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At least 19 recordsLinked to original sources
PubMed2000

Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells.

The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.

Animals↗
PubMed1987

Restriction analysis of additional "non-producing" cell clones obtained after transformation of Japanese quail fibroblasts with viruses rescued from cryptovirogenic H-19 cells.

The proviral structure of two transforming viruses rescued from cryptovirogenic H-19 hamster cells harbouring the LTR, v-src, LTR proviral structure was analysed by restriction mapping. It was established that the rescued G8 virus keeps the same restriction pattern as that observed in the cryptic H-19 provirus and that it is integrated into the cell genome at a different position characteristic of each "non-producing" (NP) cell clone. In these features it corresponds to the previously described F6 rescued virus. In studying the E6 rescued virus we found again that it is integrated at a position characteristic of the NP cell line studied. Using a series of restriction enzymes it was specified that the E6 proviral unit acquired at least 0.25 kb from the left part of the gag gene and 0.85 kb of an unidentified DNA. Because this DNA structure was not digested with the enzymes employed that cut in RSV provirus and did not hybridize with the viral gene probes, it might be of cellular origin. The recombinational events involved in E6 virus genesis, the nature of an additional DNA structure, and the functional significance of the acquired part of the gag gene are discussed.

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PubMed1998

Rapid and efficient recovery of Sendai virus from cDNA: factors influencing recombinant virus rescue.

In a comparative study the factors influencing the recovery of recombinant Sendai viruses (SeV) from plasmid based cDNA were analysed systematically in order to establish an efficient and robust method for virus rescue. The amounts and ratios of transfected helper plasmids encoding the viral N, P and L proteins proved to be crucial for virus rescue, and they were optimised step-by-step for enhanced virus release. When the C open reading frame from the P gene was expressed at low level, virus rescue was generally possible but virus release could be improved when C gene expression was abolished completely. SeV particle formation could be increased greatly when the transcription initiation site for T7 polymerase in the cDNA was modified or when the genomic ribozyme instead of the antigenomic ribozyme of hepatitis delta virus was used for processing the 3'end of the viral RNA transcript. Heterologous helper viruses vTF7-3 and MVA-T7, which are necessary for T7 polymerase production in transfected cells, were compared for their use in SeV recovery and subsequent elimination of the helper virus from recombinant SeV. Interference with SeV replication was less severe with MVA-T7, and MVA-T7 was eliminated efficiently without the need for any inhibitors by serial passages in Vero cells. Optimal combination of all parameters led to a highly efficient generation of recombinant SeV from cDNA. Titres of the released virus particles are high enough to enable analysis of the recombinant SeV directly on test cells or propagation in cell cultures without the need for amplification in embryonated chicken eggs. The system is very robust and allows rapid generation of defined SeV mutants that require specialised host cells for propagation.

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PubMed1984

Acquisition of host cell genetic information by avian sarcoma virus rescued from rat cells transformed by B77 virus.

The properties of avian sarcoma virus B77 grown in chick cells were compared with those of virus obtained by fusion of rat cells transformed by B77 virus ( RB77 cells) with chick cells ( FB77 virus). Differences in the B77 and FB77 viral genomes were demonstrated by the fingerprint technique. The rescued FB77 virus contained sequences homologous to the normal cell genome. These results suggest that the rescued FB77 virus is a recombinant between B77 virus and rat genomic elements. The RNAs from B77 and FB77 viruses were found to have similar molecular weights. The migration rates of the structural proteins and the large glycoprotein (gp 85) in polyacrylamide gels were the same for the B77 and FB77 viruses, but the small glycoprotein (gp) of the FB77 virus was found to be slightly larger than the gp37 of the B77 virus.

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PubMed2006

Establishment of a Nipah virus rescue system.

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans and incurred a high fatality rate in humans. Fruit bats, living in vast areas extending from India to the western Pacific, were identified as the natural reservoir of the virus. However, the mechanisms that resulted in severe pathogenicity in humans (up to 70% mortality) and that enabled crossing the species barrier were not known. In this study, we established a system that enabled the rescue of replicating NiVs from a cloned DNA by cotransfection of a constructed full-length cDNA clone and supporting plasmids coding virus nucleoprotein, phosphoprotein, and polymerase with the infection of the recombinant vaccinia virus, MVAGKT7, expressing T7 RNA polymerase. The rescued NiV (rNiV), by using the newly developed reverse genetics system, showed properties in vitro that were similar to the parent virus and retained the severe pathogenicity in a previously established animal model by experimental infection. A recombinant NiV was also developed, expressing enhanced green fluorescent protein (rNiV-EGFP). Using the virus, permissibility of NiV was compared with the presence of a known cellular receptor, ephrin B2, in a number of cell lines of different origins. Interestingly, two cell lines expressing ephrin B2 were not susceptible for rNiV-EGFP, indicating that additional factors are clearly required for full NiV replication. The reverse genetics for NiV will provide a powerful tool for the analysis of the molecular mechanisms of pathogenicity and cross-species infection.

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PubMed1986

Characterization of transforming viruses rescued from a hamster tumour cell line harbouring the v-src gene flanked by long terminal repeats.

The organization of proviruses derived from infecting transforming viruses rescued from hamster tumour cells was studied. Southern blot analysis indicated that the provirus from the F6 cell line was organized as long terminal repeat (LTR)-src-LTR, and S1 mapping experiments suggested that it was probably derived by reverse transcription of src mRNA followed by integration. In the E6 cell line, the provirus unit was arranged as LTR-delta gag-src-LTR, indicating a recombination event between the rescued transforming virus and the helper virus. These results suggest that transforming defective viruses containing only the src gene can be rescued from nonpermissive mammalian cells.

Animals↗
PubMed1983

Characterization of exogenous proviral sequences in hamster tumor cell lines transformed by Rous sarcoma virus rescued from XC cells.

Alterations in viral structural genes have been studied in five cell lines derived from Syrian hamster tumors which had been induced by the virus rescued from XC cells by transfection. Two cell lines, H-18 and H-20, have all the viral structural genes expressed, but a new EcoRI recognition site appeared in the region of the pol gene sequence. Provirus present in H-12 lacks the 3' part of the gag gene sequences as well as the pol gene, therefore, it gives rise to an anomalous 1.8 Md EcoRI fragment. This line also does not synthesize viral RNA of genomic size, and none of the subgenomic RNAs found hybridized with the DNApol probe. The H-19 cell line harbors only the src gene and LTR sequences, the U3 part of which seems incomplete or different from that of PR-RSV. The cryptic proviral structure in H-19 is transcribed into src mRNA. The degree of transcription of the src gene is about 25 viral RNA equivalents per cell. The H-9 cells harbor the complete provirus and, in addition, proviral structures having the deletion in gag-pol genes. The possible ways of development of provirus alterations and the role of cryptic proviral sequences in oncogenesis are discussed.

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PubMedDate not supplied

Dose-dependent inhibition of virus rescue from lymphocytes latently infected with turkey herpesvirus or Marek's disease virus.

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.

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PubMed1976

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.

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PubMed1978

Rat tracheal organ culture supports replication of parainfluenza 1 (6/94) virus and promotes 6/94 virus rescue from latently infected human brain cells.

Rat tracheal organ culture (TOC) supported replication of parainfluenza 1 (6/94) virus. Cell-associated and cell-free viruses were found after primary infection of TOC. In contrast to other mammalian systems, rat TOC was capable of maintaining 6/94 virus infectivity after primary infection. Rat TOC may be considered a potential indicator system that could be used to detect virus latent in human tissue.

Animals↗
PubMed1996

Construction and transfection of ovine adenovirus genomic clones to rescue modified viruses.

The genome of ovine adenovirus OAV287 has an arrangement which is unique among known adenoviruses. To facilitate further experimentation on the structure and function of this genome, plasmids containing a complete clone of the genome were constructed. The cloned viral genome was released from plasmids by restriction enzyme digestion as an intact linear molecule with authentic 5' termini. Transfection of the linear DNA into cells which supported replication produced infectious virus. Mutation of a unique SalI site at the right-hand end of the genome disrupted reading frames of unknown function without affecting virus rescue, identifying this region as nonessential for replication in vitro. A 20-bp oligonucleotide was also inserted into the short intergenic region between the pVIII and the fiber sequences, identifying a second site for gene insertion. These studies will facilitate the development of OAV as a gene transfer vector.

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