流行性出血热病毒在MA-104和Vero-E6传代细胞上繁殖动态的比较

用流行性出血热病毒(陈株)分別感染MA-104和Vero-E6传代细胞,结果受病毒感染的MA-104细胞荧光阴性细胞出现早,感染滴度高,胞浆内颗粒大,提示MA-104细胞用于该病毒的分离传代及抗原片的制作等方面优于Vero-E6细胞。流行性出血热病毒(EHFV)在某些原代及传代细胞上能适应增殖,国内外已有过报导。但用对轮状病毒十分敏感的恒河猴胚肾MA-104细胞培养和增殖EHFV并与通常使用分离该病毒的VeroE6细胞进行繁殖动态观察,尚未有过报导。本文用间接免疫荧光法(IFA)比较观察EHFV在两种细胞上的增殖动态,为MA-104细胞代替常规Vero-E6细胞用于该病毒的分离、传代等研究以及抗原片的制备提供依据。     

猪和牛轮状病毒的分离和鉴定

我国对动物轮状病毒的研究报道尚少。用细胞培养法分离轮状病毒又比较困难。本文报道用非洲罗猴胎肾细胞MA-104,结合胰酶处理及旋转培养技术,从北京地区腹泻仔猪和犊牛的粪便中成功地分离到6株猪轮状病毒(暂名为PRV1,PRV2,PRV8,PRY11,PRV15和PRV17)和4株牛轮状病毒(暂名为BRV6555,BRV6551,BRV6571和BRV6576)及其初步鉴定结果。     

重组胰酶在口服轮状病毒活疫苗制备中的初步应用

目的探究重组胰酶替代猪源胰酶在轮状病毒疫苗制备中的可行性。方法确立3种重组胰酶的最佳工作浓度;观察3种重组胰酶和猪源胰酶消化MA104细胞并连续传10代(P1~P10),根据消化时间、消化后细胞总数、细胞活率、细胞生长状况、细胞形态变化等方面进行考量比较;观察轮状病毒LH9株经4种胰酶活化,分别接种对应胰酶传代的MA104细胞上培养,通过病毒滴度(lg CCID_(50)/mL)及RNA-PAGE检测,分析重组胰酶替代猪源胰酶在轮状病毒疫苗制备中的应用。结果连续P1~P10代后3种重组胰酶和猪源胰酶在细胞总数、细胞活率及细胞生长状况,差异均无统计学意义(P0.05),显微镜观察细胞形态无明显改变。轮状病毒LH9株经不同胰酶活化,接种MA104细胞P1~P10代培养后,病毒滴度差异均无统计学意义(P0.05),且RNA-PAGE电泳图谱未见差异。结论初步应用试验表明3种重组胰酶均可替代猪源胰酶用于轮状病毒疫苗的制备。     

从一次新生小牛腹泻流行中分离到牛轮状病毒

1985年3月下旬,合肥市某牛奶场发生了一次新生牛非细菌性腹泻流行。用MA104细胞从牛_1粪便标本中分离到一株牛轮状病毒。电镜检查为典型轮状病声形态。在MA104细胞上第三代开始呈现胞浆内颗粒状荧光并产生细胞病变(CPE)。PAGE核酸电泳图型与牛轮状病毒NCDV株完全一致。经ELISA鉴定属A群轮状病毒。     

人轮病毒的细胞培养分离及其血清型的鉴定

<正>本轮报导从73份轮状病毒阳性粪便中,用MA-104细胞或非洲绿猴(AGMK)原代细胞培养方法分离到39株人轮状球毒。用噬斑抑制试验和中和试验法鉴定结果可分为4个血清型,其中3个型别以前已报导过。 病毒分离方法,取10％粪便悬液,先经10μg/ml胰酶处理37℃1小时,接种细胞后吸附37℃1小时,洗一次,加入含0.5μg/ml胰酶的MEM,其中含DEAE-dext-     

用细胞培养分离人轮状病毒

<正>过去许多人试图用细胞培养繁殖高效价的轮状病毒都告失败了。最近,Wyatt等曾将人工适应2型人轮状病毒(Wa株)经既定菌丛的新生小猪传11代后,在非洲绿猴原代细胞培养中得到相当高的效价。本研究中用MA104细胞(一种恒河猴胚肾的细胞系)分离培养,得到三株人轮状病毒。     

新的人轮状病毒株的分离

<正>人轮状病毒在任何培养系统中繁殖是很困难的。1980年Whyatt等经过在悉生小猪传代几次后获得了适应组织培育生长的人轮状病毒“Wa”株。1981年Sato和Urasawa等在细胞维持液中加入胰酶并使用旋转培养,直接从人粪便中分离到轮状病毒。1983年Birch等报导采用胰酶预处理粪样提取液,维持液中加入胰酶的旋转培养。成功地在HA—104和CV—1细胞上适应几株人轮状病毒。     

检测轮状病毒结构多肽的一种简易方法

本实验以猪轮状病毒为对象,利用放线菌素D抑制细胞蛋白,结合SDS-PAGE和硝酸银染色,检测轮状病毒结构多肽。方法简易灵敏,重复性好。现简要报告如下。 将MA-104细胞(恒河猴肾传代细胞)按照文献方法培养成单层(5×7cm~2,约48小时),用Hanks液洗2次,加入含5μg/ml放线菌素D(Actinomycin D Fluka)的Eagle’s MEM 5ml,预作用12小时,用Hanks液洗细胞2次,按文献方法接毒吸附1小时。选用的毒株为本室自天然病猪分离并适应于     

细胞工厂生产口服轮状病毒活疫苗的应用研究

为了确定用细胞工厂生产口服轮状病毒活疫苗的工艺参数和质控点,将原代牛肾细胞按20×104个/cm2接种到细胞工厂培养,待形成良好单层后按4×104个/cm2连续传代2次后接种轮状病毒,并与转瓶作对照.结果显示,用细胞工厂生产口服轮状病毒活疫苗与转瓶培养无差异,细胞工厂可用于改进口服轮状病毒活疫苗的生产.     

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。     

猪和牛轮状病毒抗原和抗体的检测

应用ELISA直接双抗体夹心法检查轮状病毒抗原,24份仔猪和29份犊牛的腹泻粪样,分别有12和16份阳性。用病毒RNA电泳分析检查阳性粪样,各出现两种病毒RNA电泳型,用中和试验检查17份成年牛和16份成年猪血清,分别有16和15份病毒抗体阳性。将其与ELISA间接法和结合法进行了比较。     

Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine.

Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.     

Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype.

Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.     

RNA of rotavirus: comparison of RNAs of human and animal rotaviruses.

Single-stranded RNA (ssRNA) was transcribed in vitro from inner-shell particles of human rotavirus strain Wa (HRV-Wa) and a bovine rotavirus (neonatal calf diarrhea virus [NCDV]) by virion-associated RNA polymerase activity. The ssRNA product consisted of 11 RNA segments which were separated by polyacrylamide gel electrophoresis. In vitro-transcribed 32P-labeled ssRNA was used to study the genetic relatedness between rotaviruses by annealing with genomic double-stranded RNA (dsRNA) of homologous or heterologous rotavirus. All segments of HRV-Wa ssRNA were hybridized with dsRNA of HRV TK80, collected from the feces of a gastroenteritis patient, at the level of 88 to 100% of the homologous reaction. On the other hand, no segments of ssRNA from HRV-Wa hybridized with dsRNA of NCDV or simian rotavirus (simian agent 11). Similarly, ssRNA from NCDV did not hybridize with dsRNA of HRV-Wa, but hybridized with dsRNA of simian agent 11 at the level of 30% of the homologous value.     

Recombinant porcine norovirus identified from piglet with diarrhea

ABSTRACT: BACKGROUND: Noroviruses (NoVs) are members of the family Caliciviridae and are emerging enteric pathogens of humans and animals. Some porcine NoVs are genetically similar to human strains and are classified into GII, like most epidemic human NoVs. So far, PoNoV have been exclusively detected in fecal samples of adult pig without clinical signs. METHODS: We collected 12 fecal samples from piglets with diarrhea and no accurate diagnosis of etiology from three commercial pig farms in Shanghai suburb. We tested for PoNoV, porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine sapovirus, and porcine epidemic diarrhea virus using RT-PCR method. The full-genome sequence of the PoNoV was then determined and analyzed. Experimental infection of miniature pigs with fecal suspensions was performed to make sure if this strain can cause gastroenteritis in piglets. RESULTS: Result showed that 2 of the 12 evaluated fecal samples were positive for PoNoVs, one of which was positive for PoNoV alone, and the other was coinfected with porcine circovirus and PoNoV. Phylogenetic and recombination analysis showed that the PoNoV positive alone strain was a recombinant new genotype strain. Experimental infection of miniature pigs with fecal suspensions confirmed that this strain can cause gastroenteritis in piglets. CONCLUSION: This is the first report that recombinant new genotype PoNoV exised in pig herd of China, which cause diarrhea in pigs in nature condition. This find raised questions about the putative epidemiologic role of PoNoV.