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At least 19 recordsLinked to original sources

C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage.

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

Animals↗

In vitro analysis of proliferating epithelial cell populations from the mouse mammary gland: fibroblast-free growth and serial passage.

Normal and neoplastic mouse mammary epithelial cells were cultured in nutrient medium containing D-valine substituted for L-valine. Fibroblast overgrowth was prevented and epithelial cell functions and morphology were retained in cultures maintained in D-valine medium up to 2 months. A nonenzymatic technique was devised to dissociate epithelial cell monolayers. The combined use of this dissociation buffer and D-valine nutrient medium made it possible to passage serially normal and neoplastic mammary epithelial cells. Normal cells were derived from mammary glands of animals stimulated with exogenous hormones for various periods. The period of in vivo hormonal stimulation influenced the ability of normal mammary epithelial cells to attach and proliferate in primary and serially passaged cultures. A greater proportion of cells derived from glands following 2 to 4 weeks of hormonal stimulation were recovered after replating and showed higher labeling indices during serial passage than cells from unstimulated or 5- to 7-week stimulated groups.

Adenocarcinoma↗

Virulence and in vitro growth of a cell-adapted strain of equine infectious anemia virus after serial passage in ponies.

Five serial passages of a cell-adapted strain of equine infectious anemia (EIA) virus were conducted in Shetland ponies. The 13 recipient ponies became agar-gel immunodiffusion test-positive by 25 days after they were inoculated. The virulence of the cell-adapted strain of EIA virus markedly increased through 3 serial passages, although individual variation within passages was high. The 1st serial-passage recipient remained afebrile through 200 days, whereas a febrile episode occurred about every 185, 44, 35, and 33 days in the 2nd, 3rd, 4th, and 5th serial-passage recipients, respectively. Severe clinical signs of EIA were noted in the ponies at each serial passage, but the mean virulence rating of each passage, graded on frequency of febrile episodes and number of clinical signs evident within 200 days after ponies were inoculated, increased from 0 through 4, 21, 24, and 29 for the 1st through 5th serial passages, respectively. Isolates of EIA virus, made in fetal equine kidney cells, were obtained from plasma of 75% of the samples of blood collected during febrile episodes and from 45% of the samples collected during afebrile periods, indicating that the cell culture growth capacity of this strain of EIA virus may be relatively stable through 5 serial passages in Shetland ponies.

Animals↗

Properties of a unique mutant of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus that exhibits a partial many polyhedra and few polyhedra phenotype on extended serial passaging in suspension cell cultures.

Serial passaging of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) in H. zea (Hz-AM1) insect cell cultures results in rapid selection for the few polyhedra (FP) phenotype. A unique HaSNPV mutant (ppC19) was isolated through plaque purification that exhibited a partial many polyhedra (MP) and FP phenotype. On serial passaging in suspension cell cultures, ppC19 produced fivefold more polyhedra than a typical FP mutant (FP8AS) but threefold less polyhedra than the wild-type virus. Most importantly, the polyhedra of ppC19 exhibited MP-like virion occlusion. Furthermore, ppC19 produced the same amount of budded virus (BV) as the FP mutant, which was fivefold higher than that of the wild-type virus. This selective advantage was likely to explain its relative stability in polyhedra production for six passages when compared with the wild-type virus. However, subsequent passaging of ppC19 resulted in a steep decline in both BV and polyhedra yields, which was also experienced by FP8AS and the wild-type virus at high passage numbers. Genomic deoxyribonucleic acid profiling of the latter suggested that defective interfering particles (DIPs) were implicated in this phenomenon and represented another undesirable mutation during serial passaging of HaSNPV. Hence, a strategy to isolate HaSNPV clones that exhibited MP-like polyhedra production but FP-like BV production, coupled with low multiplicities of infection during scale-up to avoid accumulation of DIPs, could prove commercially invaluable.

Animals↗

Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures.

The serial passaging of baculoviruses in cell lines numerous times can result in a variety of mutations or defective viral populations becoming predominant in the cultures. The generation of these mutants during cell culture passage, also known as "the passage effect," can seriously hinder the use of in vitro methods for large-scale production of baculoviruses for use as biopesticides. In an effort to develop a large-scale in vitro method of producing Helicoverpa armigera singly enveloped nucleopolyhedrovirus (HaSNPV), it was essential to determine whether or not the passage effect was evident when this virus is serially passaged in cell cultures. An isolate of HaSNPV was serially passaged in Helicoverpa zea cell cultures up to 10 times. The production of occlusion bodies decreased with increasing passage number and there was evidence of defective viruses becoming predominant in cultures after 5 passages. The number of virions present within cross sections of passage 3 occlusion bodies was 1.5 times higher than those from passage 10 occlusion bodies when quantified using electron microscopy. A laboratory bioassay showed that potencies of passage 3 isolates against H. armigera larvae were 8 times higher than potencies of passage 10 isolates. This study indicated that changes typical of the passage effect were evident when HaSNPV was serially passaged in H. zea cell cultures up to 10 times.

Animals↗

Maintenance of biological and biochemical characteristics of human colorectal tumours during serial passage in immune-deprived mice.

The effect of serial passage in immune-deprived mice on certain biological and biochemical parameters has been studied in a series of 6 human colorectal tumour xenografts. Histological integrity is maintained for up to 10 serial passages, together with production of epithelial mucins and carcinoembryonic antigen. Passaged tumours retain human lactate dehydrogenase and glucose-6-phosphate dehydrogenase isoenzyme patterns and a human chromosome constitution. The induction of a murine tumour has been identified in this system, and the importance of routine checks for the presence of human tissue during serial passage is stressed.

Animals↗

Structure and origin of defective genomes contained in serially passaged herpes simplex virus type 1 (Justin).

Restriction enzyme and hybridization analyses have revealed that high-density DNA prepared from passage 15 of serially passaged herpes simplex virus type 1 (Justin) contains three major classes of modified viral DNA molecules, each composed of distinct but closely related types of repeate units. The DNA sequences within the three types of repeat units are colinear with the DNA sequences located at the right end (between coordinates 0.94 and 1.0) of the parental herpes simplex virus type 1 genome. Thus, the three types of repeat units each contain the entire repeat sequence (ac) (which brackets the unique sequences of the small [S] component of herpes simplex virus type 1 DNA) and differ only with respect to the amount of unique S sequences which they contain. The three classes of high-density DNA molecules were found to be stably propagated between passages 6 and 15 of this series.

Base Sequence↗

Study of biologic attributes of Cuban dengue 2 virus after serial passage in primary dog kidney cells.

OBJECTIVE: The serial passage of dengue viruses in primary dog kidney (PDK) cells has resulted in selection of attenuated viruses. However, the molecular changes responsible for loss of virulence are not well characterized. This article describes the isolation and biologic attributes of one dengue 2 virulent strain as a first step to allow the study of determinants of virulence at the molecular level. METHODS: A15 dengue 2 Cuban strain was isolated from the viremic plasma of a patient with uncomplicated dengue fever during the 1981 epidemic. This was then subjected to serial passage in PDK cells. Viruses resulting from several PDK passages were compared to the parent strain for plaque size and temperature sensitivity, neurovirulence in newborn mice, and cytopathogenic effects on LLC-MK(2) and C6/36-HT cell lines. RESULTS: A15 dengue 2 Cuban strain was successfully propagated in PDK cells. Primary dog kidney 52 to 53 viruses exhibited several biologic attributes, such as small plaques, temperature sensitivity, reduced mouse neurovirulence, and cytopathic effect in permissive cell lines. CONCLUSIONS: These results represent the first step to allow attenuation of this strain of dengue 2 virus.

Animals↗

Acquisition of sequences homologous to host DNA by closed circular simian virus 40 DNA. II. Further studies on the serial passage of virus clones.

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.

Animals↗

Propagation of fetal human RPE cells: preservation of original culture morphology after serial passage.

The permissive effects of extracellular matrix (ECM) on in vitro growth and differentiation of fetal human retinal pigment epithelial (RPE) cells have been studied. Factors which enhanced the effect of ECM to support cell division were also examined, including growth factors, culture media, and serum requirement. Under the specific culture conditions we have defined, it is possible to propagate these RPE cells at low density (less than 20 cells/mm2) with excellent growth properties for greater than 72 doublings (fourteen passages) in serial culture. Later-passaged cells maintained the morphological appearance of early-passaged cultures. ECM produced by bovine corneal endothelial cells was by far the most predominant factor in promoting rapid cell proliferation and viability over repeated passaging. Basic fibroblast growth factor (bFGF) exerted a substantial effect on the rate of cell division at different serum concentrations on plastic dishes. In addition, this factor showed profound synergistic effect when RPE cells were maintained on ECM, both in the preservation of cell morphology and also in long term viability. Other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-beta (TGF-B), were also tested, but EGF effects were less prominent than those observed with bFGF, and TGF-B had an inhibitory effect at high concentrations. The ability to obtain a relatively large number of human RPE cells in vitro which preserve the appearance of early passage cells may provide useful opportunities to study the physiological properties and pathological alterations involving this important cell type.

Cell Division↗

Attenuation of Borrelia anserina by serial passage in liquid medium.

Borrelia anserina (Sakharoff) was successfully grown in a liquid medium (Barbour-Stoenner-Kelly) for 39 passages. By the 12th serial passage in medium, infectivity of B anserina for chicks was lost. Electron microscopy did not reveal structural differences between non-infective and infective cultured organisms. Changes in the protein profiles were found by electrophoresis as the organisms were passed in culture.

Animals↗

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium.

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.

Adult↗

Oncogenes in laryngeal cancer: serial passage of transformed cellular DNA.

DNA originally extracted from squamous cell cancer of the larynx has been serially passaged through transformed populations of NIH/3T3 mouse fibroblasts. The transformed foci were then harvested, cloned to volume, and incubated with a fresh population of NIH/3T3 cells in a second passage. Transforming efficiencies were enhanced by serial passage. In addition, Southern Blot analysis of the transformed foci revealed hybridization between transformant DNA and human probe DNA from the Alu family of conserved human DNA sequences. In the first passage this hybridization took the form of diffuse homology throughout the entire molecular weight distribution. The second-passage DNA showed "narrow bands" indicating the possibility that an oncogene has been identified in laryngeal cancer and that serial passage has eliminated contaminating human sequences. Repetitive transfection in third- and fourth-passage studies is now being completed.

Animals↗

Elevated virulence of Newcastle disease virus strains following serial passages in kidney cells in vitro.

Serial passage of two lentogenic Newcastle disease virus (NDV) strains in kidney cell lines increased virulence and changed viral biological properties. In two cell lines (BHK and MDBK), elevation in virulence was demonstrated by plaque formation under an overlay with no additives, decrease in mean death times, elevated intracerebral pathogenicity index, and cytopathic effect in chicken embryo fibroblasts. Some other markers not directly correlated to virulence, such as heat inactivation of hemagglutinin and neuraminidase, were not influenced by passage in kidney cells. In addition, all strains were slow eluters. This observation emphasizes the importance of preventing the virus from reaching the viscera.

Animals↗

Serial passage through human glioma xenografts selects for a Deltagamma134.5 herpes simplex virus type 1 mutant that exhibits decreased neurotoxicity and prolongs survival of mice with experimental brain tumors.

Previous studies have described in vitro serial passage of a Deltagamma(1)34.5 herpes simplex virus type 1 (HSV-1) strain in SK-N-SH neuroblastoma cells and selection of mutants that have acquired the ability to infect and replicate in this previously nonpermissive cell line. Here we describe the selection of a mutant HSV-1 strain by in vivo serial passage, which prolongs survival in two separate experimental murine brain tumor models. Two conditionally replication-competent Deltagamma(1)34.5 viruses, M002, which expresses murine interleukin-12, and its parent virus, R3659, were serially passaged within human malignant glioma D54-MG cell lines in vitro or flank tumor xenografts in vivo. The major findings are (i) viruses passaged in vivo demonstrate decreased neurovirulence, whereas those passaged in vitro demonstrate a partial recovery of the neurovirulence associated with HSV-1; and (ii) vvD54-M002, the virus selected after in vivo serial passage of M002 in D54-MG tumors, improves survival in two independent murine brain tumor models compared to the parent (unpassaged) M002. Additionally, in vitro-passaged, but not in vivo-passaged, M002 displayed changes in the protein synthesis profile in previously nonpermissive cell lines, as well as early U(S)11 transcription. Thus, a mutant HSV-1 strain expressing a foreign gene can be selected for enhanced antitumor efficacy via in vivo serial passage within flank D54-MG tumor xenografts. The enhanced antitumor efficacy of vvD54-M002 is not due to restoration of protein synthesis or early U(S)11 expression. This finding emphasizes the contribution of the in vivo tumor environment for selecting novel oncolytic HSV specifically adapted for tumor cell destruction in vivo.

Animals↗

Emergence of simian virus 40 variants during serial passage of plaque isolates.

Three serial passage series of simian virus 40 (SV40) in CV-1 cells were initiated by infection directly from the same wild-type plaque isolate, three series were initiated by infection with another plaque isolate, and two series were initiated with each of two other plaque isolates. Aberrant SV40 genomes were not detected in any of the passage series until after the fifty undiluted passage, and each series generated a different array of variant genomes. The results show that the variants were not present in the original plaque isolates but, instead, were randomly generated during subsequent high-input multiplicity passages. Although many of the aberrant viral genomes in each passage series contained reiterations of the SV40 origin of replication and some also contained host cell sequences, there was no indication that SV40 is predisposed toward generating any particular variant.

Animals↗

Restriction endonuclease analysis of the pseudorabies (Aujeszky's disease) virus before and after serial passage in vivo and in vitro.

The deoxyribonucleic acid (DNA) of pseudorabies virus (PRV) was examined by restriction endonuclease analysis before and after various treatments: namely 1. plaque purification of stock virus, 2. serial passage of virus in cell culture at high (H) and low (L) multiplicity of infection (MOI), and 3. serial passage of virus in swine. The objective of the study was to determine if such treatments would either select or induce virus populations with a different predominant number or location of enzyme cleavage sites. Heterogeneity of the DNA of stock virus was revealed by differences among restriction patterns of 10 plaque-purified populations. All of these populations were then relatively stable through an additional 9 plaque purifications and passages in cell culture. Without plaque purification, heterogeneity was not evident. The predominant restriction pattern of stock virus appeared unaltered by 10 serial passages in cell culture at either HMOI or LMOI, except for the appearance in the HMOI series of several minor bands. Conversely, 10 serial passages of stock virus in swine resulted in either selection or induced changes or both. Most differences were slightly altered migration rates of relatively small (less than or equal to 5 Kbp) fragments. However, after 10 passages in swine, there were also changes in the migration rates of 2 large fragments (greater than 10 Kbp) of the viral genome.

Animals↗

Covariation in the capsid protein of hibiscus chlorotic ringspot virus induced by serial passaging in a host that restricts movement leads to avirulence in its systemic host.

Hibiscus chlorotic ringspot virus (HCRSV) from naturally infected Hibiscus rosa-sinensis L. loses virulence in its experimental systemic host Hibiscus cannabinus L. (kenaf) after serial passages in a local lesion host Chenopodium quinoa. Here we report the genetic changes responsible for the loss of virulence at the molecular level. A remarkable covariation of eight site-specific amino acids was found in the HCRSV capsid protein (CP) after serial passages in C. quinoa: Val(49)-->Ile, Ile(95)-->Val, Lys(270)-->Arg, Gly(272)-->Asp, Tyr(274)-->His, Ala(311)-->Asp, Asp(334)-->Ala, and Ala(335)-->Thr. Covariation of at least three of the eight amino acids, Val(49), Ile(95), and Lys(270), caused the virus to become avirulent in kenaf. Interestingly, the nature of the covariation was consistent and reproducible at each serial passage. These data indicate that the nonsynonymous substitutions of amino acids in the HCRSV CP after serial passages in C. quinoa are not likely to be random events but may be due to host-associated positive selection or accelerated genetic drift. The observed interdependence among the three amino acids leading to avirulence in kenaf may have implications for structural or functional relationships in this virus-host interaction.

Amino Acid Sequence↗