BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Open Access
- 2 November 2010
- journal article
- research article
- Published by Springer Science and Business Media LLC in Retrovirology
- Vol. 7 (1), 91
- https://doi.org/10.1186/1742-4690-7-91
Abstract
Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals. Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. Using our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.Keywords
This publication has 46 references indexed in Scilit:
- Phylogenetic analysis of bovine leukemia viruses isolated in South America reveals diversification in seven distinct genotypesArchiv für die gesamte Virusforschung, 2010
- Risk factors associated with within-herd transmission of bovine leukemia virus on dairy farms in JapanBMC Veterinary Research, 2010
- Bovine leukemia virus can be classified into seven genotypes: evidence for the existence of two novel cladesJournal of General Virology, 2009
- Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load DeterminationJournal of Clinical Microbiology, 2009
- Sensitive and Broadly Reactive Reverse Transcription-PCR Assays To Detect Novel ParamyxovirusesJournal of Clinical Microbiology, 2008
- Recent developments in the MAFFT multiple sequence alignment programBriefings in Bioinformatics, 2008
- Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in humanRetrovirology, 2007
- Emerging viral diseases and infectious disease risksHaemophilia, 2006
- Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sitesCell, 1978
- Bovine leukemia virus specific antibodies among French cattle. I. Comparison of complement fixation and hematological testsInternational Journal of Cancer, 1977