Non-nucleoside inhibitors of the measles virus RNA-dependent RNA polymerase complex activity: Synthesis and in vitro evaluation

High-throughput screening has identified 1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)phenyl]-1H-pyrazole-5-carboxamide 16677 as a novel and potent (IC(50)=35-145 nM) inhibitor against multiple primary isolates of diverse measles virus (MV) genotypes currently circulating worldwide. The synthesis of 16677 and several analogs together with effects on MV replication is described. The most potent analog displays nanomolar inhibition against the MV and a selectivity ratio (CC(50)/IC(50)) of ca. 16,500.     

中国大陆水稻黄矮病与台湾省水稻暂黄病的血清学鉴定

以前的文献报道我国大陆水稻黄矮病和台湾省的水稻暂黄病的病状、传毒介体和病毒形状均相似或相同,被认为是同一病害,但没有做过血清学反应的鉴定比较。本试验采用琼脂扩散法和双抗体夹心法(PAS-ELISA),对上述两病原的抗血清与黄矮病株提取液和带毒黑尾叶蝉研磨液进行了琼脂双扩散和酶联免疫反应的比较研究。黄矮病毒提取液与暂黄病毒抗血清的琼脂双扩散产生清晰的沉淀带,在ELISA试验中均为典型的阳性反应;黄矮病毒抗血清和暂黄病毒抗血清对生物测定虫的同一头黑尾叶蝉研磨液的测定结果,阳性虫附合率98％,病原田捕捉虫的符合率为100％。根据以上结果可以认为两种抗血清同源,即中国大陆水稻黄矮病与台湾省水稻暂黄病为同一病害。     

基于微粒群-马尔科夫复合模型的生态空间预测模拟——以长株潭城市群为例

生态空间具有重要的生态功能,对生态空间进行科学预测模拟可为保护国土空间生态安全提供决策依据。利用Arc GIS及MATLAB软件,在生态空间风险评价的基础上构建了微粒群-马尔科夫复合模型,并以长株潭城市群为研究区,基于2013年土地利用现状数据,对2020年的生态空间进行了预测模拟,最后在此基础上提出了生态空间重构的基本思路。结果表明:1)微粒群-马尔科夫复合模型(PSO-Markov)构建的基本步骤为:第一步:粒子的选择与设计,以2000 m×2000 m的正方形单元作为基本粒子。第二步:粒子的初始化设定,根据生态空间风险由低到高的原则进行选择。第三步:适应度函数的建立,用生态空间的风险值来确定生态空间的空间格局。第四步:空间位置的更新,根据自身的历史最优值及粒子群的全局最优值进行速度和位置更新。2)微粒群-马尔科夫复合模型(PSO-Markov)是一种土地利用格局预测的新途径,生态空间的数量规模可以通过改进后的马尔科夫模型进行预测,生态空间的格局可以通过微粒群模型进行预测。3)微粒群-马尔科夫复合模型具有4个特点:第一、数量预测较为合理。第二、搜索范围大、较好地考虑到局部对全局的影响。第三、受问题维数变化影响小,在求解多目标问题时具有明显优势。第四、收敛时间短、运算速度快、易于实现。4)2020年,长株潭城市群的生态空间总体数量减少,其中林地和未利用地面积变化最明显,空间变化主要集中分布在西南部地区。生态空间总面积减小的主要原因是建设用地的扩张。因此,要控制城市群的人口密度,优化城市群生产—生活—生态的数量结构及空间布局,尤其要合理规划与利用城市建设用地,充分发挥水体与未利用地的生态价值,重点保护好生态源地、廊道及关键结点,构建结构合理、功能齐全的生态网络系统,提高系统的生态服务价值功能,要在规划的指导下合理调整城市群的城乡局部空间结构,保护生态环境,提高生境质量和景观多样性。这是今后一段时期面临的主要任务。     

汀棠湖萼花臂尾轮虫姊妹种组成和生活史特征的时间变化

于2011年7月对汀棠湖水体中的萼花臂尾轮虫(Brachionus calyciflorus)进行了每周1次的采集、实验室克隆培养、DNA提取、COI基因扩增、序列测定和分析,研究了萼花臂尾轮虫种复合体结构的时间变化;参照采样期间自然水温的波动范围(28—32℃)和处于轻度富营养状态的汀棠湖水体中藻类和有机碎屑等的丰富程度,选择了在28℃和32℃等2个温度以及1.0×106、3.0×106和5.0×106个细胞/m L等3个较高的斜生栅藻(Scenedesmus obliquus)密度下研究了轮虫姊妹种生命表统计学参数(包括生命期望、平均寿命、世代时间、净生殖率、种群内禀增长率和后代混交率)的时间变化。结果表明,59条序列共定义了38个单倍型,系统发生分析将38个单倍型分为2个支系,2支系间的COI基因序列差异百分比为13.9%—15.6%,2支系应为2个姊妹种(姊妹种T1和姊妹种T2)。姊妹种T1的相对丰度由第1采集批次向第3采集批次快速降低,至第4采集批次时从水体中消失;与此相反,姊妹种T2的相对丰度却随着时间的推移而逐渐升高,但8月初从水体中消失。姊妹种T1各生命表统计学参数以及姊妹种T2的世代时间、净生殖率、内禀增长率和后代混交率均随着采集批次的增加而发生显著的变化。在较低(20个/L)的种复合体密度下,两姊妹种之间不会因为食物或空间资源等产生竞争;两姊妹种在种群内禀增长率等主要适合度参数之间的无显著性差异(P0.05)是它们能够共存于汀棠湖水体中的另一主要原因。而姊妹种T1和姊妹种T2在7月29日和8月5日依次从水体中消失则与它们在1周前的净生殖率和种群内禀增长率等显著降低(P0.05)有关。消失时间上的差异可能在于姊妹种T1的所有生命表统计学参数均不受温度的显著影响(P0.05),而姊妹种T2的所有生命表统计学参数均随着温度的升高而显著地增大(P0.05)。     

NbEXPA1, an α‐expansin,is plasmodesmata‐specific and a novel host factor for potyviral infection

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD‐enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2‐fold increase) and 48 (≥2‐fold reduction) are significantly differentially accumulated in the PD‐enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α‐expansin designated NbEXPA1, a cell wall loosening protein, is PD‐specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA‐dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD‐proteome dataset that is useful in future studies to expound PD biology and PD‐mediated virus–host interactions but also characterizes NbEXPA1 as the first PD‐specific cell wall loosening protein and its essential role in potyviral infection.     

Genome duplication effects on pollen development and the interrelated physiological substances in tetraploid rice with polyploid meiosis stability

The breeding of polyploid rice made no breakthrough for a long time because of low seed set. The discovery and application of polyploid meiosis stability (PMeS) material played a pivotal role in solving this problem. Our results indicated that genome duplication led to different outcomes in different rice cultivars in terms of pollen fertility, viability, and the accumulation of important physiological substances such as free proline and endogenous hormones. Pollen from the PMeS HN2026-4X lines showed a high fertility and viability similar to those of HN2026-2X (4X indicates tetraploid while 2X indicates the diploid), whereas both rates decreased dramatically in Balilla-4X. The results of pollen microstructure and ultrastructure investigations suggested that the pollen development pattern in HN2026-4X appeared normal at all stages, but a lot of changes were discovered in Balilla-4X. Stable meiosis, timely tapetum degradation, and normal mitochondria development were critical factors insuring the high frequency pollen fertility of PMeS rice. The free proline content increased markedly in HN2026-4X as compared to HN2026-2X, but it was decreased for Balilla-4X. Genome duplication effects on regulating endogenous hormones accumulation in pollen were evident, resulting in the clear difference between PMeS HN2026-4X and Balilla-4X. The accumulation of IAA, ZR, and GA in mature pollen distinguished HN2026-4X from Balilla-4X, which was linked to normal pollen development. In particular, the excessive accumulation of ABA at the meiosis stage may be correlated to disorganized meiosis in Balilla-4X. All the results provided unequivocal evidence that genome duplication played specific roles in the normal pollen development of PMeS HN2026-4X.     

Turnip mosaic virus co-opts the vacuolar sorting receptor VSR4 to promote viral genome replication in plants by targeting viral replication vesicles to the endosome

Accumulated experimental evidence has shown that viruses recruit the host intracellular machinery to establish infection. It has recently been shown that the potyvirus Turnip mosaic virus (TuMV) transits through the late endosome (LE) for viral genome replication, but it is still largely unknown how the viral replication vesicles labelled by the TuMV membrane protein 6K2 target LE. To further understand the underlying mechanism, we studied the involvement of the vacuolar sorting receptor (VSR) family proteins from Arabidopsis in this process. We now report the identification of VSR4 as a new host factor required for TuMV infection. VSR4 interacted specifically with TuMV 6K2 and was required for targeting of 6K2 to enlarged LE. Following overexpression of VSR4 or its recycling-defective mutant that accumulates in the early endosome (EE), 6K2 did not employ the conventional VSR-mediated EE to LE pathway, but targeted enlarged LE directly from cis-Golgi and viral replication was enhanced. In addition, VSR4 can be N-glycosylated and this is required for its stability and for monitoring 6K2 trafficking to enlarged LE. A non-glycosylated VSR4 mutant enhanced the dissociation of 6K2 from cis-Golgi, leading to the formation of punctate bodies that targeted enlarged LE and to more robust viral replication than with glycosylated VSR4. Finally, TuMV hijacks N-glycosylated VSR4 and protects VSR4 from degradation via the autophagy pathway to assist infection. Taken together, our results have identified a host factor VSR4 required for viral replication vesicles to target endosomes for optimal viral infection and shed new light on the role of N-glycosylation of a host factor in regulating viral infection.     

Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broad-spectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para- and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.