脾及淋巴结细胞对单纯疱疹病毒Ⅱ型(HSV—2)复制的抑制作用

为寻找激活细胞免疫的制剂及定量分析免疫细胞对HSV-2复制的影响,作者先以不同干扰素处理及HSV-2感染C_(57)BL/6J小鼠,而后取其脾及淋巴结制成细胞悬液,加至HSV-2感染的Vero单层细胞中,37℃混合培养48小时后,大多数Vero细胞无异常形态改变,但在此单层中,可见一些大小不等,形态不一的无细胞区。在此区边缘,可见脾及淋巴结细胞包绕有病理形态改变的Vero细胞;来加脾及淋巴结细胞的感染性单层则显示细胞变大、变园、融合等病理性形态学变化,但仍维持完整的单层状态。将以上培养物冻溶后测病毒效价发现,不同来源、不同数量的脾及淋巴结细胞对HSV-2的复制均有一定抑制作用,但以经HSV-2感染及γ干扰素处理的小鼠之脾细胞作用最强。在此细胞与靶细胞比例为100:1时,病毒复制受抑制的Log_(10)数可达4以上。该抑制作用与非特异性可溶性细胞毒性物质、营养代谢,pH、干扰素等因素无关。以上结果提示:采取适当方法激活并吸引免疫细胞抵达病毒感染部位,溶解感染细胞或破坏其与正常细胞的联系,可能会更有效地对抗通过细胞途径扩散的病毒性感染。     

人中性粒细胞多肽HNP1，3体外抗单纯疱疹病毒Ⅰ型的作用

体外观察人中性粒细胞多肽1,3(Humanneutrophilpeptide,HNP1,3)及阿昔洛韦(Acyclovir,ACV)对单纯疱疹病毒-Ⅰ型(Herpessimplexvirus1,HSV-1)的抑制作用。以Vero细胞为靶细胞,用各种浓度HNP1,3与游离病毒颗粒(直接失活组)及感染病毒后的靶细胞(复制抑制组)进行相互作用,镜下观察各药物对HSV-1致细胞病变效应的抑制作用,并采用ELISA法测定感染48h后药物对HSV-1囊膜糖蛋白分泌的抑制作用。MTT法检测各药物对细胞的毒性作用。结果显示直接失活组中,HNP1,3可使HSV-1的致细胞病变效应减轻,对HSV-1直接失活的50%有效浓度(EC50)为8.1μg/mL、10.03μg/mL;复制抑制组中,ACV使HSV-1的致细胞病变效应减轻,EC50为0.68μg/mL。MTT检测结果表明HNP1,3在治疗浓度范围内无明显细胞毒性。以上结果表明HNP1,3除具有较强的抗菌作用和抗人类免疫缺陷病毒Ⅰ型(Humanimmunodeficiencyvirus1,HIV-1)活性外,还能失活HSV-1病毒颗粒,从而逆转病毒及其蛋白的病毒效应(致细胞病变)和抑制病毒蛋白质的合成。     

人中性粒细胞多肽HNP1,3体外抗单纯疱疹病毒Ⅰ型的作用

体外观察人中性粒细胞多肽1,3(Human neutrophil peptide,HNP1,3)及阿昔洛韦(Acyclovir,ACV)对单纯疱疹病毒Ⅰ型(Herpes simplex virus 1,HSV-1)的抑制作用.以Vero细胞为靶细胞,用各种浓度HN1,3与游离病毒颗粒(直接失活组)及感染病毒后的靶细胞(复制抑制组)进行相互作用,镜下观察各药物对HSV-1致细胞病变效应的抑制作用,并采用ELISA法测定感染48h后药物对HSV-1囊膜糖蛋白分泌的抑制作用.MTT法检测各药物对细胞的毒性作用.结果显示直接失活组中,HNP1,3可使HSV-1的致细胞病变效应减轻,对HSV-1直接失活的50%有效浓度(ECs0)为8.1μg/mL、10.03μg/mL;复制抑制组中,ACV使HSV-1的致细胞病变效应减轻,EC5o为0.68μg/mL.MTT检测结果表明HNP1,3在治疗浓度范围内无明显细胞毒性.以上结果表明HNP1,3除具有较强的抗菌作用和抗人类免疫缺陷病毒Ⅰ型(Human immunodeficiency virus 1,HIV-1)活性外,还能失活HSV-1病毒颗粒,从而逆转病毒及其蛋白的病毒效应(致细胞病变)和抑制病毒蛋白质的合成.     

褐藻裙带菜孢子叶粗提物体外抗单纯疱疹病毒Ⅱ型活性研究

从药物对细胞的保护、对HSV-2增殖的影响及对HSV-2感染细胞的综合作用三个方面研究不同稀释度的裙带菜孢子叶粗提物抑制单纯疱疹病毒Ⅱ型对Vero细胞的感染作用,细胞病变效应法(Cytopathogenic effect,CPE)观察和MTT法测定裙带菜多糖抗HSV-2活性,结果表明裙带菜多糖能明显抑制HSV-2对Vero细胞的致病变作用,使细胞存活率升高,其水提醇沉法所得裙带菜多糖的IC50为6.49μg/mL,并初步推测其抗HSV-2活性是作用在HSV-2和受体结合,侵入Vero细胞阶段,为筛选新型抗病毒药物、研究海藻多糖抗HSV-2活性机理及优化裙带菜孢子叶的提取工艺提供参考依据。     

HSV-2LAT ORF1在Vero细胞中的表达特性及其对细胞活性的影响

目的:研究单纯疱疹病毒Ⅱ型(HSV-2)潜伏相关转录体(LAT)开放读码框1(ORF1)的表达特点及其对Vero细胞活性的影响.方法:双酶切和测序验证本实验室构建的HSV-2 LAT ORF1真核表达载体pEGFP-ORF1,并以转染试剂盒Xfect介导其转染至Vero细胞,通过RT-PCR和绿色荧光蛋白检验其在细胞中的表达,用MTT法进行细胞活性分析.结果:重组质粒表达的融合蛋白主要集中细胞核,而空质粒表达的绿色荧光蛋白在细胞核和细胞质中分布均匀;重组质粒对Vero细胞没有损伤作用.结论:HSV-2 LAT ORF1影响了绿色荧光蛋白的分布,可降低空质粒对细胞的损伤作用;其作用位点可能主要定位在细胞核中,为阐明HSV-2 LAT ORF1在潜伏复发中的功能奠定了实验基础.     

HSV-1 HF株扩增子载体的构建及其在不同HSV血清型间的通用性研究

旨在构建HSV-1HF株的扩增子载体,研究其在不同血清型HSV辅助下的包装通用性。经酶切HF株的BAC-HSV-1,获得oriS和pac元件并测序。以pSilencer2.0-U6为骨架,以DsRed为报告基因构建HSV-1HF株的扩增子载体,利用脂质体2000转染扩增子载体至Vero细胞,分别应用HSV-1HF株和HSV-2HG52辅助HSV-1扩增子载体进行包装,待产生细胞病变效应后取上清,再次感染Vero细胞,观察Vero细胞内红色荧光蛋白表达情况。本研究首次构建了HSV-1HF株的扩增子载体,鉴定了HSV-1HF株oriS和pac元件,HSV-1HF株扩增子载体可以被HSV-1HF株和HSV-2HG52株包装并扩增。     

携带GFP绿色荧光标记的重组BAC-HSV-1HF株的构建及其子代病毒的特性研究

本研究旨在构建由细菌人工染色体(Bacteria artificial chromosome,BAC)携带的单纯疱疹I型病毒质粒及携带绿色荧光蛋白(Green fluorescent protein,GFP)的重组型BAC-HSV-1感染性子代病毒。构建了携带HSV-1同源臂的质粒C223-UL43左臂-UL47右臂。将该质粒线性化后与HSV-1基因组共转染至Vero细胞,通过真核细胞内同源重组产生了含有GFP报告基因的BAC-HSV-1重组病毒,噬斑纯化筛选出阳性重组病毒,并再次感染Vero细胞,Hirt法提取BAC-HSV-1环形基因组并将其电穿孔入DH10B感受态细胞,由PCR和酶切法鉴定BAC-HSV-1质粒。为研究BAC-HSV-1子代病毒的生物学特性,将实验组和对照组细胞分别给予BAC-HSV-1质粒和HSV-1基因组DNA,收取病变细胞的上清液,以MOI=0.1再次感染Vero细胞,半数组织培养感染剂量(50% tissue culture infective dose,TCID50)法测定两组的病毒滴度。PCR和酶切法分别鉴定BAC-HSV-1,结果示BAC-HSV-1构建成功。TCID50法测定实验组和对照组病毒滴度,经统计学分析两组病毒滴度间差异无统计学意义(P>0.05)。本研究成功地构建了真核细胞和原核细胞间穿梭的HSV-1-BAC重组病毒/质粒。     

螺旋藻多糖抗单纯疱疹病毒作用的实验研究

在体外进行了钝顶螺旋藻多糖(polysaccharides fromSpirulina platensis,PSP)抗单纯疱疹病毒活性的研究。以不同剂量的PSP分别作用于HSV-1及HSV-2病毒复制周期的各个环节,以病毒半数感染量(TCID50),细胞病变效应(CPE),蚀斑形成单位(PFU),MTT染色细胞保护率(MTT法)作为评价指标,判断PSP的抗病毒效果;FQ-PCR检测PSP抗病毒作用的时效关系。结果表明PSP对Vero细胞毒性极低(TC50为1750μg/mL),对HSV-1及HSV-2均无直接灭活作用,可阻滞HSV-1及HSV-2病毒吸附和抑制感染细胞内病毒的复制,但不影响病毒的释放;FQ-PCR结果显示随着PSP浓度及作用时间的增加,PSP对HSV-1病毒DNA的抑制作用明显增强,具有良好的剂量和时效关系。提示PSP抗HSV-1及HSV-2病毒作用的机制与抑制病毒吸附和感染细胞内病毒的生物合成有关。     

栀子提取物ZG对单纯疱疹病毒1型细胞吸附的影响

采用负染技术,借助高倍电子显微镜观察栀子提取物ZG作用后,病毒颗粒及其病毒吸附蛋白(virus attach-ment protein,VAP)的变化,考察药物是否直接改变或破坏病毒包膜蛋白的结构,使其失去感染性;采用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记病毒,以肝素钠为参照,借助冷却慢扫描电荷耦合器件荧光成象技术,用Aquacomos软件进行图象分析,以探讨栀子提取物ZG不同加药方式对HSV-1吸附量的影响。结果表明栀子提取物ZG对HSV-1包膜表面的VAP无直接破坏作用,不影响病毒对Hep-2细胞的感染性;先加入肝素钠再进行病毒吸附及肝素钠病毒同时加入培养细胞这两种用药方式可明显减少细胞表面病毒的吸附量;栀子提取物ZG各种不同加药方式均能阻止HSV-1对Hep-2细胞表面的吸附,使病毒吸附量减少。     

疱疹病毒Ⅱ型感染SH-SY5Y细胞的生物学效应

目的：探讨疱疹病毒Ⅱ型（HSV-2）感染人神经母细胞瘤细胞株SH-SY5Y的生物学效应。方法：病毒液接种SH-SY5Y细胞后,用相差和电子显微镜观察感染细胞的形态变化,RT-PCR检测病毒在细胞中的增殖,MTT法检测病毒感染对细胞增殖的影响,流式细胞仪测定感染后的细胞凋亡状况。结果：相差显微镜显示细胞病变,从24～72h,细胞变性、坏死的程度和数量随感染时间延长而增加;电镜结果显示感染24h后,细胞核染色质固缩,出现多核巨细胞,线粒体内嵴紊乱、断裂,出现不同程度的自噬化、溶酶体化、空泡化,并可见大量鹰眼样已包装成熟的病毒颗粒及正在包装的病毒粒子;HSV-2LAT基因RT-PCR扩增表明,病毒能在SH-SY5Y细胞中增殖;凋亡检测显示HSV-2在体外细胞感染中并未使细胞出现凋亡现象;感染后24、48及72h,SH-SY5Y细胞的抑制率分别为11.3%、31.2%和63.1%,与对照组相比均存在显著性差异（P〈0.05）;分别用0.1、1、10MOI的病毒感染SH-SY5Y细胞,上述不同组在24、48、72h时细胞形态变化基本一致,感染结果相似,各组之间病毒毒力无明显差异（P〉0.05）。结论：初步在人神经母细胞瘤细胞株SH—SY5Y中建立了HSV-2感染的细胞模型,并研究了感染对细胞生物性状的影响,为探讨HSV-2的潜伏与激发机制、了解HSV-2的致病机制打下基础。     

HSV-1感染大鼠骨髓间充质干细胞及形成潜伏感染的初步研究

在体外培养大鼠骨髓间充质干细胞(BMSCs),观察单纯疱疹病毒1型感染骨髓间充质干细胞情况.分离并鉴定BMSCs;HSV-1感染BMSCs,观察细胞病变(CPE);建立BMSCs的HSV-1潜伏感染模型.提取总DNA,PCR法扩增BMSCs内的HSV-1特异性片段,检测HSV-1感染BMSCs及潜伏感染.结果显示骨髓间充质干细胞经14d诱导后,碱性磷酸酶含量增高、形成钙结节,表现出成骨细胞特性.HSV-1感染BMSCs,出现典型的CPE,PCR法证实BMSCs内存在HSV-1的特异性片段.HSV-1潜伏感染的BMSCs,未出现明显的CPE,细胞传至7代,仍可测到HSV-1的基因片段,表明BMSCs有可能形成HSV-1的潜伏感染.大鼠骨髓间充质干细胞在体外可以向成骨细胞方向分化,可作为组织工程学的种子细胞.HSV-1可以在体外感染骨髓间充质干细胞并有形成潜伏感染的趋势.     

靶向UL27shRNA抑制Ⅱ型单纯疱疹病毒在HEK293细胞中的复制

按照shRNA(small hairpin RNA)设计原则,针对Ⅱ型单纯疱疹病毒(herpes simplex virus type 2, HSV-2)的UL27基因序列保守区域筛选设计、合成4条干扰靶序列并构建表达UL27序列特异性siRNA(short interfering RNA)的质粒载体pGPU6/GFP/Neo．通过脂质体介导重组表达载体转染HEK293细胞 (human embryonic kidney 293 cell)再接种HSV-2．采用实时荧光定量PCR(real-time fluorescent quantitative PCR)技术检测UL27各组的mRNA转录水平,终点滴定法检测细胞上清液中的病毒滴度,四甲基偶氮唑盐(four methyl thiazolyl tetrazolium, MTT)法测定细胞存活率,Western印迹法检测蛋白表达效果．结果显示,UL27shRNA75组对UL27基因mRNA表达抑制效果最佳,同时能显著抑制感染细胞的CPE(cytopathic effect, CPE),降低上清液中的病毒感染滴度,提高细胞的生存率,抑制UL27基因的蛋白表达．提示本研究构建的pGPU6/GFP/Neo-UL27表达载体能在细胞水平上不同程度地干扰HSV-2 UL27基因表达,抑制HSV-2在HEK293细胞中复制．     

Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) may prove useful as vectors for gene transfer, particularly to nondividing cells. Cgal delta 3 is an immediate-early gene 3 (IE 3) deletion mutant of HSV-1 that expresses the lacZ gene of Escherichia coli from the human cytomegalovirus immediate-early control region but does not express viral early or late genes. This vector was able to efficiently infect and express lacZ in cells refractory to traditional methods of gene transfer. However, 1 to 3 days postinfection, Cgal delta 3 induced cytopathic effects (CPE) in many cell types, including neurons. In human primary fibroblasts Cgal delta 3 induced chromosomal aberrations and host cell DNA fragmentation. Other HSV-1 strains that caused CPE, tested under conditions of viral replication-inhibition, included mutants of the early gene UL42, the virion host shutoff function, single mutants of IE 1, IE 2, and IE 3, and double mutants of IE 3 and 4 and IE 3 and 5. Inhibition of viral gene expression by UV irradiation of virus stocks or by preexposure of cells to interferon markedly reduced the CPE. We conclude from these studies that HSV-1 IE gene expression is sufficient for the induction of CPE, although none of the five IE gene products appear to be solely responsible. After infection of human fibroblasts with Cgal delta 3 at a low multiplicity of infection, we were able to recover up to 6% of the input virus 2 weeks later by a superinfection-rescue procedure, even though the virally transduced human cytomegalovirus-lacZ transgene was not expressed at this time. It is therefore likely that inhibition or inactivation of viral IE gene expression, either for establishing latency or for the long-term transduction of foreign genes by HSV-1 vectors, is essential to avoid the death of infected cells.     

敲减单纯疱疹病毒Ⅱ型（HSV-2）UL30蛋白表达可抑制HSV-2复制

按照shRNA（small hairpin RNA）设计要求,选择编码单纯疱疹病毒Ⅱ型DNA多聚酶催化亚单位的U_L30（unique long 30,U_L30）基因序列保守区域,设计、合成并构建表达U_L30序列特异性siRNA（short interfering RNA）的质粒载体pUL30．通过磷酸钙转染法将其转染入HEK（human embryonic kidney）293细胞中,用蛋白印迹法检测对HSV-2 UL30蛋白表达的影响,观察受染细胞病变效应（cytopathic effect,CPE）,终点滴定法测定细胞上清液中病毒感染滴度（50% tissue culture infective dose,TCID₅₀）．结果表明,针对U_L30基因的siRNA能有效抑制UL30蛋白表达,同时显著抑制受染细胞的CPE,降低上清液中病毒感染滴度．提示本研究建立的针对U_L30基因特异性siRNA能有效阻断HSV-2在HEK293细胞内的复制,U_L30基因是一个潜在的抗HSV-2复制的药物靶标．     

Herpes simplex virus type 2 glycoprotein gF and type 1 glycoprotein gC have related antigenic determinants.

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.     

Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors.

Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number (greater than 4 x 10(4] of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD (approximately 5 x 10(3) particles per cell) were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cells pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.     

Persistence of herpes simplex virus type-2 genome in a human leukemic cell line

To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three K562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCl density gradients; but was readily identified by Southern blot hydridization of virus-infected intracellular DNA with purified viral DNA. Viral DNa polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.     

Comparative susceptibility of a canine cell line and bluetongue virus susceptible cell lines to a bluetongue virus isolate pathogenic for dogs

Summary Recently, bluetongue virus (BLU) serotype 11 was detected in diseased dogs that had been inoculated with live attenuated vaccine contaminated with this serotype of bluetongue virus (Akita et al., 1994). For various laboratory tests, BLU can be propagated in different cell cultures. No information was found in the literature about the possibility of propagating this virus in canine cells. To determine whether the BLU isolate from the contaminated canine vaccine (BLU-vac) is unique in its ability to replicate in canine cells, this virus was studied in parallel with U.S. prototype strains of BLU (serotypes 2, 10, 11, 13, and 17), in hamster lung (HmLu-1) and canine kidney (MDCK) cell cultures. In HmLu-1 cell cultures, the BLU-vac produced cytopathic effect (CPE) of the same type as the U.S. prototype BLU strains by 4 to 6 d postinoculation. In MDCK cell cultures, all of the BLU strains tested were able to replicate but did not produce CPE. The BLU-inoculated MDCK cells became persistently infected, and these cultures continued to produce infectious BLU even after six serial passages over 2 1/2 mo. In none of these cultures was CPE observed. In mixed cultures containing both HmLu-1 and MDCK cells, CPE first affected the HmLu-1 islands; subsequently, CPE spread also to the areas with MDCK cells. The silent persistent infection of the MDCK cells with BLU indicates that more stringent screening of the cells used in the production of live vaccines for various contaminating viruses is necessary.     

Virus Type-Specific Thymidine Kinase in Cells Biochemically Transformed by Herpes Simplex Virus Types 1 and 2

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.