Development of full-length infectious cDNA clones and host range identification of an echinacea strain of tobacco streak virus.

Tobacco streak virus induces severe diseases on a wide range of plants and becomes an emerging threat to crop yields. However, the infectious clones of TSV remain to be developed for reverse genetics studies. Here, we obtained the full genome sequence of a TSV-CNB isolate and analyzed the phylogenetic characteristics. Subsequently, we developed the full-length infectious cDNA clones of TSV-CNB driven by 35 S promoter using yeast homologous recombination. Furthermore, the host range of TSV-CNB isolate was determined by Agrobacterium infiltration and mechanical inoculation. The results reveal that TSV-CNB can infect 10 plant species in 5 families including Glycine max, Vigna radiate, Lactuca sativa var. Ramosa, Dahlia pinnate, E. purpurea, Calendula officinalis, Helianthus annuus, Nicotiana. Benthamiana, Nicotiana tabacum and Chenopodium quinoa. Taken together, the TSV infectious clones will be a useful tool for future studies on viral pathogenesis and host-virus interactions.

A Comparison of the Immunostimulatory Effects of Polysaccharides from Tetraploid and Diploid Echinacea purpurea.

Polyploidization is an effective means of improving the active components and quality of secondary metabolism in medicinal plants. In the present study, we compared the immunostimulatory effects of crude polysaccharides from tetraploid and diploid Echinacea purpurea. The results showed that the carbohydrate contents of crude polysaccharide of tetraploid E. purpurea (CPE4) and diploid E. purpurea (CPE2) were 85.51% and 44.65%, respectively. 1H-nuclear magnetic resonance (NMR) spectroscopy and gel-permeation chromatography (GPC) analyses showed no major differences in the overall structure and molecular weight of polysaccharides between CPE4 and CPE2. However, some differences in the relative content of the same polysaccharides group were observed between CPE4 and CPE2. In in vitro tests, EP4 could stimulate lymphocyte proliferation and secretion of cytokines maximally at the concentration of 0.0312 mg/mL, and EP2 could stimulate lymphocyte proliferation and secretion of cytokines maximally at the concentration of 0.125 mg/mL. In in vivo tests, EP4 was more effective at promoting the proliferation of lymphocytes and secretion of cytokines in mice immunosuppressed by cyclophosphamide than EP2 at the same concentration. Taken together, these data demonstrated that the relative content of the partial polysaccharides group is increased, and the immunoregulatory effect is enhanced in tetraploid E. purpurea.

Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate.

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4(+) and cytotoxic CD8(+)- T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes.

Induction of tetraploids from petiole explants through colchicine treatments in Echinacea purpurea L.

Petiole explants were obtained from in vitro grown diploid (2x = 22) Echinacea purpurea plantlets. Shoots were regenerated by culturing the explants on MS basal medium containing 0.3 mg/L benzyladenine (BA), 0.01 mg/L naphthaleneacetic acid (NAA) and four concentrations (30, 60, 120, and 240 mg/L) of colchicine for 30 days, or 120 mg/L of colchicine for various durations (7, 14, 21, and 28 days). The regenerated shoots were induced to root on MS basal medium with 0.01 mg/L NAA, and then the root-tips of the regenerated shoots were sampled for count of chromosome number. It was found that a treatment duration of >7 days was necessary for induction of tetraploid (4x = 44) shoots, and treatment with 120 mg/L colchicine for 28 days was the most efficient for induction of tetraploids, yielding 23.5% of tetraploids among all the regenerated shoots. Chimeras were observed in almost all the treatments. However, the ratio of tetraploid to diploid cells in a chimeric plant was usually low. In comparison with diploid plants, tetraploid plants in vitro had larger stomata and thicker roots with more root branches, and had prominently shorter inflorescence stalk when mature.

Cytokine- and interferon-modulating properties of Echinacea spp. root tinctures stored at -20 degrees C for 2 years.

Echinacea spp. phytomedicines are popular for treating upper respiratory infections. The purpose of this investigation was to examine the immunomodulatory properties of Echinacea tinctures from seven species after being stored at -20 degrees C for 2 years. Two experimental techniques were employed using human peripheral blood mononuclear cells (PBMC). In the first set of experiments, PBMCs were stimulated in vitro with tinctures alone and assayed for proliferation and production of interleukin-10 (IL-10), IL-12, and tumor necrosis factor-alpha (TNF-alpha). In the second set of experiments, subjects were immunized with influenza vaccine. PBMCs from vaccinated individuals were stimulated in vitro with Echinacea tinctures and influenza virus; cytokine production (IL-2, IL-10, and interferon-gamma [IFN-gamma]) was compared prevaccination and postvaccination. In the first experiments, (1) tinctures from E. angustifolia, E. pallida, E. paradoxa, and E. tennesseensis stimulated proliferation and tended to increase IL-10, (2) E. sanguinea and E. simulata stimulated only proliferation, (3) E. purpurea stimulated only IL-10, and (4) none of the extracts influenced IL-12 or TNF-alpha. In the second experiments, (1) tinctures from E. pallida, E. paradoxa, E. sanguinea, and E. simulata diminished influenza-specific IL-2, and (2) none of the extracts influenced influenza-specific IL-10 or IFN-gamma. For in vitro models using Echinacea, immune response may vary based on stimulus (Echinacea alone vs. Echinacea + recall stimulation with virus).