Exogenous application of melatonin improves plant resistance to virus infection

In this study, melatonin (MEL)-mediated plant resistance to tobacco mosaic virus (TMV) was examined to study local infection in Nicotiana glutinosa and systemic infection in Solanum lycopersicum. Exogenous application of 100 µm MEL increased anti-virus infection activity to 37.4% in virus-infected N. glutinosa plants. The same treatment significantly reduced relative levels of virus RNA analysed by qRT-PCR and virus titres measured by dot-ELISA, and increased the relative expression levels of the PR1 and PR5 genes analysed by qRT-PCR, in virus-infected S. lycopersicum. MEL treatment induced considerable accumulations of salicylic acid (SA) and nitric oxide (NO) but did not significantly affect production of hydrogen peroxide (H₂O₂) in the virus-infected S. lycopersicum plants. Transgenic nahG N. tabacum was used to determine whether MEL-induced TMV resistance was dependent on the SA pathway. The results showed that the relative RNA level of the TMV analysed by qRT-PCR and virus titres analysed by dot-ELISA were not reduced by the MEL treatment in the nahG transgenic N. tabacum seedlings treated twice with 100 µm MEL. The increased relative expression levels of PR1 and PR5 were greatly reduced when cPTIO, an NO scavenger, was included in the MEL treatment. A working model of MEL-mediated plant resistance to TMV is proposed. MEL-mediated plant resistance to viruses provides a new avenue to control plant viral diseases.     

云南和广东省流行性出血病病毒血清6型毒株(EHDV-6)的分离与遗传特性

【目的】分离流行于我国南方地区的流行性出血病病毒血清6型毒株(EHDV-6),掌握分离毒株的遗传特征。【方法】对设立于我国云南和广东省的哨兵动物定期采血,进行EHDV感染情况监测与病毒分离培养。通过血清中和试验确定分离EHDV的血清型,采用一步法RT-PCR对分离的EHDV-6毒株基因节段2、3、6(Seg-2、-3、-6)进行扩增与序列分析。【结果】2012—2016年,从云南省和广东省的哨兵动物中分离出25株EHDV毒株,其中,11株为EHDV-6型。中国EHDV-6型毒株的Seg-2、-3与-6均属Eastern型,核酸序列相似性分别为99.1%、98.9%和98.8%,在系统发生树上分别聚为1个独立的中国支系。在日本引起疫病暴发的EHDV-6型毒株与中国EHDV-6型毒株具有最近的亲缘关系。日本EHDV-6型毒株在系统发生树上处于中国支系内部,Seg-2、Seg-3与中国毒株的核酸序列相似性分别为98.5%和93.9%。【结论】云南和广东省流行的EHDV-6型毒株核酸与氨基酸序列高度相似;日本和中国的EHDV-6型毒株具有最近的亲缘关系,提示中日之间可能存在EHDV的流动。研究结果为进一步开展我国EHDV-6型毒株流行病学分析、致病性、病原学诊断与疫苗制备等研究提供了基础。     

不同番茄材料黄化曲叶病毒病抗性评价及抗性基因检测

以15份自育的无限生长型番茄材料为试材，抗番茄黄化曲叶病毒（Tomato yellow leaf curl virus,TYLCV）品种‘齐达利’和感病品种‘Money Maker’(MM)为对照，采用苗期农杆菌接种法，研究接种黄化曲叶病毒后的病情指数，幼苗叶片防御酶活性的变化，不同基因型番茄材料的抗性水平。结果表明：853、857、867表现为免疫；817、842表现为高抗；805、818、819、820、841表现为中抗；801、802、803、822为感病。853、857、867的防御酶(PAL、PPO、POD)活性在发病初期均达到峰值，且整体高于其他接种材料，其他接种材料酶活性峰值则出现较晚。含抗性基因的接种材料均表现出不同程度的抗性，含有纯合 Ty-1和 Ty-3基因的853、857、867对 TYLCV抗性更高。经综合评价，853、857、867可作为抗番茄黄化曲叶病毒病的优良番茄材料，817、842可作为备选材料。     

PEDV N蛋白的原核表达纯化及B细胞抗原表位预测分析

本研究旨在对猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)N基因进行克隆构建、表达与其B细胞抗原表位性质的预测。将PEDV的N基因进行PCR扩增,克隆到原核表达载体pET-28a(+)中,经酶切验证获得阳性克隆,将阳性克隆在表达菌E. coli BL21(DE3)中表达。通过对表达条件及纯化条件的优化,获得高纯度表达蛋白。利用生物信息同源模型化软件Swiss-Pdb Viewer建立PEDV-N蛋白的3D结构,并根据生物信息学软件DNA-Star预测PEDV-N蛋白的二级结构、抗原性、亲水性和表面可及性分析,综合分析预测其B细胞抗原表位。经预测PEDV-N蛋白氨基酸序列中存在13个B细胞优势抗原表位区域。研究结果将进一步为PEDV-N蛋白体外表达产物的应用及研制基因工程疫苗提供理论基础。     

PVX病毒载体介导2种马铃薯Y病毒科病毒VPg蛋白表达诱导烟草叶片系统坏死的研究

芜菁花叶病毒（Turnip mosaic virus, TuMV）和槟榔坏死环斑病毒（Areca plam necrotic ringspot virus, ANRSV）是马铃薯Y病毒科（Potyviridae）中分别可诱导十字花科作物和槟榔产生坏死病症的2种不同属病毒。VPg（Viral protein genome linked）作为与马铃薯Y病毒科病毒基因组5′端的结合蛋白,在病毒复制、翻译和运动等侵染过程中发挥重要作用。本研究利用In-Fusion克隆策略分别构建了可表达3′端融合Strep蛋白标签序列的ANRSV和TuMV VPg全长编码基因的马铃薯X病毒（Potato virus X, PVX）载体pPVX-VPg-AR和pPVX-VPg-Tu。通过农杆菌渗透注射接种本生烟（Nicotiana benthamiana）,利用RT-PCR、Strep蛋白标签亲和层析和Western blot等方法证明了PVX病毒载体能够介导VPg-AR和VPg-Tu蛋白在本生烟中系统表达,且发现这2种VPg蛋白的过量表达诱导本生烟叶片产生了不同程度的系统性坏死病症。进一步采用DAB和NBT染色,在产生坏死症状叶片中检测到大量活性氧（reactive oxygen species, ROS）的积累。因此,推测VPg可能是TuMV和ANRSV诱发植物病症产生的一个决定因子,为后续研究这2种病毒诱导症状形成机制奠定了基础。     

鸭坦布苏病毒E蛋白具有独特交叉反应性中和表位的鉴定

利用抗鸭坦布苏病毒(DTMUV)中和单抗1G2,结合E蛋白多肽扫描技术,鉴定了最小抗原表位²²⁷GSSAGTWQN²³⁵。Western blot显示,残基²³¹G或²³³W的突变后,表位完全失去了与1G2抗体识别的功能,表明²³¹G或²³³W是抗体1G2识别的关键氨基酸位点。通过免疫荧光分析(IFA)显示,MAb 1G2与JEV、WNV和ZIKV的E蛋白发生交叉反应,表明该表位是黄病毒的交叉反应性表位。蛋白质和病毒模型显示,表位定位于成熟病毒粒子可接近的表面,在E蛋白结构域Ⅱ的hi环中。本研究首次证明,中和抗体1G2靶向交叉反应表位定位在E蛋白结构域Ⅱ的hi环,该表位的鉴定有助于提高对黄病毒血清诊断中存在交叉反应问题的认识和理解。     

太子参须提取物对鸭流感病毒体外试验初探

为尝试性探究太子参须提取物对鸭H9N2亚型流感病毒体外生长的抑制作用,本文基于TCID50试验检测鸭H9N2亚型流感病毒体外感染不同处理组的MDCK细胞。结果表明,太子参须提取物高低剂量组(75%、25%)和黄芪多糖阳性对照组均对鸭H9N2亚型流感病毒体外生长产生抑制,且太子参须提取物高剂量组(75%)的抑制作用最优。结论:太子参须提取物对鸭H9N2亚型流感病毒在MDCK细胞上的体外增殖具有一定抑制作用,且呈相应剂量关系。     

Management of Bemisia tabaci biotype B with botanical and mineral oils

While the toxic effects of neem, Azadirachta indica A. Juss, on Bemisia tabaci Genn. are well documented, few studies have evaluated other oils. We compared neem, sesame, citrus, castor, vegetable and mineral oils (1% v/v) to a chemical standard thiamethoxam (0.17 g A.I./L) against B. tabaci biotype B life stages on dry bean plants Phaseolus vulgaris L. under screenhouse conditions. Oils and thiamethoxam exhibited low ovicidal activity (<10% egg mortality). However, significant mortality occurred due to the residual activity to 1st instars that emerged from treated eggs. Overall, impacts of egg treatments were greatest for thiamethoxam (77% total mortality for eggs and 1st instars) compared with oils which were statistically similar (22–29% mortality). Larvicidal effect of oils (against 2nd instars) was greater than ovicidal effects. Highest nymphal mortality (>81%) was achieved with castor, sesame, citrus and neem oils, which was significantly greater than for thiamethoxam (65% mortality). Adult whiteflies were exposed to fresh and aged spray residues, rather than being sprayed directly. In this case, comparatively lower efficacy was achieved from oil treatments compared with thiamethoxam. While some mortality was observed from fresh residues of slow drying oils (up to 41% for castor oil), no significant control from any oil residues >3 days old was observed in our tests. The different route of exposure against adults likely reduced the effectiveness of oil treatments which act directly on the cuticle. In trials with viruliferous adult whiteflies exposed to fresh residues, none of the tested products completely prevented transmission of bean golden mosaic virus (BGMV). However, we noted reduced virus severity ratings from plants pre-treated with castor and citrus oil. We conclude that castor, sesame, citrus and neem oils have the potential to be used in whitefly management programs.     

条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性

前期研究初步表明非洲猪瘟病毒(ASFV)编码的D1133L基因对ASFV复制至关重要,本研究拟进一步探究D1133L在ASFV复制中的作用。利用同源重组技术结合大肠杆菌lac阻遏操作系统实现条件性敲除D1133L基因,以pUC118为骨架重组转移载体ASFVΔi130,将重组转移载体转染骨髓源巨噬细胞(BMDMs),以ASFV CN/GS/2018为亲本毒株感染BMDM,在β-D-硫代半乳糖苷(IPTG)存在的条件下,经绿色荧光和PCR鉴定,获得条件性敲除D1133L重组毒株vD1133Li。利用荧光显微镜观察该重组毒株与亲本毒株在猪肺泡巨噬细胞(PAMs)中的复制差异,利用qPCR技术比较重组病毒与亲本毒株的复制差异,分析vD1133Li在无IPTG情况下回补D1133L蛋白后与在IPTG诱导情况下的复制差异。结果显示：本研究成功构建了条件性敲除D1133L的ASFV重组病毒vD1133Li,重组毒株不表达D1133L,在IPTG诱导下复制能力显著低于亲本毒株;在稳定表达D1133L的MA-104细胞系中,vD1133Li复制能力恢复。综上所述,D1133L基因对于ASFV复制至关重...