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PubMedDate not supplied

The effects of the multiplicity of infection on viral subpopulations during passage of Newcastle disease viruses.

When mixtures of two clones of the Italy-Milano strain of Newcastle disease virus were inoculated so that one clone was present in quantities 20 times greater than the other, maintenance of that population ratio through six serial passages in 10-day-old embryonating chicken eggs depended on the multiplicity of infection of the first passage. The initial ratio was preserved when the eggs were inoculated at low multiplicity, 10(1) virions inoculated, but not at high multiplicity, 10(6) virions inoculated. In the latter situation, the clear plaque clone, which comprised 1/20 of the initial population of the inoculum, increased in numbers to parity with the red plaque clone after only six serial passages in embryonating eggs. Explanations for the competitive advantage of the clear plaque clone at high multiplicity of infection are discussed.

Animals↗

PubMed1988

JB/MS murine melanoma: a new model for studies on the modulation of differentiation and of tumorigenic and metastatic potential.

The recently obtained JB/MS melanoma (induced by DMBA in C57Bl/6 mice) has been successfully established in culture, and characterization of various parameters of these cells, as they have been serially passaged in vivo and in vitro, has begun. The culture lines were initially highly dendritic and melanotic, growing slowly in vitro and extremely slowly in vivo. During serial passage in vivo and in vitro the cell lines have gradually evolved into less melanotic, but more proliferative, tumorigenic and metastatic cells. We have been able to demonstrate that the JB/MS melanoma shares the common melanoma TSTA previously reported for B16, K1735 and JB/RH melanomas, but does not cross-react with the S91 melanoma or with other non-melanoma cell lines used as specificity controls. The JB/MS cells can be induced to differentiate in vitro by alpha-melanocyte stimulating hormone, a physiologically relevant agent, and studies have been initiated to detail the level at which this induction occurs. These sublines should prove to be excellent models for study of the progression of transformed cells from non-tumorigenic to tumorigenic phenotypes, and for progression through stages of varying metastatic potential, immunogenicity and differentiation.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1975

A defined medium for the growth of Clostridium tetani and other anaerobes of clinical interest.

The growth of six strains of Clostridium tetani was assessed in a chemically supplemented commercially available defined medium. All strains grew reliably even after 12 serial passages, and two strains produced demonstrable toxic activity after passage. Consistent growth of the test strains could also be obtained on a solid version of this medium ("CA109-S" medium), and the strains could be serially passaged on this medium. Preliminary evidence is presented that the medium supports the surface growth of some other test anaerobes. Such a defined solid medium might prove of value in further studies on the surface growth of C. tetani and of other anaerobes of clinical interest.

Anaerobiosis↗

PubMed2000

Genetic reflection of glioblastoma biopsy material in xenografts: characterization of 11 glioblastoma xenograft lines by comparative genomic hybridization.

OBJECT: Human tumors implanted as subcutaneous xenografts in nude mice are widely used for the study of tumor biology and therapy. Validation of these models requires knowledge of the genetic makeup of the xenografts. The aim of this study was to establish whether chromosomal imbalances in 11 xenograft lines derived from human glioblastomas multiforme (x-GBMs) are similar to those found in GBM biopsy samples. The authors also studied genetic stability during serial passaging of three xenograft lines. METHODS: Chromosomal imbalances in x-GBMs were detected using comparative genomic hybridization (CGH). The authors compared the CGH results in x-GBMs with those in the original GBMs (o-GBMs) that were used to establish three of the xenograft lines and with the GBM biopsy results reported in the literature (1-GBMs). In three xenograft lines two different passages were analyzed. CONCLUSIONS: The results show that the chromosomal imbalances in x-GBMs are similar to those in o-GBMs and 1-GBMs, indicating that the GBM xenograft lines used were valid models from a genetic point of view. The CGH analysis of two different passages of three xenograft lines indicates that x-GBMs (like 1-GBMs) show intratumoral genetic heterogeneity and do not acquire chromosomal imbalances as a result of serial passaging.

Animals↗

PubMed2004

Control of human articular chondrocyte differentiation by reduced oxygen tension.

Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real-time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes.

Adult↗

PubMed2002

Efficient rescue of gutted adenovirus genomes allows rapid production of concentrated stocks without negative selection.

Gutted adenoviral (Ad) vectors have a greater cloning capacity and elicit less immune response than conventional Ad vectors. Unfortunately, clinical use of gutted vectors has been slowed by production difficulties, including low yield and a tendency for recombinant virus to emerge. These two problems are related, because expansion of dilute vector stocks requires selective pressure against helper virus. The ability to rescue gutted virus at high titer would lessen the requirement for selective pressure, thereby limiting the advantage afforded to undesirable recombinants. We tested gutted virus rescue from plasmids and from synthetic terminal protein (TP)-DNA complexes by transfection/infection or cotransfection with various forms of helper viral DNA. Optimal rescue required cotransfection of gutted and helper genomes with identical origins of replication. Transfection/infection, which introduces unequal origins, was 30 times less efficient than cotransfection of genomes that had been released from plasmid DNA and bore identical origins. Cotransfection of TP-linked genomes was several times more efficient than that of unlinked genomes, yielding average gutted viral titers above 10(7) transducing units (TU)/ml. In addition, we found that limited expression of Cre recombinase doubled the yield of gutted virus. Using these techniques, gutted viruses can be rescued at titers greater than 3 x 10(7) TU/ml, about 100 times higher than is usually achieved. Finally, we found that high-titer lysates could be serially passaged on Cre-negative cells without loss of titer, further reducing selective pressure. These methods allow large-scale production of gutted virus in three or four serial passages, while minimizing exposure to Cre recombinase.

Adenoviridae↗

PubMedDate not supplied

Reversion to virulence of chicken-passaged infectious bronchitis vaccine virus.

Serial passage of two infectious bronchitis virus (IBV) vaccine strains in chickens enhanced their capacity to increase the incidence and severity of Mycoplasma synoviae (MS) airsacculitis. Included in this report were the mild Massachusetts-type Connaught strain and the Arkansas 99 vaccine strain of IBV. The Connaught strain and one of two Ark 99 vaccine strains passaged in chickens increased the incidence of airsacculitis markedly compared with nonpassaged virus. The other Ark 99 vaccine virus already exacerbated MS airsacculitis, before passage in chickens, and its influence did not increase on passage. All IBV strains studied to date have either possessed this trait or reacquired it on passage in the natural host.

Air Sacs↗

PubMed1977

[Changes in several properties of transplantable adenocarcinoma of the large intestine during passage].

In the course of serial passages of adenocarcinoma (AKTOL), arising from fetal colonic tissues which underwent spontaneous malignization after being grafted to syngeneic adult mice, there was an increase in the rate of growth of the subcutaneous transplants, and multiple intrapulmonary tumour nodes formed after its intravenous inoculation (the first transplant generations had no such capacity); the morphological anaplasia of the tumour increased. These changes were not directly dependent on the changes in its immunogenicity.

Adenocarcinoma↗

PubMed2003

Identification of a mutation in editing of defective Newcastle disease virus recombinants that modulates P-gene mRNA editing and restores virus replication and pathogenicity in chicken embryos.

Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5'-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5'-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5'-A(5) stretch (5'-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5'-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5'-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants.

Amino Acid Sequence↗

PubMedDate not supplied

[Persistence of type 1 human immunodeficiency virus (HIV-1) in MT-4 cells in presence of alpha-interferon].

The development of HIV infection during serial passages of HIV-1 strains with different replication capacity was studied in the presence of natural human alpha-interferon (IFN-alpha). The virus activity was evaluated by accumulation of virus-specific proteins in cells and culture fluid, determined by Western blot method and analysis of viral DNA in the cells by polymerase chain reaction. IFN-alpha suppressed replication of HIV-1/IIIB and 1974. The replication of strain 1974 (with a 10-fold lower replicative activity than strain IIIB) was inhibited during the first passage and of strain IIIb during the second passage. After the fourth passage IFN-2 alpha completely suppressed the replication of strain 1974, and the virus activity did not manifest after 4 consecutive co-culturings of cells from fourth viral passage with intact cells without IFN-alpha. A population of viral particles whose replicative activity was virtually undetected in the presence of IFN-alpha seemed to persist during serial passages of strain IIIB in MT-4 cells. On the other hand, the virus replication was restored during the first co-culturing of cells from the virus subculture with intact cells in the absence of IFN-alpha. Presumably, an increase in the level of IFN-alpha in the blood of HIV-infected patients at late stages of AIDS is not accidental and is caused by appearance of more virulent variants of the virus.

Antiviral Agents↗

PubMed1983

Functional consequence of variation in melanoma antigen expression.

Melanoma cells have been shown to express melanoma-associated antigens and, in many cases, the histocompatibility antigen, HLA-DR. We questioned whether the expression of these antigens was quantitatively altered during the serial passage of melanoma cells in culture. Therefore, we measured the binding of monoclonal antibodies specific for a melanoma-specific antigen and the HLA-DR antigen to melanoma cells from serial passages. Three cell lines were studied. We found that although both the melanoma-associated antigen and the HLA-DR antigen were qualitatively conserved, significant quantitative differences were seen. To study the functional consequences of these differences, we used fluorescence-activated cell sorting to create DR-enriched and DR-depleted populations from a single melanoma cell line heterogeneous for DR expression. We found that the proliferation of allogeneic T cells (measured by the 3H-TdR uptake) cultured with the DR-enriched and -depleted melanoma cell populations was directly related to the amount of the HLA-DR antigen expressed. These results indicate that in performance of experiments using melanoma cell lines quantitative assessment of antigenic expression is important, particularly if the function of a specific antigen is under examination. Further, our data clearly identify the HLA-DR antigen on melanoma cells as a participant in allogeneic lymphocyte stimulation.

Antibodies, Monoclonal↗

PubMed2000

Changes in a murine leukemia virus (MLV) receptor encoded by an alphavirus vector during passage in cells expressing the MLV envelope.

We constructed alphavirus vectors encoding the ecotropic murine leukemia virus (MLV) receptor mCAT1. Cells electroporated with these vector RNAs expressed mCAT1 protein and fused with cells that expressed a fusogenic form of the MLV envelope on the cell surface. Electroporated cells also released submicron particles that were infectious in envelope-expressing cells. Infection resulted in giant syncytia that could be enumerated by plaque assay. Cell-free supernatants could be serially passaged and contained up to 10(4) infectious units/ml. To determine whether repeated passage would select for functional variants of the receptor, we analyzed mCAT1 amplified by RT-PCR after 12 serial passages. Several amino acid substitutions were identified that encoded functional receptor variants. In independent experiments, variants containing an isoleucine or a leucine instead of a phenylalanine at position 224 in the third extracellular domain of the receptor arose spontaneously and outgrew the parental vector, indicating that mutations at this site are highly selected in this system. This region of the receptor has previously been implicated in the envelope-receptor interaction. This alphavirus vector system provides a novel method for generating and selecting functional variants of viral receptors

Alphavirus↗

PubMed1979

Variation in susceptibility of HeLa cell lines to coxsackievirus A9.

In the course of serial passages for several years a line of uncloned HeLa cells (A line) showed a gradual decrease in plaquing efficiency by coxsackievirus A9 (CA9 virus), while subcultures prepared from the same line kept frozen at an early passage level (A original line) did not show any change. However, it was observed later that the plaque-forming ability of the A original line (A orig. line) also decreased after serial passages as was observed with the A line. Comparing the characteristics of the same cell line at two different passage levels, it was found that the efficiency of adsorption of virus to cells was nearly the same, while virus yield at 8 hours after infection was different. The activity of alkaline phosphatase of cells was also different between these two passage levels, suggesting that a high enzymatic activity is associated with the susceptibility of cell cultures to CA9 virus. Magnesium chloride at 25 mM enhanced plaque formation by CA9 virus in highly passaged less susceptible cell cultures, and a possible role of the chemical as a stabilizer of alkaline phosphatase was discussed.

Alkaline Phosphatase↗

PubMed1991

Characterization of the attenuating M and NP gene segments of the avian influenza A/Mallard/78 virus during in vitro production of avian-human reassortant vaccine viruses and after replication in humans and primates.

A unique requirement for live attenuated reassortant influenza vaccines is the need to generate new reassortant vaccine viruses with the appearance of each new antigenic variant. Thus, the attenuation phenotype conferred by the attenuated donor influenza virus must remain genetically stable during the generation of each new reassortant vaccine virus. In this study we used nucleotide sequence analysis to evaluate the genetic stability of the attenuating M and NP genes of the avian influenza A/Mallard/NY/6750/78 attenuated donor virus during the in vitro generation and subsequent in vivo replication of avian-human (AH) influenza A reassortant vaccine viruses in monkeys and humans. Nucleotide sequence changes in the M and NP genes occurred at a rate of approximately 0.61 substitutions/1000 nt/reassortant during in vitro generation of four AH reassortant viruses. Only two nucleotide sequence changes occurred in the M and NP gene segments of four isolates of H1N1 or H3N2 AH vaccine viruses following 6-8 days of replication in seronegative children, and neither change affected amino acids previously identified as playing a potential role in attenuation. In addition, there were no changes in the nucleotide sequence of the M and NP genes of single gene AH reassortant viruses following five serial passages in squirrel monkeys. Finally, there was no change in the level or duration of replication of the single gene reassortant viruses in the upper or lower respiratory tract of monkeys following serial passage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

In vitro antistaphylococcal activity of dalbavancin, a novel glycopeptide.

OBJECTIVES: Dalbavancin is a novel, semi-synthetic glycopeptide antibiotic. The aim of this study was to further explore its activity against staphylococci. METHODS: The bactericidal activity of dalbavancin was studied using MBC and time-kill methods. The potential for resistance to dalbavancin was examined using single-step and serial-passage experiments. RESULTS: Dalbavancin was bactericidal against methicillin-susceptible and -resistant Staphylococcus aureus, in both the presence and absence of human serum. No resistance was seen with any isolate tested. After serial passage, bacterial populations were more homogeneous in their susceptibility to dalbavancin than to vancomycin or teicoplanin. CONCLUSION: Dalbavancin is bactericidal for staphylococci. Resistance to this semi-synthetic glycopeptide is not readily developed in vitro.

Anti-Bacterial Agents↗

PubMed1987

Biological evidence that scrapie agent has an independent genome.

There are many distinct strains of scrapie agent, identified by their relative incubation periods and quantitative and qualitative neuropathological properties in inbred mice of particular genotypes. When serially passaged under specified conditions of mouse strain, route of infection and dose of infectivity these properties are stable. However, they may change in a predictable manner if the passage strategy is altered. The scrapie strain 87A shows what has previously been defined as Class III stability; it is stable when passaged at low dose in C57BL mice, but often suddenly changes its properties in the course of a single passage if high doses are used, always resulting in the same new strain. The latter, designated 7D, has shorter incubation periods and more extensive pathology than 87A, properties which are subsequently stable on serial passage even at high dose. This phenomenon has been seen repeatedly using scrapie isolates from six different natural cases in five different breeds of sheep. These isolates are closely similar in all their properties, showing them to be independent isolations of the 87A strain; there have been no isolations of 87A in which the phenomenon did not occur. On the other hand, none of the many other scrapie strains used in the same laboratory have shown this change. 87A brain samples consistently behave as if they contain 87A together with a smaller amount of 7D. This is so even after 87A has previously been passaged at high dilution, well beyond the limiting dilution for 7D, a procedure which would eliminate any minor agent strain originally present in the isolate. Therefore it is highly likely that the 7D in tissues of mice infected with 87A is generated de novo at each passage by mutational change from 87A during the incubation period. The established fact that many different strains exist and the considerable evidence that mutation can occur lead to the conclusion that scrapie agent has its own independently replicating genome.

Animals↗

PubMed1969

Some studies on the selection and efficiency of live influenza vaccine viruses.

Live influenza virus vaccination has potential advantages and disadvantages compared with killed virus vaccine given by intramuscular injection.Attempts have been made to achieve complete attenuation of influenza A2 and B strains by up to 30 serial passages in the allantoic cavity of eggs at 33 degrees C. No attenuation occurred. The method of passing viruses in the presence of an increasing concentration of horse serum, thus selecting inhibitor-resistant variants, has also been tested. It was shown that serial passage induced an increasing degree of inhibitor resistance in the virus and an increasing degree of attenuation when tested in volunteers. This was observed with both an Asian strain and a Hong Kong strain. The Hong Kong virus was also passed serially at 25 degrees C in eggs but did not seem much further attenuated as a result; however, a virus strain supplied by H. F. Maassab with a much higher degree of adaptation to low temperature is being tested.Partly or completely attenuated strains have been used to induce or detect protection. Influenza B strains gave partial protection after 1 dose and almost complete protection after 2 doses and protection persisted for 7 months. A comparative study showed that there was protection after vaccination with killed virus by the parenteral route, and with live virus by the nasal route.

Genetic Variation↗

PubMed1988

The natural history of a family of transplantable melanomas in hamsters.

We have characterized a family of transplantable melanomas in Syrian (golden) hamsters, which originated in 1959 as a spontaneous melanoma of hamster skin, and which has been maintained since then by serial passage. Emphasis has been placed on using the same method of transplantation, keeping strict records on all passages, and applying the same investigative techniques, in order to trace tumor behavior over long periods of time. This tumor family consists of five variants linked by common origin, but which differ with respect to differentiation level, malignancy, intermediary metabolism, chromosome number, and cell surface properties. Once established, these melanomas possessed a considerable degree of phenotypic stability over decades of passaging. One tumor line in this family is emphasized. The Ab amelanotic melanoma lost its differentiated functions (the ability to synthetize melanin) a quarter of a century ago, and since then has remained dedifferentiated in serial passage in hamsters. After transfer to primary cell culture, the Ab melanoma cells differentiate readily and lose much of their proliferative potential. This process is reversible by reimplantation of the cells into a hamster. Inasmuch as this hamster melanoma system meets many of the conditions required for an experimental tumor model, five melanoma variants are characterized concisely and compared to other melanomas in humans and animals. Mechanisms by which new melanoma variants arise are discussed and compared to some phenomena in the evolution of the species.

Animals↗