Adaptation of hepatitis A virus strain (JaM-55) originally pathogenic for monkeys to a cell culture

The results of adaptation of hepatitis A viral strain JaM-55 to the culture of embryo kidney cells FRhk-4 from macaque Rhesus are presented. The viral strain was isolated from a M. fascicularis suffering from spontaneous hepatitis. Before inoculating the cell culture the virus was passaged twice in the M. arctoides capable of reproducing hepatitis. FRhk-4 cell line inoculation by the monkey liver extract, containing the strain HAV-YaM-55, resulted in isolation of single viral particles of hepatitis A in the preparations obtained at the first 3 passages by the 28-31 day of cultivation. Beginning from the fourth passage the abrupt increase in the number of viral particles and hepatitis A antigen was registered. There were no traces of cytopathogenic effect at any level of viral passages in the inoculated cell culture. The adapted virus contains hepatitis A viral RNA identified by spot hybridization with the cloned cDNA of hepatitis A virus.     

Tracking micrometastasis to multiple organs with lacZ-tagged CWR22R prostate carcinoma cells.

Metastasis to organs other than lung is rarely observed in animal model systems of human prostate carcinoma (PCA), with the exception of already metastatic isolates of human PCA cultured for long periods of time. To analyze more directly the evolution of metastatic variants from primary PCA tumor isolates, the lacZ histochemical marker gene was transfected into the CWR22Rv1 cell line isolated from the CWR22R xenograft (primary tumor). Three clones of varying lacZ-expression stability were analyzed for tumorigenicity and progression in athymic nude mice. Clones B and D were highly tumorigenic in the subcutis; however, lacZ expression was highly unstable. In contrast, clone H demonstrated highly stable lacZ expression for >25 passages in culture or in animals. Clone H, injected sc in a PBS vehicle, gave a 15-40% tumorigenic take. All primary tumor-bearing animals exhibited micrometastases in lung and other organs. Clone H injected in a Matrigel vehicle gave 100% tumorigenicity, with all animals displaying micrometastases in lung, liver, and/or bone (lower frequency in brain and kidney). Overall, the relative frequency of micrometastasis to multiple organs was lung>liver=bone>brain>kidney. Overt metastases were never observed in the lung or bone but were occasionally found in liver. lacZ-transfected clone H CWR22Rv1 cells represent a much more accurate model of metastasis of PCA to the organs normally involved in progression of the human disease. Use of marker gene-tagged cells and other high-resolution molecular techniques will now permit analyses of the earliest events in PCA progression and micrometastasis.     

Viremia in a Recipient of HPV-77 Rubella Virus Vaccine

A live rubella virus vaccine, HPV-77 (High Passage Virus - 77 tissue culture passages) was administered subcutaneously to eight rubella-susceptible children housed in an isolation ward. One blood specimen, taken on the tenth day after vaccination, from one of the eight vaccines, yielded a rubella virus. This virus had laboratory markers which were “vaccine-like.” To our knowledge, this represents the first isolation of rubella virus from the blood of a recipient of HPV-77 vaccine. However, the consistent antibody responses among vaccinees and the regular presence of rubella virus in their pharynges argue that viremia occurs in almost every susceptible recipient. The most logical explanation for the failure to document viremia in other recipients of HPV-77 vaccine is that the viremia is ordinarily low grade or transient or both.     

登革热Ⅳ型病毒在地鼠肾细胞培养内减毒的研究

本文报道登革热Ⅳ型病毒Ban18株通过原代地鼠肾细胞连续传代后的适应和减毒过程。 Ban18原株对原代地鼠肾细胞无病变或仅有极轻微病变,对乳小白鼠及断乳鼠的脑内毒力高达LD_(50)为Log6—7。通过连续传20—70代后,病变逐渐加重,出现时间缩短,二天即可达到细胞完全破坏。培养液内的病毒含量也随之提高达10~6—10~7/ml TCID_(50)。但随着传代代数增加,病毒对乳小白鼠的嗜神经毒力逐渐降低,脑内接种后仅个别发病或完全不致死。脑组织病理变化也较原株明显减轻,接种树鼩不产生病毒血症。以上结果符合弱毒株的减毒指标,是一株有希望的活疫苗毒株。     

气管插管法接种H5N1病毒感染恒河猴及系统组织病毒检测

目的测试气管插管法接种高致病性禽流感病毒H5N1感染恒河猴的优势效果及疾病分析,为有效感染恒河猴、制备H5N1疾病模型提供实验依据。方法使用人源H5N1病毒液经气管插管滴入恒河猴上呼吸道进行感染,观察感染恒河猴的临床表现,每天采集咽拭子、鼻灌洗液,在感染前2d感染后第3、5、7天采血,感染后第3和7天分别解剖1只恒河猴,取支气管淋巴结、肠淋巴结、鼻甲、心、肝、脾、肺、肾、肠、气管、脑及血液进行病毒分离、核酸载量检测和血常规测定。结果感染后第2天恒河猴出现食欲下降,活动减少,并伴有一过性体温升高,白细胞数和淋巴细胞数下降。咽拭子、鼻灌洗液、肺、心、气管、脑、肝、肾、肠和血液中都能分离到H5N1病毒。结论气管插管法接种H5N1病毒能有效感染恒河猴,并在猴体内多组织中分离、检测到病毒,为制备完善的H5N1模型和检测指标确定、进一步研究H5N1病毒的致病机制等奠定了基础。     

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.     

Establishment and characterization of a continuous cell line from pupal ovaries of Japanese oak silkworm Antheraea yamamai Guerin-Meneville

Pupal ovaries of the wild oak silkworm Antheraea yamamai Guerin-Meneville were cultured in MGM-448 (Modified Grace Medium-448) medium containing 10% fetal bovine serum. After the primary culture was set up in 1988, a continuous cell line was obtained in 1991, designated as NISES-Anya-0611 (Anya-0611). The population doubling time was 54 hrs. and 19 min. at 96 passages and 88 hrs. and 29 min. at 387 passages. Spindle-shaped and spherical cells coexisted in the cell group. The cell line karyotype line was typical of lepidopteran cell lines, consisting of numerous small chromosomes. The cell line was distinguished from other lepidopteran cell lines by comparing malic enzyme, phosphoglucose isomerase, phosphoglucose mutase, and isocitric dehydrogenase isozyme patterns. The cell line was highly infected to the Antheraea yamamai nuclear polyhedrosis virus (Anya NPV). The luciferase gene of recombinant Bm NPV (BmNPVP6ETL) was able to express in the cell line, too, so that luciferase recombinant products were able to be detected in the cell body and in supernatant. The Anya NPV clone group was isolated on the cell seat using plaque purification.     

Virulence and Pathogenesis of Yellow Fever Virus Serially Passaged in Cell Culture

Viscerotropic virulence of the Asibi strain of yellow fever virus (YFV) for monkeys has been known to be lost after serial passage in HeLa cell monolayers. This phenomenon was investigated in several other mammalian and insect tissue cell lines. Assay in monkeys of original seed virus and of virus after 7 and 11 passages in a porcine kidney cell line (PK) indicated essentially equal infectivity and mortality. Moreover, monkeys receiving the passaged virus exhibited more rapid onset of disease and death than animals infected with original seed virus. Histological changes in animals inoculated with passaged virus were identical to those in animals receiving the seed virus. Virus from later passages in PK cells was also lethal for approximately 50% of the monkeys; however, evidence for progressive attenuation was seen in these preparations. Similar results were obtained with a mosquito (Aedes aegypti) cell line. In contrast to results obtained in PK and mosquito cells, YFV became essentially avirulent (nonlethal and less infective) for monkeys after only seven passages in HeLa cell cultures.     

修饰的痘苗病毒安卡拉株（MVA）基因组中高频的同源重组

痘苗病毒由于其外源基因容量大,表达产物后加工完善等优势而广泛用于基因工程的研究以及基因治疗,痘苗病毒基因组的同源重组现象为其基因操作带来了方便,也被用于很多痘苗病毒基因结构和功能的研究,痘苗病毒安卡拉株（MVA）是一种修饰的复制限制的痘苗病毒,由于极高的安全性,正在实验室和临床应用的很多领域取代普通的痘苗病毒,为提高重组MVA系统的安全性以及筛选重组MVA的效率,发展了一种暂时选择系统,此系统利用分子内2段同向的相同序列发生同源重组去除选择标记k1l基因,从而消除选择标记对宿主可能的危害。利用此暂时表达系统构建了4个携带编码不同长度外源多蛋白质序列的重组MVA,并估算了每次传代的重组频率,结果显示,MVA同源重组频率虽然比其他痘苗病毒株要低,但仍然是较斋的,将带有k1l基因的重组MVA经3-4次盲传（blind passage),即可获得完全去除选择标记的重组MVA。进一步证明上述利用暂时选择标记k1l基因构建重组MVA的系统具有十分可靠的安全性,适合作为人体活疫苗开发和基因治疗的载体,而且,通过盲传进行筛选,能大大提高去除选择标记的效率,降低鸺建重组MVA的成本。     

Primary culture and immortalization of human fallopian tube secretory epithelial cells

Primary human fallopian tube secretory epithelial cell (FTSEC) cultures are useful for studying normal fallopian tube epithelial biology, as well as for developing models of fallopian tube disease, such as cancer. Because of the limited ability of primary human FTSECs to proliferate in vitro, it is necessary to immortalize them in order to establish a cell line that is suitable for long-term culture and large-scale in vitro experimentation. This protocol describes the isolation of FTSECs from human fallopian tube tissue, conditions for primary FTSEC culture and techniques for establishing immortal FTSEC lines. The entire process, from primary cell isolation to establishment of an immortal cell line, may take up to 2 months. Once established, immortal FTSECs can typically be maintained for at least 30 passages.     

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.     

Comparison of infectious haematopoietic necrosis virus (IHNV) isolation on monolayers and in suspended cells

A cell culture virus isolation procedure for infectious haematopoietic necrosis virus (IHNV) in the epithelioma papulosum cyprini cell line (EPC) is described. Ovarian fluid samples were collected from fish and tested for IHNV at 9 farms. The samples were inoculated in parallel on 24 h old EPC cell monolayers and in freshly trypsinized cells. The titre of the initial virus isolation and of first passages were compared using the 2 methods for each sample. Titres were consistently higher in suspended cells and this method also proved more sensitive for isolation of IHN virus from ovarian fluids of infected fish.     

Susceptibility of continuous lines of monkey kidney cells to influenza and parainfluenza viruses in the presence of trypsin

LLC-MK2, GMK AH-1, BSC-1, and Vero cells were compared in titrations of recent isolates and laboratory strains of influenza A and B and parainfluenza types 1, 2, and 3 viruses. About the same titres, as determined by haemadsorption in cell cultures, were obtained in LLC-MK2, GMK AH-1, and BSC-1 cells when trypsin had been added to the medium, whereas the Vero cells were less sensitive to the influenza virus strains tested. Virus titres were usually low in the absence of trypsin. A laboratory strain of parainfluenza 2 virus reached about the same titres in medium without as in medium with trypsin, possibly owing to prior adaptation by passages in Vero cells. Comparative titrations of influenza A, and parainfluenza 1 and 3 viruses suggested the same susceptibility of LLC-MK2 cells with trypsin as of primary monkey kidney cells. Re-isolation experiments from 38 clinical specimens showed LLC-MK2 cells to be as efficient as primary monkey kidney cells for isolation of influenza and parainfluenza viruses, whereas the susceptibility of the other cell lines to clinical material has not yet been tested on a larger scale. It is concluded that a continuous line of monkey kidney cell culture may be acceptable as an alternative to primary monkey kidney cells for the isolation of influenza and parainfluenza viruses from patients.     

Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Establishment and characterization of a unique 1 μm diameter liver-derived progenitor cell line

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.     

Growth Curve and Distribution of Rous Sarcoma Virus (Bryan) in Japanese Quail

On primary infection with the Bryan strain of Rous sarcoma virus (RSV), the growth curve of the virus in the brain of Japanese quail was similar to that observed in chicks and turkey poults. Infectious virus disappeared from the brain after inoculation. After an eclipse period during which no virus was detectable, infectious virus began to appear at 2 days and reached maximal titers in the brain samples at 7 days after inoculation. When Japanese quail were infected intracerebrally with RSV, relatively high titers of virus were recovered from brain tissue but not from liver, lung, kidney, or blood of moribund birds. Only tumors produced in the wing web of quail infected subcutaneously yielded high titers of virus. Other tissues yielded no virus, even though wing web tumors appeared as early as in chicks similarly infected. RSV could be propagated in the wing web of quail for at least 14 passages without any loss of infectivity. On the other hand, serial passage in quail brain resulted in a progressive loss of infectivity until virus was completely lost.     

鸭群中REV感染的流行病学调查

[目的]了解网状内皮组织增生病病毒(REV)在鸭群中的感染状态.[方法]从山东省不同地区送检的病(死)鸭中,随机采集法氏囊、脾脏和肝脏等220份样品.细胞培养分离病毒,以提取的组织DNA为模板进行特异性斑点杂交、PCR和nest-PCR检测.从不同地区阳性样品中任选一个进行克隆测序、同源性比较和进化树分析.[结果]从35/39份法氏囊、54/84份脾脏和32/97份肝脏DNA样品中检出REV(121/220).其中法氏囊的检出率最高,显著高于肝脏、脾脏(P<0.01),但用细胞培养分离病毒、常规PCR、组织DNA直接点杂交检测时,均未检出REV.YN-1和BZ-1株env基因片段与美国分离的鸭源SNV株同源性高达99.8%,LQ-1株env基因片段与美国鸡源分离株的同源性为100%,均高于近几年中国鸡源分离株.[结论]在检测REV感染时,应加强对法氏囊的检测,但由于REV在感染鸭的组织中含量很低,应采用更为敏感的nest-PCR.同源性和进化树分析表明,我国鸭源REV很可能是在引进未经对REV检疫的种鸭时引入的.     

Isolation of canine rotavirus and study of its immunobiological properties]

Canine rotavirus was isolated in MA104 roller culture of rhesus macaque cells. Two passages in gnotobiotic puppies and two in colostrum-free puppies resulted in isolation of strain P of canine rotavirus. After 20 passages in MA104 culture the virus was adapted to MDCK culture. Optimal conditions for accumulation of canine rotavirus and its antigen (9.01 g TCD50/ml) in MDCK culture are trypsin pretreatment of the virus inoculate in the final concentration of 50 mcg/ml for 30 min at 37 degrees C, presence of trypsin (10 mcg/ml) in the maintenance medium, multiplicity of infection 0.1 TCD50/ml, and incubation in roller culture at 37 degrees C during 24-30 h. After 60 passages in cell culture, canine rotavirus completely lost its virulence for colostrum-free puppies but retained antigenic activity and induced manifest seroconversion in infected.