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Recombinational joints in a simian virus 40 variant generated in a persistent infection.

SP1, a viable simian virus 40 (SV40) variant isolated from a persistent infection of rhesus monkey kidney cells, contains sequence rearrangements in the untranslated region of the SV40 genome which are transcribed into late mRNA leader sequences and in the region which encodes the large T antigen. Nucleotide sequences about the recombinational junctions in SP1 were determined. The sequence data show that in most instances there was not extensive homology between recombining sequences. The recombinant sequences are discussed with respect to the mechanisms by which they might have been generated.

Animals↗

Monoclonal antibody immunotherapy in nude mice persistently infected with Cryptosporidium parvum.

Three groups of congenitally athymic nude mice were persistently infected following oral administration of 2 x 10(7) Cryptosporidium parvum oocysts. Two groups were treated once daily for 10 days with either neutralizing monoclonal antibody (MAb) 17.41 or an isotype control MAb. The third group received no treatment. Intestinal-infection scores were significantly decreased in nude mice treated with MAb 17.41 compared with isotype control MAb-treated and nontreated control groups (P less than 0.005). Biliary and pancreatic cryptosporidial-infection scores were similar for the MAb 17.41-treated and isotype control MAb-treated groups (P greater than 0.05).

Animals↗

Enhancement of Borna disease virus transcription in persistently infected cells by serum starvation.

Transcription of Borna disease virus (BDV) in persistently infected MDCK (MDCK/BDV) cells increased in the fetal bovine serum free media as detected by Northern blot analysis. Especially, the amount of 1.9-kb RNA without cap formation at the 5' end and polyadenylation at the 3' end, increased as compared to other mRNA molecules of BDV. Growth arrest of MDCK/BDV cells observed in the condition of serum starvation might be important for increasing viral transcription. Since N-cadherin is the responsible factor for cell-to-cell contact, MDCK/BDV cells were cultured in calcium free medium which inhibits the interaction of N-cadherin. However, inhibition of cell-to-cell contact by N-cadherin is not effective on up regulation of viral transcription. Our finding in this study indicates that enhancement of BDV transcription by serum starvation is a useful technique for further investigation in understanding of mechanisms of BDV transcription.

Animals↗

Ultrastructure of porcine circovirus in persistently infected PK-15 cells.

The ultrastructure of porcine circovirus was examined in persistently infected porcine kidney (PK)-15 cells. Virus-infected PK-15 cells had large numbers of intracytoplasmic inclusions, and a few cells had intranuclear inclusions. Intracytoplasmic inclusions were dispersed throughout the cytoplasm but were most numerous in the perinuclear cytoplasm. Inclusion were of various sizes, round to oval, and electron dense and were of two general types. Inclusions of the first type were small (0.1-0.5 microm diameter), not surrounded by trilaminar membranes, and granular with indistinct margins that blended with surrounding cytoplasm. Some contained 12+/-2-nm-diameter icosahedral virions in loose aggregates or rarely forming paracrystalline arrays. Small inclusions could be sites of viral assembly or maturation. Intracytoplasmic inclusions of the second type were larger (0.5-5.0 microm diameter) and more numerous and had abrupt margins surrounded by trilaminar membranes. They were more electron dense than small inclusions and were heterogeneous, containing various proportions of aggregated virions, electron-dense crystalline lamellae of 5 nm periodicity, and/or whorls of myelinoid membranes. Virions usually formed paracrystalline arrays and occasionally were loosely aggregated. Larger inclusions were typical of autophagolysosomes. Intranuclear inclusions were not membrane bound and were often associated with reticulated nucleoli or aggregates of heterochromatin. Some inclusions were irregularly shaped aggregates of indistinct, circular 10-12-nm-diameter viruslike particles. Others were 0.1-1.0 microm in diameter, round or ring shaped, dense, and finely granular, with sharply demarcated margins.

Animals↗

Five hours to identify immunotolerant cattle, persistently infected with bovine virus diarrhoea virus.

Detection of animals which are persistently-infected with bovine virus diarrhoea virus (BVDV) is of prime importance in the control of pestivirus infections in cattle, as these animals constitute the main reservoir of the virus. Identification of such animals can be readily performed using crude whole blood samples with a sandwich enzyme-linked immunosorbent assay (ELISA) requiring only approximately five hours. This ELISA uses a combination of monoclonal antibodies as the capture agent and an immunological amplification step of the specific signal for detecting the non-structural 80/120 kDa protein of BVDV. The degree of correlation between this ELISA and virus isolation as the reference method is 100% for animals older than six months.

Animals↗

Vaccination to prevent persistent viral infection.

Persistent virus infections are increasingly being recognized as a significant cause of human morbidity and mortality. To establish persistence, a virus must establish infection and evade eradication by the host immune response, in particular by cytotoxic T lymphocytes (CTL). We have studied a virus that establishes persistence in part by suppressing the CTL response of the infected host. The virus persists in many cell types, including lymphocytes and macrophages. We show that prior immunization with a vaccine designed to induce CTL (in the absence of antiviral antibody) confers complete protection against subsequent establishment of persistence in all tissues analyzed. The vaccine can be designed to express as few as 10 amino acids of a viral protein that comprise the CTL epitope. Further, two CTL epitopes for two discrete MHC haplotypes can be successfully used in a single vaccine that protects both strains of mice. Hence, a "string of CTL epitopes" (beads) concept for vaccination is feasible. Finally, the CTL vaccine provided protection against the establishment of persistence by an immunosuppressive virus.

Amino Acid Sequence↗

Coexistence in lactate dehydrogenase-elevating virus pools of variants that differ in neuropathogenicity and ability to establish a persistent infection.

Neuropathogenic isolates of lactate dehydrogenase-elevating virus (LDV) differ from nonneuropathogenic isolates in their unique ability to infect anterior horn neurons of immunosuppressed C58 and AKR mice and cause paralytic disease (age-dependent poliomyelitis [ADPM]). However, we and others have found that neuropathogenic LDVs fail to retain their neuropathogenicity during persistent infections of both ADPM-susceptible and nonsusceptible mice. On the basis of a segment in open reading frame 2 that differs about 60% between the neuropathogenic LDV-C and the nonneuropathogenic LDV-P, we have developed a reverse transcription-PCR assay that distinguishes between the genomes of the two LDVs and detects as little as 10 50% infectious doses (ID50) of LDV. With this assay, we found that LDV-P and LDV-C coexist in most available pools of LDV-C and LDV-P. For example, various plasma pools of 10(9.5) ID50 of LDV-C/ml contained about 10(5) ID50 of LDV-P/ml. Injection of such an LDV-C pool into mice of various strains resulted in the rapid displacement in the circulation of LDV-C by LDV-P as the predominant LDV, but LDV-C also persisted in the mice at a low level along with LDV-P. We have freed LDV-C of LDV-P by endpoint dilution (LDV-C-EPD). LDV-C-EPD infected mice as efficiently as did LDV-P, but its level of viremia during the persistent phase was only 1/10,000 that observed for LDV-P. LDV-permissive macrophages accumulated and supported the efficient replication of superinfecting LDV-P. Therefore, although neuropathogenic LDVs possess the unique ability to infect anterior horn neurons of ADPM-susceptible mice, they exhibit a reduced ability to establish a persistent infection in peripheral tissues of mice regardless of the strain. The specific suppression of LDV-C replication in persistently infected mice is probably due in part to a more efficient neutralization of LDV-C than LDV-P by antibodies to the primary envelope glycoprotein, VP-3P. Both neuropathogenicity and the higher sensitivity to antibody neutralization correlated with the absence of two of three N-linked polylactosaminoglycan chains on the ca. 30-amino-acid ectodomain of VP-3P, which seems to carry the neutralization epitope(s) and forms part of the virus receptor attachment site.

Amino Acid Sequence↗

Characteristics of a persistent infection in Madin-Darby canine kidney cells with influenza C virus.

Influenza C/Ann Arbor/1/50 was used to establish a persistent infection in Madin-Darby canine kidney cells. The persistent state has been stable for more than 4 years (over 66 passages), with infected cells differing from controls in morphology and growth characteristics. In addition, virus recovered from the media of persistent cultures (passage 58) differed from parental wild-type virus C/Ann Arbor/1/50 in (i) antigenicity by the hemagglutination-inhibition test; (ii) its ability to produce plaques in different host cells; (iii) hemagglutination of chicken erythrocytes at different temperatures; (iv) receptor-destroying enzyme activity, and (v) by sensitivity to hemagglutination inhibitors present in rat serum.

Animals↗

Analysis of ehrlichial p28 gene expression in a murine model of persistent infection.

Historically, ehrlichioses were tick-borne diseases of veterinary medical importance and are now important emerging infectious diseases in humans. p28s are encoded by multigene families with ORFs tandemly arranged with intergenic spaces of variable lengths. We reported initial sequencing of the Ehrlichia muris p28 locus. A model of persistent infection was described and provided tools for study of persistent ehrlichial infection. We completed the sequence of the E. muris p28 locus and examined mRNA expression.

Animals↗

Expression and translocation of chlamydial protease during acute and persistent infection of the epithelial HEp-2 cells with Chlamydophila (Chlamydia) pneumoniae.

Chlamydial protease-like activity factor (CPAF) is secreted to the cytoplasm of the infected cells where it proteolytically cleaves eukaryotic transcription factor RFX5. Here, we determined the localization pattern of CPAF during the course of an acute and persistent in vitro infection of the epithelial cell line HEp-2 with Chlamydophila pneumoniae strain VR1310. Using immunoblotting, confocal microscopy and electron microscopy, we found CPAF in the inclusion lumen or associated with bacteria during the first 48 h of an acute infection. Seventy-two hours and later, CPAF was present predominantly in the cytoplasm of the infected cells. Translocation of CPAF into cytoplasm correlated in time with degradation of the transcription factor RFX5, as confirmed by immunoblotting. Interestingly, during the persistent infection induced by either IFN-gamma or iron limitation CPAF translocation to the cytoplasm was inhibited resulting in unaffected or only partially reduced levels of RFX5. Based on presented findings, we propose that CPAF translocation to the cytoplasm is separated from its production. The translocation mechanism appears to be fully active during an acute infection; however, it is fully or partially inhibited during persistent infection induced by IFN-gamma or by iron limitation respectively. Consequently, this work demonstrates the importance of subcellular localization of CPAF for the characteristics of chlamydial acute and persistent infection in epithelial HEp-2 cells.

Biological Transport, Active↗

Persistent infection of mice with the virus of lymphocytic choriomeningitis: virus-specific immunological tolerance.

Lymphocytic choriomeningitis (LCM) virus-infected culture cells as targets were markedly reduced in numbers when incubated in vitro with spleen cells from LCM virus-immune mice, even if the cells were taken months after a subcutaneous immunizing infection of the donor animal. Spleen cells from mice persistently infected with LCM virus had no effect on target cells. Also, mitomycin C-blocked LCM virus-infected culture cells stimulated the incorporation of radiolabeled thymidine into spleen cells from LCM virus-immune mice. No stimulation was observed with spleen cells from LCM virus carrier mice. It was concluded that cell-mediated immunity directed against LCM virion surface antigen(s) is absent in LCM virus carrier mice.

Journal Article↗

Persistent infection of BHK-21 cells with pneumonia virus of mice.

Trypsinization of BHK-21 cells 72 h after primary infection with pneumonia virus of mice yielded clones of persistently infected cells which specifically adsorbed murine erythrocytes. We describe one clone of cells, the progeny of which, after more than 100 passages, still bore viral antigen demonstrable by immunofluorescence and immune electron microscopy, but produced little or no detectable infectious virus.

Animals↗

Response of cells persistently infected with arenaviruses to superinfection with homotypic and heterotypic viruses.

Vero cell cultures persistently infected with the arenaviruses Junin, Pichinde, Tacaribe, and Tamiami were established and designated Vero-Jun, Vero-Pic, Vero-Tac, and Vero-Tam, respectively. Two types of carrier cultures could be easily distinguished: Vero-Jun and Vero-Tac systems were characterized by a lack of infectious virus production after a few cell transfers, whereas a more productive state with continuous release of virus was observed in Vero-Pic and Vero-Tam cultures. These differences appeared to be related to resistance of the culture to viral superinfection. In fact, Vero-Jun and Vero-Tac cultures totally excluded only the replication of the serologically more closely related arenaviruses Amapari, Junin, or Tacaribe, while the refractoriness of Vero-Pic and Vero-Tam cultures was extended to most of the virus group members. The resistance of Vero-Jun cells to superinfection by Junin or Tacaribe virus could be ascribed to the production of specific uv-resistant Junin interfering particles, which showed a specific range of interference against Junin and Tacaribe viruses. Interfering particles against homotypic and heterotypic arenaviruses were isolated from Vero-Pic cultures. However, the degree of interference developed by these Pic-interfering particles was not enough to fully explain reinfecting virus exclusion from Vero-Pic cultures. Viral susceptibility of persistent cultures is proposed as a useful tool to examine relationships of members of the arenavirus group.

Animals↗

Persistent infection with a nontransforming RNA virus leads to impaired growth factor receptors and response.

The potential role of viral persistence with nontransforming viruses on cellular growth and cellular function has received little attention. We found that when infected with type 3 reovirus (five plaque-forming units (PFU/cell), balb/C 3T3 cells (a mouse embryo fibroblast cell line) undergo a limited lytic phase. The surviving cells, about 90% of the original cells, appear morphologically normal by light microscopy and exhibit normal growth patterns in serum-supplemented medium but are persistently infected by electron microscopy. These persistently infected cells shed infectious virus in the culture medium (1.6-60 X 10(6) PFU per 10(6) cells per 24 h). In comparison to control uninfected 3T3 cells, the persistently infected cells exhibit a 70-90% decrease in receptor number for epidermal growth factor (EGF). This occurs without production of any EGF-like material and is associated with a parallel decrease in EGF-stimulated DNA synthesis. By contrast, insulin receptors are increased in number three-fold and insulin and serum stimulated DNA synthesis are comparable to control uninfected cells. These results suggest that persistent infection with a nontransforming virus may lead to major alteration in control of cell growth by specific growth factors.

Animals↗

Surface antigens on HeLa cells persistently infected with HVJ (Sendai virus).

Surface antigens of HeLaHVJ cells, a cell line persistently infected with HVJ, were studied by fluorescent antibody staining. After absorption with concentrated HVJ virions and HeLa cells, anti-HeLaHVJ antiserum was able to demonstrate specific surface fluorescence on HeLaHVJ cells, while this serum no longer reacted with original HeLa cells nor with HVJ virions. During cytolytic infection of HeLa cells with HVJ, this specific surface antigen appeared at an early stage of infection prior to the appearance of newly synthesized HVJ viral antigens and moreover appeared in spite of the inhibition of viral protein synthesis. This antigen was detected neither on HeLa cells infected with other myxoviruses except HVJ nor on various other kinds of cells infected with HVJ. The specific surface antigen was still found on the HeLaHVJ cell surface after incubation at 38 degrees C for two days, while HVJ structural antigens on the cell surface no longer could be detected. Mild short-term treatment of HeLa cells with trypsin, neuraminidase from vibrio cholerae, phospholipase-C and hyaluronidase failed to expose specific antigen. The antigen was distinguishable from the Forssman and human blood type antigens. The mechanism of appearance of a new antigen on the surface of HeLaHVJ cells remains unclear.

Antigens↗

Expression of human immunodeficiency virus type 1 (HIV-1) gag antigens on the surface of a cell line persistently infected with HIV-1 that highly expresses HIV-1 antigens.

MT-4 cells persistently infected with human immunodeficiency virus type 1 (HIV-1) (MT-4/HIV-1) were recently isolated (K. Ikuta, C. Morita, M. Nakai, N. Yamamoto, and S. Kato, Japan. J. Cancer Res. (Gann), 79, 418-423, 1988). Mouse hybridoma cell clones producing monoclonal antibodies (MoAbs) to HIV-1 gag p24 and p18, and pol reverse transcriptase (RT) were isolated by using this MT-4/HIV-1 cell line for the screening of MoAb production by the immunofluorescence (IF) test. By indirect IF tests of acetone-fixed cells with these MoAbs, the IF intensities in MT-4/HIV-1 cells were found to be higher than those in the other HIV-1 infected cells, such as MOLT-4/HIV-1, HL-60/HIV-1, and U937/HIV-1 cells. Cell surface expression of the HIV-1 gag p24 and p18 antigens examined by IF and radioimmune techniques with these MoAbs revealed the p24 and p18 antigens to be expressed strongly on the cell surface of MT-4/HIV-1 cells and faintly on the cell surface of MOLT-4/HIV-1 cells, respectively. However, monoclonal antibody isolated in the present study failed to detect pol RT antigen on the surface of MT-4/HIV-1 cells. These results indicate that the gag p24 and p18 antigens are expressed, at least in part, on the surface of HIV-1-infected cells.

Antibodies, Monoclonal↗

Description of a BHK/21 cell line persistently infected with Junin virus: its use in diagnostic procedures.

A BHK/21 cell line persistently infected by an arenavirus is described. During four consecutive passages, 30-45% of the cells showed granular cytoplasmic antigen by indirect immunofluorescence, employing both Argentine hemorrhagic fever convalescent sera and sera from animals immunized with Junin virus. Virus isolated from the cells killed suckling mice but not adult mice and protected guinea pigs against further challenge with the virulent prototype strain of Junin virus. Neutralization tests showed that the virus isolated from the cells was neutralized by anti-Junin virus antisera. The usefulness of this cell line in rapid immunofluorescent serological procedures is described.

Animals↗