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PubMed2006

Serial passage of the parasite Crithidia bombi within a colony of its host, Bombus terrestris, reduces success in unrelated hosts.

In the wild, Bombus spp. bees may contract infections of the trypanosome parasite Crithidia bombi from their nestmates or from others while foraging on contaminated flowers. We expected that as C. bombi is transmitted repeatedly among related workers within a colony, the parasite population would become more successful in this relatively homogeneous host population and less successful in individuals from unrelated colonies of the same or different species. To test our prediction, we serially passaged cocktails of C. bombi strains through workers from the same colony, taking the intensity of infection in related versus unrelated workers as a measure of parasite success at each step in the serial transfer. Using a repeated measures ANOVA, we found the ability of C. bombi to exploit Bombus spp. hosts did not increase within a colony, but did decrease for infections in workers from unrelated colonies. This reduction in success is most likely due to a gradual loss of appropriate C. bombi strains from the infecting the population as the cocktail is 'filtered' during the serial passage within a given colony, without a corresponding increase in overall intensity of the surviving strains.

Alleles↗

PubMed1984

Replication and serial passage of a singly enveloped baculovirus of Orgyia leucostigma in homologous cell lines.

A singly enveloped nuclear polyhedrosis virus (SNPV) of the white-marked tussock moth, Orgyia leucostigma, was successfully grown in four continuous cell lines developed from minced neonate larvae of this insect. Level of infection in two cell lines, IPRI-OL-12 and IPRI-OL-13, was 65-90%, but in the other two, IPRI-OL-4 and IPRI-OL-9, it was about 3%. Polyhedral inclusion bodies (PIBs) appeared in the nuclei of cells within 24 h postinoculation. Cytopathological changes and morphogenesis of the virus, as revealed by light and electron microscopy, were in general typical of an SNPV. However, some of the PIBs contained very few virions, and some were fractured. Rate zonal centrifugation of alkali-released occluded virions further confirmed the singly enveloped characteristics of the virus. The SNPV was serially passaged 60 times each in OL-12 and OL-13 cells. Percentage infected cells and PIB production stayed generally high throughout serial passaging in OL-13 cells, but declined sharply after the 41st passage in OL-12 cells. PIBs from the 4th passage of the virus in OL-12 cells were tested and found to be pathogenic to O. leucostigma larvae.

Animals↗

PubMed1983

Serial passage of Eimeria tenella and E. necatrix in turkey embryos.

The serial passage of 3 strains of Eimeria from the chicken in embryonating turkey embryos is reported here. All 3 strains increased their reproduction in turkey embryos; reproduction being equal to or better than the same lines maintained in chicken embryos. Pathogenicity and immunogenicity trials conducted in chickens using these 3 strains of Eimeria showed that all 3 turkey embryo-adapted lines were significantly less pathogenic than the parent lines. Only 1 of the lines (E. tenella (PS) turkey embryo-adapted) maintained its immunogenic potential.

Adaptation, Physiological↗

PubMed2000

Enhanced infectivity of an R5-tropic simian/human immunodeficiency virus carrying human immunodeficiency virus type 1 subtype C envelope after serial passages in pig-tailed macaques (Macaca nemestrina).

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating strategies against the transmission of the virus. A chimeric simian/human immunodeficiency virus (SHIV), SHIV(CHN19), was generated with a primary, non-syncytium-inducing HIV-1 subtype C envelope from a Chinese strain in the background of SHIV(33). Unlike R5-tropic SHIV(162), SHIV(CHN19) was not found to replicate in rhesus CD4(+) T lymphocytes. SHIV(CHN19) does, however, replicate in CD4(+) T lymphocytes of pig-tailed macaques (Macaca nemestrina). The observed replication competence of SHIV(CHN19) requires the full tat/rev genes and partial gp41 region derived from SHIV(33). To evaluate in vivo infectivity, SHIV(CHN19) was intravenously inoculated, at first, into two pig-tailed and two rhesus macaques. Although all four animals became infected, the virus replicated preferentially in pig-tailed macaques with an earlier plasma viral peak and a faster seroconversion. To determine whether in vivo adaptation would enhance the infectivity of SHIV(CHN19), passages were carried out serially in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. The passages greatly enhanced the infectivity of the virus as shown by the increasingly elevated viral loads during acute infection in animals with each passage. Moreover, the doubling time of plasma virus during acute infection became much shorter in passage 4 (P4) animals (0.2 day) in comparison to P1 animals (1 to 2 days). P2 to P4 animals all became seropositive around 2 to 3 weeks postinoculation and had a decline in CD4/CD8 T-cell ratio during the early phase of infection. In P4 animals, a profound depletion of CD4 T cells in the lamina propria of the jejunum was observed. Persistent plasma viremia has been found in most of the infected animals with sustained viral loads ranging from 10(3) to 10(5) per ml up to 6 months postinfection. Serial passages did not change the viral phenotype as confirmed by the persistence of the R5 tropism of SHIV(CHN19) isolated from P4 animals. In addition, the infectivity of SHIV(CHN19) in rhesus peripheral blood mononuclear cells was also increased after in vivo passages. Our data indicate that SHIV(CHN19) has adapted well to grow in macaque cells. This established R5-tropic SHIV(CHN19)/macaque model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.

Animals↗

PubMed1991

Adaptation and serial passage of porcine group C rotavirus in ST-cells, an established diploid swine testicular cell line.

A porcine group C rotavirus (strain Cowden-AmC-1) was adapted and serially passaged in an established diploid swine testicular cell line (ST cells). Growth of group C rotavirus in ST cells, which in maintenance medium required trypsin, but not pancreatin resulted in cytopathic effect characterized by cellular stranding and subsequent cell lysis. Active replication and assembly were confirmed by RNA profile analysis, immune electron microscopy and immunofluorescence. Adaptation of non-group A rotavirus to a continuous cell line has not previously been reported and should facilitate progress in diagnostic procedures and vaccine development.

Adaptation, Physiological↗

PubMed2001

Deletions in the Ac-iap1 gene of the baculovirus AcMNPV occur spontaneously during serial passage and confer a cell line-specific replication advantage.

The PstI-I region of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome was previously shown to be a frequent target of spontaneous deletions during serial virus passage in TN-368 cells (Kumar and Miller, Virus Res. 7 (1987) 335). Analysis of two of these serial passage mutants showed that a portion of the Ac-iap1 gene was deleted. To directly test the effect of loss of Ac-iap1, three different deletions in Ac-iap1 were introduced into recombinant viruses and the ability of these viruses to replicate was examined in two cell lines, TN-368 and SF-21, as well as in two species of insect larvae, Trichoplusia ni and Spodoptera frugiperda. The mutant viruses were indistinguishable from wild type or control revertant virus in their ability to infect larvae of either species. Moreover, no effect was seen on the rate of replication or the overall amounts of budded or occluded virus produced in cultured cells. However, in co-infection experiments using TN-368 cells, it was consistently observed that mutants lacking a functional Ac-iap1 gene out-competed control viruses carrying Ac-iap1. Interestingly, this replication advantage was only evident in the TN-368 cell line, the cell line used for the original serial passage experiments, and not in SF-21 cells.

Animals↗

PubMed2005

Molecular study of the quasispecies evolution of a typical pigeon paramyxovirus type 1 after serial passages in pigeons by contact.

The quasispecies nature of a typical pigeon paramyxovirus type 1 (pPMV-1) was, for the first time, studied under conditions close to the natural infectious environment. The virus was serially passaged in pigeons by successive contacts. Viral heterogeneity was analysed in the kidneys and brain of five pigeons from the last contact, by reverse transcriptase-polymerase chain reactions performed on RNA directly extracted from the organ and targeting the P and HN genes of the virus. The viral diversity following in vivo passage was found to be different from that in the inoculum, but demonstrated the reality of the quasispecies concept for pPMV-1 strains. Moreover, some aberrant genomic RNAs comprising insertions in the P gene editing site or deletions in the HN gene were also detected, with possible consequences for the pathogenicity and infectivity of the virus.

Animals↗

PubMed1984

Stability of the pseudorabies virus genome after in vivo serial passage.

Restriction endonuclease patterns of pseudorabies virus (PRV) DNA were examined after each of 11 serial passages of the virus through pigs. Minor variations in the electrophoretic mobility of certain restriction enzyme fragments were observed by the sixth passage. This variability was similar to some of the minor variability observed in field isolates. The variable fragments were mapped to three locations on the PRV genome: the junction between the short unique sequences and the repeat sequences, the terminus of the long unique region, and an internal area of the long unique region.

Animals↗

PubMed1985

Tumor progression in serial passages of the Dunning R3327-G rat prostatic adenocarcinoma: growth rate response to endocrine manipulation.

Serial passages of the poorly differentiated, androgen-sensitive R3327-G prostatic adenocarcinoma were used to study the progressive changes that occur in tumor growth rate and androgen sensitivity. Different in vivo transplant generations (21st to 28th) were compared. The tumor doubling and animal survival times resulting from the implantation of the 21st to 22nd generation (21-22G) tumor cells in intact male rats were significantly greater than those resulting from the implantation of 23-28G tumor cells. The most dramatic difference between early (21-23G) and late (26-28G) tumor generations, however, was in androgen sensitivity. The 26-28G tumors displayed androgen sensitivity only when implanted into animals castrated 2 to 7 days previously. Tumors grown in the pretreated castrates grew at a significantly slower rate than those in intact rats and the pretreated castrates had longer survival times than the intact rats. When 26-28G tumors were allowed to grow in intact rats to approximately 1 cu cm and then the rats were castrated, no significant difference in the growth rate between these tumors and tumors grown in intact rats was observed. In contrast, the androgen sensitivity of 21-23G tumors could be demonstrated, regardless of whether treatment was started before or after implantation. The fact that androgen sensitivity was still evident under certain conditions in late-generation R3327-G tumors demonstrates that the basic mechanism involving androgen response was still present, although functioning at a much reduced level.

Adenocarcinoma↗

PubMed2004

Genetic stability of the attachment glycoprotein of human respiratory syncytial viruses during serial passages in cell cultures.

Thirteen isolates of human respiratory syncytial viruses (HRSV) of groups A and B were isolated in HEp-2 cells from nasopharyngeal aspirates (NPA) from the children with acute respiratory infections. Three isolates of HRSV of group A were propagated in HEp-2 cells in 20 serial passages. Nucleotide sequences of the products obtained by RT-PCR from the glycoprotein (G) hypervariable region of the original virus isolates in NPA and those after one or several passages were compared. All the isolates analyzed showed no changes during passaging in HEp-2 cells.

Cell Line↗

PubMed1953

The experimental production of combination forms of virus. II. A study of serial passage in the allantoic sac of agents that combine the antigens of two distinct influenza A strains.

Double infection of the allantoic sac with Melbourne and WSN viruses induced the formation of a combination virus, which had some of the antigenic properties of both parents and which maintained itself through serial passage in the chorioallantoic sac. In the course of prolonged passage in the egg, three varieties of combination virus were found. The first (X(1)) occurred in ten passages and was characterized as follows: X(1) was produced in and could be successfully passed from those chick embryos that had received very large inocula of virus. The X(1) hemagglutinin was efficiently inhibited by both M and W antisera. Each passage fluid, containing X(1) as the predominant strain, also contained large amounts of the parent forms M and W. It is very likely that X(1) reverted to parent types of virus at a high rate. The second variety (X(2)) arose from the passage series of X(1) and was carried for approximately 20 passages without definite alteration in its character. It differed from X(1) in that it had the ability to appear as the predominant form occasionally in embryos that had received a limiting infective dilution of virus. Because of this, it seems probable that X(2) reverted to parent types at a slower rate than X(1). Like X(1), this virus was never obtained in pure form and suspensions in which it predominated contained large amounts of M and W, especially the former. Good evidence was obtained that X(2) reverted to M and W virus while multiplying in the chorioallantoic sac. X(3) was derived from passage of X(2) virus after 32 transfers and can be characterized in several ways: (a) X(3) yielded the only X(3) fluids on passage in eggs. This was in striking contrast to X(2) which could not be passed without giving rise to some fluids in which M and W predominated, (b) The X(3) hemagglutinin was weakly inhibited by all but the highest concentrations of M serum, but was strongly inhibited by W serum. X(2) was readily inhibited by relatively small amounts of both sera. (c) X(3) virus, after several passages at limiting dilutions, was neutralized to a highly significant degree by specific M and W sera. This is not necessarily an essential difference from the behavior of X(2) since technical difficulties (large amounts of M and W in X(2) fluids) may have prevented the demonstration of equally striking double neutralization of this virus, (d) After five limiting dilution passages, the X(3) virus retained the characteristics listed under (a) to (c). It is felt that the foregoing facts justify the conclusion that X(3) is a stable virus which combines some of the specific antigens of two parent types.

Animals↗

PubMed2000

Human bone cell cultures in biocompatibility testing. Part I: osteoblastic differentiation of serially passaged human bone marrow cells cultured in alpha-MEM and in DMEM.

Well-characterised human osteoblastic bone marrow cell cultures are a useful in vitro tool to analyse bone tissue/biomaterials interactions. In this work, human bone marrow was cultured in experimental conditions described to favour osteoblastic differentiation and, serially passaged cells were cultured in two widely used culture media, minimum essential medium Eagle, alpha modification (alpha-MEM) and Dulbecco's modified Eagle's medium (DMEM). Cultures were grown for 35 d and compared concerning morphologic appearance on scanning electron microscopy (SEM), cell viability/proliferation, total protein content, activity of alkaline phosphatase (ALP) and ability to form calcium phosphate deposits. Results showed that cell proliferation was similar in cultures grown in the two media but ALP activity and ability to form mineralised deposits were lower in DMEM cultures. In both experimental situations, osteoblastic parameters were strongly reduced on cell passage, particularly from the first to the second subculture. In the experimental conditions used (presence of ascorbic acid, sodium beta-glycerophosphate and dexamethasone in the primary and secondary cultures), osteoblastic differentiation was observed in the first and second subcultures grown in alpha-MEM and in the first subculture grown in DMEM. These results underline the importance of the definition of the experimental conditions in studies involving bone cell cultures.

Adult↗

PubMed1985

The alterations in pathogenicity and immunogenicity of a Kenya sheep and goat pox virus on serial passage in bovine foetal muscle cell cultures.

A Kenyan sheep and goat pox virus was attenuated by serial passage in bovine foetal muscle cell cultures. The pathogenicity of the strain was lost between the 15th and 20th passages. Serum-neutralizing antibody developed after inoculation with passages tested up to the 50th. These passages appeared to protect animals against laboratory challenge by intra-dermal titration. The 18th passage was successfully and extensively used to control the disease under field conditions.

Animal Diseases↗

PubMed1986

Growth and ploidy of human osteosarcoma xenografts in serial passage in nude mice.

Six human osteosarcoma tumors have been transplanted to and maintained as xenografts in serial passage in nude mice. The xenografts, characterized by growth rate, histologic appearance, and DNA flow cytometric data, are now in the 6-17 passages in mice. Histologic appearances and growth rates of the xenografts were unchanged through all passages except for one xenograft in which the growth rate doubled in passage 3. All six tumors had hyperploid DNA contents. The ploidy levels were unchanged, compared to their original tumors, in all passages of five xenografts. One xenograft, concomitantly with the increase in growth rate, also doubled the ploidy from 2.8 to 5.6C. There was a good agreement between the proportion of S-phase cells and mitotic indices of the six xenografts. The growth rate, depending also on cell loss, was less related to the proliferative activity of the xenografts. It was concluded that osteosarcomas have a stable aneuploid DNA content in nude mice, but polyploidization can occur after a number of passages.

Animals↗

PubMed1992

Identification of murine thymocyte populations capable of extensive proliferation in serially passaged thymic organ cultures.

Using the approach of titrating precursor cells into mouse thymus organ cultures and serial passage, we have sought to compare the proliferative capacities of cells derived from adult and embryonic thymus and related haemopoietic tissues. We find that cells derived from the liver and thymus of day 14 embryos are capable of extensive proliferation in such cultures (surviving for at least 12 weeks) whereas cells derived from adult sources (blood and thymus) display a much more restricted lifespan and potential for division. Analysis of sequentially passaged thymic lobes shows that cells lacking CD4 and CD8 (CD4- and CD8-) and a subset of single CD8 positives are selectively expanded in these cultures. A preliminary study of the CD4-CD8- populations in these lobes suggest that these include cells expressing surface CD3 (in association with either TCR alpha beta or TCR gamma delta) and a subset of CD4-CD8-CD3-. These findings suggest that sequential passage of thymocytes in organ culture may be a useful alternative strategy for characterising cells with high proliferative potential, resident in the thymus and also for probing their lineage relationships.

Animals↗

PubMed1978

The DNA of serially passaged herpes simplex virus: organization, origin, and homology to viral RNA.

High-density DNA prepared from serially passaged herpes simplex virus contains three major classes of modified viral DNA molecules. The altered DNA molecules are composed of multiple repetitions of sequences derived from the right-hand side of the S region of the parental plaque-purified viral DNA. The repeat units contained in the three types of high-density DNA share most of their DNA sequences but differ with respect to a small region derived from the unique sequences of the S component of HSV-1 DNA. Hybridization of the defective DNA to HSV-infected cell RNA shows that the high-density DNA contains sequences complementary to both early and late viral transcripts.

Base Sequence↗

PubMedDate not supplied

Heterozygous ras mutations are preserved in serially passaged human tumor xenografts and established cell lines.

We examined c-K-ras gene point mutations in human tumor xenografts and established cell lines as markers of genetic stability. Our previous study demonstrated the stability of c-K-ras gene mutations in human primary neoplasms and their tumor xenografts through serial passages in mice. In this study, we established 27 human cell lines derived from various human tumor xenografts in nude mice. Point mutation of the c-K-ras gene at codon 12 was found in 29.6% (8/27) of the cell lines, as well as in 29.6% (8/27) of the xenografts. The eight ras-mutated cell lines were derived from corresponding tumor xenografts carrying the ras mutation. Heterozygous ras gene mutation was confirmed in seven of the eight ras-mutated cell lines, as well as their corresponding xenografts. The incidence, type and heterozygosity of the c-K-ras gene mutation showed no discrepancies between the original xenografts and the established cell lines. From these findings, we concluded that point mutation of the c-K-ras gene was very stable in human tumor xenografts and established cell lines derived from the xenografts.

Animals↗

PubMed1996

The restoration of the functions of serially passaged calf hepatocytes by spheroid formation.

A serial cultivation system of hepatocytes was established for the first time using calf liver as a cell source and, repeating passage of more than 30 cumulative population doublings (PDs), was obtained in the presence of long-acting ascorbic acid derivative (L-ascorbic acid 2-phosphate) and epidermal growth factor. The complete purification of hepatocytes was achieved by repeating ethylenediaminetetraacetic acid (EDTA) treatment, by which hepatocytes were easily detached from the culture dish, leaving most of the nonparenchymal cells on the dish. As the population cumulatively doubled, the cell density and albumin-synthesizing ability decreased gradually, and doubling time has exceeded 120 h at about 30 cumulative PDs. In serially passaged cells, the hepatocyte-specific histochemical and biochemical markers-including glucose-6-phosphatase, ornithine carbamoyltransferase, glutamate dehydrogenase, and ammonia-metabolizing activities-have been lost after 20 cumulative PDs. However, when these passaged cells were allowed to form spheroids, the morphologic and biochemical characteristics of hepatocytes have rapidly been restored to levels comparable to those in younger generations. Because no extrinsic factor was needed for this restoration, three-dimensional cell-cell interaction would be indispensable for the differentiation of the hepatocytes. The routine serial cultivation of hepatocytes and their redifferentiation by spheroid formation will be useful for studying metabolism, gene regulation, and transplantation of hepatocytes.

Animals↗