methoxycarbonylβ-carboline from methanolic extract of P. quassioides wood [136], drupacine and cephalotaxine from methanolic extract of C. sinensis leaves and branches [140], Opuntin B from aqueous extract of prickly pear cladode [142,144]; flavonoids: Liquiritin from methanolic extract of G. glabra root [137,150]; phenolics: gramniphenol C from a chromatographic fraction of A. graminifolia whole plant extract [138] and salicylic acid from aqueous extract of S. alba leaves and bark [48]; terpenoids: 1β-methoxydiversifolin-3-0-methyl ether and tagitinin C isolated from methanol extract of T. diversifolia whole plant part [139], rhodoxanthin from ethanol extract of T. orientalis leaves and fruits [151,152], spathodic acid-28-O-β-D-glucopyranoside and 5-methoxycoumarin-7-O-β-D-glycosidase (coumarin) from ethyl International Journal of Agronomy acetate extract of A. chinensis root bark [141]; and a variety of phytochemicals have been detected in methanol extract from T. vulgare flower [143] and ethanol extracts of S. speciosus leaves and fruits [145]. e use of microbes as effective producers of antiviral agents has also been exploited in the eradication of plant viruses. ...
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The increase in demand for agricultural produce necessitates the continuous search for affordable, ecofriendly, readily available crop protectors, and food preservatives. Historically, the use of various chemicals was employed in controlling plant diseases and to maintain food quality. In the past few decades, several natural product-based alternatives have been discovered and projected as better alternatives to synthetic pesticides and other synthetic agrochemicals. Recent studies focusing on the application of different botanicals in crop protection and food preservation were carefully selected and reviewed. The application of plant extract in the biogenic preparation of nanoparticles was also reviewed. This review confirms that several natural products can be used as a safe replacement for synthetic agrochemicals. Different plant extracts have also served as feed for the synthesis of nanoparticle, which is increasingly applicable in crop protection and food preservation.
... Opuntia ficus extract also reported as being antiviral especially inhibits intracellular replication of DNA and RNA viruses such as HIV-1, Influenza virus, Pseudorabies virus, Equine herpes virus, Herpes simple virus [156,157]. Rasoulpour et al. (2018) isolated an antiviral protein from Opuntia ficus indica and confirmed its antiviral activity against cucumber mosaic virus, zucchini yellow mosaic virus, squash mosaic virus and tobacco mosaic virus [158]. Kumar et al. (2014) reported that methanol extract of Opuntia dellenii flowers possesses antiviral activity against vaccinia and herpes simplex virus type 1 and 2 [154]. ...
... Opuntia ficus extract also reported as being antiviral especially inhibits intracellular replication of DNA and RNA viruses such as HIV-1, Influenza virus, Pseudorabies virus, Equine herpes virus, Herpes simple virus [156,157]. Rasoulpour et al. (2018) isolated an antiviral protein from Opuntia ficus indica and confirmed its antiviral activity against cucumber mosaic virus, zucchini yellow mosaic virus, squash mosaic virus and tobacco mosaic virus [158]. Kumar et al. (2014) reported that methanol extract of Opuntia dellenii flowers possesses antiviral activity against vaccinia and herpes simplex virus type 1 and 2 [154]. ...
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Background: Opuntia species, locally known as prickly pear was used for various purpose as food, medicine, beverage, source of dye and animal food. Many studies revealed its pharmacology activity time to time. This review is a collection of chemistry, pharmacognosy, pharmacology and bioapplications of cactus family. Method: Many source were used to collect the information about Opuntia species such as Pub med, Google scholar, Agris, science direct, Embase, Merk index, Wiley online library, books and other reliable sources. This review contains studies from 1812 to 2019. Result The plants from cactus family were offer various pharmacological active compound including phenolic compounds, carotenoids, betalains, vitamins, steroids, sugar, amino acids, minerals and fibers. These bioactive compound were served various pharmacological activity such as anticancer, antiviral, anti-diabetic, Neuroprotective, anti-inflammatory, antioxidant, Hepatoprotective, antibacterial, antiulcer and alcohol hangover. According to various studies Opuntia species were offer many bioapplications such as fodder for animal, soil erosion, prevention, human consumption and waste water decontamination. Finally different parts of plants were used in various formulation that offer many biotechnology applications. Conclusion Different parts of Opuntia plant (fruits, seeds, flowers and cladodes) were used in various health problems include wound healing, anti-inflammatory and urinary tract infection from ancient time. Nowadays, researches were extended several pharmacological and therapeutic used of Opuntia species as discussed in this review. Many in-vitro and in-vivo model were also discussed in this review as the proofs of research finding. Various research gaps were observed in current studies that need attention in future.
... Our results showed that application of alligator plant extract can significantly inhibit TMV activity in five plant species. Similar to the previous studies emphasizing the investigation of antiviral effects in both systemic and local lesion hosts (Rasoulpour et al., 2017;Rasoulpour et al., 2018), antiviral effects were assesed using two viruses. Results indicate infection rate in Nicotiana tabacum var. ...
... Reduced TMV inhibition was associated with reduced concentration of alligator plant extract which has been previously reported about other antiviral extracts (Rasoulpour et al., 2017;Rasoulpour et al., 2018). The virus inhibition, however, was found to be different among the plant hosts which might be due to variations in host response to the viral infection. ...
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Here we showed that aqueous extract of alligator plant, Bryophyllum daigremontianum L., is able to inhibit systemic and local infection of Tobacco mosaic virus (TMV: family Virgaviridae) in broad bean, Vicia faba L., nettle-leaved goosefoot, Chenopodium murale L., and tobacco, Nicotiana tabacum var. Turkish, N. tabacum var. Xanthi and N. glutinosa L., hosts. Antiviral activity of the extract was retained for a period of 8 days on treated broad bean and tobacco leaves. This activity was negatively correlated with the extract concentration, and it was completely lost by washing the treated leaves of tobacco plants 2 h post application, and was ineffective when applied 24 h post inoculation. No inhibitory effect was found against agro-inoculated strain of Beet curly top virus (BCTV: family Geminiviridae) on sugarbeet, Beta vulgaris L., seedlings. To determine the antiviral agent, Bryophyllum bulk protein designated BBP was isolated from the extract. BBP exhibited RNase activity against total RNA of TMV-infected tobacco tissues and genomic RNA of TMV while it failed to degrade genomic DNA of BCTV. Additionally, BBP completely inhibited TMV on N. glutinosa leaves at concentration of 40 μg/ml. These results suggest that a ribonuclease is mainly responsible for antiviral activity of alligator plant extract. To our knowledge, this is the first report on inhibitory effect of alligator plant extract on a plant virus. This plant species can be considered as a promising source for antiviral proteins in order to develop plant-derived compounds for effective control of plant mosaic diseases caused by TMV.
... It is cultivated in dry regions as a crucial source of nutrients (El Mannoubi et al. 2009). Over centuries, prickly pear was a medicinal plant endemic to Mexico and currently being introduced to several parts of the planet (Rasoul Rasoulpour et al. 2018), including North Africa, Europe, Mediterranean countries, and also the Middle East (Coşkuner and Tekin 2003). Thanks to its ability to adapt in several environmental conditions, the cactus grows in plains, coastal regions, and plateaus (Lahsasni et al. 2004). ...
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... The exponential increase after the 1960s, however, was related to the purification of PAP (Phytolacca antiviral protein), peaking in the 1970s/1980s with several studies related to AVP properties, purification and activity [22,24,27,29,50,55,57,62,105]. Despite a sharp decline in the 1990s, the discovery of AVP inhibitory activity at ribosome inactivation led to the isolation and characterization of numerous ribosome-inactivating proteins (RIPs) [34,45,89], and in the 2000s and 2010s, researchers endeavored in cloning of RIPs, as well as to understand the mode of action of AVP, focusing primarily on the N-glycosidase activity of RIPs [37,38,46,52,65,66,84]. Plant extracts from different species were tested in several pathosystems; however, the role of these antiviral proteins (AVPs) in the induction of resistance/protective mechanisms has yet to be elucidated [108]. ...
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... Overloading gibberellins during plant growth, inhibits the growth of other predatory herbs, and acts as the enhanced, new generation herbicides (Dayan and Duke 2014). These are also used as pesticides, enhance seed germination, and as antibacterial, antifungal and antiviral agents to protect plants from microbes, as well as to elevate nutrient uptake in soil and in soil remediation (Liu et al. 2013;Altunok et al. 2015;Rasoulpour et al. 2018). ...
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The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.
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The infectivity of three RNA plant viruses belonging to the genera Cucumovirus, Potyvirus and Tobamovirus was inhibited by an aqueous extract of prickly pear (Opuntia ficus-indica (L.) Miller) cladode. This antiviral activity was not dialyzable, and not related to isolated plant polysaccharides, but was sensitive to heating and extract treatment with trichloroacetic acid and sodium dodecyl sulfate. An antiviral protein with MW of 28.3 kDa was purified from cactus extract by ammonium sulfate fractionation followed by ion exchange chromatography. The isolated protein inhibited Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus and Tobacco mosaic virus at a concentration of 40 μg mL⁻¹. An IC50 of 2.8 μg mL⁻¹ was calculated for isolated protein for CMV inhibition on faba bean plants. It was demonstrated that the antiviral protein has RNase, but not DNase and protease activity. It interacted with viral nucleic acid in far Western dot blot analysis, was resistant to 2-mercaptoethanol and ethylenediaminetetraacetic acid, and was active in the pH range of 4–10. To our knowledge, this is the first report on inhibitory effect of prickly pear extract on members of Tobamovirus, Potyvirus and Cucumovirus, as well as isolation and characterization of an antiviral protein from prickly pear cladode. This plant can be regarded as an encouraging source of antiviral protein to develop environmentally-friendly compounds for management of plant viral diseases.
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A nonphytotoxic, systemic resistance-inducing agent present in Clerodendrum aculeatum leaves was purified. A specific basic protein (C. aculeatum-systemic resistance inducing [CA-SRI]) with a molecular mass of 34 kDa was observed consistently in leaf extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of plants with the purified protein preparation induced a very high level of systemic resistance against virus infection. Resistance was detectable a few hours after challenge-inoculation with virus and resulted in lesions that were fewer in number or totally absent. The minimum time required for induction of systemic resistance in nontreated leaves of susceptible host plants was 5 to 30 min depending on the host. The resistance-inducing activity of CA-SRI was not affected by protease treatment. After digestion of CA-SRI with endoproteinase Arg-C, the eluted protein fragments from SDS-PAGE were biologically active. An antiserum to the 34-kDa protein was highly specific for CA-SRI, and Western blots of the purified protein recognized the 34-kDa protein band. The isoelectric point of the protein was 8.65. Treatment of susceptible healthy test hosts with purified CA-SRI consistently resulted in the accumulation of a virus inhibitory agent in the resistant leaves. An extract prepared from resistant leaves reduced the infectivity of added virus, with an average reduction in the number of lesions by more than 90%. The specific 34-kDa protein was observed consistently in the leaves of plants with induced resistance and was highly active in reducing the infectivity of the virus. There seems to be a causal relationship between induced resistance and accumulation of the 34-kDa protein based on gel electrophoresis. The protein was present naturally in very low amounts in nontreated healthy plants.
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Cactus (Opuntia ficus-indica) belongs to the family Cactaceae. Family Cactaceae is reported to contain about 130 genera and nearly 1500 species. This plant is native of Mexico and it is widely distributed in Mexico and in all American hemispheres as well as in Africa and in the Mediterranean basin. It has been used in traditional folk medicine because of its role in treating a number of diseases and conditions, including anti-inflammatory effects hypoglycemic effects inhibition of stomach ulceration, neuroprotective effects Through antioxidant actions and also used for treating diabetes, burns, bronchial, asthma and indigestion in many countries over the world. It is also used in Pharma industry as a pharmaceutical agent. The fruit, as well as cactus stem are used to prepare value-added products, such as jam, squash, wine, pickle, body lotions, shampoo, creams, etc. It also has several medicinal and industrial uses. Its seeds can be used as flavouring agents. Due to the remarkable biological activity of Opuntia and its constituents, it will be appropriate to develop them as a medicine.
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Quantitation of the amount of protein in a solution is possible in a simple spectrometer. Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore the A 280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 for some tyrosine-rich wool proteins, although most values are in the range 0.5–1.5 [1]). The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be determined. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.
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Determination and location of cystine residues are essential to protein characterization since their disulfide bonds stabilize chainfolding and multichain structure responsible for physical and biological properties. Reduction with mercaptans, such as dithiothreitol, in dissociating solvents is the choice procedure for attaining disulfide cleavage, since other methods may cause undesirable side reactions. Under selected conditions the resulting sulfhydryls can be specifically alkylated with α,β-unsaturated compounds to prevent their reoxidation. Determination of alkylated cysteine residues supplements the cysteic acid method for cystine-cysteine analysis. Reoxidation studies on reduced proteins indicate that positions of formation of both intra- and interchain disulfide bonds are governed by amino acid sequences as well as by concentration, solvent, and pH. Location of disulfide bonds in peptides obtained by chromatography of proteolytic digests of proteins has been accelerated by automatic analysis with specific reagents. Disulfide interchange may change physical properties of proteins and must be avoided in characterization work.
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Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
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An extract of the cactus plant Opuntia streptacantha inhibited intracellular virus replication and inactivated extracellular virus. Inhibition of virus replication also occurred following pre-infection treatment--a favourable finding in terms of in-vivo limitation of virus disease. There was inhibition of both DNA and RNA virus replication, for example, herpes simplex virus, equine herpes virus, pseudorabies virus, influenza virus, respiratory syncytial virus and human immunodeficiency virus, with normal protein synthesis in uninfected cells at extract concentrations which were 15-fold in excess of 50% viral inhibitory concentrations (1 mg/ml). The active inhibitory component(s) of the extract appeared to be protein in nature and resided mainly in the wall of the plant rather than in the cuticle or inner sap. The extract was non-toxic on oral administration to mice, horses and human patients; the non-toxicity of intravenous administration of 70 mg to a mouse representing at least fifty tissue culture 50% viral inhibitory dosages encourages clinical trial of this extract in virus disease of human and veterinary species.
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Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.
Infectivity of the cloned genome of Iranian isolate of Beet severe curly top virus in experimental hosts
  • Ebadzad Sahraei
  • G Behjatnia
  • S A A Izadpanah
Ebadzad Sahraei, G., Behjatnia, S.A.A., Izadpanah, K., 2008. Infectivity of the cloned genome of Iranian isolate of Beet severe curly top virus in experimental hosts. Iran. J. Plant Pathol. 44, 176-183.
Potency of Plant Products in Control of Virus Diseases of Plants. in: Natural Products in Plant Pest Management. CAB International
  • H N Verma
  • V K Baranwal
Verma, H.N., Baranwal, V.K., 2011. In: Dubey, N.K. (Ed.), Potency of Plant Products in Control of Virus Diseases of Plants. in: Natural Products in Plant Pest Management. CAB International, pp. 293.