Complete genome sequence of a novel monopartite geminivirus identified in mulberry (Morus alba L.).

The genome sequence of a novel geminivirus from mulberry samples exhibiting crinkle leaf symptoms is reported. The sequence consisted of 2952 nt, containing four open reading frames (ORFs) in the viral-sense strand and two ORFs in the complementary-sense strand. The size of the genome and the conserved origin of replication are similar to those of members of the family Geminiviridae, but the genomic organization, number of ORFs, and especially five contiguous GAAAAA repeats positioned upstream of ORF1 distinguish it from other geminiviruses. Phylogenetic analysis coupled with ORF analysis suggests that this is a novel virus that does not fit into the established seven genera of the family Geminiviridae. The virus, found in Zhenjiang, Jiangsu province, China, is tentatively named mulberry crinkle leaf virus isolate Jiangsu (MCLV-js).

A new nepovirus identified in mulberry (Morus alba L.) in China.

An isometric virus was identified in mulberry leaves showing symptoms of mulberry mosaic leaf roll (MMLR) disease. Its genome consists of two (+)ssRNAs. RNA1 and RNA2 have 7183 and 3742 nucleotides, excluding the 3'-terminal poly(A) tail. Based on phylogenetic analysis of the RNA1-encoded polyprotein and CP amino acid sequences, the properties of the the 3'-UTR of RNA1 and RNA2, and <75 % identity in the CP amino acid sequence, this virus is proposed to be a new member of the genus Nepovirus, subgroup A. Since a causal relationship between this virus and MMLR has not been established, it is tentatively referred to as MMLR-associated virus.

[Sequence variation and protein structure of pipo gene in Potato virus Y].

The objectives of this study were to understand the sequence variation and the putative protein structure of pipo gene in the Potato virus Y (PVY) collected from Solanum tuberosum. The pipo gene in PVY was cloned using a pair of degenerate primers designed from its conserved region and its sequences were used to re-construct phylogenetic tree in Potyvirus genera by a Bayesian inference method. An expected fragment of 235 bp was amplified in all 20 samples by RT-PCR and the pipo genes in the 20 samples assayed shared more than 92% nucleotide sequence similarity with the published sequences of PVY strains. Among the 20 pipo gene sequences, 13 polymorphic sites were detected, including 4 parsimony informative sites and 9 singleton variable sites. These results indicate that PVY pipo gene is highly conserved but some sequence variations exist. Further analyses suggest that the pipo gene encodes a hydrophilic protein without signal peptide and transmembrane region. The protein has theoretical isoelectric points (pI) ranging from 11.26 to 11.62 and contains three highly conserved regions, especially between aa 10 and 59. The protein is likely located in the mitochondria and has a-helix secondary structure. Bayesian inference of phylogenetic trees reveals that PVY isolates are clustered in the same branch with high posterior probability, while Sunflower chlorotic mottle virus (SoCMoV) and Pepper severe mosaic virus (PepSMV) are closely related, consisting with the classification of Potyvirus genera using other approaches. Our analyses suggest that the pipo gene can be a new marker for phylogenetic analysis of the genera. The results reported in this paper provide useful insights in the genetic variation and the evolution of PVY and can stimulate further research on structure and function of the PIPO protein.

Identification and expression of caspase-1 gene under heat stress in insecticide-susceptible and -resistant Plutella xylostella (Lepidoptera: Plutellidae).

A caspase gene in Plutella xylostella (DBM) was identified firstly and named Px-caspase-1. It had a full-length of 1172 bp and contained 900 bp open reading frame that encoded 300 amino acids with 33.6 kDa. The deduced amino acid of Px-caspase-1 had two domain profile including caspase_p20 (position 61-184) and caspase_p10 (position 203-298) (i.e. the big and small catalytic domains), and the highly conserved pentapeptide QACQG in caspase_p20 domain (the recognized catalytic site of caspases). Being highly homologous to effector caspase genes in other insect and mammalian species, Px-caspase-1 was thought to be an effector caspase gene. Heat stress could result in significant mortality increase on adult DBM. Px-caspase-1 mRNA expression and caspase-3 enzyme activity (a effector caspase) were elevated with age and heat treatment. And, heat stress facilitated the procession of Px-caspase-1 expression. Significantly higher mRNA transcription levels were found in a chlorpyrifos-resistant DBM strain, as compared to those in insecticide-susceptible DBM. The results indicated that high temperature could significantly promote apoptosis process resulting in an the increased DBM mortality rate, and that insecticide-susceptible DBM had a significantly higher physiological fitness at high temperatures than insecticide-resistant DBM.

A new phenolic glycoside and cytotoxic constituents from Celosia argentea.

A new phenolic glycoside, 4-O-ß-D-apifuranosyl-(1→2)-ß-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone (2) and 11 known compounds were isolated from the MeOH extract of the plant Celosia argentea. The structures of the compounds were elucidated on the basis of spectroscopic analysis and chemical methods. Among the isolated compounds, stigmasterol (10) showed moderate inhibitory activities against SGC-7901 and BEL-7404 cells.

Antiphytoviral activity of bruceine-D from Brucea javanica seeds.

BACKGROUND: Brucea javanica (L.) Merr. is widely distributed throughout the southern parts of China and has been used in traditional medicine to treat a variety of diseases. The objective of the present study was to identify the active antiphytoviral compound in the seeds of B. javanica and evaluate the inhibitory activity of the compound against plant virus. RESULTS: Bioassay-guided fractionation of the most active extract from the seeds led to the isolation of an antiphytoviral compound which was identified as bruceine-D by conventional spectroscopy methods. The compound exhibited significant inhibitory activity against the infection and replication of tobacco mosaic virus (TMV), with IC(50) values of 13.98 and 7.13 mg L(-1) respectively. The compound also showed a strong inhibitory effect on the infectivity of potato virus Y (PVY) and cucumber mosaic virus (CMV). Furthermore, the compound could effectively inhibit systemic TMV infection in the host tobacco plant under glasshouse conditions. CONCLUSION: The results suggested that bruceine-D from Brucea javanica may have the potential to be used as a natural viricide, or a lead compound for new viricides.

Insecticide toxicity and synergism by enzyme inhibitors in 18 species of pest insect and natural enemies in crucifer vegetable crops.

The toxicities of three enzyme inhibitors and their synergistic effects on four insecticides were studied by using the dry film method on field populations of 18 species of insects collected in Jianxin and Shanjie, China, from 2003 to 2005. Meanwhile, the inhibitory effects of these enzyme inhibitors on the activities of acetylcholinesterases (AChE), carboxyesterases (CarE) and glutathione-S-transferases (GST), in vivo, were also studied. In general, triphenyl phosphate (TPP) and diethyl maleate (DEM) showed low toxicities to six herbivorous pest insects, four ladybirds and eight parasitoids. Piperonyl butoxide (PB) exhibited low toxicities to the herbivorous pest insects and ladybirds, but high toxicities to the eight parasitoids. The tolerance to the insecticides in 11 pest insects and natural enemies was mainly associated with the tolerance to PB. PB showed the highest synergism on methamidophos, fenvalerate, fipronil and avermectin in nine species of pest insects and natural enemies. In general, TPP and DEM showed significant synergisms to these four insecticides in four parasitoid species. However, in contrast to their effects on the parasitoids, the synergistic effects of TPP and DEM on the four insecticides by TPP and DEM against four pest insects and one ladybird varied depending on the insect species and enzyme inhibitor. Activity of AChE, CarE or GST could be strongly inhibited, in vivo, by PB, TPP or DEM, depending on the insect species and enzyme inhibitors. From the results obtained in this study, mixed-function oxidase (MFO) was thought to play the most critical role in insect tolerances to the tested insecticides in the field. Low competition existed in the evolution of insecticide resistance in the field populations of parasitoids, as compared with herbivorous pest insects and ladybirds. Possible causes of the high synergistic effects of PB on the four classes of insecticides, based on multiattack on the activity of CarE, GST or AChE in the insect species, are also discussed.

[Effects of microcystins on expression of apoptosis-associated hepatocyte genes].

OBJECTIVE: To deeply explore the effects of microcystins (MC-LR) on Bax and Bcl-2 during the course of MC-LR promoting liver tumor. METHODS: applied to set up the animal model, and the effect of MC-LR promoting liver tumor was evaluated by the Albertgamma-GT methods. And then, the immunohistochemical technique, RT-PCR and image analysis were used to study the expression of the Bcl-2 and Bax during the course of promoting tumor. RESULTS: (1) MC-LR might enhance the positive reaction rate of GGT. The positive reaction rate of GGT in DEN + pure toxin group was 100%, it was significantly higher than the DEN control group 22.22% (P < 0.05). (2) The intension and areas of the protein expression of Bcl-2 in DEN + pure toxin group were 0.0977 and 0.0315, and in DEN control group were 0.0460 and 0.0205, respectively. The expression level of Bcl-2 protein in DEN + pure toxin group were significantly higher than in DEN control group (P < 0.05). Simultaneously, the protein expression of Bax was significantly decreased by MC-LR (P < 0.05). The intension and areas of the expression of Bax in DEN + pure toxin group were 0.0283 and 0.0073, and in DEN control group were 0.0655 and 0.0244 respectively. (3) The mRNA expression of Bcl-2 was significantly increased by MC-LR. The intension of Bcl-2 mRNA expression in DEN + pure toxin group was 2.244, being significantly higher than in the other groups (P < 0.05). However, the mRNA expression of Bax showed no significant difference between DEN + pure toxin and the other groups. CONCLUSION: The expression change of Bcl-2 and Bax should possibly play an important role in the course of MC-LR promoting liver tumor.

[Current situation and prospect of studies on genus Umbravirus].

Umbraviruses are a group of imperfectly characterized plant viruses, which are distinguished from most other viruses by their genomes lack of a gene for coat protein (CP) , and as a result umbraviruses do not form conventional virus particles. Umbraviruses are mechanically transmissible, and can be aphid transmitted in the persistent manner by an unrelated assistor virus, which is always a member of the family Luteoviridae . In nature, each umbravirus depends for survival on one particular luteovirus. The genus Umbravirus comprises seven distinct virus specieses and three tentative members. Only Tobacco bushy top virus (TBTV) has been reported in China as an umbravirus. Tobacco bushy top disease, caused by TBTV and its helper, Tobacco vein distorting virus(TVDV), which resulted in severe tobacco losses in western of Yunan. Umbraviruses had a restricted host range in nature, and their infectivity and longevity in vitro are not so stable. Plants infected umbraviruses contain abundant double-stranded RNA (dsRNA), and some umbraviruses possess one or more additional dsRNA species associated with the presence of a satellite RNA. The genomes of the umbraviruses consist of one linear segment of positive sense single-stranded RNA(ssRNA), and the nucleotide sequences possess ORFs for four potential non-structural protein products. The umbravirus-encoded ORF3 proteins play essential roles in stabilization of viral RNA and mediation of its long-distance movement. The current research progresses have been reviewed detailly, and the future research tendency and research fields about umbraviruses and umbravirus-caused diseases are put forward.

Viruliferous rate of small brown planthopper is a good indicator of rice stripe disease epidemics.

Rice stripe virus (RSV), its vector insect (small brown planthopper, SBPH) and climatic conditions in Jiangsu, China were monitored between 2002 and 2012 to determine key biotic and abiotic factors driving epidemics of the disease. Average disease severity, disease incidence and viruliferous rate of SBPH peaked in 2004 and then gradually decreased. Disease severity of RSV was positively correlated with viruliferous rate of the vector but not with the population density of the insect, suggesting that the proportion of vectors infected by the virus rather than the absolute number of vectors plays an important role in RSV epidemics and could be used for disease forecasting. The finding of a positive correlation of disease severity and viruliferous rate among years suggests that local infection is likely the main source of primary inoculum of RSV. Of the two main climatic factors, temperature plays a more important role than rainfall in RSV epidemics.

[Advance in researches of drugs derived from marine bacteria].

Marine bacteria are capable of producing a lot of unique bioactive substances, and therefore provide a luxuriant resource for screening new drugs. Bioactive substances derived from marine bacteria have bright prospect in marine drugs development and research.

Purification and Characterization of AAVP, a Protein Inhibitor of TMV Infection, from the Edible Fungus, Agrocybe aegerita.

A protein with activity of inhibiting TMV infection on Nicotiana glutinosa, named AAVP, was isolated and purified from the fruiting bodies of edible fungus Agrocybe aegerita by precipitation of 40%--80% saturation of (NH(4))(2)SO(4) followed by DEAE-Fast Flow and S-200 column chromatography. The protein was proved to be homogeneous by SDS-PAGE and IEF. Analysis of the composition of amino acids showed that this protein contained no cysteine, poor in methionine and phenylalanine and rich in acidic and hydroxyl amino acids. The inhibition of TMV was 84.32% when the concentration of AAVP was 200 mg/L. N-terminus of this protein was blocked by pyroglutamyl, and the N-terminal sequence was QGVNIYNIVAGA. On potato-sucrose-agar medium, the purified protein did not inhibit the growth of three plant pathogen fungi, Trichoderma viride, Colletotrichum musae and Fusarium oxysporum.

[Sequence analysis of RNA3 of Rice stripe virus isolates found in China: evidence for reassortment in Tenuivirus].

The RNA3 segments of four isolates of Rice stripe virus (RSV), isolated from endemic sites at Panjin (PJ), Liaoning Province, Kunming (KM) and Yiliang (YL), Yunnan Province, as well as from outbreak sites at Hongze (HZ), Jiangsu Province, were determined. RNA3 of these four isolates were 2480 bp, 2509 bp, 2489 bp and 2497 bp in length, respectively. Compared with RNA3 of T and M isolates from Japan and Y isolate from Yunnan Province of China, that had been previously reported, these seven isolates could be divided into two groups. KM and YL isolates formed group one, and PJ, HZ, Y, T and M isolates belonged to another group. The two groups shared 97%-98% and 93%-94% sequence homology in viral RNA3 (vRNA3) and viral complementary RNA3 (vcRNA3) at the nucleotide levels, respectively, and there was no significant difference between the two groups at the amino acid levels. In the first group, Y isolate was significantly different from HZ,PJ and two Japan isolates in their RNA4 segment. These results show that there were two subgroups in RSV natural population related with geographical location, and reassorment may be the main factor leading to different segments of Y isolate belonging to different subgroups. The results may provide another evidence for reassortment variation in Tenuivirus.

[Construction of rice stripe virus NS2 and NS3 Co-RNAi transgenic rice and disease-resistance analysis].

NS2 and NS3 are two post-transcriptional gene silencing suppressors that are encoded by Rice stripe virus. Gene silencing suppressors are always related to the pathogenicity of viruses. In this study, the cDNA of NS2 and NS3 were recombined by overlapping PCR assays, ligated to the RNAi vector, and inserted into the PXQ expression vector using Pst I; the expressed vector was transferred into calluses induced from seeds of the japonica rice cultivar, 'Nipponbare', using an Agrobacterium-mediated method. Thirty-one T0 transgenic plants were selected by G418 screening. PCR and southern blot analyses confirmed that the target gene was transformed into transgenic rice successfully, and different transgenic plants contained various copies of the gene. The disease resistance assay revealed that T0 transgenic rice had a delayed onset of RSV for approximately 10-20 d, and the accumulation of virus in the transgenic plants was reduced by 30%-50%. This was related to the delayed onset of disease.

Protein from red cabbage (Brassica oleracea) seeds with antifungal, antibacterial, and anticancer activities.

A 30 kDa antifungal protein was purified from red cabbage ( Brassica oleracea ) seeds. It exhibited a molecular mass and N-terminal amino acid sequence disinct from those of previously isolated Brassica antifungal proteins. The protocol used entailed ion exchange chromatography on Q-Sepharose and SP-Sepharose followed by fast protein liquid chromatography on Mono S. The protein hindered mycelial growth in Mycosphaerella arachidicola (with an IC50=5 µM), Setospaeria turcica, and Bipolaris maydis. It also inhibited the yeast Candida albicans with an IC50=96 µM. It exerted its antifungal action by permeabilizing the fungal membrane as evidenced by staining with Sytox green. The antifungal activity was stable from pH 3 to 11 and from 0 to 65 °C. It manifested antibacterial activity against Pseudomonas aeruginosa (IC50=53 µM). Furthermore, after 48 h of culture, it suppressed proliferation of nasopharyngeal cancer and hepatoma cells with IC50=50 and 90 µM, respectively.

Expression of rice gall dwarf virus outer coat protein gene (S8) in insect cells.

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Genetic diversity and population structure of rice stripe virus in China.

Rice stripe virus (RSV) is one of the most economically important pathogens of rice and is repeatedly epidemic in China, Japan and Korea. The most recent outbreak of RSV in eastern China in 2000 caused significant losses and raised serious concerns. In this paper, we provide a genotyping profile of RSV field isolates and describe the population structure of RSV in China, based on the nucleotide sequences of isolates collected from different geographical regions during 1997-2004. RSV isolates could be divided into two or three subtypes, depending on which gene was analysed. The genetic distances between subtypes range from 0.050 to 0.067. The population from eastern China is composed only of subtype I/IB isolates. In contrast, the population from Yunnan province (southwest China) is composed mainly of subtype II isolates, but also contains a small proportion of subtype I/IB isolates and subtype IA isolates. However, subpopulations collected from different districts in eastern China or Yunnan province are not genetically differentiated and show frequent gene flow. RSV genes were found to be under strong negative selection. Our data suggest that the most recent outbreak of RSV in eastern China was not due to the invasion of new RSV subtype(s). The evolutionary processes contributing to the observed genetic diversity and population structure are discussed.

Hydrolysis of acetylthiocholine iodide and reactivation of phoxim-inhibited acetylcholinesterase by pralidoxime chloride, obidoxime chloride and trimedoxime.

The hydrolysis of acetylthiocholine iodide (ATCh) by pralidoxime chloride (2-PAM Cl), trimedoxime (TMB(4)) and obidoxime chlpride (LUH(6)) was studied at pH 5.8-8.0 and incubation temperature from 5 to 40 degrees C in vitro. Significant ATCh hydrolysis by 2-PAM Cl, TMB(4) and LUH(6) was found, with the exceptions of those at pH 7.0, 6.2 and 5.8 at 5 degrees C and those at pH 6.2 and 5.8 at 15 degrees C. The hydrolysis by TMB(4) and LUH(6) was significantly stronger than that by 2-PAM Cl. The hydrolysis increased with increasing pH, incubation temperature and three oxime or ATCh concentration. Significant hydrolysis of ATCh by the three oximes could be found when the terminal concentration of oxime was higher than 0.01 mM at pH 7.0 and 7.4 at 30 and 37 degrees C. However, no hydrolysis of natural substrate (acetylcholine iodide) by the three oximes was found when very high terminal concentrations of oximes were used. In addition, the three oximes displayed an extraordinary efficiency in the reactivation of phoxim-inhibited acetylcholinesterase (AChE) from fish (Carassius auratus) or rabbit (Oryctolagus cuniculus domestic) brain in vitro. Parallel to the level of ATCh hydrolysis by the oximes, TMB(4) and LUH(6) displayed significantly higher reactivation efficiency than 2-PAM Cl to phoxim-inhibited AChE. And, the extent of reactivation by 2-PAM Cl was also lower than the other two. Plausible antidotal actions of the oximes against organophosphate poisoning AChE and erroneously high estimation of AChE activity by the Ellman method were discussed.

Structure-activity relationship of triterpenes and triterpenoid glycosides against tobacco mosaic virus.

A new activity of triterpenes and triterpenoid glycosides against phytovirus has been found. Based on the anti-TMV replication activity in vivo, 47 triterpenes and triterpenoid glycosides were screened through the leaf-disk method together with indirect ELISA. Structure-activity relationships were analyzed according to the results of bio-activity screening. Besides, the EC(50) values of those compounds having higher activities were measured.

The quantification of monacolin K in some red yeast rice from Fujian province and the comparison of the other product.

When used as a dietary supplement to achieve and maintain healthy cholesterol levels,Chinese red yeast rice has significant potential to reduce health care costs and contribute to public health by reducing heart disease risk in individuals with moderate elevations of circulating cholesterol levels. Red yeast rice is typical product in Mingbei area of Fujian province. Nine products from different area were measured, using high-performance chromatography (HPLC) with photodiode array detector (PDA) and tandem mass spectrometry (MS) and the results show that the contents of monacolin K in these products were considerably different, and more than the other product.