Discovery of Resveratrol and its Derivatives as Novel Antiviral and Anti- Phytopathogenic-Fungus Agents.

BACKGROUND: Plant diseases caused by viruses and pathogens have posed a serious threat to global agricultural production and are difficult to control. Natural products have always been a valuable source for lead discovery in medicinal and agricultural chemistry. The natural product resveratrol was found to have good antiviral activity against the tobacco mosaic virus (TMV) and fungicidal activities against 14 kinds of phytopathogenic fungi. OBJECTIVE: The aim of this work was to design, synthesize a series of derivatives of resveratrol, and evaluate their antiviral and fungicidal activities systematically. METHODS: Novel resveratrol sulfonate derivatives were prepared by a convenient synthesis method from resveratrol, alkyl sulfonyl chloride, aryl sulfonyl chloride, and heterocyclic sulfonyl chloride. Their structures were also identified by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). RESULTS: Most of the targets were obtained at a high yield. Compounds I-2, I-5, I-10, II-2, and II-4, with excellent antiviral activities, showed higher anti-TMV activities than those of lead compounds and commercial ribavirin (inhibitory rates of 38, 37, and 38% at 500 µg/mL for inactivation, curative, and protection activities in vivo, respectively). In particular, compounds I-5, I-10, II-2, and II-4 displayed similar inhibitory effects as ningnanmycin (inhibitory rates of 54, 56, and 58% at 500 µg/mL for inactivation, curative, and protection activities in vivo, respectively), the best antiviral agent at present, thereby emerging as new antiviral pilot compounds. Further fungicidal activity tests showed that resveratrol derivatives also displayed broad-spectrum fungicidal activities. CONCLUSION: The anti-TMV activities of these compounds were discovered for the first time. Some of these simply structured compounds showed higher TMV inhibitory effects than ribavirin. The current study provided valuable insights into the antiviral and fungicidal activities of resveratrol derivatives, but more modification of the structure should be conducted.

Natural phytoalexin stilbene compound resveratrol and its derivatives as anti-tobacco mosaic virus and anti-phytopathogenic fungus agents.

Plant diseases caused by plant viruses and pathogens seriously affect crop yield and quality, and it is very difficult to control them. The discovery of new leads based on natural products is an important way to innovate pesticides. Based on the resveratrol is a kind of natural phytoalexin, but it cannot be used as candidate for the development of new drug due to its poor druggability. The phenolic hydroxyl groups in the resveratrol structure are easily destroyed by oxidation, in order to improve its stability, ester formation is the most commonly used modification method in drug design. Their structures were characterized by 1H NMR, 13C NMR and HRMS. The activity against tobacco mosaic virus (TMV) of these ester derivatives has been tested for the first time. The bioassay results showed part of the target compounds exhibited good to excellent in vivo activities against TMV. The optimum compounds III-2 (inhibitory rates of 50, 53, and 59% at 500 µg/mL for inactivation, curative, and protection activities in vivo, respectively), III-4 (inhibitory rates of 57, 59, and 51% at 500 µg/mL, respectively), and II-5 (inhibitory rates of 54, 52, and 51% at 500 µg/mL, respectively) displayed higher activity than commercial plant virucide ribavirin (inhibitory rates of 38, 37, and 40% at 500 µg/mL, respectively). Compounds I-9 and I-10 also showed excellent activities. The systematic study provides strong evidence that these simple resveratrol derivatives could become potential TMV inhibitors. The novel concise structure provides another new template for antiviral studies.

Multiscale correlations of iron phases and heavy metals in technogenic magnetic particles from contaminated soils.

Technogenic magnetic particles (TMPs) are carriers of heavy metals and organic contaminants, which derived from anthropogenic activities. However, little information on the relationship between heavy metals and TMP carrier phases at the micrometer scale is available. This study determined the distribution and association of heavy metals and magnetic phases in TMPs in three contaminated soils at the micrometer scale using micro-X-ray fluorescence (µ-XRF) and micro-X-ray absorption near-edge structure (µ-XANES) spectroscopy. Multiscale correlations of heavy metals in TMPs were elucidated using wavelet transform analysis. µ-XRF mapping showed that Fe was enriched and closely correlated with Co, Cr, and Pb in TMPs from steel industrial areas. Fluorescence mapping and wavelet analysis showed that ferroalloy was a major magnetic signature and heavy metal carrier in TMPs, because most heavy metals were highly associated with ferroalloy at all size scales. Multiscale analysis revealed that heavy metals in the TMPs were from multiple sources. Iron K-edge µ-XANES spectra revealed that metallic iron, ferroalloy, and magnetite were the main iron magnetic phases in the TMPs. The relative percentage of these magnetic phases depended on their emission sources. Heatmap analysis revealed that Co, Pb, Cu, Cr, and Ni were mainly derived from ferroalloy particles, while As was derived from both ferroalloy and metallic iron phases. Our results indicated the scale-dependent correlations of magnetic phases and heavy metals in TMPs. The combination of synchrotron based X-ray microprobe techniques and multiscale analysis provides a powerful tool for identifying the magnetic phases from different sources and quantifying the association of iron phases and heavy metals at micrometer scale.

Design, synthesis, antiviral activity and mode of action of phenanthrene-containing N-heterocyclic compounds inspired by the phenanthroindolizidine alkaloid antofine.

BACKGROUND: The phenanthroindolizidine alkaloid antofine and its analogues have excellent antiviral activity against tobacco mosaic virus (TMV). To simplify the structure and the synthesis of the phenanthroindolizidine alkaloid, a series of phenanthrene-containing N-heterocyclic compounds (compounds 1 to 33) were designed and synthesised, based on the intermolecular interaction of antofine and TMV RNA, and systematically evaluated for their anti-TMV activity. RESULT: Most of these compounds exhibited good to reasonable anti-TMV activity. The optimum compounds 5, 12 and 21 displayed higher activity than the lead compound antofine and commercial ribavirin. Compound 12 was chosen for field trials of antiviral efficacy against TMV, and was found to exhibit better activity than control plant virus inhibitors. Compounds 5 and 12 were chosen for mode of action studies. The changes in fluorescence intensity of compounds 5 and 12 on separated TMV RNA showed that these small molecules can also bind to TMV RNA, but the mode is very different from that of antofine. CONCLUSION: The compounds combining phenanthrene and an N-heterocyclic ring could maintain the anti-TMV activity of phenanthroindolizidines, but their modes of action are different from that of antofine. The present study lays a good foundation for us to find more efficient anti-plant virus reagents.

Design, Synthesis, and Acaricidal/Insecticidal Activities of Oxazoline Derivatives Containing a Sulfur Ether Moiety.

On the basis of etoxazole, a series of novel 2-(2,6-difluorophenyl)-4-(4-substituted phenyl)-1,3-oxazolines containing a sulfur ether moiety were designed and synthesized via the key intermediate N-(1-(4-(bromomethyl)phenyl)-2-chloroethyl)-2,6-difluorobenzamide. The bioassay results showed that most of these designed target compounds exhibited excellent acaricidal activity against both the eggs and larvae of Tetranychus cinnabarinus, especially the eggs. Among compounds with high activity against the eggs of mites, the LC50 values of 2, 11, 17, and 19 were 0.0003, 0.0002, 0.0005, and 0.0008 mg L(-1), respectively, much lower than that of etoxazole (0.0089 mg L(-1)). Compound 2 was chosen to evaluate the acaricidal activity in the field, and the results displayed that at a concentration of 22 mg kg(-1), 2 had a much better control effect than etoxazole against both T. cinnabarinus and P. latus on eggplant. Some compounds also showed good insecticidal activities against oriental armyworm and mosquito. On the basis of our research, the newly found structure-activity relationship may guide the development of new acaricides/pesticides that are required in the agriculture market.

RNAi-based inhibition of porcine reproductive and respiratory syndrome virus replication in transgenic pigs.

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease causing heavy losses to the swine industry worldwide. Many studies have shown that transient delivery of small interfering RNA (siRNA) or adenovirus-mediated RNA interfere (RNAi) could potentially inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in vivo and in vitro. Here, we applied RNAi to produce transgenic (TG) pigs that constitutively expressed PRRSV-specific siRNA derived from small hairpin RNA (shRNA). First, we evaluated siRNA expression in the founding and F1 generation pigs and confirmed stable transmission. Then, we detected the expression of IFN-ß and protein kinase R (PKR) and found no difference among TG, non-transgenic (NTG), and wild-type pigs. Lastly, the F1 generation pigs, including TG and NTG piglets, were challenged with 3×104·5 TCID50 of JXA1, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Our results showed that the in vivo siRNA expression substantially reduced the serum HP-PRRSV titers and increased survival time by 3 days when TG pigs were compared with the NTG controls. These data suggested that RNAi-based genetic modification might be used to breed viral-resistant livestock with stable siRNA expression with no complications of siRNA toxicity.

Overexpression of cytoglobin gene inhibits hypoxic injury to SH-SY5Y neuroblastoma cells.

A plasmid for cytoglobin expression, pAcGFP1-C1-cytoglobin, was transfected into SH-SY5Y cells. Cobalt chloride was used to establish a model of hypoxia. Western blotting indicated that cytoglobin was overexpressed and there was low expression of hypoxia-inducible factor-1α in SH-SY5Y cells after transfection. Following cobalt chloride-induced hypoxia, cytoglobin and hypoxia-inducible factor-1α expression gradually increased in SH-SY5Y cells. Flow cytometry showed that with increasing duration of hypoxia, the proportion of normal cells significantly diminished in the transfected and non-transfected groups. The proportion of cells in the early stages of apoptosis increased. However, the proportion of apoptotic cells was significantly lower in the transfected group compared with the non-transfected group. These results demonstrate that cytoglobin and hypoxia-inducible factor-1α are strongly up-regulated by hypoxia, and that there is a strong relationship between hypoxia-inducible factor-1α and cytoglobin during hypoxic injury.

The epidemic status and genetic diversity of 14 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) isolates from China in 2009.

A high-mortality swine disease, the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS), reappeared in some regions of China in 2009. To explore the possible mechanisms underlying the emergence of HP-PRRSV and more fully understand the extent of the genetic diversity of this virus in China, the complete genome of 14 isolates from 10 provinces in China from 2009 were analyzed. Full-length genome sequencing analysis showed that the 14 isolates were closely related to HP-PRRSV, with 98.0-98.9% nucleotide similarity, although 2 of the 14 strains exhibited a new, discontinuous 29-amino acid deletion in the Nsp2 gene. Furthermore, amino acid analysis of the GP5 protein indicated that the 14 isolates had a concurrent mutation in a decoy epitope and different mutations in glycosylation sites. Additionally, the antigenic drift in GP3 and a 1-nucleotide deletion in both the 5'-UTR and 3'-UTR, which are found in almost all highly pathogenic Chinese PRRSV isolates, were examined in all 14 isolates. The phylogenetic analysis showed that the 14 strains belonged to the North American genotype and were clustered in a subgroup with other HP-PRRSV isolates that have been found in China since 2006. However, compared with other Chinese HP-PRRSV isolates collected in 2006-2008, the phylogenetic tree showed that the 14 isolates had a closer relationship with each other. These results indicated that HP-PRRSV remained an extensive pandemic, affecting swine farms in China in 2009 and revealed new genetic diversity.

Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China.

Porcine reproductive and respiratory syndrome (PRRS) has a major negative economic impact on the swine industry worldwide. During the investigation of PRRS virus (PRRSV) in mainland China, European genotype (EU, type 1) PRRSV isolates were detected in swine herds both with and without clinical symptoms. Two complete genome sequences for Chinese type 1 PRRSV isolates were identified from viruses isolated from lung tissue and sera. The two viruses, designated BJEU06-1 and NMEU09-1, produced cytopathic effects in primary porcine alveolar macrophages but not in Marc-145 cells, and had a mean diameter of 55 nm, as measured by transmission electron microscopy . Comparative sequence analysis revealed that they shared 87.0-91.5% and 58.0-58.2% identity with the EU and North American genotype (NA, type 2) prototypic strains LV and VR-2332, respectively. Remarkably, these isolates, characterized by concomitant deletions within non-structural protein 2 (Nsp2) and ORF3 hypervariable regions, have never been described. Phylogenetic trees showed that all of the novel Chinese isolates of European genotype are in the pan-European subtype 1 that is predominant in Europe. However, they evolved from different ancestors. These novel viruses are predicted to be products of the divergent evolution of ancestor PRRSV isolates introduced from Europe. This is the first report of type 1 PRRSV wild isolates being in mainland China. Our findings confirm that the Chinese type 1 PRRSV isolates originated from diverse progenitors and the type 1 and type 2 PRRSV isolates, having different biological properties, have coexisted on the Chinese mainland for several years.

Detection of Neospora caninum from farm-bred young blue foxes (Alopex lagopus) in China.

Neospora caninum has been detected in several wild mammalian species, i.e., deer, coyotes, dingoes, and foxes. Farm-bred foxes were rarely reported to be affected by the parasite. In this study, we detected for the first time the infection of N.caninum in farm-bred young blue foxes (Alopex lagopus) in China. Neospora-like tissue cysts were observed in brains and kidneys of the foxes by histopathological and immunohistochemical examinations. One hundred and three sera from the clinically normal vixens were tested for the presence of N.caninum and Toxoplasma gondii antibodies by two commercial ELISA test kits. Twenty-eight of 103 (27.2%) sera were positive for N. caninum and 1 serum (0.97%) was positive for T. gondii. A portion of the Nc5 gene of N. caninum was amplified from the DNA extracted from the fox brains by semi-nested PCR, further confirmed the existence of N. caninum among the farm-bred fox herd in China.