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At least 343 records · Page 19Linked to original sources

PubMedDate not supplied

Attenuation of a strain of Eimeria mivati of U.S. origin by serial embryo passage.

A strain of Eimeria mivati (FS50) isolated in Georgia was purified and serially passaged in groups of developing chicken embryos. Starch gel electrophoresis using glucose phosphate isomerase and lactate dehydrogenase showed the parasite to be similar to another strain of E. mivati isolated in the U.S. The embryo-passaged line of E. mivati (FS50) was less pathogenic than the parent line but retained its immunogenicity. This strain may be suitable for inclusion in an improved coccidiosis vaccine. The status of E. mivati and E. mitis is discussed.

Animals↗

PubMed2003

Molecular analysis of the capsid coding region of a virulent encephalomyocarditis virus isolate after serial cell passages and assessment of its virulence.

EMCV has been recognized as a worldwide-shared pathogen in many host species with natural differences of virulence among strains. Differences of virulence were also demonstrated experimentally after serial passages of particular EMCV strains in cell culture. In order to evaluate the genetic variability of the CCR of an highly virulent Belgian EMCV isolate after 210 passages in cell culture, this region has been molecularly characterised by RT-PCR and sequencing. The analyses enlightened the stability of the isolate as only 3 mutations appeared within the CCR and this was observed between passage 200 and 210. One of the mutations is adjacent to the amino acid described as the differentiation amino acid between diabetogenic and non-diabetogenic variants of EMCV. An experimental inoculation of pigs with the passage 210 of the virus enlightened its attenuation and the challenged pigs were protected against the wild-type virus. Results confirm the ability of EMCV to become attenuated after passages in cell culture.

Animals↗

PubMedDate not supplied

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture.

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of time of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can be reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the BV formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Animals↗

PubMed2001

Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells.

Defective interfering baculoviruses (DIs) lack considerable portions of the genome, interfere with the replication of helper virus, and cause the so-called "passage-effect" during serial passaging in insect cells and in bioreactor configurations. We investigated their origin by (nested) PCR and demonstrated that DIs lacking approximately 43% (d43) of their DNA are present in low-passage Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)-E2 virus stocks and in polyhedra, but not in the authentic AcMNPV isolate obtained prior to passage in cell culture. To investigate whether DIs are rapidly generated de novo in Sf21 insect cells, a genetically homogeneous AcMNPV bacmid was serially passaged, resulting in the generation of d43 DIs within two passages. AT-rich sequences of up to 66 nucleotides of partly unknown origin were found at the deletion junctions in the d43 DI genomes. These data suggest that the rapid generation of DIs is an intrinsic property of baculovirus infection in insect cell culture and involves several recombination steps.

Animals↗

PubMed1995

[Establishment and characteristics of a model of nude mouse xenograft of human retinoblastoma].

The authors reported the establishment and biological characteristics of a model of nude mouse xenograft of human retinoblastoma (RB). Immediately after the enucleation of the human globe with RB, the tumor tissue was transplanted subcutaneously into a nude mouse. The tumor grew constantly and was passaged serially for 14 passages till now. The results of the following examinations of the xenograft and primary tumor were compared: light and electron microscopy, immunohistochemistry, flow cytometry and chromosome analysis. The comparisons reveal that the main biological characteristics of the xenograft model are consistent with those of the primary tumor and also maintain throughout the serial passages. The model can be used for further laboratory studies.

Animals↗

PubMed2002

[Generation and analysis of a repeat DNA fragment of SeMNPV by serial undiluted passage in Se301 insect cells].

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from insect hosts have been previously identified. In order to gain insight into DNA replication of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis-acting DNA segments were studied. A strain, named Se-4, was plaque-purified from SeMNPV isolate US1. PCR, ELT-PCR and REN showed that Se-4 was genetically relatively homogeneous and retained the full-length of a hypervariable region which is usually prone to deletion from SeMNPV genome. To study the stability of this isolate in vitro, Se-4 was serially passaged in the Se301 cell line up to 10 times without dilution. Intracellular viral DNA extracted from every passage was analyzed by REN. A novel 3.5 kb PstI fragment was observed in passage 7 and the relative intensities of the bands increased with subsequent passages. In passage 10, the molar ratio of the fragment was much higher than those of any other viral DNA fragments. This fragment was thus expected to contain an important cis-acting element for SeMNPV DNA replication. The fragment was cloned and sequenced and it was found that it overlapped 3 525 bp with the published SeMNPV genome sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained the SeMNPV non-hr origin (ori) of DNA replication and some ORFs including partial vlf-1, partial p26, Se84 as well asSe83, Se85, Se86, which are unique to SeMNPV. As compared to Autographa californica MNPV (Groups I), which also generated hypermolar DNA fragments containing the non-hr ori by serial undiluted passage in IPLB-SF-21 cell culture, our results provided in vitro evidence that the non-hr ori may also play an important role in the viral infection cycle of Groups II NPVs.

Animals↗

PubMed2004

Analysis of murine leukemia virus replication complemented by yeast tRNA(Phe) reveals inherent preferences for the tRNA primer selected for reverse transcription.

The replication of murine leukemia virus (MuLV) requires the capture of a cellular tRNA(Pro) as a primer for reverse transcription. To further study the specificity of primer selection, we have utilized a defective MuLV in which the primer-binding site (PBS) has been altered to be complementary to a nonmammalian tRNA, yeast tRNA(Phe). Infectivity of the defective MuLV is dependent upon co-expression of yeast tRNA(Phe) in the cell. Defective MuLV genomes have been constructed in which the PBS was altered to be complementary to tRNA(Phe) that also encoded the cDNA for tRNA(Phe). Transfection of these defective proviral genomes into cells resulted in the production of infectious MuLV as determined by a single-round assay. The amount of infectious virus produced using this complementation system, though, was approximately 6-fold lower than that produced following transfection of defective proviral genomes with a wild-type PBS complementary to tRNA(Pro). The lower infectivity was not due to reduced expression of tRNA(Phe) in the transfected cells as compared to endogenous tRNA(Pro) or tRNA(Lys,3). Serial passage of the MuLV genome with a PBS complementary to tRNA(Phe) that encoded tRNA(Phe) resulted in amplification of the virus. Using this rescue system, we have passaged the virus for four serial passages, after which time a revertant genome in which the PBS was altered to be complementary to tRNA(Gln) was detected that grew to high titers following subsequent serial passage. The results of these studies suggest that MuLV has preferences for the tRNA primer used in reverse transcription and are discussed with respect to the mechanism of primer selection.

Base Sequence↗

PubMed2001

Serial human passage of simian immunodeficiency virus by unsterile injections and the emergence of epidemic human immunodeficiency virus in Africa.

There is compelling evidence that both human immunodeficiency virus (HIV) types emerged from two dissimilar simian immunodeficiency viruses (SIVs) in separate geographical regions of Africa. Each of the two HIVs has its own simian progenitor and specific genetic precursor, and all of the primates that carry these SIVs have been in close contact with humans for thousands of years without the emergence of epidemic HIV. To date no plausible mechanism has been identified to account for the sudden emergence in the mid-20th century of these epidemic HIVs. In this study we examine the conditions needed for SIV to complete the genetic transition from individual human SIV infections to epidemic HIV in humans. The genetic distance from SIV to HIV and the mutational activity needed to achieve this degree of adaptation to human hosts is placed within a mathematical model to estimate the probabilities of SIV completing this transition within a single SIV-infected human host. We found that the emergence of even one epidemic HIV strain, following a single human exposure to SIV, was very unlikely. And the probability of four or more such transitions (i.e. HIV-1 groups M, O and HIV-2 subtypes A and B) occurring in a brief period is vanishingly small. We conclude that SIV cannot become a zoonosis, but requires adaptive mutations to become HIV. Some modern event must have aided in the transition of SIV to HIV. Our research indicates that serial passage of partially adapted SIV between humans could produce the series of cumulative mutations sufficient for the emergence of epidemic HIV strains. We examined the rapid growth of unsterile injections in Africa beginning in the 1950s as a biologically plausible event capable of greatly increasing serial human passage of SIV and generating HIV by a series of multiple genetic transitions. We conclude that increased unsterile injecting in Africa during the period 1950-1970 provided the agent for SIV human infections to emerge as epidemic HIV in the modern era.

Africa↗

PubMed1983

Naturally arising recombinants that are missing portions of the simian virus 40 regulatory region.

When simian virus 40 (SV40) is serially passaged at high multiplicity, a heterogeneous collection of naturally arising variants is generated. Those which are the most abundant presumably have a selective replicative advantage over other defective and wild-type helper SV40s. Two such naturally arising host-substituted variants of SV40 have been characterized in terms of complete nucleotide sequence determination. Evolutionary variant ev-1101 (previously isolated by Lee et al., Virology 66:53-69, 1975) is from undiluted serial passage 13, whereas ev-2101 is newly isolated from undiluted serial passage 6 of an independently-derived evolutionary series. Both variants contain a five-times tandemly repeated segment of DNA consisting of viral Hin C and Hin A sequences that have recombined with a segment of host DNA that is not highly reiterated in the monkey genome. The monkey segment differs in the two variants as does the size of the viral segment retained. In two additional host-substituted variants, ev-1102 (previously isolated from serial passage 20 by Brockman et al., Virology 54:384-397, 1973) and ev-1108 (newly isolated from serial passage 40), the SV40 sequences derived from the replication origin are present as inverted repetitions. The inverted repeat regions of these two variants have been analyzed at the nucleotide sequence level and are compared with SV40 variant ev-1104 from passage 45 (previously characterized by Gutai and Nathans, J. Mol. Biol. 126:259-274, 1978). The viral segment containing the regulatory signals for replication and viral gene expression is considerably shortened in later serial passages as demonstrated by these five variants. It is of interest that the variants presumably arose due to their enhanced replication efficiency, yet are missing some of the sequence elements implicated in the regulation of replication. Furthermore, a comparison of the structure of the replication origin regions indicates that additional changes occur in the SV40 regulatory region with continued undiluted serial passage.

Base Sequence↗

PubMedDate not supplied

Effect of serial embryo passage of an Arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity.

Infectious bronchitis virus (IBV) Arkansas-type DPI strain (Ark DPI) was attenuated by serial passage in chicken embryos. Virus of passage 50 was less pathogenic for day-old maternally immune broiler-type chickens than virus of passage 10 or 25 as determined by clinical response to vaccination, virus isolation in respiratory (trachea) and nonrespiratory (kidney and cloaca) tissues, and weight-gain studies. Chickens vaccinated with virus of passage 10, 25, or 50 were at least 80% resistant to homologous virus challenge of the upper respiratory tract 4 and 6 weeks postvaccination. Serum antibody production by passage-50-vaccinated chickens was comparatively low at 4 weeks but at 6 weeks was similar to that in chickens vaccinated with virus of passage 10 or 25.

Animals↗

PubMed1977

Equine rhinopneumonitis virus (herpesvirus type 1): attenuation in stable monkey cell line.

An isolate of virulent equine herpesvirus (EHV) type 1 was adapted to Vero stable cell line by 13 serial passages at 37 C and 50 serial passages at 26 C. Characteristics of the attenuated EHV-1 were found to be avirulent, but immunogenic in horses if injected intramuscularly. The attenuated virus was regularly isolated from peripheral leukocytes in inoculated horses, but was not recovered from nasal turbinate tissues. A mild leukopenia was noticed. The attenuated virus produced characteristic large syncytia on primary isolation in rabbit kidney (RK13) or Vero cells at 37 C in contrast to cell rounding observed with virulent EHV-1. The syncytial marker was stable through 20 serial passages in Vero cells at 37 C. New application of double immunodiffusion test for distinguishing between EHV-1 and EHV-2 also is described.

Animals↗

PubMed1987

Defective interfering particles of human parainfluenza virus 3.

A cyclic pattern of virus production was observed when human parainfluenza virus 3 (HPIV3) was serially passaged nine times in LLC-MK2 cells. Viruses produced from serial passages 8 and 9 interfered with the replication of standard HPIV3. Three subgenomic RNA species (DI-1, DI-2, and DI-3) and virus genomic RNA were detected in the progeny virions produced from cells mixedly infected with standard virus and virus from either serial passages 5 or 8. Northern blot analysis with probes representing all six HPIV3 structural protein genes revealed that DI-1 and DI-2 RNAs contain sequences from the 5' end of the standard virus genome. DI-1 RNA contains L, HN, and F specific sequences, while DI-2 RNA contains only L and HN sequences. DI-3 RNA did not hybridize with any of the probes used. The possibility that DI-3 RNA contains sequences from the 5' end of the standard virus genome is discussed. These results demonstrate that 5' defective interfering particles are generated during serial passage of HPIV3.

Defective Viruses↗

PubMed1993

Ross River virus variants selected during passage in chick embryo fibroblasts: serological, genetic, and biological changes.

Serial passage of Ross River virus in chick embryo fibroblasts selected for virus variants altered in their reactions to neutralizing monoclonal antibodies. The E1 and E2 genes of each antigenic variant were sequenced; single nucleotide changes were found in E2 leading to amino acid substitutions at either residue 4 (Glu-->Lys) or 218 (Asn-->Lys); no changes were found in the E1 gene. Variants with the substitution at E2 residue 218 replicated less efficiently in 1-day-old mice than did the parental strain. The variant changed at E2 residue 4 showed little alteration in replication efficiency in mice. Similar genotypic or phenotypic changes were not found in virus passaged serially in human or mosquito cell lines.

Animals↗

PubMed1998

Demonstration of the specificity of poliovirus encapsidation using a novel replicon which encodes enzymatically active firefly luciferase.

The specificity of poliovirus encapsidation has been studied using a novel chimeric genome in which the gene encoding firefly luciferase has been substituted for the VP2-VP3-VP1 genes of the poliovirus capsid (P1) gene. Transfection of RNA transcribed in vitro from this genome resulted in a VP4-luciferase fusion protein which retained luciferase enzyme activity. Since the detection of enzyme activity was dependent upon replication of the transfected RNA genome, we refer to these genomes as replicons. The replicon encoding luciferase was encapsidated upon transfection of the genomic RNA into cells previously infected with a recombinant vaccinia virus, VV-P1, which encodes the poliovirus type 1 capsid proteins (P1). Infection of cells with each serial passage, followed by analysis of luciferase enzyme activity, revealed that encapsidated replicons could be detected at the first passage with VV-P1. Amplification of the titer of encapsidated replicons occurred upon serial passage with VV-P1, as evidenced by the high expression levels of luciferase enzyme activity following infection. Serial passage of the luciferase replicons with poliovirus type 1, 2, or 3 resulted in the trans encapsidation into the type 1, 2, or 3 capsids, respectively. In contrast, serial passage with bovine enterovirus, Coxsackievirus A21 or B3, or enterovirus 70 did not result in trans encapsidation, even though co-infection of cells with the replicon and different enteroviruses resulted in high-level expression of luciferase. The results of this study highlight the specificity of poliovirus encapsidation and point to the use of encapsidated replicons encoding luciferase as a reagent for dissecting elements of replication and encapsidation.

Animals↗

PubMed1995

Trace amount of satellite RNA associated with tobacco ringspot virus: increase stimulated by nonaccumulating satellite RNA mutants.

The small satellite RNA of tobacco ringspot virus (sTRSV RNA) is dependent on tobacco ringspot virus (TRSV) for replication and encapsidation. sTRSV RNA has appeared during serial passage of certain TRSV strains in some hosts. Co-inoculation of bean with TRSV and either of two related, nonaccumulating mutants of sTRSV RNA induced the appearance of sTRSV RNA in a single passage (van Tol et al., 1991, Virology 180, 23-30). The sTRSV RNA obtained after serial passage and after co-inoculation have the same nucleotide sequence, designated the endogenous sequence. The endogenous sTRSV RNA nucleotide sequence differs from that of each of the nonaccumulating sTRSV RNA at three positions. In order to detect possible trace amounts of endogenous satellite RNA in virion RNA preparations, RNA from two TRSV isolates was subjected to reverse transcription and polymerase chain reaction of the transcript (RT-PCR), using primers with sTRSV RNA terminal sequences. The yield of RT-PCR product suggests that the virion RNA preparations contained approximately 0.1 fg of sTRSV RNA per microgram of virion RNA. The nucleotide sequence of the RT-PCR product corresponded to that of the endogenous sTRSV RNA. The endogenous sTRSV RNA of TRSV inocula appears to be latent, being maintained in very small amounts during serial passage of TRSV in some hosts but capable of dramatic increase during serial passage in other hosts or when TRSV was co-inoculated with either of two specific sTRSV RNA mutants. Ten other nonaccumulating sTRSV RNA mutants did not induce a detected increase in sTRSV RNA.

Base Sequence↗

PubMed1975

Establishment and characteristics of a hamster lung adenocarcinoma in vivo and in vitro.

Several cell lines designated HLAC were derived from primary lung carcinomas induced in Syrian hamsters by polonium-210 or benzo[a]pyrene. Primary tumor nodules were initially transplanted into cheek pouches, and the tumors that grew were passed into tissue culture and into the cheek pouches of other hamsters for continued in vivo passage. By serial passage and cloning, cell lines were isolated with plating efficiencies of 20-50% in vitro and 10-25% when cultured directly from solid tumors. These cells formed adenocarcinomas in vivo. The radiosensitivities in vitro of HLAC-4 and HLAC-14 varied; observed D0 (the inverse of the slope of the exponential portion of the survival curve) values were 80 and 155 rads, respectively; n (the dose at which the exponential portion of the survival curve extrapolates to 100% survival) values were approximately 1.8. Survival curves obtained following in situ irradiation of 4- to 5-mm3 HLAC-4 tumors showed a D0 of 80 rads and an n of 7. Morphology and growth characteristics of two HLAC cell lines in vivo and in vitro were described.

Adenocarcinoma↗

PubMed1988

Characterization of lymphoid tumors induced by a recombinant murine retrovirus carrying the avian v-myc oncogene. Identification of novel (B-lymphoid) tumors in the thymus.

Lymphoid tumors induced by a recombinant murine retrovirus carrying the v-myc oncogene of avian MC29 virus were characterized. The Moloney murine leukemia virus myc oncogene (M-MuLV (myc], carried by an amphotropic MuLV helper, induced tumors in NIH Swiss and NFS/N mice after a relatively long latency (8 to 24 wk). Tumor masses appeared in the thymus, spleen, and lymph nodes. Flow cytometry of the tumor cells indicated that approximately 50% were positive for Thy 1.2. Most of these tumors also expressed one or more other cell surface markers of thymocytes and mature T cells (CD4, CD8). Southern blot hybridization revealed genomic rearrangements for the TCR beta genes. The TCR beta analysis suggested that the M-MuLV(myc)-induced Thy 1.2+ tumors were derived from somewhat less mature cells than tumors induced by M-MuLV, which is a classical non-acute retrovirus lacking an oncogene. The remainder of the M-MuLV(myc)-induced tumors were Thy 1.2-, but they were positive for Ly-5 (B220) and also for MAC-2. The Thy 1.2- tumors were characteristically located in the thymus. However, they were negative for TCR beta gene rearrangements. Some, but not all, of the Thy 1.2- tumors contained rearrangements for Ig genes. Additionally, they typically expressed mRNA specific for B but not for T cells. Thus, these thymic tumors had characteristics of the B cell lineage. Tumor transplantation experiments demonstrated that the Thy 1.2- tumor cells could reestablish in the thymus and spleen of irradiated hosts, and low level expression of the Thy 1 molecule was observed in the thymus but not the spleen on the first passage. After serial passage, one Thy 1- tumor altered its cell surface phenotype to Thy 1low B220-.

Animals↗

PubMed1973

Reassortment of simian virus 40 DNA during serial undiluted passage.

Alterations occur in the supercoiled form of viral DNA after the serial undiluted passaging of simian virus (SV) 40. We have identified a portion of the viral genome which is amplified during this process. These SV40 DNA sequences represent about 30% of the viral genetic information and are present in a reiterated form in twisted circular molecules prepared from purified virions. In addition, reiterated and unique green monkey DNA sequences are incorporated into supercoiled viral DNA. The cellular DNA appears to be inserted at numerous locations in the DNA I molecules.

Base Sequence↗