不同代次牛肾原代细胞培养轮状病毒的比较研究

口服轮状病毒活疫苗(LLR株)生产用细胞基质为新生小牛肾原代细胞。原始的初代细胞(P0)产量小,一对牛肾平均生产7瓶细胞。将原始的初代细胞传代可使细胞产量显著增加,传代后(P2代)细胞产量可由7瓶增加为96~112瓶,细胞核型检查传至P5代的细胞染色体数目与初代细胞一致。细胞培养物均一性提高。P0代与P2代细胞病毒培养物滴度分别在6.2±1.5和6.5±0.5lgCCID50/ml,使用P2代细胞培养病毒,产量增加10~15倍。提高了疫苗生产的可控性和质量,生产规模显著放大,经济效益明显。     

应用细胞工厂建立人用狂犬病疫苗连续培养多次收获工艺

目的为提高生产效率、增加原代地鼠肾细胞单产量及狂犬病病毒产量,建立人用狂犬病疫苗（地鼠肾细胞）连续培养多次收获工艺。方法选用12-14日龄SPF地鼠,无菌取肾经消化,制备成细胞悬液,分装到40层细胞工厂并培养细胞成单层;接种狂犬病病毒固定毒aG株,连续培养病毒并多次收获。分别对同一细胞批制备的多个单次病毒收获液的免疫原性、病毒滴度和地鼠肾细胞蛋白质含量进行检测。结果用40层细胞工厂培养原代地鼠肾细胞和狂犬病病毒,细胞接种浓度为1．0×10。～1．5x10。cells／mL,（36±1）℃培养72h成致密单层;按0．1MOI病毒接种,可进行6次收获病毒;多个单次病毒收获液病毒滴度均不低于6．0lgLD50／mL;免疫原性检查保护指数不低于100;地鼠肾细胞蛋白质残留量随着收获次数的增加而不断降低。结论用细胞工厂建立了人用狂犬病疫苗连续培养多次收获工艺,能显著提高地鼠。肾单产量,增加产能。     

两种传代细胞系对轮状病毒D36株(P[4]G2)敏感性的比较

目的探讨轮状病毒D36株在MDCK细胞和Vero细胞上培养的适应性,确定其培养的最佳细胞基质及培养条件。方法将D36株以MOI 1.0按不同培养瓶分组接种MDCK细胞和Vero细胞,补充含有不同浓度胰酶的维持液,于不同时间观察两种细胞病变的情况,同时抽样检测病毒滴度,分析两种细胞对D36株的敏感性。结果D36株病毒感染MDCK细胞后第6天病毒滴度达到最高,为(5.00～5.50)lgCCID50/mL;而D36株病毒感染Vero细胞后病毒滴度于第8天达高峰,为(4.50～4.75)lgCCID50/mL。另外,在两种细胞维持液中加入约0.8μg/mL的胰酶均可提高病毒滴度。结论两种细胞系在同等条件下感染D36株病毒后,MDCK细胞比Vero细胞出现病变的时间早,每一批MDCK细胞培养物病毒滴度高于同批次试验的Vero细胞培养物。     

应用细胞工厂传代原代地鼠肾细胞培养狂犬病病毒

目的应用细胞工厂建立原代地鼠肾(primary hamster kidney, PHK)细胞传代培养工艺,培养狂犬病病毒,为PHK细胞和人用狂犬病疫苗(地鼠肾细胞)的规模化放大奠定基础。方法选用SPF级地鼠,无菌取肾,经消化分散接种到细胞工厂中,在37℃下培养筛选出PHK细胞静置培养的最适细胞接种密度;在最适细胞接种密度下,从0.20%水解乳蛋白MEM培养基和改良的DMEM/F12培养基中筛选出更适宜地鼠肾细胞静置培养的培养基;同时对消化液A(含0.02%EDTA的0.25%胰蛋白酶溶液)和消化液B(含0.30%柠檬酸钠的0.25%胰蛋白酶溶液)的消化效果进行比对,选择适宜的消化液;最后,使用最佳培养基和消化液对PHK细胞进行传代,观察细胞生长情况并分析代谢水平;对同一细胞批不同代次的单层细胞(P0代、P1代和P2代)接种狂犬病病毒aG株,进行多次收获,检测单次病毒收获液病毒滴度。结果 PHK细胞(P0代)在细胞工厂中适宜的接种密度为0.30×10~6～0.50×10~(6 )cells/cm~2;改良的DMEM/F12培养基和消化液B更适用于PHK细胞的培养和消化传代;在40层细胞工厂(CF40)中使用最佳培养基和消化液传代地鼠肾细胞至第4代(P4代),随着代次的增加,收获的细胞密度降低、葡萄糖消耗减少和乳酸生成下降;P0代、P1代和P2代细胞均有较佳的细胞状态和增殖能力;用P0代、P1代和P2代细胞培养狂犬病病毒,能有效收获4次,且单次病毒收获液病毒滴度结果符合要求,差异无统计学意义(P0.05)。结论成功建立了PHK细胞传代培养工艺,为PHK细胞的规模化放大,冻干人用狂犬病疫苗(地鼠肾细胞)生产工艺的改进、变更以及产能的扩大提供了参考。     

无血清细胞培养在发热伴血小板减少综合征布尼亚病毒生长中的应用

目的建立无血清培养基培养Vero细胞制备发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的工艺。方法分别采用含10%牛血清的MEM(10%MEM培养基)和无血清M2培养基(SF-M2培养基)在方瓶中培养Vero细胞制备SFTSV,比较无血清与含血清培养基培养Vero细胞制备SFTSV在病毒滴度及病毒繁殖曲线之间的差异。在生物反应器里用无血清培养的方式进行工艺放大,收获病毒原液并进行检定。结果无血清培养的Vero细胞能够满足SFTSV培养需求,与含血清细胞培养相比,单位细胞病毒产量没有降低,达到30~60个活病毒/细胞。可以实现在生物反应器的工艺放大,病毒高峰时病毒滴度均在7.0lg PFU/m L以上。结论无血清细胞培养可以应用于SFTSV的培养,有利于降低疫苗生产过程中的纯化难度,提高疫苗安全性。     

由恒河猴腎上皮细胞分离的隐性病毒

1．两年来,我们从未曾接种过病毒的正常恒河肾上皮细胞培养物分离到6株自发性隐性病毒,并对其中的III一32毒株比驶系就地进行了捆胞病变特征,繁殖特性,理化性质,细胞敏感性和动物救病性等方面的研究和观察。 2．所有6株病毒在猴腎上皮细胞培养物上都产生相同的特异性病变,即产生胞浆内泡沫样空泡和多核融合细胞。未见有核内或胞浆内包涵体。 3．III一32毒株对恒河、平顶、红面、藏西及熊猴的腎上皮细胞培养物都有敏感性,对兔肾,人胚腎,皮肤,肌肉,人羊膜,恒河猴睾九的糸阳胞培养物以及乳鼠和家兔都不敏感。在猴腎细胞培养物的繁殖滴度都很低,接种14天以后,最高滴度只有10“。TCD50／毫升。在猴肾细胞培养物上经过十多次传代,洋度仍未见提高。 4．III一32毒株未能被脊髓灰质炎1、2、3型,coxsackie‘A9、Bl—B5型,EcHo 1—9型特异免疫血清以及典型麻疹患者恢复期血清所中和。 5．III一32毒株对乙醚有抵抗性。该病毒在22℃保存不超过1周,4℃不超过1个月,一20℃不超过牛年。用37℃连续传代培养的病毒液,接种猴腎细胞培养物时,细胞病变出现时间比4℃或一20℃保存者为早。     

轮状病毒LH9株在Vero细胞中的培养条件优化研究

为优化轮状病毒株在Vero细胞上的培养条件,将轮状病毒基因重配株LH9按0.1MOI分别接种于不同规格的细胞培养瓶(100ml、2000ml、3L、15L)。病毒接种采用吸附与未吸附两种方式、病毒收获采取低温冻融后离心与直接离心两种方法,观察分析对病毒滴度的影响。实验中,用CASY细胞计数仪分析活细胞率,病毒接种后逐日观察细胞病变(CPE)并取样,采用细胞半数感染量测定病毒滴度。结果表明,使用不同规格细胞培养瓶经吸附法培养接种、低温破碎法收获的LH9株病毒滴度高,其中以15L立瓶培养滴度最高(6.0～7.0 logCCID50/ml)。     

细胞工厂培养轮状病毒基因重配株Ls(G3型)条件的优化研究

目的以细胞工厂代替转瓶培养轮状病毒基因重配株Ls的可行性研究。方法采用细胞工厂与相应的转瓶培养工艺作对比,比较两种容器内细胞生长状态与病毒收获液滴度,并对细胞工厂培养条件进行了优化。结果以相同浓度接种细胞时,细胞工厂4 d长成单层,转瓶却需要7 d,经细胞仪计数后单位面积内细胞密度相当;以相同MOI接种病毒后,转瓶内的病毒于第7天病毒滴度达到峰值,细胞已完全脱落;细胞工厂于第3天病毒滴度达到峰值,并实现了3次收获。细胞工厂每次收获的病毒液滴度都稳定在一定范围,与转瓶相当。另外,细胞工厂培养条件优化结果表明,Vero细胞最佳接种浓度为3.0×104细胞/cm2,接种病毒的最适MOI为0.02~0.04。结论使用细胞工厂培养Ls株病毒不仅提高了效率,而且减少了培养空间,可替代转瓶规模化生产轮状病毒疫苗。     

细胞工厂在轮状病毒基因重配株LD9培养中的应用初探

为了探索用细胞工厂代替转瓶培养轮状病毒基因重配株LD9及收获高滴度的LD9病毒原液和提高产量的可行性,分别在2层4、层细胞工厂和3L1、5L转瓶培养Vero细胞,比较两种容器内细胞的生长状态。结果显示,以相同活细胞数2.5×104/ml同时接种两种不同培养容器时,细胞工厂培养3d已长成单层,而转瓶培养需5d;对两种容器长满单层时的细胞经胰酶消化后通过细胞仪计数、分析,结果显示,两种容器培养细胞长成单层时的单位面积细胞密度相当;对长成致密单层细胞的两种容器以相同的MOI(MOI=0.1)接种LD9病毒,转瓶培养的病毒于第8d病毒滴度达到高峰,为6.0～6.5 lgCCID50/ml;细胞工厂第5d病毒滴度达高峰,为6.5 lgCCID50/ml,并于第9d病毒滴度再次达到峰值,为6.0～6.5 lgCCID50/ml,实现二次收获病毒。     

重组逆转录病毒介导的转蚓激酶基因兔

目的通过重组逆转录病毒介导获得转蚓激酶基因家兔。方法将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,利用脂质体转染PA317包装细胞,G418进行筛选得到了阳性细胞克隆,克隆扩大培养后病毒上清直接注射雄性兔的睾丸组织转染精原干细胞。结果病毒上清经NIH3T3细胞进行滴度测定,最高滴度为1×104CFU/mL。病毒注射3月龄兔睾丸组织,过1.5个月取其睾丸、肾、脾、肝、肺进行组织切片观察,结果正常。提取交配所得F0、F1代仔兔基因组,经PCR、Southern检测,转基因阳性率F0代为6.3%(2/32)、F1代为15.3%(2/13)。结论通过重组逆转录病毒直接注射3月龄兔睾丸组织可获得转基因兔,病毒对兔的脏器无损害。     

风疹病毒松叶株衣壳蛋白C基因稳定性研究

目的研究风疹病毒(Rubella virus,RV)疫苗松叶株(Matsuba)衣壳蛋白(Capsid Protein)的基因稳定性及遗传特征,探讨其功能结构及生物学活性在病毒传代过程中的变化特点。方法利用RT-PCR方法扩增风疹病毒Matsuba株14、16、17、18、23代次C基因序列,测序后进行序列比对分析,并将各代次病毒的C基因与风疹病毒疫苗株Matsuba(GenBank登陆号:AB588193)及其他风疹病毒株C基因序列进行同源性分析。结果风疹病毒Mat-suba株传代病毒C基因在传代过程中核苷酸及氨基酸均未发生变异;各代次病毒与AB588193核苷酸及氨基酸序列完全一致,各关键功能区未发生变异;Matsuba株与18株风疹病毒核苷酸相似性在90.2%～99.9%之间。结论风疹病毒Matsuba疫苗株C基因遗传特性非常稳定,与生物学作用相关的区域未发生传代改变。从分子水平证明Matsuba疫苗株毒种及其生产的疫苗具有安全性。     

鸡胚细胞的传代培养和麻疹病毒的增殖

为了建立原代鸡胚细胞的传代培养工艺,探究传代鸡胚细胞对麻疹病毒的敏感性和适应性,本研究将原代鸡胚细胞进行传代培养,分别采用原代鸡胚细胞和传代鸡胚细胞培养麻疹病毒沪-191(Shanghai-191,S-191)株毒种,并对病毒收获液进行滴度检测和基因序列测定。结果显示,原代鸡胚细胞可稳定传代培养至第10代,各代次细胞生长趋势相似;第5代鸡胚细胞染色体检查为正常染色体核型;第8代鸡胚细胞成瘤性检查未见成瘤;采用第3、5代鸡胚细胞制备的麻疹病毒滴度水平高于原代鸡胚细胞,但无显著性差异(n＝3,P＞0.05),编码病毒核蛋白(nucleoprotein,N)和血凝素蛋白(hemagglutinin,H)的基因序列与S-191株完全一致,未发生变异。本研究证实,原代鸡胚细胞可进行传代培养,各代次鸡胚细胞对麻疹病毒的敏感性不变,产毒水平无显著差异,可用于培养麻疹病毒。     

Alteration of the In Vitro Host Range of Rabies Virus after Serial Chick Embryo Cell Passage Using Alkaline Maintenance Medium

HEP Flury strain of rabies virus maintained by 7-day chicken egg passage (parent line) and the same strain serially passaged in primary chick embryo (CE) cells using alkaline maintenance medium (AM line) were inoculated to cells of various species. Growth was negative in primary mouse embryo, L and HeLa cells, and positive in primary hamster kidney and BHK21 cells with both lines. An all-or-none difference between the two lines was observed in primary monkey kidney and Vero cells. The parent line did not multiply in these monkey cells, whereas the AM line grew to high titers. In the case of Vero cells a unique cytopathic effect (CPE) was induced by the AM line. After five consecutive passages in Vero cells, the CPE-inducing agent was identified as rabies virus by a neutralization test. It was infective to intracerebrally inoculated suckling mice but not to adult mice, and its Vero cell-infective titer determined by CPE induction was about 1 log lower than the baby mouse-infective and CE plaque-forming titers. In contrast to the AM line, HEP Flury strain receiving 150 CE cell passages under neutral maintenance medium and three other strains receiving similar CE cell passages all failed to grow in Vero cells.     

风疹减毒活疫苗主种子批毒种(松叶株)的安全性和免疫原性检测

评价兰州生物制品研究所用风疹病毒松叶株主种子批毒种制备的冻干风疹减毒活疫苗的安全性和免疫原性。采用自身对照、开放性的免疫原性临床观察试验,对100名8～10月龄筛选后符合条件的健康易感儿童,皮下接种1剂风疹减毒活疫苗,观察其免疫后的局部和全身反应并采集每个受试者免前和免后35d的血清标本,检测风疹HI抗体,计算阳转率和几何平均滴度。试验中所有受试者在系统观察期内均未观察到注射部位局部的不良反应;总的发热率为5%,且均为轻度发热;有1例在观察期内出现腹泻和咳嗽并持续5d,发生率为1%,属中度全身反应;血清风疹病毒抗体(HI)阳转率为100%,GMT为1:638.7±1.7。该疫苗与国内、外其它种类的风疹疫苗一样具有良好的安全性和免疫原性。     

Markers of Rubella Virus Strains in RK13 Cell Culture

When tested on RK(13) cell cultures, strains of rubella virus could be differentiated by their ability to form small or large plaques. Large plaques were produced by the HPV-77 and Cendehill strains, and also by a laboratory stock strain (West Point), after only 14 passages in RK(13) culture. Five wild-type rubella viruses, isolated and passaged only a few times in African green monkey kidney tissue culture, grew well in RK(13) cell culture, but they were sensitive to agar inhibitors and, therefore, formed small plaques. On the other hand, RA27/3, an attenuated strain grown in WI-38 human fibroblast cells, developed low titers in RK(13) cells and also produced small plaques. We concluded that the morphological differences between small-plaque and large-plaque viruses depended on their sensitivity to agar inhibitors and on the pH of the medium during plaque formation.     

Beagle狗肾细胞及用于制备乙脑活疫苗的研究

Ｂｅａｇｌｅ乳狗（５－１０日龄）肾块用热消化法制成细胞批放液氮保存,检定合格后做下列试验：（１）复苏培养长满单层后更换维持液,其细胞能维持４天以上,克服了常规消化培养细胞维持时间短（３６小时）的缺陷。（２）传代１４－２株病毒时,１代病毒的滴度即达６．０４－６．２４ＬｏｇＰＦＵ／ｍｌ,１代后的各代（至１１代）病毒滴度无明显提高。（３）该细胞接种１４－２株病毒时不产生明显的破坏性病变（ＣＰＥ）,在收毒前采取冻溶１次比冻溶前的病毒滴度提高０．２７－０．５ＬｏｇＰＦＵ／ｍｌ。以ＢＤＫ细胞传１代的１４－２株病毒作生产毒种,收毒前冻溶１次等工艺法制备五批疫苗,全面检定结果均符合《乙脑活疫苗规程》的质量标准。其中病毒滴度为６．０９－６．２４ＬｏｇＰＦＵ／ｍｌ;免疫效价（ＩＤ５０＝ｍｌ）为１．９－４．０×１０－５,与相同滴度的原毒株１４－２ＰＨＫ苗对照无明显差异（ｔ＝０．９６８Ｐ＞０．０５）,表明其免疫原性与原毒株相似。     

Rubella virus interference and interferon production

An interference technique for the titration of rubella virus on primary rabbit kidney (PRK) cells using either vaccinia or vesicular stomatitis virus (VSV) as challenge is described.Rubella virus passed and propagated on PRK-cells interfered with a challenge dose of either vaccinia virus or VSV given on the 7th day after inoculation. Concomitantly, interferon was demonstrated in these cultures in amounts up to 100 units per ml.The same strain of rubella virus passed on a continuous rabbit kidney cell line (RK13) induced no interferon in PRK-cells. Yet it interfered equally well with a challenge of vaccinia virus or VSV.Under experimental conditions where no interferon could be detected, rubella virus growth inhibited the one-cycle multiplication of poliovirus-RNA, suggesting that interference resides at the intracellular level.Under the described experimental conditions the multiplication of rubella virus in PRK-cells was not inhibited by an amount of interferon 20 times higher than that necessary to inhibit vaccinia virus cytopathic effect in the same cell system.Aspirant of the Belgian N.F.W.O.Navorsingsstagiair of the Belgian N.F.W.O.     

Characterization of recombinant rabies virus carrying double glycoprotein genes

A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UVinactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.     

Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons—the possible effects of the multiple passages of EHV-1 on its neurovirulence

Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.