从随机噬菌体肽库中筛选抗草鱼出血病病毒多肽的研究

从随机噬菌体九肽表位库中筛选出抑制草鱼出血病病毒（ＧｒａｓＣａｒｐＨｅｍｏｒｈａｇｅＶｉｒｕｓ,ＧＣＨＶ８７３）感染的多肽分子。采用完整、生物素化的草鱼出血病病毒颗粒作为受体,与以融合蛋白形式在丝状噬菌体ｆＵＳＥ５的外壳蛋白Ⅲ的Ｎ端表达的随机九肽库作用。经三轮体外亲和筛选（Ｂｉｏｐａｎｎｉｎｇ）及ＥＬＩＳＡ法检测后,从肽库中筛选出１６个与病毒高亲和力结合的噬菌体克隆,其中六个阳性克隆能有效地抑制病毒在ＣＩＫ细胞中的复制,并使病毒的半数组织培养感染剂量（ＴＣＩＤ５０）下降五个数量级。表明噬菌体肽库技术完全能够应用于抗病毒研究,为研制抗病毒短肽制剂打基础。     

草鱼出血病病毒多肽的免疫原性

采用中和试验比较了草鱼出血病病毒ＧＣＨＶ８７３的１１个多肽的免疫原性,确定了主要中和抗原。纯化的单个病毒多肽免疫家兔获得抗血清,多太ＶＰ１、ＶＰ５、ＶＰ６和ＶＰ９的抗血清具有中和效价,而ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ８、ＶＰ１０和ＶＰ１１则不能诱生中和抗体。其中ＶＰ５的抗血清中和效价最高,因此,ＶＰ６极可能是病毒多肽中的主要中和抗原。     

草鱼出血病病毒多肽的基因定位

用聚丙烯酰胺凝胶电泳分离纯化的草鱼出血病病毒ＧＣＨＶ８７３的基因组ｄｓ－ＲＮＡ的１１个片段,分别在麦胚无细胞翻译体系中进行翻译。其翻译产物经ＳＤＳ－ＰＡＧＥ系统分析。结果表明,基因组片段１、２、３、４、５、和１０分别编码病毒核心衣壳的结构多肽ＶＰ１、ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ５和ＶＰ１０,片段６和７分别编码病毒外层衣壳的结构多肽ＶＰ６和ＶＰ７。片段８和９分别编码５２ｋＤ和４１ｋＤ的多肽,片段１１编码两种多肽,分子量分别为２９ｋＤ和１９．５ｋＤ,它们与病毒结构多肽无明显对应关系。病毒基因组与多肽大体是一一对应的关系。     

稀有鮈鲫对草鱼出血病病毒敏感性的初步研究

本文报道稀有鲫对草鱼出血病病毒（ＧＣＨＶ）的敏感性。ＧＣＨＶ是草鱼出血病的病原,用ＧＣＨＶ人工感好１－６月龄的稀有鲫,在水温２２－３２℃时能导致稀有鲫出现出血病症状。在水温２８℃时,病鱼在ｌｄ内死亡,潜伏期为５ｄ,发病高峰期在感染后第６－８ｄ。ＧＣＨＶ能在稀有鲫体内传代,并诱导８０％以上的稀行鲫患病死亡。将人工感染ＧＣＨＶ的稀有鲫病鱼组织超薄切片,电镜观察,发现在肠道、脾脏、肾脏等组织中存在大小与形态和ＧＣＨＶ相似的病毒颗粒。从稀有鲫出血病病鱼组织中纯化病毒,免疫电镜观察,发现病毒颗粒能被ＧＣＨＶ的特异性抗体聚集成团。由上可知,稀有鲫出血病是由ＧＣＨＶ感染所致,稀有鲫对ＧＣＨＶ是敏感的,可以作为草鱼抗出血病育种的模型。     

草鱼出血病病毒多肽的荧光染色

将草鱼出血病病毒（ＧｒａｓｓＣａｒｐＨｅｍｏｒｒｈａｇｅＶｉｒｕｓ,ＧＣＨＶ）置于还原性的溶液中,然后加入等体积的ＮａＨＣＯ３配制的异硫氰酸荧光索溶液进行多肽的标记,再经ＳＤＳ－ＰＡＧＥ分析,在紫外灯下即可检测到ＧＣＨＶ全部的１１个结构多肽的荧光带。该方法最小检测量为５００ｎｇ,由该方法回收的多肽具有抗原活性,可作为抗原进行免疫学实验。     

利用噬菌体随机肽库筛选抗柯萨奇病毒B3多肽的研究

本实验将柯萨奇病毒B3型(CVB3)大量扩增,应用蔗糖密度梯度离心法纯化病毒.利用噬菌体随机9肽库进行筛选,3轮淘洗后,测定噬菌体克隆抗病毒复制能力.提取阳性克隆DNA并进行测序,推导外源多肽的氨基酸序列.结果表明3个具有明显抗病毒复制能力的噬菌体阳性克隆被筛选出来,使TCID50由10-7.5SFU/mL分别降至10-5.25、10-6、10-5.5SFU/mL,由此证明可以应用噬菌体肽库来筛选具有抗病毒作用的多肽,本研究为抗病毒多肽制剂的研究奠定了基础.     

利用噬菌体随机肽库筛选抗柯萨奇病毒B3多肽的研究

本实验将柯萨奇病毒B3型(CVB3)大量扩增,应用蔗糖密度梯度离心法纯化病毒。利用噬菌体随机9肽库进行筛选,3轮淘洗后,测定噬菌体克隆抗病毒复制能力。提取阳性克隆DNA并进行测序,推导外源多肽的氨基酸序列。结果表明:3个具有明显抗病毒复制能力的噬菌体阳性克隆被筛选出来,使TCID50由10-7.5SFU/mL分别降至10-5.25、10-6、10-5.5SFU/mL,由此证明可以应用噬菌体肽库来筛选具有抗病毒作用的多肽,本研究为抗病毒多肽制剂的研究奠定了基础。     

噬菌体展示的豆蔻酰转移酶抑制肽序列结构和功能研究

对一个具有很强的抑制豆蔻酰转移酶活性的抑制肽噬菌体所展示的随机区１５肽序列ＴＷＰＶＶＨＧＡＣＲＡＨＧＨＣ进行关键氨基酸残基的单点,双点和缺失等一系列突变研究,以确定其功能区段。结果显示;Ｗ２Ａ,Ｈ６Ｎ和Ｈ６Ｒ突变抑制活性明显下降,Ｐ３Ａ,Ｖ４Ｖ５→Ｖ４Ａ５双点突变对活性也有一定的影响,而Ｖ４Ｖ５→Ｗ４Ｗ５,Ｈ１２Ｎ,Ｈ１４Ｎ,Ｃ９Ｓ,Ｃ１５Ｓ,ΔＣ１５,ΔＨ１４Ｃ１５和Δ９－Ｃ１５等突变对则基本上     

两例丙型肝炎病毒(HCV)和乙型肝炎病毒(HBV)重叠感染者的HCV核心区cDNA序列分析

从临床肝病患者中选择两例ＨＣＶ和ＨＢＶ重叠感染者ＨＳＱ和ＳＺＨ,他们血清中的生化指标丙氨酸转氨酶（ＡＬＴ）持续异常,肝活检病理示有严重的肝损伤。在ＡＬＴ异常期,血清学检测结果为ＨＢｓＡｇ、ＨＢｅＡｇ阳性,抗ＨＣＶＩｇＧ（包括Ｃ２２、Ｃ３３ｃ）阴性,但套式ＰＣＲ检测ＨＣＶＲＮＡ阳性,核心区ｃＤＮＡ序列分析发现该区有１个密码子（ＧＧＣｎｔ３８５—３８７）缺失,对应缺失的氨基酸是甘氨酸（ＧＬＹ）,从血清学检测和序列分析结果推测,在ＨＣＶ和ＨＢＶ重叠感染中,ＨＢＶ和ＨＣＶ均可处于持续复制状态,抗ＨＣＶＩｇＧ抗体阴性可能是ＨＣＶ的多蛋白前体翻译和病毒颗粒装配受到ＨＢＶ干扰的结果。     

利用噬菌体随机肽库筛选可结合猪繁殖与呼吸综合症病毒的多肽序列

【目的】采用完整的猪繁殖与呼吸综合症病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)颗粒筛选噬菌体肽库,以获得能高亲和力结合并能抑制该病毒复制的特异性多肽。【方法】用纯化的病毒粒子包被ELISA板,再用M13噬菌体随机12肽库进行筛选。经过3轮淘筛,ELISA鉴定噬菌体单克隆与PRRSV的亲和力,选取与PRRSV具有高亲和力的噬菌体单克隆进行DNA测序,据此推导多肽的氨基酸序列。通过TCID50检测其抗病毒复制能力,同时人工合成FITC标记的展示肽用于PRRSV的检测。【结果】经筛选和鉴定得到17个阳性噬菌体克隆能与PRRSV呈高亲和力结合,DNA测序发现各克隆之间有部分共有基序,其中2个克隆体外能明显抑制PRRSV的复制,使TCID50由10-7.3/0.1mL分别降至10-3.2、10-3.6/0.1mL,而FITC标记该展示肽能够在5mg/L工作浓度检测PRRSV。【结论】通过噬菌体肽库能够筛选到具有抗病毒作用的阳性噬菌体克隆,为进一步开发高效PRRSV的诊断和治疗试剂奠定基础。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.