单增李斯特菌毒力因子溶血素关键氨基酸位点在感染过程中的作用研究

【目的】探究单增李斯特菌溶血素O (listeriolysin O, LLO)中D3区域β8折叠片上第253位氨基酸(谷氨酰胺,Q)和第254位氨基酸(异亮氨酸,I)对单增李斯特菌(Listeria monocytogenes)感染生物学功能的影响。【方法】构建LLO_Q253A和LLO_I254A突变蛋白的原核表达菌株,以及利用同源重组方法构建hly_Q253A和hly_I254A突变株;通过表达纯化突变蛋白,测定溶血活性;比较LLO第253位Q和第254位I均突变成丙氨酸(A)后,对细菌体外生长能力、黏附侵袭、胞内迁移和增殖能力的影响。【结果】相应位点突变后,LLO蛋白均能够正常表达。在pH 6.5条件下,所有突变蛋白和突变株的溶血活性丧失。然而,在pH 5.5条件下,LLO_I254A和hly_I254A恢复了溶血活性。与野生株相比,突变株的体外生长、黏附能力和胞内增殖能力均无明显差异;突变株的侵袭能力和胞间迁移能力显著低于野生株。【结论】本研究证实第253位Q和第254位I均突变成A后,单增李斯特菌在pH 6.5条件下丧失溶血活性,并降低了感染宿主细胞的能力,但具体机制还有待进一步探索。本研究为深入探究LLO结构对单增李斯特菌生物学功能的影响奠定基础,对单增李斯特菌点突变株的构建具有一定参考意义。     

表达新冠病毒S基因重组狂犬病病毒的构建及其免疫原性

【背景】新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)在全球流行已近3年,除对人类造成了巨大伤害,也影响了人类的伴侣动物。人的COVID-19疫苗已在全球应用,但动物用的新冠病毒疫苗却鲜有报道。【目的】研制兽用新冠病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)和狂犬病病毒(rabies virus,RABV)的二联苗。【方法】将合成的SARS-CoV-2 S基因和S1基因分别克隆至RABV弱毒疫苗株rHEP-Flury基因组G与L基因间,并将2个重组质粒分别与辅助质粒共转染至BHK-21细胞中,拯救重组病毒rHEP-nCOV-S和rHEP-nCOV-S1。通过RT-PCR、Western blotting和荧光抗体染色,验证重组病毒、确证S和S1蛋白在RABV中成功表达。再将重组病毒接种NA细胞及成年小白鼠,测定病毒的体外生长特性、重组病毒的致病性及免疫原性。【结果】免疫荧光结果显示,转染7d后细胞上清均出现了绿色免疫荧光,表明已成功拯救嵌合SARS-CoV-2S和S1基因的重组病毒RABV rHEP-nCOV-S和rHEP-nCOV-S1,并且rHEP-nCOV-S1的增殖和扩散能力强于亲本株rHEP-Flury,但rHEP-nCOV-S与亲本株无显著差异。Western blotting结果显示,在目的位置处均出现72kDa和144kDa特异性条带,表明S和S1蛋白在重组RABV中高效表达。重组病毒免疫6周KM小鼠后,小鼠的体重变化与亲本RABV基本一致,重组病毒诱导小鼠产生狂犬中和抗体。【结论】本研究拯救出了嵌合SARS-CoV-2 S/S1基因的重组RABV,为动物COVID-19载体疫苗的研发奠定了基础。     

高山被孢霉中二酰甘油酰基转移酶2同源基因的克隆、表达和活性分析

[背景] 高山被孢霉（Mortierella alpina）是一种可积累大量花生四烯酸（Arachidonic Acid,AA）的产油丝状真菌,其所产脂肪酸主要被组装到甘油骨架上以三酰甘油（Triacylglycerol,TAG）形式存在。二酰甘油酰基转移酶（Diacylglycerol Acyltransferase,DGAT）是TAG生物合成途径的关键酶,对于高山被孢霉TAG的生产具有重要意义。[目的] 通过探究高山被孢霉DGAT2在TAG生物合成方面的功能特点,以期为提高产油真菌的TAG产量及改善TAG的脂肪酸组成提供参考。[方法] 利用序列比对在高山被孢霉ATCC32222基因组中筛选出2个编码DGAT2的候选基因MaDGAT2A/2B,在酿酒酵母（Saccharomyces cerevisiae）中异源表达后进行功能分析,并在外源添加AA条件下通过检测TAG产量进一步分析MaDGAT2A/2B的活性,最后在高山被孢霉中同源过表达MaDGAT2A/2B,通过检测重组菌总脂肪酸产量及组分以分析MaDGAT2A/2B的体内活性。[结果] MaDGAT2A在S. cerevisiae中异源表达时,重组酵母菌TAG的产量达到细胞干重的3.06%,为对照组的4.91倍;而MaDGAT2B未明显提高重组酵母菌TAG的产量。在外源添加AA时,MaDGAT2A/2B均可显著促进重组酵母菌中TAG合成,表达MaDGAT2A的重组酵母菌TAG含量为对照组的3.67倍,表达MaDGAT2B的重组酵母菌TAG含量为对照组的2.61倍。MaDGAT2A/2B在高山被孢霉中过表达对其总脂肪酸产量无显著影响,但可显著提高总脂肪酸中AA的含量,AA占总脂肪酸比例最高达到39.15%,相比对照组提高16.14%。[结论] MaDGAT2A/2B可以参与TAG的生物合成,表明2个候选基因编码的蛋白具有DGAT活性,并且可提高高山被孢霉脂肪酸中AA的含量,对于改善产油真菌的脂肪酸组成从而提高其应用价值具有重要意义。     

gbpC和gbpD基因在盘基网柄菌细胞趋化性和趋电性运动中的差异研究

目的 趋化性和趋电性是细胞定向迁移的主要方式，并在生物有机体的生理和病理过程中发挥重要作用，但二者存在差异。本文对盘基网柄菌gbpC和gbpD基因在细胞趋电性和趋化性中的作用进行对比研究，以寻找两种迁移方式之间的新差异。方法 将gbpC基因突变株gefT-和gbpD基因突变株gefU-分别置于场强为12 V/cm的直流电场中，分析细胞在电场中的运动方向及运动速度，探讨细胞的趋电性变化；利用电穿孔技术将标记F-actin的Lifeact-GFP质粒转化进入细胞，用荧光显微镜观察活细胞运动时F-actin的分布；用蛋白质印迹技术定量分析细胞的肌球蛋白调节轻链（RLC）在受直流电场刺激后的磷酸化变化情况。结果 gefT-突变株细胞极化消失，但保持与野生型类似的趋电性；gefU-突变株细胞发生超极化，但趋电性显著降低。在直流电场中，突变株细胞和野生型细胞的F-actin主要分布在伪足部位。在电场作用下，细胞株的肌球蛋白RLC磷酸化变化情况存在差异，即野生型细胞以时间依赖的方式发生磷酸化，gefT-突变株细胞先急剧下降，然后再上升，gefU-突变株细胞却以时间依赖方式脱磷酸化。结论 本研究表明gbpC和gbpD基因在盘基网柄菌趋化性和趋电性中的作用不同，暗示了电信号与化学信号确实通过不同的机理指导细胞的定向迁移。     

新生儿科院内感染的原因分析与应对策略研究

目的 了解新生儿院内感染发生的现状，分析其原因，并提出政策建议。方法 采用文献分析法，掌握新生儿发生院内感染的特殊原因，分别从新生儿自身的特殊性、新生儿科室的特殊性、新生儿治疗的特殊性3方面探讨，归纳得出新生儿科易发生院内感染的原因。结果 新生儿免疫力低，新生儿病房布局合理性差、护理人员数量不足、防感意识差，新生儿治疗时侵入性操作多、医护人员与患儿接触频繁、滥用抗生素等。结论 应对新生儿院内感染应从预防、发生、结束3个方面来采取相应措施。     

SARS-CoV-2 Delta变异株：从结构变异到强传染力

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)变体Delta变异株(B.1.617.2毒株),以极强的传播力迅速成为了目前世界范围的2019冠状病毒病(COVID-19)主要致病毒株。刺突蛋白作为冠状病毒的关键结构蛋白质，介导了SARS-CoV-2与靶细胞识别、结合及融合过程，与Delta变异株的高传染性密切相关。Delta变异株中刺突蛋白基因突变位点从其空间构象、表面亲水性与自由能等方面加以改变，影响了刺突蛋白与靶细胞膜的识别与融合过程，加速了结合进程。Delta变异株的分子结构及其功能研究对于COVID-19的预防及治疗至关重要。本文简述了SARS-CoV-2 Delta变异株相较于野生株的分子结构特点，着重以冠状病毒刺突蛋白为基础，回顾了中和抗体与血清之于Delta变异株的效力改变，并总结了SARS-CoV-2 Delta变异株的病毒动力学及临床特征，以期为拓宽Delta变异株分子结构功能变异研究方向，为未来COVID-19疫苗设计与应用提供思路。     

RNA沉默手段分析烟草悬浮细胞中线粒体分裂蛋白NtFIS1A/1B功能

为了解烟草悬浮细胞中的线粒体分裂蛋白的功能,将拟南芥(Arabidopsis thaliana)的线粒体分裂复合体成员FIS1A和FIS1B在烟草(Nicotiana tabacum)表达序列数据库中进行序列比对,鉴定到烟草中的同源基因NtFIS1A 和NtFIS1B (NtFIS1A/1B).以烟草悬浮细胞为材料,通过RNA 干扰和人工microRNA 干扰技术抑制NtFIS1A/1B 的表达, RT-PCR 分析结果表明,RNA 沉默细胞系中NtFIS1A/1B 的转录受到抑制.观察RNA 沉默细胞系中的线粒体形态可见,当NtFIS1A/1B 的表达被抑制后,单个线粒体的平均面积显著增加.这些表明NtFIS1A/1B 参与了烟草悬浮细胞中线粒体分裂的调控,有助于了解烟草中线粒体的形态调控.     

严重急性呼吸综合征冠状病毒2膜蛋白对宿主细胞pre-mRNA 3"UTR加工的影响

目的 研究严重急性呼吸综合征冠状病毒2（SARS-CoV-2）膜蛋白对宿主细胞mRNA前体（pre-mRNA）3"非翻译区（UTR）加工的影响。方法 本研究以人肺上皮细胞系A549为模型，利用瞬时转染在细胞内过表达SARS-CoV-2膜蛋白；利用RNA-Seq测序技术及生物信息学分析方法，系统性描绘宿主细胞选择性多聚腺苷酸化（alternative polyadenylation，APA）事件；Metascape数据库对发生显著APA变化的基因进行功能富集分析；RT-qPCR验证靶基因3"UTR长度变化；蛋白质免疫印迹（Western blot）检测目的蛋白表达水平。结果 SARS-CoV-2膜蛋白外源表达后宿主细胞内共813个基因发生显著APA变化。GO和KEGG分析显示，差异APA基因广泛参与有丝分裂细胞周期、调节细胞应激等生物过程，涉及病毒感染和蛋白质加工等。从中进一步筛选出AKT1基因，在IGV软件中显示3"UTR延长；RT-qPCR验证AKT1基因的3"UTR长度变化趋势；Western blot结果显示AKT1蛋白磷酸化水平增加。结论 SARS-CoV-2膜蛋白潜在影响宿主pre-mRNA的3"UTR加工，其中参与多种病毒性生物过程的AKT1基因 3"UTR延长，且其编码的蛋白质功能在细胞内被激活。     

新型冠状病毒(SARS-CoV-2)SNV分型检测技术

新型冠状病毒肺炎(COVID-19)的全球大流行对整个人类社会造成了重大影响,人类面临着财政刺激、金融压力、债务重整等挑战。在特效治疗药物与方法出现之前,大规模的人群筛查隔离成为现在疫情治理的最有效方法。然而,这一次的新冠病毒(SARS-CoV-2)展示出了极高的遗传变异性,截至2022年3月31日统计突变率超过了2.3‰,迄今为止高传染性的新病毒株不断出现,被世界卫生组织正式警告的变异株就达到了7个。因此,在接下来的病毒防控与研究中,不但需要检测SARS-CoV-2,更需要精准、实用的单核苷酸变异(single nucleotide variation, SNV)基因分型技术,特别针对大规模人群筛查中,不仅需要获得SRAS-CoV-2的信息,还需要精准快速区分具有更高传染性与毒性的变异株感染。对病毒的感染和突变机制进行了简要介绍,并着重对现有主要的SARS-CoV-2 SNV分型技术进行了分类综述,希望为新型检测技术的开发提供参考。     

乙型脑炎病毒NS1′蛋白表达对小鼠毒力的影响

为探究乙型脑炎病毒NS1′蛋白表达对小鼠神经毒力和神经侵袭力的影响，以乙脑病毒疫苗株SA14-14-2为模板，对NS2A基因进行A66G定点突变，并构建全长感染性克隆。体外转录后，将病毒RNA导入BHK21细胞，获得突变病毒SA14-14-2（A66G）。同样以乙脑病毒野毒株SA14为模板，采用相同方法构建并获得突变病毒SA14（G66A）。经测序和间接免疫荧光鉴定，证明突变病毒构建成功。Western blot检测发现疫苗株SA14-14-2和SA14(G66A)不表达NS1′蛋白，而野毒株SA14和SA14-14-2（A66G）能表达NS1′蛋白。生长曲线显示突变病毒的生长特性无明显改变。蚀斑实验发现SA14(G66A)的蚀斑较SA14更小，SA14-14-2(A66G)的蚀斑较SA14-14-2更大。细胞增殖活性实验显示NS1′蛋白表达对BHK21细胞的增殖有明显抑制作用 (P＜0.01)。疫苗株SA14-14-2与SA14-14-2（A66G）对1周龄小鼠脑内接种的LD50分别为297.9 PFU(0.02 mL)和143.4 PFU(0.02 mL)，野毒株SA14和SA14(G66A)对3周龄小鼠脑内接种的LD50分别为0.8 PFU(0.03 mL)和2.3 PFU(0.03 mL)。疫苗株SA14-14-2与SA14-14-2(A66G)对2周龄小鼠腹腔接种的LD50分别为＞5.65×10⁶ PFU(0.5 mL)和＞1.46×10⁶ PFU(0.5 mL)。野毒株SA14和SA14(G66A)对3周龄小鼠腹腔接种的LD50分别为1.3×10³ PFU (0.5 mL)和1.2×10⁴ PFU(0.5 mL)。结果表明，乙脑病毒NS2A中第66位碱基是影响NS1′蛋白表达与否的关键位点，且NS1′蛋白表达能提高病毒对小鼠的神经毒力与神经侵袭力，其中对侵袭力的影响更为显著。     

严重急性呼吸综合征冠状病毒2变异体对全球疫情防控的影响分析

由严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引起的新型冠状病毒肺炎（COVID-19，简称新冠肺炎）的出现，对国际公众健康构成了严重威胁，伴随COVID-19大流行而来的是SARS-CoV-2基因组的不断突变，尤其是受关注的变异体（variants of concern，VOCs）给全球COVID-19疫情防控带来了挑战。本文综述了SARS-CoV-2的突变情况和现阶段主要流行的VOCs的特征，总结了现有及潜在的COVID-19预防、诊断和治疗手段，并通过分析SARS-CoV-2变异体对全球COVID-19疫情防控措施的影响，提出合理的建议，以期为今后可能爆发的大范围流行病的防控提供理论依据。     

新冠肺炎疫情防控常态化背景下医院信息化建设的实践与探索

摘要：近年来随着信息化技术的深入发展,其在人们的生产生活中的作用日益突出,对提高工作效率方面作用显著。2020年新年伊始,湖北省武汉市爆发新冠肺炎（COVID-19）疫情并迅速蔓延全国,国家高度重视,医务人员迅速行动,积极投入到这场与病毒作战的没有硝烟的战争中。经过全国上下艰苦努力,我国新冠肺炎疫情防控向好态势进一步巩固,防控工作已从应急状态转为常态化防控状态。医院信息化建设是医院现代化建设和新冠疫情防控的客观要求,在新冠肺炎疫情防控常态化背景下有重要意义。本文从新冠肺炎疫情背景下医院信息化建设概述,医院信息化建设存在的问题,医院信息化建设实践和医院信息化建设探索四个方面进行阐述,致力于探索出疫情防控背景下医院信息化建设的整体思路。     

Transmission dynamics and control of COVID-19 in Chile,March-October, 2020

Since the detection of the first case of COVID-19 in Chile on March 3^rd, 2020, a total of 513,188 cases, including ~14,302 deaths have been reported in Chile as of November 2^nd, 2020. Here, we estimate the reproduction number throughout the epidemic in Chile and study the effectiveness of control interventions especially the effectiveness of lockdowns by conducting short-term forecasts based on the early transmission dynamics of COVID-19. Chile’s incidence curve displays early sub-exponential growth dynamics with the deceleration of growth parameter, p, estimated at 0.8 (95% CI: 0.7, 0.8) and the reproduction number, R, estimated at 1.8 (95% CI: 1.6, 1.9). Our findings indicate that the control measures at the start of the epidemic significantly slowed down the spread of the virus. However, the relaxation of restrictions and spread of the virus in low-income neighborhoods in May led to a new surge of infections, followed by the reimposition of lockdowns in Greater Santiago and other municipalities. These measures have decelerated the virus spread with R estimated at ~0.96 (95% CI: 0.95, 0.98) as of November 2^nd, 2020. The early sub-exponential growth trend (p ~0.8) of the COVID-19 epidemic transformed into a linear growth trend (p ~0.5) as of July 7^th, 2020, after the reimposition of lockdowns. While the broad scale social distancing interventions have slowed the virus spread, the number of new COVID-19 cases continue to accrue, underscoring the need for persistent social distancing and active case detection and isolation efforts to maintain the epidemic under control.     

A mechanistic and data-driven reconstruction of the time-varying reproduction number: Application to the COVID-19 epidemic

The effective reproduction number R_eff is a critical epidemiological parameter that characterizes the transmissibility of a pathogen. However, this parameter is difficult to estimate in the presence of silent transmission and/or significant temporal variation in case reporting. This variation can occur due to the lack of timely or appropriate testing, public health interventions and/or changes in human behavior during an epidemic. This is exactly the situation we are confronted with during this COVID-19 pandemic. In this work, we propose to estimate R_eff for the SARS-CoV-2 (the etiological agent of the COVID-19), based on a model of its propagation considering a time-varying transmission rate. This rate is modeled by a Brownian diffusion process embedded in a stochastic model. The model is then fitted by Bayesian inference (particle Markov Chain Monte Carlo method) using multiple well-documented hospital datasets from several regions in France and in Ireland. This mechanistic modeling framework enables us to reconstruct the temporal evolution of the transmission rate of the COVID-19 based only on the available data. Except for the specific model structure, it is non-specifically assumed that the transmission rate follows a basic stochastic process constrained by the observations. This approach allows us to follow both the course of the COVID-19 epidemic and the temporal evolution of its R_eff(t). Besides, it allows to assess and to interpret the evolution of transmission with respect to the mitigation strategies implemented to control the epidemic waves in France and in Ireland. We can thus estimate a reduction of more than 80% for the first wave in all the studied regions but a smaller reduction for the second wave when the epidemic was less active, around 45% in France but just 20% in Ireland. For the third wave in Ireland the reduction was again significant (>70%).     

Traditional Chinese herbal medicine at the forefront battle against COVID-19: Clinical experience and scientific basis

BackgroundThroughout the 5000-year history of China, more than 300 epidemics were recorded. Traditional Chinese herbal medicine (TCM) has been used effectively to combat each of these epidemics’ infections, and saved many lives. To date, there are hundreds of herbal TCM formulae developed for the purpose of prevention and treatment during epidemic infections. When COVID-19 ravaged the Wuhan district in China in early January 2020, without a deep understanding about the nature of COVID-19, patients admitted to the TCM Hospital in Wuhan were immediately treated with TCM and reported later with >90% efficacy.ApproachWe conducted conduct a systematic survey of various TCM herbal preparations used in Wuhan and to review their efficacy, according to the published clinical data; and, secondly, to find the most popular herbs used in these preparations and look into the opportunity of future research in the isolation and identification of bioactive natural products for fighting COVID-19.ResultsAlthough bioactive natural products in these herbal preparations may have direct antiviral activities, TCM employed for fighting epidemic infections was primarily based on the TCM theory of restoring the balance of the human immune system, thereby defeating the viral infection indirectly. In addition, certain TCM teachings relevant to the meridian system deserve better attention. For instance, many TCM herbal preparations target the lung meridian, which connects the lung and large intestine. This interconnection between the lung, including the upper respiratory system, and the intestine, may explain why certain TCM formulae showed excellent relief of lung congestion and diarrhea, two characteristics of COVID-19 infection.ConclusionThere is good reason for us to learn from ancient wisdom and accumulated clinical experience, in combination with cutting edge science and technologies, to fight with the devastating COVID-19 pandemic now and emerging new coronaviruses in the future.     

Plant Derived Antiviral Products for Potential Treatment of COVID-19: A Review

COVID-19 caused by SARS-CoV-2 is declared global pandemic. The virus owing high resemblance with SARS-CoV and MERS-CoV has been placed in family of beta-coronavirus. However, transmission and infectivity rate of COVID-19 is quite higher as compared to other members of family. Effective management strategy with potential drug availability will break the virus transmission chain subsequently reduce the pressure on the healthcare system. Extensive research trials are underway to develop novel efficient therapeutics against SARS-CoV-2. In this review, we have discussed the origin and family of coronavirus, structure, genome and pathogenesis of virus SARS-CoV-2 inside human host cell; comparison among SARS, MERS, SARS-CoV-2 and common flu; effective management practices; treatment with immunity boosters; available medication with ongoing clinical trials. We suggest medicinal plants could serve as potential candidates for drug development against COVID-19 infection.     

Coronavirus disease 2019 (COVID-19): diagnosis and prognosis

Coronavirus disease 2019 (COVID-19) has become a global pandemic with a high rate of transmission. Currently, there is a lack of vaccines and specific drugs for this newly-emerged virus. Timely diagnosis and treatment, as well as isolation of patients and virus carriers, contribute to the effective prevention and control of this epidemic. This review focuses on early stage COVID-19 diagnosis methods and strategies, highlighting the guiding role of laboratory indicators on treatment strategy formulation, and prognosis assessments.     

Ocular transmissibility of COVID-19: possibilities and perspectives

Since the initial outbreak of coronavirus disease 2019 (COVID-19), extensive research has emerged from across the globe to understand the pathophysiology of this novel coronavirus. Transmission of this virus is a subject of particular interest as researchers work to understand which protective and preventative measures are most effective. Despite the well understood model of aerosol-respiratory mediated transmission, the exact mechanism underlying the inoculation, infection and spread of COVID-19 is currently unknown. Given anatomical positioning and near constant exposure to aerosolized pathogens, the eye may be a possible gateway for COVID-19 infection. This critical review explores the possibility of an ocular-systemic or ocular–nasal–pulmonic pathway of COVID-19 infection and includes novel insights into the possible immunological mechanisms leading to cytokine surge.

     

Prevention and treatment of COVID-19 using Traditional Chinese Medicine: A review

BackgroundA novel coronavirus (SARS-CoV2) outbreak in more than 200 countries recently caused viral pneumonia that was extremely infectious and pathogenic. The Chinese government proposes that both Traditional Chinese medicine (TCM) and Western medicine can be used in combination to treat pneumonia caused by SARS-CoV2, and TCM effectively provides continuous prevention and treatment.MethodsThe present review analyzes and summarizes the prevention and treatment of the novel coronavirus disease (COVID-19) with TCM. A classified analysis of the efficacy and advantages of TCM for the prevention and treatment of COVID-19 was performed, and the mechanisms of TCM in treating COVID-19 are summarized.ResultsTCM is effective in preventing COVID-19, and medical staff can prevent an iatrogenic infection by taking a decoction made based on the principles of TCM. As of March 13, 2020, new cases of COVID-19 in China have decreased in number to single digits. TCM's curative effect was outstanding, with a national participation rate of over 90%. More than 70,000 people were cured of COVID-19 and discharged from the hospital. Only approximately 10,000 patients are currently being treated, and the total treatment time is approximately 2 months.ConclusionsTCM is currently the best choice for the treatment and prevention of COVID-19, and it is expected that it will be promoted by countries around the world.