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At least 19 recordsLinked to original sources
PubMed2000

Quantitative analysis of viral RNA kinetics in coxsackievirus B3-induced murine myocarditis: biphasic pattern of clearance following acute infection, with persistence of residual viral RNA throughout and beyond the inflammatory phase of disease.

Although the association remains controversial, enteroviruses have been implicated in the aetiology of several chronic diseases of humans. To further understand the mechanism of enterovirus persistence and its relationship to organ pathology, virus infectivity and viral RNA kinetics in the heart and other target organs during acute and persistent phases of murine coxsackievirus B3 infection were investigated. These studies revealed a biphasic pattern of virus clearance. Thus, there was a rapid but incomplete clearance of viral RNA from the myocardium following the acute phase of virus replication, which paralleled the elimination of virus infectivity. The mean half-life of viral RNA between days 5 and 14 post-infection (p.i.) was 13.4 h. In contrast, a much slower rate of decline in viral RNA levels was observed during the post-infectious inflammatory phase of myocarditis. The mean half-life of viral RNA between days 14 and 90 p.i. was 14.1 days. Viral RNA persisted in the myocardium beyond the resolution of inflammation and was still detectable in a proportion of animals 90 days after infection. Clearance of viral RNA from other target organs occurred more rapidly, but the rate of clearance was largely independent of the level of viral RNA present during the acute phase of infection. Thus, while antiviral immune responses effectively eliminated infectious virus, clearance of residual viral RNA from the myocardium and other target organs was significantly delayed, despite a prolonged inflammatory response. These findings suggest that clearance of persistent enterovirus infection requires mechanisms different from those responsible for the elimination of virus infectivity.

Animals↗
PubMed1997

Kinetics of viral RNA synthesis following cell-to-cell transmission of human immunodeficiency virus type 1.

The temporal appearance and levels of human immunodeficiency virus type 1 (HIV-1) tat, rev, nef, env and gag mRNA species were examined using a synchronized, one-step, cell-to-cell HIV-1 infection model involving HUT-78 cells and HIV- 1 persistently infected H3B cells. Individual mRNAs were quantified by RT-PCR using RNA standards transcribed in vitro from cDNA clones. Consistent with an infection that produces high yields of virus, significant levels of env and gag mRNAs were detected in the cytoplasm of infected cells late in the infection cycle. However, at no time after infection did levels of tat, rev and nef mRNA, which encode the regulatory proteins of HIV-1, exceed their levels present in the persistently infected virus donor H3B cells. The absence of early phase induction of these mRNAs is in contrast to what is observed in cell-free HIV-1 infections or in PMA-stimulated HIV-1 chronically infected cell lines. Our results suggest that tat and rev mRNAs are already present in the cytoplasm of the persistently infected virus donor cells at levels sufficient for initiation and establishment of a highly productive infection in HIV-1 fusion-mediated infected cells. Thus, lack of sufficient Tat and Rev proteins is not likely to be the limiting factor for virus production in H3B cells, nor is increased production of these proteins likely to be the cause of the increased virus production seen following cell-to-cell transmission.

Cell Line↗
PubMed1990

In vitro transcription of the double-stranded RNA genome of maize rough dwarf virus (Reoviridae).

An RNA-dependent RNA polymerase associated with particles of maize rough dwarf virus, a Fijivirus, was characterized using two in vitro assays differing in their energy regeneration systems. Optimum reaction rates occurred at pH 8.0 to 8.5 at 20 degrees C. The presence of virus and Mn2+ or Mg2+ was essential for enzyme activity; Mn2+ stimulated more incorporation events than Mg2+, at optimum concentrations of 2 to 4 mM and 4 mM, respectively. Incorporation was not affected by alpha-amanitin, actinomycin D or rifampicin. The products synthesized in vitro were single-stranded RNAs which hybridized specifically with the double-stranded genomic RNAs of five other reoviruses. The in vitro transcripts were also used to detect maize rough dwarf virus RNA in plants and in vector insects.

DNA-Directed RNA Polymerases↗
PubMed1977

Immunologic selection against simian virus-40 transformed cells: concomitnat loss of viral antigens and early viral gene sequences.

A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.

Antigens, Viral↗
PubMed2005

Distribution of viral RNA in mouse tissues during acute phase of coxsackievirus B5 infection.

OBJECTIVE: To investigate histopathological changes and distribution of coxsackievirus B5 (CVB5) RNA in mouse heart, liver, and pancreas during the acute phase of infection. METHODS: C3H/HeJ male mice, aged 3-4 weeks, were inoculated intraperitoneally with 5 x 10(5) plaque-forming units of CVB5 and sacrificed at 1, 2, 3, 4, 7 and 10 days postinfection (p.i.). Inflammation of the heart, liver, and pancreatic tissue sections was evaluated by hematoxylin and eosin staining, and virus was detected using antibody to viral coat protein VP1. A quantitative real-time RT-PCR method, using primers and probe targeted to the highly conserved sequences in the 5'-untranslated region of the virus, was used to evaluate the kinetics of CVB5 RNA during the development of myocarditis or pancreatitis. RESULTS: Marginal inflammatory changes were observed in the heart tissues although viral RNA was constantly present between 1 and 10 days p.i., peaking at 4 days p.i. The pancreatic tissues displayed massive lymphocyte infiltration and loss of acinar cells at day 4 p.i. and viral RNA was detected between 1 and 10 days p.i., peaking at 2-3 days p.i. In the liver, viral RNA was detected between 1 and 7 days. No mortality was observed. CONCLUSIONS: CVB5 induced acute pancreatitis without subsequent development of myocarditis. Clearance of CVB5 RNA from the pancreas and heart was slower than clearance from the liver. Our real-time RT-PCR method, which is more sensitive than conventional plaque assay, may provide valuable insight into viral RNA kinetics during CVB5 infection.

Acute Disease↗
PubMed2006

Quantitative analysis of viral RNA in the murine heart and pancreas with different concentration of coxsackievirus B3.

OBJECTIVES: We investigated the clinical features, pathologic changes, and viral RNA kinetics in the course of acute and subacute experimental coxsackievirus B3 (CVB3) infection in a murine model. METHODS: Five-week-old A/J inbred male mice were divided into 5 groups. Four of those groups were inoculated intraperitoneally with 5 x 10(4) (group 1), 1 x 10(5) (group 2), 5 x 10(5) (group 3), or 1 x 10(6) (group 4) PFU of CVB3. Control mice were inoculated with uninfected Vero cell lysate in DMEM. Mice from each group were sacrificed on days 7 or 14 after inoculation. RESULTS: Bloody diarrhea, earlier weight loss, perianal swelling, and death were correlated with higher viral load. One of ten mice in group 3 and 5 of 10 mice in group 4 died spontaneously between days 4 and 12 after inoculation. All of the remaining 34 mice of infected groups demonstrated extensive pancreatic inflammation. Focal myocarditis developed in only 4 (11.8%) of those 34 subjects. Amylase and creatine kinase activities in the serum were increased in the mice of infected groups. CVB3 RNA was detected in the heart and pancreatic tissue in all subjects. The CVB3 RNA copy number in pancreatic tissue was not correlated with the severity of inflammation. CONCLUSIONS: In the murine model, viral loading dose determines the clinical features of CVB3-induced infection, and the severity of pancreatitis is not correlated with the viral loading dose or tissue level of viral RNA. .

Amylases↗
PubMed1988

Delayed formation of defective interfering particles in vesicular stomatitis virus-infected cells: kinetic studies of viral protein and RNA synthesis during autointerference.

The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.

Animals↗
PubMed1984

Cell cycle-dependent expression of early viral genes in one group of simian virus 40-transformed rat cells.

SV40-transformed FR 3T3 rat cells were previously shown to exhibit different patterns of accumulation of the virus-coded T-antigen. One group of transformants accumulates T-antigen throughout the cell cycle, whereas in another group, only the cells in the G2 phase of the cell cycle are stained by immunofluorescence with anti-T antigen antibodies. We investigated the mechanism involved by determining the amounts of early SV40 RNA during the cell cycle. Cells in the various phases of the cell cycle were sorted from an asynchronously growing population using a flow cytofluorimeter. Determination of the amounts of viral RNA in the different nuclear and cytoplasmic RNA fractions showed that in transformants with a G2-restricted accumulation of T-antigen, viral RNA was present in G2, to some extent in S, but could not be detected in cells in G1. In contrast, equivalent amounts of viral RNA were detected in all the phases of the cell cycle in the other group of transformants. Cell sorting, performed after pulse-labeling the cells for 2 h with [35S]methionine, confirmed that translation of the viral mRNAs occurred only in G2 in the first group of transformants, and throughout the cell cycle in the second group.

Animals↗
PubMed1990

Selection of genome segments following coinfection of chicken fibroblasts with avian reoviruses.

Two avian reoviruses (883 and 176) shown to have distinct growth kinetics were used to coinfect chicken embryonic fibroblasts asynchronously to generate reassortants. More than 300 plaque-derived clones were obtained from passage 3 of two separate coinfections made at different m.o.i. and time intervals between infection and superinfection. The genome electropherotype of each plaque-derived clone was determined, and a diverse group of reassortants were detected. Genome segments 883 M2 and 176 S1 were shown to be preferentially selected. The preferential selection of the 176 S1 segment was shown to be a virus growth-determined nonrandom event conferred by the function of 176 S1 segment, whereas the data suggest that a factor(s) other than viral growth properties was involved in the preferential selection of 883 M2 segment.

Animals↗
PubMed1983

Physiological properties of a T7-T3 recombinant bacteriophage that productively infects strains of Escherichia coli that harbor the F plasmid.

The T7-T3 recombinant phage BO2, whose isolation and physical properties are described in the accompanying paper (Molineux et al., J. Virol. 46:881-894, 1983), has been characterized by physiological means after infection of male (F plasmid-containing) and female strains of Escherichia coli. Single-step growth analyses have shown that the hybrid phage gives a burst about two-thirds that of T7 or T3 on females and about one-half that of T3 on males. This reduced burst size can be correlated with altered kinetics of macromolecular synthesis, probably at the level of transcription. The T3 insertions of BO2, designated M1 and M2, that are essential for growth of the hybrid phage on male strains, have been shown to be trans acting and can rescue T7 from F-mediated restriction. The nature of the gene products encoded by the M1 and M2 regions and their role in overcoming the abortive infection of males by T7 are discussed.

DNA, Viral↗
PubMed2001

Principles of selective inactivation of a viral genome. Comparative kinetic study of modification of the viral RNA and model protein with oligoaziridines.

Comparative kinetic analysis of inactivation of bacteriophage MS2 infectivity and aminoalkylation of a model protein (trypsin inhibitor) with oligoaziridines was performed in order to evaluate the selectivity of viral RNA modification with oligocationic reagents. The transition from ethyleneimine monomer to di-, tri-, and tetramer leads to a sharp increase in the rate constant of infectivity inactivation, whereas the rate constant of protein modification changes insignificantly. The selectivity coefficient of the phage RNA aminoalkylation relative to trypsin inhibitor modification increases in this series by more than an order of magnitude. This effect is probably associated with the strengthening of the reagent binding to the nucleic acid, which implies a reaction mechanism that involves the formation of a reactive intermediate. The latter might be an electrostatic complex of the oligocationic reagent and RNA, the only polyanion in the virion. A pronounced decrease in the rate constant of infectivity inactivation in the presence of multiply charged anions (in phosphate buffer) and a biogenic polyamine (spermine) favors this hypothesis. Increasing the reaction temperature increases the rate constant of infectivity inactivation and decreases selectivity of the viral RNA modification.

Alkylation↗
PubMed1975

RNAs of simian virus 40 in productively infected monkey cells: kinetics of formation and decay in enucleate cells.

We demonstrate here the usefulness of cytochalasin B enucleate cells for the study of the metabolism of cytoplasmic mRNA and for determining its half-life in animal cells. Simian virus 40 infected monkey cells in which the RNA had been labeled with [3H]uridine were enucleated, and the decay of the two prominent RNAs of simian virus 40, the 19S and 16S species, was measured by analysis on sucrose gradients. The results of these experiments, together with kinetic analysis of nuclear and cytoplasmic viral RNA, indicate a precursor-product relationship between the 19S and 16S cytoplasmic viral RNA species, which decay by first-order kinetics with a mean half-life of about 3 hr and 6 hr, respectively.

Animals↗
PubMed1987

Synthesis of plus- and minus-strand RNA in rotavirus-infected cells.

The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus- and minus-strand RNA and double-stranded RNA in simian rotavirus SA11-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and minus-strand RNAs was detected initially at 3 h postinfection. The steady-state levels of plus- and minus-strand RNAs increased from this time until 9 to 12 h postinfection, at which time the levels were maximal. Pulse-labeling of infected cells with [3H]uridine showed that the ratio of plus- to minus-strand RNA synthesis changed during infection and that the maximal level of minus-strand RNA synthesis occurred several hours prior to the peak of plus-strand RNA synthesis. No direct correlation was found between the levels of plus-strand and minus-strand RNA synthesis in the infected cell. Pulse-labelling studies indicated that both newly synthesized and preexisting plus-strand RNA can act as templates for minus-strand RNA synthesis throughout infection. Studies also showed that less than 1 h was required between the synthesis of minus-strand RNA in vivo and its release from the cell within virions.

Animals↗
PubMed2004

[Comparison of hepatitis C viral RNA and core antigen kinetics in the therapeutic follow up of hepatitis C virus and human immunodeficiency virus co-infected patients, treated by bitherapy interferon-ribavirin, within the framework of RIBAVIC protocol].

AIM OF STUDY: The RIBAVIC protocol, established by ANRS in 2001 and closed in 2003, compared the efficacy and the tolerance of two bitherapy anti-Hepatitis C Virus for HIV-HCV co-infected patients: IFN-ribavirin and PEG-IFN-ribavirin for 48 weeks. Two hundred patients from protocol were tested for hepatitis C virus core antigen, to study this viral marker kinetics, before and under treatment, in comparison with hepatitis C virus RNA evolution. MATERIAL AND METHODS: The available samples for the 204 patients of our study were tested for RNA detection (COBAS AMPLICOR v2.0, Roche Diagnostics) and quantification (VERSANT HCV RNA v3.0, Bayer Diagnostics) and for quantification of core antigen (Ortho trak-C Assay, Ortho Clinical Diagnostics). The viral kinetics were established from samples quantified at D0, W2, W4, W12, W24, W48, W52, W72 (W =week), according to virological response assessed by PCR, six month after the end of treatment (non responders, sustained responders, relapsers et breakthroughs). RESULTS: We obtained, for each type of response, similar evolution of both viral markers. Trak-C assay show to be enough sensitive, with similar results whatever genotype of hepatitis C virus. The Pearson's correlation is excellent (R =0.94; P <0.001). The intergenotype correlation is correct too, whatever HCV genotype (1, 2, 3, 4). CONCLUSIONS: The HCV core antigen quantification by trak-C assay is a new tool for the follow-up of the treatment of patients with chronic hepatitis C and HIV co-infected.

Antigens, Viral↗
PubMed1986

Molecular cloning and analysis of three cDNA clones homologous to human cytomegalovirus RNAs present during late infection.

Three virus-specific clones were isolated from a cDNA library synthesized from human cytomegalovirus (AD169)-infected cell RNA and cloned into the expression vector lambda gt11. These clones, designated C3, D10, and H10 were each found to express a human cytomegalovirus/beta-galactosidase fusion protein that was reactive with antibody prepared against purified virions. By using the cloned cDNA, we were able to identify the transcripts that code for each gene product and study the kinetics of expression during permissive infection. Our results suggest that at least two of the RNAs undergo posttranscriptional processing and appear in infected cells at immediate-early times. The authentic C3 gene product was identified by probing Western blots with antibody prepared against fusion protein fpC3.

Cell Line↗
PubMed1983

Identification of adenovirus genes that require template replication for expression.

The relationship between adenovirus type 2 DNA replication and expression of intermediate stage viral genes was investigated. The 1.03-kilobase mRNA from early region 1b (E1b) and the mRNAs coding for proteins IX and IVa2 were first detected between 6 and 8 h postinfection. Inhibition of viral DNA replication with hydroxyurea prevented expression of the IX and IVa2 mRNAs, but not of the E1b mRNA. Pulse-labeling experiments demonstrated that the block of IX and IVa2 expression in hydroxyurea-treated cells was at the level of transcription. By a series of superinfection experiments, it was determined that the viral and cellular factors present during the late stage of adenovirus infection are insufficient to activate IX gene expression. The viral DNA template must first replicate before IX transcription can begin.

Adenoviruses, Human↗