非洲猪瘟 (ASF) 是家猪和野猪的一种毁灭性疾病。目前没有有效的商品化疫苗，疾病的控制主要依赖于对感染猪的监测和早期检测。ASF是由非洲猪瘟病毒 (ASFV) 引起的疾病。以前的血清学检测方法（如 ELISA）主要基于ASFV的重组结构蛋白，包括 p72、p54和p30等。然而，猪感染 ASFV后产生的针对此类蛋白的抗体不能为其提供有效的免疫保护。因此，仍然需要可用于临床诊断和评估疫苗免疫猪体液免疫反应的新血清学检测方法。在此项研究中，我们表达并纯化了重组pB602L蛋白。然后将纯化的pB602L蛋白作为抗原进行间接 ELISA 检测。该方法对猪瘟病毒、伪狂犬病病毒、猪细小病毒等15种最常见的猪病原的抗血清无交叉反应。然后使用该方法与商品化ELISA试剂盒同时对60头猪的血清样本进行检测，其中包括数量未知的抗ASFV血清，两种方法检测的符合率为95%。此外，使用基于pB602L蛋白的iELISA对7基因缺失的ASFV株（HLJ/18-7GD）免疫猪体内的抗体水平进行检测。结果表明，接种10 天以后，在所有接种猪体内都检测到抗体水平持续增加。我们的结果表明，这种基于 pB602L的间接ELISA检测可用于ASFV的临床诊断。

重组病毒活载体疫苗是一种能够有效激活特异性和非特异性免疫、可多联多价、安全性好的新型疫苗。动物α疱疹病毒拥有较大的基因组，含有多个不影响病毒复制的非必需区，能够插入接受外源基因并表达相应抗原蛋白；同时具有较广泛的宿主谱，能够在宿主体内复制并持续刺激动物产生对抗相应病原的免疫力，是作为重组病毒活载体疫苗的理想载体。随着基因编辑技术的发展，可通过多种方法构建能够表达外源基因的重组病毒。目前以动物α疱疹病毒为载体的重组病毒活载体疫苗研究已经涉及禽类、猪、牛、羊、伴侣动物等，目前成功构建的多株重组动物α疱疹病毒能免疫后可使动物同时获得对多种疾病的免疫。本文总结了重组动物α疱疹病毒构建方法、外源基因的引入和表达以及动物α疱疹病毒活载体疫苗免疫作用三个方面的内容，包括了最新的基因编辑技术、不同的构建策略及其优缺点、外源基因的选择、插入形式和位点等，并介绍了各种动物α疱疹病毒活载体疫苗的最新研究进展，旨在为新型动物α疱疹病毒活载体疫苗的研究和开发提供一定的参考。

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.

    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.

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.

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.