Detection of bovine leukemia virus proviral DNA in individual cells.

Mirsky, Michael; Da, Yang; Lewin, Harris A.

doi:10.1101/gr.2.4.333

Cited by 25 publications

(34 citation statements)

References 35 publications

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“…Approximately 30% of infected cattle develop persistent lymphocytosis, a polyclonal expansion of B lymphocytes (31,32). The development of persistent lymphocytosis, in which the absolute number of lymphocytes (33) and the percentage of infected lymphocytes are dramatically increased (34), markedly enhances the probability of transmission (35). The critical importance ofpersistent lymphocytosis in BLV transmission was shown by experiments in which <0.3 ,ul ofblood from cattle with persistent lymphocytosis could transmit BLV, while >1 ml of blood was necessary to transmit from infected but nonlymphocytotic cattle (35).…”

Section: Discussionmentioning

confidence: 99%

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Cantor

McElwain

Birkebak

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammerhead catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves >80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.Bovine leukemia virus (BLV), a retrovirus structurally similar to human T-cell leukemia viruses I and II (HTLV-I and -II), causes persistent lymphocytosis and B-lymphocyte lymphoma in cattle and sheep (1). After initial infection, BLV expresses a doubly spliced transcript encoding the regulatory proteins Rex and Tax (2). Tax trans-activates the viral long terminal repeat and also cellular promoters, including c-fos and somatostatin (3). Cotransfection experiments have shown that Tax is necessary for viral expression in vitro (4). Rex regulates the transition from early expression of the doubly spliced transcript encoding regulatory proteins to the later expression of singly spliced or unspliced transcripts that express the env gene or the gag and pol genes (5). Recently, the 3' region of HTLV-I and BLV has been shown to encode RNA with alternative splice patterns that may express other regulatory proteins (6-8).Because of the critical role of regulatory proteins such as Tax and Rex in the HTLV/BLV group of viruses, we hypothesized that the rex/tax mRNA would be a rational target for ribozyme-mediated inhibition. The hammerhead motif, first identified in plant RNA pathogens (9, 10), cleaves the phosphodiester bond downstream of a GUC triplet (9,10). By flanking the hammerhead motif with antisense sequences, Haseloff and Gerlach (11) demonstrated cleavage of specific target RNAs. In this study, we demonstrate a ribozyme that cleaves rex/tax mRNA and, when transfected into BLV-infected cells, markedly inhibits viral expression.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact...

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Section: Discussionmentioning

confidence: 99%

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Cantor

McElwain

Birkebak

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…2,17,34 Polymerase chain reaction (PCR) is an alternative that aims at the detection of integrated proviral DNA 1,7,9 and is considered to be highly sensitive. 16,25,28 The readily obtained results and relative ease of implementation make PCR an attractive alternative to serology. 3 A diagnostic test is a tool to indicate the presence or absence of a specific disease or condition in an individual from a specific population.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a New Antibody-Based Enzyme-Linked Immunosorbent Assay for the Detection of Bovine Leukemia Virus Infection in Dairy Cattle

et al. 2005

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Abstract. The objective of this study was to validate a new blocking enzyme-linked immunosorbent assay (ELISA) (designated M108 for milk and S108 for serum samples) for detecting bovine leukemia virus (BLV) infection in dairy cattle. Milk, serum, and ethylenediaminetetraacetic acid-blood samples were collected from 524 adult Holstein cows originating from 6 dairy herds in Central Argentina. The M108 and S108 were compared with agar gel immunodiffusion (AGID), polymerase chain reaction and a commercial ELISA. Because there is currently no reference test capable of serving as a gold standard, the test sensitivity (SE) and specificity (SP) were evaluated by the use of a latent class model. Statistical inference was performed by classical maximum likelihood and by Bayesian techniques. The maximum-likelihood analysis was performed assuming conditional independence of tests, whereas the Bayesian approach allowed for conditional dependence. No clear conclusion could be drawn about conditional dependence of tests. Results with maximum likelihood (under conditional independence) and posterior Bayes (under conditional dependence) were practically the same. Conservative estimates of SE and SP (with 95% confidence intervals) for M108 were 98.6 (96.7; 99.6) and 96.7 (92.9; 98.8) and for S108 99.5 (98.2; 99.9) and 95.4 (90.9; 98.1), respectively. The ELISA 108 using either milk or serum to detect BLV-infected animals had comparable SE and SP with the official AGID and a commercial ELISA test, which are currently the most widely accepted tests for the serological diagnosis of BLV infection. Therefore, ELISA 108 can be used as an alternative test in monitoring and control programs.

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“…PGE 2 suppresses antigen-specific PBMC proliferation. To determine if PBMC proliferation and BLV expression were correlated, different concentrations of PGE 2 were added to the PBMC cultures with or without BL3* supernatant as a viral antigen source (23). Following incubation of PBMCs for 3 to 5 days, cells were harvested for further experiments.…”

mentioning

confidence: 99%

Prostaglandin E₂Increases Bovine Leukemia VirustaxandpolmRNA Levels via Cyclooxygenase 2: Regulation by Interleukin-2, Interleukin-10, and Bovine Leukemia Virus

Pyeon¹,

Diaz

Splitter³

2000

J Virol

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Prostaglandin E 2 (PGE 2 ), produced by macrophages, has important immune regulatory functions, suppressing a type 1 immune response and stimulating a type 2 immune response. Type 1 cytokines (interleukin-2 [IL-2], IL-12, and gamma interferon) increase in freshly isolated peripheral blood mononuclear cells (PBMCs) of animals with an early disease stage of bovine leukemia virus (BLV) infection, while IL-10 increases in animals with a late disease stage. Although IL-10 has an immunosuppressive role in the host immune system, IL-10 also inhibits BLV tax and pol mRNA levels in vitro. In contrast, IL-2 stimulates BLV tax and pol mRNA and p24 protein expression in cultured PBMCs. The inhibitory effect of IL-10 on BLV expression depends on soluble factors secreted by macrophages. Thus, we hypothesized that PGE 2 , a cyclooxygenase 2 (COX-2) product of macrophages, may regulate BLV expression. Here, we show that the level of COX-2 mRNA was decreased in PBMCs treated with IL-10, while IL-2 enhanced the level of COX-2 mRNA. Addition of PGE 2 stimulated BLV tax and pol mRNA levels and reversed the IL-10 inhibition of BLV mRNA. In addition, the specific COX-2 inhibitor, NS-398, inhibited the amount of BLV mRNA detected. Addition of PGE 2 increased BLV tax mRNA regardless of NS-398 addition. PGE 2 inhibited antigen-specific PBMC stimulation, suggesting that stimulation of BLV tax and pol mRNA levels by PGE 2 is independent of cell proliferation. These findings suggest that macrophage-derived COX-2 products, such as PGE 2 , regulate virus expression and disease progression in BLV infection.

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Detection of bovine leukemia virus proviral DNA in individual cells.

Cited by 25 publications

References 35 publications

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Evaluation of a New Antibody-Based Enzyme-Linked Immunosorbent Assay for the Detection of Bovine Leukemia Virus Infection in Dairy Cattle

Prostaglandin E₂Increases Bovine Leukemia VirustaxandpolmRNA Levels via Cyclooxygenase 2: Regulation by Interleukin-2, Interleukin-10, and Bovine Leukemia Virus

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Detection of bovine leukemia virus proviral DNA in individual cells.

Cited by 25 publications

References 35 publications

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Evaluation of a New Antibody-Based Enzyme-Linked Immunosorbent Assay for the Detection of Bovine Leukemia Virus Infection in Dairy Cattle

Prostaglandin E2Increases Bovine Leukemia VirustaxandpolmRNA Levels via Cyclooxygenase 2: Regulation by Interleukin-2, Interleukin-10, and Bovine Leukemia Virus

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Prostaglandin E₂Increases Bovine Leukemia VirustaxandpolmRNA Levels via Cyclooxygenase 2: Regulation by Interleukin-2, Interleukin-10, and Bovine Leukemia Virus