甲型肝炎病毒与水痘病毒感染2BS细胞的超微结构变化

目的观察甲型肝炎病毒(Hepatitis A virus,HAV)、水痘带状孢疹病毒(Varicella zoster virus,VZV)分别感染与共同感染二倍体细胞2BS株时细胞超微结构的变化。方法电镜观察HAV单独感染21、23、25 d,VZV单独感染1、3、5 d及HAV感染21 d后再以VZV感染1、3、5、7 d的2BS细胞的超微结构。结果与正常对照2BS细胞相比,单独感染HAV的2BS细胞超微结构无明显变化;单独感染VZV的2BS细胞的超微结构主要出现核膜破坏;HAV与VZV共同感染的2BS细胞核内出现VZV包涵体,核膜缺失,胞浆中可见HAV与VZV形态,且细胞粗面内质网和滑面内质网膨胀,内质网小池内有致密颗粒。结论HAV与VZV共同感染的2BS细胞的超微结构与两种病毒单独感染的细胞的超微结构存在不同的变化规律和形态特征,先感染HAV后感染VZV的2BS细胞比单独感染VZV的2BS细胞受到的破坏程度轻。     

甲型肝炎病毒血凝及血凝抑制试验的进一步研究

<正> 龙甲_(25)株甲肝病毒(HAV)适应生长于2BS株人二倍体细胞,培养5周后收获病毒,经冻融一超声处理3次后,以等量氯仿抽提,在PH5.4～6.4的酸性条件下,于4℃～10℃时可使鹅红血球凝集,其血凝素滴度可达1:128。逐周观察组织培养中HAV的增殖动态,发现以ELISA法和血凝法检测HAV,增殖高峰均在培养第3周,以后开始下降,二法检测结果平行,提示HAV血凝试验有望用于HAV的检测。采集甲肝急性期血清30份,恢复期血清32份,非甲肝病人血清18份,以25％白陶土处理1次,再以鹅红血球吸附,选用8个单位HAV血凝素,按常     

人胚肺二倍体细胞2BS株的不同细胞系对甲肝病毒的敏感性

目的　筛选人胚肺二倍体细胞 2BS株中对HAV敏感的细胞系。方法　在相同条件下 ,用同一批HAV感染 2BS株的 4个细胞系细胞 ,采用免疫荧光法 (IFA)每周检测细胞中HAV的荧光强度 ,Ridit分析法计算各细胞系对HAV的敏感性系数 ,即Ridit值。结果　 4个细胞系对HAV敏感性不同 ,且差异有显著意义。结论　选择人胚肺二倍体细胞 2BS株中敏感的细胞系可提高HAV增殖强度     

不同材质细胞培养容器培养甲型肝炎病毒的比较

目的比较中性玻璃转瓶和聚苯乙烯细胞工厂两种材质的培养容器培养人二倍体细胞和甲型肝炎病毒(hepatitis A virus,HAV)的差异。方法采用相同的培养条件,在中性玻璃转瓶和聚苯乙烯细胞工厂中分别培养人胚肺二倍体细胞KMB17,并接种HAV H2株,倒置光学显微镜下观察细胞的形态变化;细胞接种病毒后第0、7、14、18、22、26、30天时取样,ELISA法测定病毒的感染性滴度,分析病毒的增殖动力学。结果两种材质的容器培养的KMB17细胞的生长状态和形态无明显差异;HAV H2株增殖动力学相似;单位体积病毒收获液中的病毒产量无显著差异;聚苯乙烯细胞工厂培养的HAV的单位培养容积产量是中性玻璃转瓶的7.28倍。结论聚苯乙烯细胞工厂和中性玻璃转瓶培养的人二倍体细胞和HAV均能良好生长,细胞工厂可替代转瓶成为新的疫苗规模化生产的细胞培养技术。     

用重组杆状病毒表达甲型肝炎病毒外壳蛋白

应用分子生物学技术 ,将编码甲型肝炎病毒 (HAV )外壳蛋白的开放读码框架 (ORF)全序列插入杆状病毒多角体启动子下游 ,构建成重组杆状病毒 (r BHAV ) ,并在昆虫细胞 SF9中表达。实验证明 ,在该重组杆状病毒中含有完整的 HAV编码序列 (HAV ORF) ;表达的重组蛋白具有良好的抗原性和免疫原性 ;电镜检查被 r BHAV感染的 SF9细胞中有与甲肝病毒颗粒类似的球形结构 ;经氯化铯梯度离心测得该重组蛋白的浮力密度与 HAV空壳一样。     

甲型肝炎病毒SYX1株在人二倍体细胞MRC-5上的适应性及遗传稳定性

目的 探讨甲型肝炎病毒(hepatitis A virus,HAV)SYX₁株在人二倍体细胞MRC-5上的适应性及遗传稳定性。方法 将来源于甲肝患者粪便的HAV SYX₁株接种至MRC-5细胞，连续传代至第28代，检测第1～26代病毒的抗原含量和病毒滴度；镜下观察第6代病毒的形态，并检测其理化性质。取第13～15代病毒进行病毒增殖动态研究，确定病毒增殖高峰期。选择第8、12、18、20、22、25、26和28代病毒，提取病毒基因组RNA进行序列测定，分析其遗传稳定性。建立HAV SYX₁株主种子批和工作种子批，按照《中国药典》三部(2020版)要求进行检定。结果 HAV SYX₁株在MRC-5细胞上连续传至8代后，抗原含量稳定在160～320 EU/mL之间，病毒滴度维持在7.3～8.3 lgCCID₅₀/mL；病毒颗粒有空心和实心两种类型，直径为27～32 nm，呈球形，无包膜，表面无突起；可耐受低pH及乙醚。确定病毒增殖高峰期为10 d，抗原含量达160 EU/mL以上，病...     

采用一批细胞基质同时收获甲肝和麻疹病毒

目的 采用一批细胞基质同时收获甲肝和麻疹病毒。方法 应用甲肝 L-A-1株和麻疹 27D3株,间隔3周先后感染同一批人胚肺二倍体细胞2BS株,待两种病毒同时达到增殖高峰期时收获病毒液（以下简称HAM）,并分别进行病毒滴定、特异性检查、猴体安全性和免疫效果试验。结果HAM的甲肝和麻疹病毒滴度与同批单价甲肝和麻疹疫苗病毒滴度,差异均无显著意义。结论 该方法用于制备甲肝-麻疹联合疫苗,操作简便,省时省力,并可显著降低疫苗生产成本。     

人巨细胞病毒ISCOMs对小鼠细胞免疫应答的调节作用

应用人巨细胞病毒被膜蛋白制备实验型免疫刺激复活物 (ISCOMs)疫苗能诱发细胞免疫反应。对小鼠 1次接种ISCOMs未能诱生明显细胞毒性T细胞 (Tc)及自然杀伤细胞 (NK)细胞活性增加 ,但能使L3T4及其与LyT2的比值增加。第 2次免疫后小鼠Tc及NK细胞活性明显增加。提示该疫苗有可能在预防人巨细胞病毒 (HC MV)原发感染中发挥作用。     

甲型肝炎病毒在细胞培养时的最佳收获期

为获得较高滴度的甲型肝炎病毒抗原（以下简称HAAg），除有优良的毒种外，选择最佳收获期是关键。本文观察了HAV在细胞内的增殖动态，以确定病毒滴度达到高峰的最佳收获期。这对制备甲肝疫苗及甲肝诊断制剂均有实用价值。实验用已适应KMB17人胚肺二倍体细胞的甲肝病毒H。减毒株。以相当于106.0TCID50病毒接种干KMB17单层细胞后，置35℃培养，于培养后第7天、14天、18天、22天、26天、28天和31天，各取2瓶细胞，奔去维持液，以0.25%胰酶消化后用PBS液收取细胞，经提取后合并提取液。同时取未感染病毒的正常细胞按同法处理作为对照。…     

泽泻提取物中萜类单体V-54对小RNA病毒增殖的抑制效应

目的探讨泽泻提取物中提取的萜类衍生物单体V-54对小RNA病毒感染增殖的抑制效应及其生物学机理。方法采用KMB17细胞,分别感染HAV-H、PV-Ⅰ和Cox-B2病毒,经V-54处理后检测病毒滴度,分析PV-Ⅰ和Cox-B2病毒增殖抑制的动力学及V-54对KMB17细胞凋亡的影响。结果V-54可抑制3种病毒在KMB17细胞上增殖,PV-Ⅰ和Cox-B2病毒感染滴度比未处理组明显降低,V-54对两种病毒的抑制作用呈剂量依赖性。作用24h内,V-54可使KMB17细胞凋亡率明显增加,随后逐渐恢复正常。结论V-54可抑制特定的小RNA病毒的感染增殖,其机制可能为V-54分子结合到细胞表面的相关受体,导致病毒感染效率降低。     

甲型肝炎病毒在FRhk-4细胞培养繁殖中的特性

本文对 HAV-FH 株病毒在 FRhk-4细胞繁殖的特性进行了观察,该株 HAV 感染 FRhk-4细胞后26至28天达繁殖高峰,病毒不自发释放于细胞外;不同日龄细胞接种同代同量病毒,其繁殖高峰随日龄不同而有所不同;同龄细胞接种不同量病毒则无明显差别。用免疫酶玻片法、免疫荧光染色法观察病毒增殖情况,在种毒后24小时胞浆内就有 HAV 存在,随培养时间延长查出感染病毒的细胞数增多,堆积在核周围表面的特异性着色的病毒颗粒增加。脱落细胞裂解后的提取液中也有相当数量的病毒。接种 HAV 后的 FRhk-4细胞提取液,通过中速、高速离心所获病毒液,用磷钨酸负染在电子显微镜下观察见有大量规则的 HAV 颗粒,其直径为27μmm。文中附有病毒繁殖动态、不同日龄 FRhk-4细胞对病毒繁殖的影响、免疫荧光染色及电镜照片。     

Impact of Medium-Sized Extracellular Vesicles on the Transduction Efficiency of Adeno-Associated Viruses in Neuronal and Primary Astrocyte Cell Cultures

(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.     

Sulfonated and Carboxymethylated β-Glucan Derivatives with Inhibitory Activity against Herpes and Dengue Viruses

The infection of mammalian cells by enveloped viruses is triggered by the interaction of viral envelope glycoproteins with the glycosaminoglycan, heparan sulfate. By mimicking this carbohydrate, some anionic polysaccharides can block this interaction and inhibit viral entry and infection. As heparan sulfate carries both carboxyl and sulfate groups, this work focused on the derivatization of a (1→3)(1→6)-β-D-glucan, botryosphaeran, with these negatively-charged groups in an attempt to improve its antiviral activity. Carboxyl and sulfonate groups were introduced by carboxymethylation and sulfonylation reactions, respectively. Three derivatives with the same degree of carboxymethylation (0.9) and different degrees of sulfonation (0.1; 0.2; 0.4) were obtained. All derivatives were chemically characterized and evaluated for their antiviral activity against herpes (HSV-1, strains KOS and AR) and dengue (DENV-2) viruses. Carboxymethylated botryosphaeran did not inhibit the viruses, while all sulfonated-carboxymethylated derivatives were able to inhibit HSV-1. DENV-2 was inhibited only by one of these derivatives with an intermediate degree of sulfonation (0.2), demonstrating that the dengue virus is more resistant to anionic β-D-glucans than the Herpes simplex virus. By comparison with a previous study on the antiviral activity of sulfonated botryosphaerans, we conclude that the presence of carboxymethyl groups might have a detrimental effect on antiviral activity.     

甲肝减毒活疫苗(H2株)接种后HAV的毒力和基因型

用普通狨猴对甲肝减毒活疫苗（H2株）接种者的粪便中排出的甲肝病毒（HAV）进行毒力／减毒水平检测，并对疫苗病毒及分离出毒株的P1和P2连接区的核苷酸片段进行序列分析。结果表明，4份疫苗病毒经人体增殖后均无毒力回升迹象，分离毒株Ⅱ、Ⅴ与K7疫苗病毒的同源性为99．7％～100％，所编码的114个氨基酸其序列完全相同，属同一亚基因型IB。提示我国至少有2个亚基因型的HAV，值得进一步展开甲肝分子流行病学研究。     

Molecule Based Materials for Quantum Cellular Automata: A Short Overview and Challenging Problems

In this review we shortly summarize in an accessible way the physical and nanoscale material science aspects of the promising field of molecular quantum cellular automata (QCA). QCA is a revolutionary paradigm in quantum electronics with promising application in the conceptually new computing scheme which can be referred to as “computing with molecules”. This scheme of devices have vitally important advantages compared with conventional schemes of quantum computing in which the binary information is stored in the eigenvectors of a two-level quantum system and therefore their practical application is strongly limited by the requirement of coherence. Molecular QCA promise nanometer-scale units with ultra-high device densities, as well as room temperature operation with extremely small heat release. Molecular materials provide at the same time options to control the key properties of the active molecules by chemical means. Hereunder we summarize the background of the electronic and vibronic problems in QCA cells represented by the tetrameric mixed-valence (MV) complexes and organic molecules proposed for implementation as four-site molecular QCA. We discuss the basic model of the molecular cell that include the following ingredients: 1) intracell Coulomb repulsion energy of the electrons in different electronic distributions among the redox sites; 2) electron transfer between redox sites which changes electronic distributions; 3) interaction of the itinerant electrons with molecular vibrations which is referred to as the vibronic interactions; 4) intercell Coulomb interaction by mean of which the binary information is transmitted from cell to cell. On the basis of this model we will study the cell polarization, adiabatic picture of the switching cycle describing interaction between the “input” and “output” molecular cells, non-linear cell-cell response function whose shape describes the action of molecular logic gates. Along with developed and current problems, we discuss new challenging trends of research in this fascinating area.     

甲型肝炎病毒Js-4株经人二倍体细胞传代后的核苷酸序列分析

目的分析甲型肝炎病毒(Hepatitis A virus,HAV)Js-4株经人二倍体细胞MRC-5传代后的核苷酸序列变异情况。方法将HAV Js-4株经Vero细胞适应14代获得的原始株Js-4-V14经人二倍体细胞MRC-5于35℃连续适应培养15代,取其中8个适应株(Js-4-M2、Js-4-M3、Js-4-M4、Js-4-M5、Js-4-M10、Js-4-M12、Js-4-M14、Js-4-M15)及原始株,提取病毒基因组RNA,通过RT-PCR扩增病毒全基因组,采用生物信息学软件DNAstar Lasergene 7进行序列分析。结果 8个适应株全基因组与Js-4-V14株的核苷酸序列同源性在99.8%～100.0%之间;第5代适应株在5′-NCR(101～200 bp)发生较大变异,表现为105～157 bp缺失,之后该突变稳定遗传;第10、12、14、15代适应株在2A(3 012～3 210 bp)区段3 122位点发生点突变;8个适应株和Js-4-V14毒株与野毒株HM175为同一基因亚型,均为IB型;9个毒株的主要抗原位点与野生株HM175完全一致。结论 HAV Js-4-V14株在MRC-5细胞传代适应过程中,主要抗原位点未发生突变,其与细胞适应性和病毒增殖有关的基因在传代早期已经变异,传代后期这些变异保持相对稳定。