SEROLOGICAL AND MOLECULAR CHARACTERISTICS OF TOMATO MOSAIC TOBAMOVIRUS COAT PROTEIN

Plant viruses cause serious disease of crop plants reducing both quality and quantity of final produce. Serological tests are used all over the world in laboratory and field based detection of plant viruses and they are of great indispensable importance in agricultural production; virus certification programs; agricultural quarantine and production of virus-free crops grown for processing or fresh market. Cross reaction between viruses and their strains antisera limits serological differentiation of viruses and their strains by enzyme-linked immunosorbent assay (ELISA). This study aims to characterize the antigenic property of Tomato mosaic virus ToMV coat protein by using some bioinformatic tools to analyze its gene. Therefore, new methods in antibody production could be used as equivalent to Mabs in its high specificity. ToMV isolate was confirmed by Transmission electron microscope and differential hosts and propagated on N. tabacum cv. Samson. Systemic infection was developed on N. tabacum cv. Samson and local infection on Datora metel; D. stramonium; N. glutinosa; Chenopodium amaranticolor; C. quinoa. ToMV was purified and used as immunizing agent for antiserum production. TEM showed rod shaped particles with 300 x 18 nm dimensions. The titer of produced antiserum was 1:1024 evaluated by microprecipitin test and indirect-ELISA. Coat protein gene was amplified by RT-PCR with expected size of (Approx. 500 bp). The PCR product was sequenced then the generated nucleotide sequence  was translated into 160 amino acids. The amino acid sequence of Five B-cell epitopes, of 14 amino acid residues each, were predicted. Identifying Bcell epitopes play an important role in vaccine design, immunodiagnostic tests, and antibody production. Therefore, computational tools for reliably predicting B-cell epitopes are studied

An obligate microsporidian parasite modulates defense against opportunistic bacterial infection in the yellow fever mosquito, Aedes aegypti

ABSTRACTThe ability of Aedes aegypti mosquitoes to transmit vertebrate pathogens depends on multiple factors, including the mosquitoes’ life history traits, immune response, and microbiota (i.e., the microbes associated with the mosquito throughout its life). The microsporidium Edhazardia aedis is an obligate intracellular parasite that specifically infects Ae. aegypti mosquitoes and severely affects mosquito survival and other life history traits critical for pathogen transmission. In this work, we investigated how E. aedis impacts bacterial infection with Serratia marcescens in Ae. aegypti mosquitoes. We measured development, survival, and bacterial load in both larval and adult stages of mosquitoes. In larvae, E. aedis exposure was either horizontal or vertical and S. marcescens was introduced orally. Regardless of the route of transmission, E. aedis exposure resulted in significantly higher S. marcescens loads in larvae. E. aedis exposure also significantly reduced larval survival but subsequent exposure to S. marcescens had no effect. In adult females, E. aedis exposure was only horizontal and S. marcescens was introduced orally or via intrathoracic injection. In both cases, E. aedis infection significantly increased S. marcescens bacterial loads in adult female mosquitoes. In addition, females infected with E. aedis and subsequently injected with S. marcescens suffered 100% mortality which corresponded with a rapid increase in bacterial load. These findings suggest that exposure to E. aedis can influence the establishment and/or replication of other microbes in the mosquito. This has implications for understanding the ecology of mosquito immune defense and potentially disease transmission by mosquito vector species.IMPORTANCEThe microsporidium Edhazardia aedis is a parasite of the yellow fever mosquito, Aedes aegypti. This mosquito transmits multiple viruses to humans in the United States and around the world, including dengue, yellow fever, and Zika viruses. Hundreds of millions of people worldwide will become infected with one of these viruses each year. E. aedis infection significantly reduces the lifespan of Ae. aegypti and is therefore a promising novel biocontrol agent. Here, we show that when the mosquito is infected with this parasite, it is also significantly more susceptible to infection by an opportunistic bacterial pathogen, Serratia marcescens. This novel discovery suggests the mosquito’s ability to control infection by other microbes is impacted by the presence of the parasite

Improved antibacterial efficacy of bacteriophage-cosmetic formulation for treatment of Staphylococcus aureus in vitro

Currently phages are used as alternative antibiotics for treating pathogenic bacteria causing skin disease. However, the efficacy of pure preparations of phage is greatly reduced due to its short longevity on surface of skin. supplemented cosmetic phages [0.5% phage conc./cosmetic] significantly increased phage longevity on skin surface. The phages were isolated by the single plaque assay from the infected skin showing edema and erythema symptoms. The isolated phages had plaques with 3–5 mm diameters and a distinct translucent spreading halo. The morphological phage particles were cubic nucleocapsid with 65–75 nm across with short contractile tails. The supplemented cosmetic phages reduced the bacterial growth to 95.45%, compared with free phages and non-supplemented cosmetic 86.1% and 77% respectively. The phage containing cosmetic was applied for disease treatment and increased the phage longevity from 24 to 100 h and preserved initial phage population. This work indicated the enhanced antibacterial efficacy of fortifying specific bacteriophage in cosmetics to be a promising formulation for efficient treatment of skin diseases

Isolation and Characterization of Two Virulent Phages to Combat Staphylococcus aureus and Enterococcus faecalis causing Dental Caries

This study aimed to isolate and characterize bacteriophages, as a biocontrol agent, against certain antibiotic-resistant bacteria causing dental caries. Here, two dental caries-causing bacteria S. aureus and E. faecalis were isolated and characterized biochemically using the automated VITEK® 2 system. Antibiotic sensitivity pattern of the isolated dental caries bacteria was assessed against selection of antibiotics. The two isolates showed resistance against most of the tested antibiotics. To overcome this problem, two lytic phages vB_SauM-EG-AE3 and vB_EfaP-EF01 were isolated, identified, and applied to control the growth of S. aureus and E. faecalis, respectively. Phages were identified morphologically using TEM and showed that vB_SauM-EG-AE3 phage is related to Myoviridae and vB_EfaP-EF01 phage belongs to Podoviridae. The two phages exhibited high lytic activity, high stability, and a narrow host range. The one-step growth curve of phages showed burst sizes of 78.87 and 113.55 PFU/cell with latent periods of 25 and 30 minutes for S. aureus phage and E. faecalis phage respectively. In addition, the two phages showed different structural protein profiles and exhibited different patterns using different restriction enzymes. The genome sizes were estimated to be 13.30 Kb and 15.60 Kb for phages vB_SauM-EGAE3, vB_EfaP-EGAE1, respectively. Complete inhibition of bacterial growth was achieved using phages with MOIs of 103, 102 and 10 after 1, 3, 5, and 24 h of incubation at 37°C. Hence, this study indicates that the isolated bacteriophages are promising biocontrol agents that could challenge antibiotic-resistant dental caries bacteria to announce new successful alternatives to antibiotics

Characterization and Full Genome Sequence of Novel KPP-5 Lytic Phage against Klebsiella pneumoniae Responsible for Recalcitrant Infection

Klebsiella pneumoniae is a hazardous opportunistic pathogen that is involved in many serious human diseases and is considered to be an important foodborne pathogen found in many food types. Multidrug resistance (MDR) K. pneumoniae strains have recently spread and increased, making bacteriophage therapy an effective alternative to multiple drug-resistant pathogens. As a consequence, this research was conducted to describe the genome and basic biological characteristics of a novel phage capable of lysing MDR K. pneumoniae isolated from food samples in Egypt. The host range revealed that KPP-5 phage had potent lytic activity and was able to infect all selected MDR K. pneumoniae strains from different sources. Electron microscopy images showed that KPP-5 lytic phage was a podovirus morphology. The one-step growth curve exhibited that KPP-5 phage had a relatively short latent period of 25 min, and the burst size was about 236 PFU/infected cells. In addition, KPP-5 phage showed high stability at different temperatures and pH levels. KPP-5 phage has a linear dsDNA genome with a length of 38,245 bp with a GC content of 50.8% and 40 predicted open reading frames (ORFs). Comparative genomics and phylogenetic analyses showed that KPP-5 is most closely associated with the Teetrevirus genus in the Autographviridae family. No tRNA genes have been identified in the KPP-5 phage genome. In addition, phage-borne virulence genes or drug resistance genes were not present, suggesting that KPP-5 could be used safely as a phage biocontrol agent

Improving Regulation of Enzymatic and Non-Enzymatic Antioxidants and Stress-Related Gene Stimulation in Cucumber mosaic cucumovirus-Infected Cucumber Plants Treated with Glycine Betaine, Chitosan and Combination

Cucumber mosaic cucumovirus (CMV) is a deadly plant virus that results in crop-yield losses with serious economic consequences. In recent years, environmentally friendly components have been developed to manage crop diseases as alternatives to chemical pesticides, including the use of natural compounds such as glycine betaine (GB) and chitosan (CHT), either alone or in combination. In the present study, the leaves of the cucumber plants were foliar-sprayed with GB and CHT—either alone or in combination—to evaluate their ability to induce resistance against CMV. The results showed a significant reduction in disease severity and CMV accumulation in plants treated with GB and CHT, either alone or in combination, compared to untreated plants (challenge control). In every treatment, growth indices, leaf chlorophylls content, phytohormones (i.e., indole acetic acid, gibberellic acid, salicylic acid and jasmonic acid), endogenous osmoprotectants (i.e., proline, soluble sugars and glycine betaine), non-enzymatic antioxidants (i.e., ascorbic acid, glutathione and phenols) and enzymatic antioxidants (i.e., superoxide dismutase, peroxidase, polyphenol oxidase, catalase, lipoxygenase, ascorbate peroxidase, glutathione reductase, chitinase and β-1,3 glucanase) of virus-infected plants were significantly increased. On the other hand, malondialdehyde and abscisic acid contents have been significantly reduced. Based on a gene expression study, all treated plants exhibited increased expression levels of some regulatory defense genes such as PR1 and PAL1. In conclusion, the combination of GB and CHT is the most effective treatment in alleviated virus infection. To our knowledge, this is the first report to demonstrate the induction of systemic resistance against CMV by using GB

Ameliorating the Adverse Effects of <i>Tomato mosaic tobamovirus</i> Infecting Tomato Plants in Egypt by Boosting Immunity in Tomato Plants Using Zinc Oxide Nanoparticles

Tomato mosaic virus (ToMV) is one of the economically damageable Tobamovirus infecting the tomato in Egypt that has caused significant losses. It is therefore of great interest to trigger systemic resistance to ToMV. In this endeavor, we aimed to explore the capacity of ZnO-NPs (zinc oxide nanoparticles) to trigger tomato plant resistance against ToMV. Effects of ZnO-NPs on tomato (Solanum lycopersicum L.) growth indices and antioxidant defense system activity under ToMV stress were investigated. Noticeably that treatment with ZnO-NPs showed remarkably increased growth indices, photosynthetic attributes, and enzymatic and non-enzymatic antioxidants compared to the challenge control. Interestingly, oxidative damage caused by ToMV was reduced by reducing malondialdehyde, H2O2, and O2 levels. Overall, ZnO-NPs offer a safe and economic antiviral agent against ToMV

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds

Molecular Characterization of the <i>Alfalfa mosaic virus</i> Infecting <i>Solanum melongena</i> in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds