Mapping the rubella virus subgenomic promoter.
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Rubella virus (RUB), the sole member of the Rubivirus genus in the Togaviridae family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3' end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5'-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) -175 to +76 relative to the SG start site, including the 3' 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5' Deletions of SGP-2 to nt -40 (9 nt beyond the 3' end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5' deletions to nt -26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt -28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt -28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5' end of SG RNA was also required. Thus, the minimal SGP maps from nt -26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus Alphavirus, the other genus in the Togaviridae family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt -48 and nt -23 with respect to the SG start site in the RUB genome. (+info)
Cytotoxic activity against rubella-infected cells in the supernatants of human lymphocyte cultures stimulated by rubella virus.
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Supernatant fluids of lymphocyte cultures from rubella-seropositive donors, stimulated with inactivated rubella virus, showed cytotoxic activity against rubella-infected target cells (NYU 32 line of human embryonic fibroblasts) but not against uninfected fibroblasts. The time of appearance of cytotoxic activity in rubella-stimulated lymphocyte cultures correlated with increased rate of DNA synthesis as measured by thymidine uptake. No such cytotoxic activity became detectable in the supernatants of lymphocyte cultures from rubella-seronegative donors cultured in the presence of rubella virus, or in unstimulated lymphocyte cultures from seropositive or seronegative donors. The cytotoxic activity was lost at 60degreesC in 30 min. In contrast to this rubella virus-induced cytotoxic activity, cytotoxin produced in mitogen-stimulated lymphocyte cultures from rubella seropositive and seronegative donors was equally cytocidal against rubella-infected and uninfected human fibroblasts. Although the nature of cytotoxic activity remains to be characterized, it is suggested that it is associated with a lymphokine released immune-specifically from rubella virus-stimulated lymphocytes. (+info)
Simple procedure for the removal of nonspecific inhibitors of rubella virus hemagglutination.
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The adsorption of serum lipoproteins onto an insoluble matrix of colloidal silicic acid results in the removal of nonspecific inhibitors of rubella virus hemagglutinin. The procedure can be performed in 15 min at room temperature. Comparative studies using both the dextran sulfate-CaCl2 and heparin-MnCl2 methods for removal of inhibitors demonstrated that the colloidal silicic acid procedure yielded identical hemagglutination inhibition titers. In addition, it is technically feasible to read titers below 1:8. (+info)
Apoptosis induction by the Therien and vaccine RA27/3 strains of rubella virus causes depletion of oligodendrocytes from rat neural cell cultures.
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The induction of cell death by the Therien strain of rubella virus (RVT), and the vaccine RA27/3 strain, was investigated in mixed glial cell cultures derived from the rat CNS. Cell death induction in Vero and rat glial cells by RVT and RA27/3 was dependent on virus replication. In both cell types and for both virus strains, cell death induction had the hallmarks of apoptosis, as detected by DNA laddering, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling staining and Annexin V staining. For rat mixed glial cells, the depletion of oligodendrocytes was due to the induction of apoptosis for both virus strains. The induction of apoptosis in H358a cells, which carry a homozygous deletion of the p53 gene, indicated that a p53-independent pathway can be involved. The induction of cell death by RVT and RA27/3 in Vero and rat glial cells was associated with caspase-3 activity. It is concluded that rubella virus (RV) induces apoptosis in oligodendrocytes in rat glial cell cultures by a caspase-dependent pathway and that similar mechanisms occur for both the RVT laboratory strain and the vaccine RA27/3 strain. The tropism of both strains of RV for oligodendrocytes and the induction of apoptosis in such cells may have important implications for the mechanism of virus neuropathogenesis. (+info)
Seroprevalence of rubella among women of childbearing age in Taiwan after nationwide vaccination.
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Taiwan initiated a nationwide program in 1986 to have all 15-year-old schoolgirls vaccinated against rubella and another program in 1992 to encourage all women of childbearing age to receive rubella vaccination. To assess the immunity among women after the implementation of these programs, we conduct a serosurvey. We recruited women who were 15-44 years old and received pre-employment health examinations at the clinic of an industrial park from January 1 to June 30, 2000. Anti-rubella antibody titers were determined by enzyme-linked immunoassays. All 1,087 women who fit the selection criteria agreed to participate, and the overall susceptible (seronegative) rate was 5.7%. The susceptible rate was much lower among women who were covered by both programs than women who were not (4% versus 23%, P < 0.001). The nationwide vaccination programs were effective, but a substantial proportion of childbearing-age women were still susceptible and need booster vaccination. (+info)
Detection of low-avidity immunoglobulin G in oral fluid samples: new approach for rubella diagnosis and surveillance.
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Low-avidity rubella immunoglobulin G (IgG) was detected in oral fluid samples from 30 of 32 rubella IgM-positive patients (sensitivity, 94%) and from 4 of 34 IgM-negative patients (specificity, 88%). Measuring IgG avidity in oral fluid samples could improve the reliability of rubella surveillance when the incidence of the disease and the positive predictive value of IgM tests are low. (+info)
Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication.
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Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs. (+info)
Rubella 1974 and its aftermath, congenital rubella syndrome.
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An epidemic of rubella reached its peak in the Atlantic provinces in 1974, subsiding in early 1975. With the exception of Quebec the remainder of Canada showed a reverse trend, with a large increase in the numbers of cases reported in the first 41/2 months of 1975. The Halifax virus laboratory reported 106 serologically proven cases of rubella in 1974, 44 of them in pregnant women. In the aftermath of the epidemic many infants were born with the congenital rubella syndrome (CRS). A study carried out from Sept. 1, 1974 through Apr. 30, 1975 showed an 80% correlation between clinical diagnosis and the presence of rubella-specific IgM antibodies in 35 of these infants. Of the 23 infants in whom the diagnosis of CRS was made by laboratory or clinical findings or both, laboratory criteria were met in 20 (87.0%), clinical criteria in 19 (82.6%) and both laboratory and clinical criteria in 16 (69.6%). (+info)