African swine fever (ASF), caused by the African swine fever virus (ASFV), is a devastating disease of domestic and wild pigs.  There is no effective vaccine, and the control of the disease relies mainly on surveillance and early detection of infected pigs.  Previously, serological assays, such as ELISA, have been developed mainly based on recombinant structural viral proteins of ASFV, including p72, p54, and p30.  However, the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.  Therefore, new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.  In this study, we expressed and purified a recombinant pB602L protein.  The purified pB602L protein was then used as an antigen to develop an indirect ELISA assay.  This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China, such as classical swine fever virus, pseudorabies virus, and porcine parvovirus.  This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples, including an unknown number of anti-ASFV sera.  The coincidence of the two assays was 95%.  Furthermore, the pB602L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7GD in pigs.  The results showed that the antibody levels in all vaccinated pigs, starting from the 10th day post-inoculation, have increased continuously during the observation period of 45 days.  Our results indicate that this pB602L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Duck hepatitis B virus (DHBV) shares many basic characteristics with hepatitis B virus (HBV) and is an attractive model for vaccine development.  In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks.  Attenuated Salmonella typhimurium (SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response.  Based on this strategy, novel DNA vaccines (SL7207-pVAX1-LC and SL7207-pVAX1-SC) were generated.  Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy.  The relative transcript levels of the CD4 and CD8 T genes and the antibody levels (IgY) in ducks receiving the vaccines were higher than those in single gene delivered groups.  Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

    The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus’s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCV Cap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pastoris GS115. The strains that displayed the phenotype of Mut+ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What’s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15–20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the results demonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

Many mammalian herpes viruses utilize heparan sulfate (HS) moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C (gC) homologues.  However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus (DPV) remains preliminary.  To study the role of gC during DPV infection, we used a gC-deleted mutant virus (DPV-ΔgC-EGFP).  Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface.  The affinity of viral glycoproteins (gB-DPV, gC-DPV, and gE-DPV) to HS was assessed using a prokaryotic expression system and HiTrap^TM Heparin HP column chromatography.  In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-ΔgC-EGFP or wild-type strain Chinese virulent duck plague virus (DPV-CHv).  The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on viral adsorption is independent of interactions between gC and heparin sulfate on cell surface.  All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses.  Future studies will be required to identify the receptors involved in gC protein binding to cells.  This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

    Thirteen kinds of steroid hormones in raw milk (cow, goat and buffalo milk) were analyzed with ultra performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) after extraction and cleanup with the modified QuEChERS method. These steroid hormones included 17β-estradiol, estriol, estrone, diethylstilbestrol, progesterone, melengestrol acetate, megestrol acetate, chlormadinone acetate, 19-nortestosterone, metandienone, boldenone, epitestosterone, and testosterone. The limits of detection for the raw milk basing on 3 times the signal to noise ratios (S/N=3) was in range of 0.07-0.51 µg kg^–1, and the limits of quantification (basing on S/N=10 method) covered the ranges from 0.23 to 1.7 µg kg^–1. With matrix external standard method, the substances presented recoveries over the range 74.2–99.7%. Qualitative analysis was also done in the mass/mass spectrum (MS/MS) mode and each debris structure of 13 kinds of steroid hormones was achieved. The methodology was then applied in real raw milk samples which were collected in several areas of China and the progesterone was detected with high level.

An in vitro rumen gas production technique was employed to determine the methane production and fermentation characteristics of Leymus chinensis and Medicago ruthenica at differing harvest dates (May 15, May 30, June 30, July 30, August 30 and September 30), which are sequential phases within a single continuous growth of two 10-year-old pastures. To quantify the rate of degradation and compare in vitro rumen fermentation characteristic, a logistic-exponential model, where initial gas volume was zero (LE0), was used to fit gas production and methane output results. Dried, milled forage samples were incubated in vitro for 72 h at 39°C and gas production was recorded intermittently throughout the incubation and gas samples were collected to measure methane production. Results showed that there were significant interactions between species and harvest for all chemical composition variables (P<0.001) and condensed tannin content (P<0.001). L. chinensis produced more total gas and methane than M. ruthenica (P<0.001). Both total gas and methane production decreased lineally (P<0.001) with advancing harvest date. The degradation rates of L. chinensis and M. ruthenica harvested on September 30 were lower than those on the other harvest dates (P<0.01). M. ruthenica fermented fluid had higher concentration of ammonia N (P<0.05) and molar proportions of isobutyrate (P<0.01), valerate (P<0.001) and isovalerate (P<0.01) in total volatile fatty acids than L. chinensis. Furthermore, concentration of isovalerate decreased cubically with advancing harvest date (P<0.05). In conclusion, M. ruthenica produced less methane than L. chinensis and the total gas and methane production decreased with advancing harvest date for both species, which may be due to the changes in contents of chemical compositions and condensed tannin in forages.

    CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for specific detection of goose CD8α (goCD8α). The results showed that the optimal coated antibody and antigen dilutions were 1:50 (the antibody titer was 1:12 800) and 1:32 (0.3 ng mL^–1), respectively, while the optimal capture antibody and horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG dilutions were 1:50 (the antibody titer was 1:51 200) and 1:4 000 (the antibody titer was 1:5 000), respectively. The optimal blocking buffer was 5% bovine serum albumin (BSA). The best incubating condition was overnight at 4°C, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10–3 ng mL^–1. Most importantly, goCD8α expression levels in goose spleen mononuclear cells (MNCs) post-Goose parvoviruse (GPV) infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of goCD8α.

    Quorum sensing (QS) is a type of microbe-microbe communication system that is widespread among the microbial world, particularly among microorganisms that are symbiotic with plants and animals. Thereby, the cell-cell signalling is likely to occur in an anaerobic rumen environment, which is a complex microbial ecosystem. In this study, using six ruminally fistulated Liuyang black goats as experimental animals, we aimed to detect the activity of quorum sensing autoinducers (AI) both in vivo and in vitro and to clone the luxS gene that encoded autoinducer-2 (AI-2) synthase of microbial samples that were collected from the rumen of goats. Neutral detergent fiber (NDF) and soluble starch were the two types of substrates that were used for in vitro fermentation. The fermented fluid samples were collected at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h of incubation. The acyl-homoserine lactones (AHLs) activity was determined using gas chromatography-mass spectrometer (GC-MS) analysis. However, none of the rumen fluid extracts that were collected from the goat rumen showed the same or similar fragmentation pattern to AHLs standards. Meanwhile, the AI-2 activity, assayed using a Vibrio harveyi BB170 bioassay, was negative in all samples that were collected from the goat rumen and from in vitro fermentation fluids. Our results indicated that the activities of AHLs and AI-2 were not detected in the ruminal contents from six goats and in ruminal fluids obtained from in vitro fermentation at different sampling time-points. However, the homologues of luxS in Prevotella ruminicola were cloned from in vivo and in vitro ruminal fluids. We concluded that AHLs and AI-2 could not be detected in in vivo and in vitro ruminal fluids of goats using the current detection techniques under current dietary conditions. However, the microbes that inhabited the goat rumen had the potential ability to secrete AI-2 signaling molecules and to communicate with each other via AI-2-mediated QS because of the presence of luxS.

Eight compounds were isolated from the fermentation cultures of rice sheath blight pathogen Rhizoctonia solani Kühn. They were identified as ergosterol (1), 6β-hydroxysitostenone (2), sitostenone (3), m-hydroxyphenylacetic acid (4), methyl m-hydroxyphenylacetate (5), m-hydroxymethylphenyl pentanoate (6), (Z)-3-methylpent-2-en-1,5-dioic acid (7) and 3-methoxyfuran-2-carboxylic acid (8) by means of physicochemical and spectroscopic analysis. Among them, 2, 3, 5–8 were isolated from R. solani for the first time. All the compounds were evaluated for their biological activities. 4–6 and 8 showed their inhibitory activities on the radical and germ elongation of rice seeds. 1, 4 and 7 showed moderate antibacterial activity to some bacteria. 4, 7 and 8 exhibited weak inhibitory activities on spore germination of Magnaporthe oryzae. 8 showed moderate antioxidant activity with the 1,1-diphenyl-2-picryhydrazyl (DPPH) and β-carotene-linoleic acid assays. This is the first time to reveal compounds 5, 6 and 8 from rice sheath blight pathogen R. solani to have in vitro phytotoxic activity.

RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the specific yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not significantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no significant impact on normal plant development.

This study explored the effects over time of lactic acid (LA) on I Bα phosphorylation and nuclear factor-kappa B (NF- B) p65 protein expression, and on tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) mRNA levels in rat intestinal mucosa microvascular endothelial cells (RIMMVECs) stimulated by lipopolysaccharide (LPS). I B , phosphorylated I B (p-I B ) and p65 protein levels were monitored by Western blot analysis, and TNF- and IL-6 mRNA levels were analyzed using real-time PCR. LA treatment reduced TNF- and IL-6 mRNA levels in LPS-stimulated RIMMVECs, with the greatest effect being after 3 h. The highest inhibitory effect of LA on I B phosphorylation to prevent activation of NFB was after 6 h. These results suggest that LA reduces TNF- and IL-6 mRNA levels through decreasing I B phosphorylation and blocking the dissociation of IKK complex, which prevents activation of NF- B.

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyses phosphoenolpyruvate (PEP) to yield oxaloacetate, which is involved in protein biosynthesis. Pyruvate kinase (PK; EC 2.7.1.40) catalyzes PEP to yield pyruvate, which is involved in fatty acid synthesis. In this study, five PEPC genes (AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5) from peanut have been cloned. Using a quantitative real-time RT-PCR approach, the expression pattern of each gene was monitored during the seed development of four peanut varieties (E11, Hebeigaoyou, Naihan 1, and Huayu 26). It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging (DAP); whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou. In Naihan 1 and Huayu 26, the expression levels of the five genes remained relatively high in the first stage. The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development. The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter. These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.

Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (mio16) was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments (DSAIFF), was employed to isolate the Mu flanking fragments (MFFs) of mio16. The target site duplications (TSDs) isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and mio16 was mapped in silico onto the physical position ranged from 229 965021 to 229 965409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of mio16 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of mio16 is alternated by Mu insertion.