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PubMed1976

Studies on a measles virus variant inducing persistent infections in cultured cells.I. Isolation and characterization of plaque purified virus clones.

Attempts were made to characterized by a plaque assay two variants of the Edmonston strain of measles virus and to obtain plaque purified virus populations. The UP non-cytocidal variant, in all the examined cell systems, mainly produced small but also large plaques; the DP cytocidal variant always large plaques. Three clones, UP-SP4, UP-LP4 and DP-LP4, were derived by plaque purfication respectively of the UP small plaque, UP large plaque and DP large plaque forming particles. The virus populations of the clones could be distinguished by some other biological and physical characters: cytopathic effect in roller tube cultures, growth potential in HeLa cells, thermal stability at 45 degrees C, stability of the properties during serial passages at different input multiplicity. The hypothesis was supported that the typical properties of the UP and DP variants are host-independent and genetically controlled viral markers.

Animals↗

PubMed1976

Survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids.

Influenza A virus, strain WSNH, propagated in bovine, human and chick embryo cell cultures and aerosolized from the cell culture medium, was maximally stable at low relative humidity (RH), minimally stable at mid-range RH, and moderately stable at high RH. Most lots of WSNH virus propagated in embryonated eggs and aerosolized from the allantoic fluid were also least stable at mid-range RH, but two preparations after multiple serial passage in eggs showed equal stability at mid-range and higher RH's. Airborne stability varied from preparation to preparations of virus propagated both in cell culture and embryonal eggs. There was no apparent correlation between airborne stability and protein content of spray fluid above 0.1 mg/ml, but one preparation of lesser protein concentration was extremely unstable at 50 to 80 per cent RH. Polyhydroxy compounds exerted a protective effect on airborne stability.

Aerosols↗

PubMed1991

Production of infectious particles from defective human immunodeficiency virus type 1 (HIV-1)-producing cell clones by superinfection with infectious HIV-1.

A total of 81 cell clones persistently infected with the LAV-1 or HTLV-IIIB strain of human immunodeficiency virus type 1 (HIV-1) was isolated from cells which were obtained by serial passage of some proliferating MT-4 cells after a drastic cytolysis of most cells by HIV-1-infection. These cell clones were classified into 8 types (I to VIII) in terms of the expression of HIV-1 antigens, syncytium formation capacity, and reverse transcriptase activity and infectivity of virus particles in the culture fluid. Type I cell clones were producers of infectious HIV-1 particles, while types II to VIII cell clones did not produce infectious HIV-1 or were producers of uninfectious defective HIV-1 particles. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (PAGE) showed that the gag precursor protein in L-2 cell clone (type IV) was not cleaved to mature gag proteins, while the env precursor protein on L-3 cell clone (type III) was not cleaved to mature env protein. H-7 cell clone (type VIII) did not express any HIV-1 antigen. All these cell clones after the superinfection with infectious HIV-1 synthesized intact gag and env proteins, which were, at least in part, related to the HIV-1 genome persistently present in the cell clones before the superinfection, resulting in production of infectious HIV-1. For example, it was found that L-2 cell clone contained a single copy of the LAV-1 genome per haploid cell and produced doughnut-shaped particles. On the other hand, the cell clone isolated from the L-2 cell clone superinfected with infectious HTLV-IIIB contained the integrated HTLV-IIIB genome in addition to the LAV-1 genome present before the superinfection, and produced intact HIV-1 particles in addition to doughnut-shaped particles from a single cell. These results indicate that complementation and/or genetic recombination events in the superinfected cells may account for the production of infectious intact HIV-1 virions.

Animals↗

PubMed1988

Antigenic variation of equine infectious anemia virus as detected by virus neutralization. Brief report.

The antigenic structure of 16 viruses isolated from four horses which were inoculated with a clone of equine infectious anemia (EIA) virus was compared by the neutralization test. The antigenic structure of viruses isolated after development of neutralizing antibody differed from virus to virus. Back mutation of the antigenic structure was also demonstrated by serial passage of the virus in horses. These results suggest that EIA virus is subject to multidirectional antigenic variation. The possibility that the variants originated in the heterologous virus population in the inoculum seems to be unlikely since the virus used for the primary inoculation was cloned by three repeated high-limiting dilutions.

Animals↗

PubMed1994

Studies on the antigenicity and nucleotide sequence of the rabies virus Nishigahara strain, a current seed strain used for dog vaccine production in Japan.

The Nishigahara strain of rabies virus, a current seed strain used for animal vaccine production in Japan, is believed to derived from the original Pasteur strain obtained from Paris in or before 1915. In Japan, the virus was serially passaged through several kinds of animals and cell cultures. Reactions with anti-nucleocapsid protein monoclonal antibodies (MAb-N) indicated the Nishigahara strain had maintained the antigenic profile of the Pasteur virus. Reactions with monoclonal antibodies to the glycoprotein (MAb-G) revealed differences between the Nishigahara strain and the Pasteur strain; however, the Nishigahara strain maintained a closer resemblance to the Pasteur virus than to other Pasteur-related viruses or to rabies strains unrelated to the Pasteur strain. Comparative amino acid sequence analysis of cloned cDNA encoding the G gene confirmed the antigenic differences among these strains and the resemblance of the Nishigahara strain to the original Pasteur strain. Comparative nucleotide sequence analysis of the noncoding pseudogene region (Tordo et al., Proc Natl Acad Sci USA 83, 3914-3918, 1986) revealed different relationships. Unlike the Pasteur strain, which encodes a transcription-terminating signal at the end of the G gene (marking the beginning of the pseudogene), a long G-L intergenic sequence in the Nishigahara strain was connected to the 3' end of the cDNA, and the transcription-terminating signal was present only at the end of, but not before, the pseudogene. These results are not inconsistent with the documented origin of the Nishigahara strain, but the genome structure around the pseudogene region suggests divergence from the Pasteur strain and a closer resemblance to other strains of rabies virus.

Amino Acid Sequence↗

PubMed1996

Ebola virus infection in guinea pigs: presumable role of granulomatous inflammation in pathogenesis.

An approach combining virology with light and electron microscopy was used to study the organs of guinea pigs during nine serial passages of Ebola virus, strain Zaire. It was observed that the wild type of Ebola virus causes severe granulomatous inflammation in the liver and reproduces in the cells of the mononuclear phagocyte system (MPS). Based on morphological characterization, two types of virus-cell interactions were demonstrated. The obtained data evidenced for heterogeneity of the population of wild type of Ebola virus. The virus accumulated in the liver of the infected animals, and the lesions became more pronounced with passage. Degenerative changes appeared, and their severity was increased with passage in the other organs as well. The set of target cells diversified and, as a result, not only the MPS cells, but also hepatocytes, spongiocytes, endotheliocytes and fibroblasts became involved in the reproduction of Ebola virus. The possible role of granulomatous inflammation in the development of the adaptive mechanism of Ebola virus to guinea pigs is discussed.

Animals↗

PubMed1988

Establishment and characterization of a cell line from a human cholangiocellular carcinoma.

A cell line, HuH-28, was established in vitro from a patient with cholangiocellular carcinoma (CCC). This cell line has grown slowly, revealing a doubling time of approximately 80 h, and the serial passages were carried out 20 times within 10 months. Light microscopy revealed spindle and polygonal morphology of the cells. Chromosome number of the cells were distributed near the hypotriploid region at passages 3 and 14. HuH-28 cells were not transplantable into nude mice, but secreted some tumor markers including alkaline phosphatase (ALP), gamma glutamyltranspeptidase (GGT), beta 2-microglobulin (BMG), ferritin, elastase-1, and tissue polypeptide antigen (TPA). This HuH-28 cell line will represent a good model for the investigation of carcinogenesis, histogenes, and diagnosis of CCC.

Adenoma, Bile Duct↗

PubMed1991

Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody.

The monoclonal antibody (mAB) L1, which binds to the nucleocapsid protein of canine distemper virus (CDV), was shown to bind to avirulent CDV obtained after serial passages in Vero cells, but not to two different virulent demyelinating CDV-strains propagated in dog glial cell cultures. However, when both virulent CDV-strains were passaged through Vero cells they expressed, after a number of passages, an epitope recognized by mAB L1. The occurrence of the L1 epitope appeared to coincide with loss of virulence in animal inoculation experiments.

Animals↗

PubMed1988

Cryopreservation of parasites.

In this review, advances in cryopreservation of helminth parasites are reported. Our own studies demonstrate that metacestodes of Echinococcus multilocularis can be maintained in a viable state for at least 1-2 years by appropriate deep-freezing and storage in liquid nitrogen. Infective larvae of the nematode Toxocara canis cryopreserved for 1 week in liquid nitrogen were maintained after thawing in vitro in a chemically defined medium for 35 weeks. Although motility of previously deep-frozen larvae was reduced they produced secretory/excretory antigens of similar immunodiagnostic quality as those from unfrozen larvae. Whereas infective larvae of several species of trichostrongy-lids can be easily cryopreserved, the infective larvae of the cattle lungworm, Dictyocaulus viviparus, and muscle larvae of Trichinella spiralis are more sensitive to damage by subzero temperatures. Therefore, survival rates after cryopreservation are low, but improvement of the cooling schedules appears to be feasible. It is concluded that cryopreservation of certain stages of helminth and protozoan parasites is a useful technique for long-term storage of defined isolates, which can contribute considerably to reducing the number of experimental animals usually required for serial passages.

Animal Testing Alternatives↗

PubMed1994

Possible prevention of in vitro selection of resistant Streptococcus pneumoniae by beta-lactamase inhibitors.

The development of resistance in vitro in five strains of Streptococcus pneumoniae (3 with full susceptibility and 2 with intermediate susceptibility to penicillin) was investigated by serial passages in the presence of subinhibitory concentrations of amoxicillin and ampicillin. At the end of passaging, MICs of antibiotics for all the strains increased by a factor of four or more, reaching at least intermediate levels. MICs of cephalosporins, ampicillin and amoxicillin increased for almost all variants obtained. Similar results were obtained with amoxicillin plus clavulanic acid at a ratio of 2:1 and at a constant concentration of 2 micrograms/ml, and with ampicillin plus sulbactam at a ratio 2:1. In contrast, no significant modification of MIC was seen with ampicillin plus sulbactam at a constant concentration of 4 micrograms/ml sulbactam. These results suggest interaction of sulbactam with penicillin binding proteins as described previously for other bacterial species, and merit further investigation.

Amoxicillin↗

PubMed1986

Studies on the adaptation of mumps virus to chick embryo.

About 15 serial passages of wild type mumps virus (Sasazaki strain) in the amnion sac of chick embryo (CE) yielded a CE-adapted strain which was poorly replicative and did not form plaques in Vero cells where the wild strain grew well. In the course of this limited replication of the CE-adapted strain in Vero cells, we have analysed the viral protein and RNA synthesis. It was found that protein synthesis took place very efficiently at least early in infection by 12 h. The subsequent rate of synthesis remained, however, at a low level without showing the progressively increasing synthesis observed with the wild strain. Furthermore, 50S genomic RNA was synthesized early in the limited infection, but the subsequent synthesis was markedly suppressed. In addition, the other virus-specific RNA species could not be detected throughout. Thus the amplified RNA synthesis observed in the permissive CE cells and in the wild strain-infection of Vero cells seemed not to occur in the limited replication. Neither interferon nor DI (defective interfering) RNA was involved in the limited virus growth. When Vero cells were infected with the wild strain 6 to 8 h before inoculation of CE-adapted strain, growth restriction was overcome and the yield of the latter virus was greatly enhanced by a factor more than 10(3). These results suggest that through adaptation to CE, mumps virus may be altered in such a way that there is a restriction, probably at a step (s) involved in amplification of the viral RNA synthesis in Vero cells and that the restriction may be overcome by the simultaneous genome expression of the prototype wild strain.

Animals↗

PubMed1987

Growth and characterization of rabbit corneal cells in vitro.

Primary organ cultures of rabbit corneal epithelium, stroma, and endothelium were established by microdissection. Secondary cultures of epithelial cells, keratocytes, and endothelial cells were established by serial passage. The doubling time for epithelial cells and keratocytes was 18 h, and endothelial cells doubled their number in 5 days. Ultrastructural studies demonstrated characteristic morphological, nuclear, and cytoplasmic features of corneal epithelial cells, keratocytes, and endothelial cells and confirmed the identity of the cell lines. The purity of the three distinct cell types was ascertained by indirect immunofluorescence techniques, using antibodies against keratin, which identified epithelial cells, and fibronectin, which identified keratocytes and endothelial cells. The indirect immunofluorescence technique represents a simple method to screen an aliquot of a cell suspension and determine the purity of corneal cells grown in vitro.

Animals↗

PubMedDate not supplied

Preparation of islets of Langerhans from rabbits and hamsters by the collagenase digestion technique.

In this report we describe some modifications of the collagenase digestion technique for the preparation of pancreatic islets, that we have found helpful in preparing isolated islets of Langerhans from rabbits and hamsters. These modifications include disrupting the pancreatic acinar tissue by directly infiltrating it with Hanks' solution, periodically monitoring the progress of the collagenase digestion of the pancreatic tissue with the dissecting microscope, and purifying the islets by serial passage of the digested tissue through three Petri dishes containing Hanks' solution. This technique results in the successful isolation of the fragile and irregular rabbit islets as well as the isolation of the sturdier and more uniformly shaped hamster islets. The isolated respond to a glucose stimulus with an increase in radioimmunoassayable insulin secretion. We also describe a filtration technique for the preparation of isolated islets for histological examination. Isolated islets are retained on Millipore filters. The filters are then fixed, stained and mounted. Finally, we present realistic photographs of rabbit and hamster islets, as they appear under the dissecting microscope, during various stages of purification and isolation.

Animals↗

PubMed1978

Initiation and characterization of a diploid cell line from larval tissues of Aedes dorsalis (Meigen).

Mosquito cell cultures were initiated from the minced tissues of newly hatched Aedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n = 6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (including Mycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation.

Aedes↗

PubMed1977

Chlamydia trachomatis in cell culture. II. Susceptibility of seven established mammalian cell types in vitro. Adaptation of trachoma organisms to McCoy and BHK-21 cells.

Trachoma organisms of serotype B were grown serially in irradiated cells (McCoy, BHK-21, Microbiological Associates, and BHK-21, Lister) and tested for infectivity in monolayers of five mammalian cell lines (BHK-21, CHO, HeLa S3, McCoy and OWMK) and two diploid strains (ST/BTL and WI-38). All cell types had low susceptibility to chlamydial infection but the number of inclusions increased when the inoculum was centrifuged onto the monolayers, or when the cells were irradiated. Infection was higher in non-irradiated CHO than in irradiated CHO in three out of a total of six experiments. Inclusion number was increased 300 times in HeLa S3 and up to three times in the other cell types after treatment with diethylaminoethyl-dextran (DEAE-D). Serial passage of Chlamydia trachomatis serotype B (strain Har-36) in CO60 McCoy and CO60 BHK-21 Lister resulted in partial adaptation of the strain to the host cell. The phenomenon of adaptation of serotype B to McCoy compensated for the lower susceptibility of this cell revealed when McCoy cells were inoculated with trachoma elementary bodies grown in BHK-21 Lister or in chick embryo yolk sac. Trachoma organisms of immunotypes A, B and C prepared in yolk sac produced more inclusion-forming units per ml in CO60 BHK-21 Lister than in CO60 McCoy.

Animals↗

PubMed1978

Evidence for clonal attenuation of growth potential in HeLa cells.

The growth of primary clones and serial subclones of HeLa cells and of diploid human fibroblast-like cells were compared both in the presence and absence of feeder layers; the latter had no significant effects upon the results. Clones and subclones of both cell types displayed great heterogeneity in growth rates, typically with a bimodality of growth distributions. Serial passages of clones selected on the basis of superior rates of proliferation showed attentuation of growth potentials; the extent of such attentuations was much less in the case of HeLa cells, suggesting at least one possible basis for the differences in long-term growth potential between these two classes of cell lines.

Cell Division↗

PubMedDate not supplied

Host effect on vesicular stomatitis virus morphogenesis and "T" particle formation in reptilian, avian, and mammalian cell lines.

The morphogenesis of vesicular stomatitis virus (VSV) in three reptilian cell lines (turtle heart, gecko lung, and viper spleen) was studied by thin section electron microscopy. Simultaneous growth studies were conducted in reptilian, chick embryo fibroblast, and BHK/21 cells to compare the yields of infectious VSV during serial passages. The mean length of gecko lung cell VSV measured from electron micrographs is statistically significantly shorter than that of VSV replicating in other reptilian systems studied. This observation, along with comparative growth studies, suggests the predilection of gecko lung cells to form "auto-interfering" truncated "T" rather than infectious "B" VS virions.

Animals↗

PubMed1983

Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells.

Supplementation of tissue culture medium with chicken egg yolk can support the proliferation of low density bovine vascular and corneal endothelial cells and vascular smooth muscle cells maintained on basement lamina-coated dishes. The optimal growth-promoting effect was observed at concentrations of 7.5 to 10% egg yolk (vol/vol). The average doubling time of bovine vascular endothelial cells during their logarithmic growth phase when exposed to egg yolk-supplemented medium was longer than that of their counterparts grown in serum-supplemented medium (21 versus 15 h, respectively). Cultures grown in egg yolk-supplemented medium on basement lamina-coated dishes could be serially passaged, but their in vitro life span (15 generations) was less than that of serum-grown cultures (50 generations). The egg white was devoid of any growth-promoting activity.

Animals↗