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At least 649 records · Page 36Linked to original sources

PubMed1991

The genes associated with trans-dominance of the influenza A cold-adapted live virus vaccine.

Segment 7 (M) of the cold-adapted live influenza A virus vaccine plays a primary role in the ability of this virus to interfere with the replication of wild-type influenza A viruses. This conclusion is based on several lines of evidence. Single gene reassortant viruses derived by crossing influenza A/Ann Arbor/6/60 (H2N2) cold-adapted donor virus with an epidemic wild-type strain, A/Korea/1/82 (H3N2), were tested for their ability to interfere with wild-type parental virus in the Madin-Darby line of canine kidney cells and embryonated eggs. It was apparent in both hosts that the single gene reassortant carrying segment 7 (M) derived from the cold-adapted virus was dominant over wild-type virus. Additional confirmation of the role of segment 7 (M) in trans-dominance of the cold-adapted vaccine virus was derived from the analysis of reassortants produced by mixed infection by a wild-type virus and its cold-adapted reassortant vaccine strain. After three serial passages, the virus yield contained a high proportion of reassortants carrying segment 7 (M) of the cold-adapted parental strain. When used in mixed infections, these reassortants were dominant over the replication of the parental wild-type virus.

Animals↗

PubMed1998

Failure of Ixodes ticks to inherit Borrelia afzelii infection.

To define conditions promoting inherited infection by Lyme disease spirochetes in Ixodes ticks, we variously infected ticks with Borrelia afzelii and examined their progenies by dark-field microscopy, immunofluorescence, PCR, and serial passage. No episode of inherited infection was evident, regardless of instar or gender infected or frequency of exposure. We suggest that these spirochetes rarely, if ever, are inherited by vector ticks.

Animals↗

PubMedDate not supplied

Pathogenicity studies of an Arkansas variant infectious bursal disease virus.

A variant infectious bursal disease virus (IBDV), IBDV-s977, was blind passaged in cell culture, plaque purified, and attenuated by serial passage at a high multiplicity of infection (MOI) in chick embryo fibroblasts (CEF). Cell culture passages of virus caused less bursal atrophy and splenomegaly than did the original isolate and retained immunogenicity; however, virus tended to persist for a longer time in the bursa and spleen of birds infected with the highest CEF passages. Antibody to both low MOI and high MOI passages of IBDV-s977 poorly neutralized virus that was isolated from bursal tissue 28 days postinfection (PI). The spleens of chickens infected with the eighteenth CEF passage were negative for virus at 3 and 7 days PI but had high titers of virus at 14 and 28 days PI. There was also more virus in the bursa of birds infected with the fifteenth and eighteenth CEF passages at 28 days PI than at 7 or 14 days PI. Defective interference (DI) was demonstrated when cell cultures were coinfected with a constant amount of low MOI virus and serial dilutions of high MOI virus. There was an increase in interference score with increased passage number in CEF, and there was more interference in virus passaged at a high MOI. There was an inverse relationship between interference score and bursal lesion score and splenomegaly at 7 days PI, indicating that DI particles may be involved in virus attenuation. There was a positive relationship between interference and viral persistence in the bursa and spleen at 28 days PI. Antiserum to s977 was shown to enhance the nonlytic replication of s977 in CEF, presumably within macrophages, providing a possible mechanism for the pathotypic variation seen in emerging strains of IBDV.

Animals↗

PubMed1995

Mutational analysis of influenza virus promoter elements in vivo.

RNA polymerase I transcription in vivo in transiently DNA-transfected cells has been used to express influenza virus vRNA molecules coding for chloramphenicol acetyltransferase (CAT) in an antisense orientation. Influenza virus superinfection provided viral RNA polymerase and other proteins required for transcriptional conversion of minus-strand vRNA into plus-strand viral mRNA molecules expressing CAT activity. This system has been used for analysis of the vRNA sequences which cooperatively constitute the vRNA promoter structure via nucleotide exchanges as well as deletions and insertions of both terminal segments. Several mutants caused greatly enhanced expression over wild-type levels, which was transmitted during serial passage of progeny virus. The data obtained for the mutations in various promoter elements support a model implicating double-stranded vRNA promoter structures in binding of viral polymerase, and in consecutive steps during initiation of RNA synthesis.

3T3 Cells↗

PubMed1994

A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus.

In addition to the seven known homologous regions (hrs) of Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) the HindIII-K fragment was also found to carry a putative ori, although this fragment does not contain an hr. Deletion analysis showed that this ori contains several segments essential for its activity and other 'auxiliary' sequences that enhance the ori activity. Sequence analysis identified several structures often found in other viral replication oris, such as palindromes and other repeated motifs. Although most of the auxiliary sequences of this ori were found to be deleted in the Bombyx mori nucleocapsid polyhedrosis virus genome, the essential part of this ori, containing the palindromes and the A/T-rich region, was retained. This and the fact that after prolonged serial passage of AcMNPV large replicating DNA molecules are found in which repeated sequences derived from the HindIII-K fragment accumulate are consistent with this region being a putative origin of AcMNPV DNA replication.

Animals↗

PubMed1997

Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein.

The agent responsible for transmissible spongiform encephalopathies (TSEs) is thought to be a malfolded, protease-resistant version (PrPres) of the normal cellular prion protein (PrP). The interspecies transmission of bovine spongiform encephalopathy (BSE) to mice was studied. Although all of the mice injected with homogenate from BSE-infected cattle brain exhibited neurological symptoms and neuronal death, more than 55 percent had no detectable PrPres. During serial passage, PrPres appeared after the agent became adapted to the new host. Thus, PrPres may be involved in species adaptation, but a further unidentified agent may actually transmit BSE.

Animals↗

PubMed1996

Generation of defective interfering particles by two vaccine strains of measles virus.

A systematic study was made to measure the generation of defective interfering particles upon up to 13 serial passages of two measles vaccine strains, Edmonston and Edmonston-Zagreb, through either simian (Vero) or human (WI-38) cell lines. Results for the Vero cell passage were nearly identical for both viruses. Infectivity titers dropped by nearly 8 logs to undetectable levels at passage 4 and cycled between maximum and minimum levels every 4 passages. Samples with the lowest infectivity titers produced the greatest reduction in titer of standard virus and contained an approximately 900-nucleotide subgenomic RNA for the Edmonston strain and two subgenomic RNAs of 4300 and 3000 nucleotides for the Edmonston-Zagreb vaccine strain. A defective interfering RNA-specific reverse transcription-polymerase chain reaction (RT-PCR) detected subgenomic RNAs at all passage levels. In contrast, samples obtained after passage of these viruses in WI-38 did not reduce the yield of standard virus and did not contain subgenomic RNAs in both Northern blot and RT-PCR assays. These results clearly show that cell type rather than virus strain affects defective interfering particle generation for measles virus.

Animals↗

PubMed1995

Characterization of a human immunodeficiency virus type 1 variant with reduced sensitivity to an aminodiol protease inhibitor.

Development of viral resistance to the aminodiol human immunodeficiency virus (HIV) protease inhibitor BMS 186,318 was studied by serial passage of HIV type 1 RF in MT-2 cells in the presence of increasing concentrations of compound. After 11 passages, an HIV variant that showed a 15-fold increase in 50% effective dose emerged. This HIV variant displays low-level cross-resistance to the C2 symmetric inhibitor A-77003 but remains sensitive to the protease inhibitors Ro 31-8959 and SC52151. Genetic analysis of the protease gene from a drug-resistant variant revealed an Ala-to-Thr change at amino acid residue 71 (A71T) and a Val-to-Ala change at residue 82 (V82A). To determine the effects of these mutations on protease and virus drug susceptibility, recombinant protease and proviral HIV type 1 clones containing the single mutations A71T and V82A or double mutation A71T/V82A were constructed. Subsequent drug sensitivity assays on the mutant proteases and viruses indicated that the V82A substitution was responsible for most of the resistance observed. Further genotypic analysis of the protease genes from earlier passages of virus indicated that the A71T mutation emerged prior to the V82A change. Finally, the level of resistance did not increase following continued passage in increasing concentrations of drug, and the resistant virus retained its drug susceptibility phenotype 34 days after drug withdrawal.

Amino Acid Sequence↗

PubMed1998

Infection of primary dermal microvascular endothelial cells by Kaposi's sarcoma-associated herpesvirus.

OBJECTIVE: To develop an in vitro model for infection of primary human cells with Kaposi's sarcoma (KS) herpesvirus (KSHV). DESIGN: The recent identification of a herpesvirus associated with KS, its successful isolation in vitro, and its complete DNA sequencing facilitates experiments on the pathogenesis of AIDS-related KS. Completed studies demonstrate that the endothelial cells lining the vascular slits in KS lesions are productively infected with KSHV and may be the principal site of virus replication. We have designed a model system to study the infection of primary human cells with KSHV. METHODS: A coculture technique was used with KS cells (KS-1) and primary dermal microvascular endothelial cells. RESULTS: We detected increasing viral DNA concentrations as well as viral mRNA suggesting that a productive virus infection occurs in the target cells. Infection of these cells is dose- and time-dependent and is inhibited by lobucavir, foscarnet and 9-(2-phosphomethoxyethyl) adenine. With a modification of the model, KSHV can be serially passaged in primary cells in excess of 16 passages. CONCLUSIONS: This novel model assay system makes new studies on the role of KSHV and KSHV-induced cellular products on the pathogenesis of KS possible. It also provides a high volume screening method to detect agents that inhibit KSHV infection of primary endothelial cells.

Animals↗

PubMed1991

Persistent infection of Vero cells by the flavivirus Murray Valley encephalitis virus.

Murray Valley encephalitis (MVE) virus strain OR2 was serially passaged on Vero cells to establish a persistent infection which was maintained for over 300 days. Supernatants from infected cells protected Vero cells from c.p.e. and caused up to a 95% reduction of wild-type virus yield. These protective and interfering effects suggest that defective interfering (DI) particles are responsible for the establishment and maintenance of the MVE virus persistent infection. The persistently infected cell supernatant preparations shared several features with DI particle preparations from other viral systems, such as their amplification to detectable levels after two to four passages of virus. However, results from this study suggest that DI particles of MVE virus differ from other studied systems in that they are able to affect only moderately the yield of infectious wild-type virus. The genetic drift of the parental virus during the course of a long term persistent infection in vitro appears to be minimal.

Animals↗

PubMed1992

[Development of resistance in bacteria interacting with subinhibitory antibiotic concentrations].

In order to determine the role of subinhibitory concentrations of antibiotics in resistance development, we performed an in vitro trial in which E. coli gained resistance after exposure to low concentrations of ampicillin in serial passages, while MIC values for amikacin and gentamicin increased 2 and 4 times, respectively. Pseudomonas also became resistant to ceftazidime whereas the MIC value for ciprofloxacin increased 10 times. There was not any significant change in susceptibility of S. aureus to vancomycin and amoxicillin + clavulanate.

Amikacin↗

PubMed2005

Glycine-amide is an active metabolite of the antiretroviral tripeptide glycyl-prolyl-glycine-amide.

The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH(2)) inhibits replication of human immunodeficiency virus (HIV) type 1 (HIV-1) in vitro, probably by interfering with capsid formation. The aim of the present study was to determine whether the metabolites glycyl-proline (GP-OH), glycine (G-OH), prolyl-glycine-amide (PG-NH(2)), proline (P-OH), and glycine-amide (G-NH(2)) from proteolytic cleavage may inhibit the replication of HIV-1 in vitro. PG-NH(2) has previously been shown to have a modest effect on HIV-1 replication. In the present study we show that G-NH(2) exhibits a pronounced inhibitory effect on HIV-1. This effect was not due to a decrease in cell proliferation or viability and could not be shown for herpes simplex virus type 1. The G-NH(2) concentration that inhibited virus replication by 50% (IC(50)) was equimolar to that of GPG-NH(2) and ranged from 3 to 41 microM. Transmission electron microscopy revealed that the effect of G-NH(2) on HIV-1 morphology was equivalent to that of GPG-NH(2) and showed disarranged capsid structures, indicating interference with capsid formation. Serial passage of HIV-infected cells with G-NH(2) for more than 20 subcultivations did not decrease the susceptibility to the compound. The results from this study suggest that GPG-NH(2) might act as a prodrug and that G-NH(2) is an active antiretroviral metabolite.

Animals↗

PubMed2006

Genetic variability of encephalomyocarditis virus (EMCV) isolates.

In order to evaluate the variability of encephalomyocarditis virus (EMCV), field isolates originating from different European regions and inducing different clinical pictures in pigs have been molecularly characterised. The regions targeted were the poly(C) tract, a part of the 5'-UTR (360 nucleotides), the Leader gene (201 nucleotides), the complete capsid coding region (2502 nucleotides), the 2A gene (403 nucleotides), the end of the 3D polymerase gene (305 nucleotides) and the 3'-UTR (123 nucleotides). Analyses have also been performed on a virulent field isolate, which had been subjected to serial passages in vivo and in vitro resulting, in the case of the in vitro passaged virus, in attenuation, as demonstrated by animal experiments. The present study shows that different clinical pictures, such as acute fatal myocarditis or reproductive failure, may not only be caused by EMCV isolates which are genetically diverse but also by the same isolate. Thus no correlation could be demonstrated between genotype and clinical disease. However, the European isolate which showed the highest genetic divergence also gave rise to a more complex clinical picture. Despite EMCV having been isolated from cases of acute fatal myocarditis in pigs in certain areas of the world for many years, clinical disease, including a variety of clinical pictures and pathogenicity, has only been recognised in Europe since 1986 and thus it can be considered an emerging disease in this region. These findings, associated with the reported phenotype changes of the virus under environmental changes (passages), along with its wide distribution among vertebrate species (including higher primates), shows the validity of considering EMCV as a potential pathogen for recipients in xenotransplantation.

3' Untranslated Regions↗

PubMed1988

An adenovirus infection of the kidney of Franklin's ground squirrels (Spermophilus franklini) in Saskatchewan, Canada.

During routine pathological studies of Franklin's ground squirrels (Spermophilus franklini) collected during a predator control program, basophilic intranuclear inclusions were found in the collecting tubule epithelium of the renal papillae in seven of 13 squirrels. This was associated with marked karyomegaly in affected cells. An inflammatory response was not seen in the adjacent tissues. Electron microscopic examination of affected cells demonstrated that the enlarged nuclei contained numerous virus-like particles. Autoculture and serial passage of renal medullary cells resulted in the isolation of virus particles producing intranuclear inclusions and cytopathic effect. The virus possessed properties typical of adenoviruses, but showed no evidence of hemagglutinating activity with a range of species of erythrocytes tested under several temperature conditions. The isolates were relatively host-cell specific; they failed to grow in hamster and rabbit kidney cell lines and in ground squirrel kidney cortical cells.

Adenoviridae Infections↗

PubMed1995

Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene.

Measles virus (MV) mRNA transcription and replication are thought to be controlled by cis-acting sequence elements contained within the terminal MV genomic noncoding nucleotides. To validate these promoter and regulatory signal assignments, cDNAs were constructed allowing synthesis of RNAs corresponding to a MV genome in which all coding and intercistronic regions were replaced by the chloramphenicol acetyl transferase (CAT) coding sequence. Transcript production by T7 polymerase starting and ending precisely with the MV genome terminal residues was achieved by fusing the T7 polymerase promoter and the hepatitis delta virus genome ribozyme followed by tandem T7 polymerase termination sequences to the MV genomic 5' and 3' ends, respectively. Transfection of these negative polarity transcripts, mimicking natural defective interfering RNAs of the internal deletion type, into MV-infected 293 cells gave rise to CAT activity which could be serially transferred and massively amplified together with progeny helper virus in fresh cells. Transfer was blocked only by antibodies able to neutralize MV infectivity, indicating that the chimeric RNA not only was encapsidated, transcribed, and replicated, but also packaged into virions. Sequence analyses confirmed that both the expected chimeric antigenome and mRNA products were transcribed and replicated with fidelity during serial passage. Minor changes introduced in the transcription promoter markedly compromised function. This system now can be exploited to examine MV genomic cis-acting regulatory elements and extended to the development of full-length MV cDNAs.

Base Sequence↗

PubMed2001

Adaptation of Leishmania cells to in vitro culture results in a more efficient reduction and transport of biopterin.

Leishmania major and Leishmania donovani cells freshly isolated from infected animals divided slowly as axenic promastigotes but the addition of biopterin in the culture medium greatly enhanced their growth. However, when cells were subjected to serial passages and adapted to culture, this growth-promoting effect of biopterin was no longer observed. Genetic analysis of these culture-adapted Leishmania cells demonstrated that the genes coding for the pterin reductase PTR1 or for the biopterin transporter BT1 were over-expressed. This suggests that Leishmania cells adapted to culture were more efficient in utilizing biopterin, an essential growth factor in Leishmania.

Adaptation, Biological↗

PubMedDate not supplied

[Production and study of hybridomas, producing monoclonal antibodies to the structural glycoprotein of Marburg virus].

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus glycoprotein were prepared by splicing of mouse myeloma cells (NSO and X.63-Ag6.653 strains) and splenocytes of immunized BALB/c mice. Cultural and MAb-producing properties of hybridomas were studied. The production of MAb during serial passages 15-30 was confirmed. MAb titers in culture fluids were 1:4096-1:8152, in immune ascitic fluids of BALB/c mice 1:409,600-1:638,400. The possibility of using MAbs as a component of ELISA test system was demonstrated. The sensitivity of ELISA-MAb method for Marburg virus was 1 x 10(3) PFU/mu.

Animals↗

PubMed2000

Effects of environment on compensatory mutations to ameliorate costs of antibiotic resistance.

Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium. This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.

Adaptation, Physiological↗