SARS-CoV感染Vero E6细胞诱导细胞凋亡

为了确定SARS冠状病毒(SARS-CoV)感染Vero E6是否引起细胞凋亡,我们利用细胞DNA琼脂糖电泳,感染细胞的间接荧光染色和Hoechst 33258细胞核染色,以及流式细胞仪分析等方法证明了SARS-CoV感染的Vero E6具有典型的凋亡细胞学和生物化学特征.实验证明具有细胞凋亡特征的所有细胞均为处于感染晚期的细胞.表现明显细胞病变(CPE)的细胞大多已经出现核质凝缩或形成凋亡小体进入细胞凋亡的过程.可以断定SARS-CoV感染Vero E6细胞诱发了细胞凋亡.     

Vero及Vero-dst细胞对犬瘟热病毒敏感性比较

目的为了更好地分离犬瘟热病毒（CDV）并确诊犬瘟热,本实验比较了Vero及Vero-dst细胞对此病毒的敏感性。方法将CDV标准毒株Snyder Hill株及临床犬瘟热阳性犬组织匀浆分别接种Vero及Vero-dst两种细胞,通过观察细胞病变、检测病毒滴度（TCID50）,并通过RT-PCR法进行比较,分析两种细胞对CDV的敏感性。结果接种病毒后Vero细胞盲传5代始终未见细胞病变,而Vero-dst细胞12 h出现了明显的合胞样细胞病变,且RT-PCR扩增出了CDV基因特异性片段。结论 Vero-dst细胞对CDV表现了良好的敏感性,是体外分离培养CDV的一个有效细胞系。而所本实验中使用的Vero细胞并不适于CDV的分离与培养。另外,本实验利用Vero-dst细胞从临床犬瘟热阳性病例中成功分离到了野毒株,并确定其毒力较标准毒株毒力强,可用于进一步的研究。     

肠道病毒71型在RD细胞和Vero细胞中的增殖动力学特征

目的:利用噬斑法比较肠道病毒71型(EV71)在RD细胞和Vero细胞中的增殖动力学特征。方法:首先探讨培养基类型、羟乙基哌嗪乙磺酸(HEPES)、胎牛血清(FBS)、牛血清白蛋白(BSA)及甲基纤维素(MC)含量对EV71噬斑形成的影响,得到最适营养覆盖物配比;进一步,EV71以感染复数(MOI)为0.1分别接种RD细胞和Vero细胞,收集接种后不同时间点的细胞培养液,噬斑法测定各时间点培养液上清中的病毒滴度,并绘制log2(病毒滴度)-时间图,对比分析EV71在2种细胞中的增殖动力学特征。结果:终浓度含1%MC和2%FBS的MEM(1×)或DMEM(1×)为EV71噬斑形成的最适营养覆盖物;EV71在RD细胞和Vero细胞中的增殖周期均约为12 h,MOI=0.1时,EV71在RD细胞中的增殖活动较Vero细胞中活跃,增殖效率比Vero细胞中高2个数量级。结论:用RD细胞扩增EV71比Vero细胞更具优势。     

狂犬病病毒Vero细胞适应株3aG-V的致病性和免疫原性

狂犬病病毒3aG—V株是3aG—5在vero细胞适应的毒株,对其在小鼠、豚鼠、家兔、犬的不同感染途径的致病性,小鼠、豚鼠、兔子上的免疫原性进行研究,同时进行纯毒及在中枢神经系统中能否形成尼氏小体试验,结果表明：狂犬病病毒3aG—V株潜伏期较长,在动物脑内不形成尼氏小体,为固定毒,其在实验动物上具有较弱的致病性和较好的免疫原性,可作为纯化Vero细胞狂犬病疫苗的生产用疫苗株。     

Vero细胞在不同微载体固定化培养中的生长和代谢

目的:比较Vero细胞在不同的商品化微载体中固定化培养的生长和代谢。方法:以Vero细胞在含1%新生牛血清的DMEM/F12中培养的细胞形态、活细胞密度和细胞活力为指标,考察Vero细胞在2D MicroHex、Biosilon、Cytodex1和Cytopore1微载体固定化培养的细胞生长;以葡萄糖比消耗速率(qglc)、乳酸比生产速率(qlac)、谷氨酰胺比消耗速率(qgln)和谷氨酸比生产速率(qglu)为指标,考察Vero细胞在不同微载体固定化培养的细胞代谢。结果:Vero细胞在2DMicroHex、Biosilon、Cytodex1和Cytopore1微载体固定化培养7d的活细胞密度分别为18.4×105cells/ml、21.9×105cells/ml、23.9×105cells/ml和16.2×105cells/ml,生长在Cytodex1表面的Vero细胞分布均匀、形态清晰;Vero细胞在不同微载体固定化培养的代谢指标基本相同。结论:Vero细胞在Cytodex1微载体固定化培养的效果优于其它商品化微载体,可作为目前用于病毒疫苗生产的Vero细胞固定化培养的首选微载体。     

HMCES蛋白促进猪流行性腹泻病毒在Vero细胞中的复制

【目的】鉴定能够调控猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)复制的关键宿主蛋白。【方法】利用LC-MS/MS技术结合串联质谱标签(tandem mass tag,TMT)，分析PEDV感染Vero细胞36 h后和未感染组的蛋白组学差异。鉴定筛选了114个显著差异表达蛋白，其中宿主胚胎干细胞特异性5-羟甲基胞嘧啶结合蛋白(5-hydroxymethylcytosine binding,ES cell-specific protein,HMCES)显著上调。进一步构建HMCES真核表达质粒，通过蛋白免疫印迹和实时荧光定量PCR检测过表达HMCES对PEDV复制的影响；合成针对HMCES基因的特异性si RNA，利用Western blotting和RT-q PCR检测si RNA对HMCES表达的干扰效果及HMCES被干扰后对PEDV复制的影响。【结果】过表达HMCES能显著促进PEDV在Vero细胞中复制，并且复制水平随着HMCES的剂量递增呈现剂量依赖式增加；si RNA-341下调内源性HMCES表达进而抑制PEDV复制。【结论】H...     

Vero细胞和脊髓灰质炎病毒在无血清条件下的培养探究

目的：探索Vero细胞和脊髓灰质炎病毒在无血清条件下的最佳培养条件,为无血清培养Vero细胞生产脊髓灰质炎疫苗奠定基础。方法选择直接适应（直降组）和序贯适应（驯化组）两种无血清培养方法,观察Vero细胞和脊髓灰质炎病毒在无血清条件下的生长情况,并检测脊髓灰质炎病毒及其病毒滴度。结果 Vero细胞在两种无血清条件下均生长良好,其中驯化组细胞生长速度更接近对照组。以脊髓灰质炎病毒Sabin株Ⅰ型分别感染直降组、驯化组和对照组细胞的病毒滴度平均值分别为8．94、8．81和8．94 LgCCID50/mL;Ⅱ型病毒滴度平均值分别为8．84、8．25和7．94 LgCCID50/mL;Ⅲ型病毒滴度平均值分别为8．91、8．57和8．63 LgCCID50/mL;且3组的变异系数（ CV）均小于10％。结论 Vero细胞在无血清条件下生长良好,无血清培养的Vero 细胞可用作脊髓灰质炎疫苗生产的基质。     

甲肝病毒在猴胚肾细胞和Vero细胞上的分离与适应

使用恒河猴胚肾(MEK)细胞从临床标本中分离和适应了甲肝病毒W和X,通过阻断实验、中和实验、免疫荧光和免疫电镜实验、RT-PCR对其进行特异性鉴定,证明是甲肝病毒.W株和X株第7代在MEK细胞上的抗原滴度分别为1∶512、1∶1024,感染滴度(logTCID50/mL)分别为8.17、8.50.只有分离株W可以适应于Vero细胞,第6代21d抗原滴度为1∶256,感染滴度(logTCID50/mL)为8.00.     

甲肝病毒在猴胚肾细胞和Vero细胞上的分离与适应

使用恒河猴胚肾 (MEK)细胞从临床标本中分离和适应了甲肝病毒W和X ,通过阻断实验、中和实验、免疫荧光和免疫电镜实验、RT PCR对其进行特异性鉴定 ,证明是甲肝病毒。W株和X株第 7代在MEK细胞上的抗原滴度分别为 1∶5 12、1∶10 2 4,感染滴度 (logTCID50 /mL)分别为 8.17、8.5 0。只有分离株W可以适应于Vero细胞 ,第 6代 2 1d抗原滴度为 1∶2 5 6 ,感染滴度 (logTCID50 /mL)为 8.0 0。     

Plaque Assay for Polyoma Virus on Primary Mouse Kidney Cell Cultures

A plaque assay for polyoma virus using primary baby mouse kidney cells is reported.     

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing ≤50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.Download video file.^{(46M, mov)}     

狂犬病毒蚀斑方法在病毒研究和疫苗研制中的应用

本文采用狂犬病毒CTN-1和4aC株,经Vero细胞传代适应后,以Vero细胞为培养基质,建立了狂犬病毒蚀斑试验和蚀斑减少试验的方法。目前已将此方法应用于病毒鉴定、病毒克隆、病毒滴定以及抗狂犬血清的检测,并取得了较好的结果。     

In Vitro Studies with Modoc Virus in Vero Cells: Plaque Assay and Kinetics of Growth, Neutralization, and Thermal Inactivation

A sensitive and quantitative assay system is described for plaquing Modoc virus in Vero cells. Neutralizing antibodies to Modoc virus could be detected by using this in vitro system by their interference with viral plaque formation. Virus was readily neutralized within 30 min at 37 C by a 1:10 dilution of hyperimmune hamster serum. The rate of neutralization and the total amount of virus neutralized was not altered significantly by the addition of 20 U of guinea pig complement to the hyperimmune hamster serum. A study of the growth of Modoc virus in Vero cells is also presented. After an initial latent period of 20 h, viral titer increased exponentially for 20 h. By 83 h after infection, 8,000 plaque-forming units of virus were detected per cell. The stability of viral infectivity in phosphate-buffered saline at pH 7.4 was evaluated. No reduction in viral titer was detected after 3 days at 7 or 22 C. A continuous decrease in infectivity at 37 C was observed, however, throughout the observation period.     

Sindbis病毒蚀斑方法的建立及其在血液制品病毒灭活实验中的应用

实验建立了Ｓｉｎｄｂｉｓ病毒在ＢＨＫ－２１细胞内蚀斑形成的方法,Ｓｉｎｄｂｉｓ病毒接种于ＢＨＫ－２１细胞内,３天半染色,结果显示蚀斑清晰可见,直径为２－４ｍｍ,病毒滴度已达高峰期,同时将此方法用于血液制品病毒灭活实验中,结果表明该方法准确、客观,Ｓｉｎｄｂｉｓ病毒在Ｓ／Ｄ低ｐＨ孵放法等灭活病毒实验中作为有脂质包膜类病毒的指示病毒具有相对稳定性,较为适宜并且能客观的体现出各种灭活方法灭活病毒的作用     

Plaque Assay of Nuclear Polyhedrosis Viruses in Cell Culture

The nuclear polyhedrosis virus of Autographa californica has been titrated in Spodoptera frugiperda cells by the plaque method, using a solid overlay which does not require either the use of modified culture medium or expensive purified agarose or the addition of culture medium as a liquid layer above the solid agarose. This assay is more sensitive than that using a viscous methyl cellulose overlay but less sensitive than the end-point dilution technique. Neither Trichoplusia ni nor Bombyx mori cells were satisfactory as indicators for the assay as described, since they failed to form a stable monolayer. Manduca sexta cells could be utilized for assay of A. californica nuclear polyhedrosis virus, but the sensitivity was lower than with S. frugiperda cells.     

Plaque Assay of Rickettsiae in a Mammalian Cell Line

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.     

狂犬病固定毒Vero细胞适应株的建立

本文报道狂犬病毒Vero细胞适应株建株经过以及对适应株的抗原和免疫原性分析的情况,并推荐“RFD”和“Hs_3”适应株作为狂犬疫苗微载体培养系统的候选毒株。     

肾综合征出血热病毒LR1株在Vero细胞上适应的特性研究

选取不同代次的ＬＲ１毒株在Ｖｅｒｏ细胞上传代适应,不同天数连续动态观察细胞病变情况,同时用免疫荧光法和ＥＬＩＳＡ进行抗原检测,收毒前细胞反复冻融及超声波破碎,细胞破碎前后用ＥＬＩＳＡ检测抗原含量,半微量空斑法检测病毒滴度。结果证明：ＬＲ１株病毒能在Ｖｅｒｏ细胞上产生细胞病变,病变程度与其毒力明显相关;ＬＲ１株病毒在Ｖｅｒｏ细胞上繁殖的最佳收毒时间为１１天左右;病毒释放性较差,冻融和超声波破碎细胞能明显提高其抗原含量和病毒滴度