scholarly journals BLV-CoCoMo-qPCR-3: Improved BLV-CoCoMo-qPCR for Bovine Leukemia Virus Detection by Mixing Probes Targeting all BLV Variants

2020 ◽  
Author(s):  
Liushiqi Borjigin ◽  
Shuji Yoneyama ◽  
Susumu Saito ◽  
Polat Meripet ◽  
Michihito Inokuma ◽  
...  
Keyword(s):  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Virus Detection ◽  
Nationwide Survey ◽  
Neoplastic Disease ◽  
Wild Type ◽  
Degenerate Primers ◽  
Assay Sensitivity ◽  
New Strategy ◽  
Bovine Leukosis

Abstract Background: The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease in cattle. The proviral load (PVL) is an important index for estimating disease progression. Previously, we developed a quantitative real-time PCR (qPCR) assay to measure the PVL of BLV using coordination of common motif (CoCoMo) degenerate primers that can amplify all known BLV strains. However, mutations, which potentially affect the detection ability, have been recently reported in the probe sequences of the long terminal regions (LTRs) of the BLV-CoCoMo-qPCR-2 assay. Here, we developed a new strategy to overcome these newly generated mutations located in the probe regions of this assay.Methods: We collected genomic DNA from 887 cows from 27 BLV-positive farms, using a nationwide survey conducted in 2011 and 2017 in Japan. BLV variants were investigated by quantifying the provirus using BLV-CoCoMo-qPCR-2 targeting the BLV LTR gene and the TaKaRa Cycleave PCR system targeting the BLV tax gene. Additionally, we sequenced the partial BLV LTR gene. The modified probes were designed to completely match the three BLV variants identified here, and the modified assay was established using mixed probes.Results: We found four single mutations within the probe region of the original BLV-CoCoMo-qPCR-2 assay, three of which negatively affected its sensitivity. Furthermore, we examined the optimum ratio of the concentration to be mixed with the wild type and three new BLV TaqMan probes were designed here using genomic DNAs extracted from cattle infected with the wild-type BLV strain and cattle infected with variants. Hence, we successfully established an improved BLV-CoCoMo-qPCR-3 assay that uses mixed probes corresponding to all three BLV variants. Conclusions: To overcome the loss of assay sensitivity due to newly emerging variants, we have established the BLV-CoCoMo-qPCR-3 assay that could amplify all BLV strains using newly designed mixed probes in addition to degenerate primers that were previously designed in our original assay. Our proposed method maintained the original sensitivity and reproducibility and can detect all mutant strains; thus, it is a useful tool to prevent the spread of BLV infections, especially those caused by newly emerging variants.

A novel real time PCR assay for bovine leukemia virus detection using mixed probes and degenerate primers targeting novel BLV strains

2021 ◽  
pp. 114264
Author(s):  
Liushiqi Borjigin ◽  
Shuji Yoneyama ◽  
Susumu Saito ◽  
Meripet Polat ◽  
Michihito Inokuma ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Virus Detection ◽  
Pcr Assay ◽  
Degenerate Primers

Strip-dried blood sampling: applicability for bovine leukemia virus detection with ELISA and real-time PCR

2019 ◽  
Vol 263 ◽  
pp. 101-104 ◽  
Author(s):  
Nikolay Yu. Saushkin ◽  
Jeanne V. Samsonova ◽  
Alexander P. Osipov ◽  
Sergey E. Kondakov
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Virus Detection ◽  
Blood Sampling

scholarly journals The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

1999 ◽  
Vol 73 (2) ◽  
pp. 1293-1301 ◽  
Author(s):  
Kazunori Inabe ◽  
Masako Nishizawa ◽  
Shigeru Tajima ◽  
Kazuyoshi Ikuta ◽  
Yoko Aida
Keyword(s):  
Viral Entry ◽  
Envelope Protein ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Transient Transfection ◽  
Viral Envelope ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Viral Envelope Protein

ABSTRACT The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

Distribution of bovine leukemia virus proviral DNA sequences in tissues of animals with enzootic bovine leukosis

1978 ◽  
Vol 2 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Richard Kettmann ◽  
Arsène Burny ◽  
Yvette Cleuter ◽  
Jacques Ghysdael ◽  
Marc Mammerickx
Keyword(s):  
Dna Sequences ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Enzootic Bovine Leukosis ◽  
Proviral Dna ◽  
Bovine Leukosis

Variations in the viral genome and biological properties of bovine leukemia virus wild-type strains

2018 ◽  
Vol 253 ◽  
pp. 103-111 ◽  
Author(s):  
Hironobu Murakami ◽  
Jumpei Uchiyama ◽  
Chihiro Suzuki ◽  
Sae Nikaido ◽  
Kaho Shibuya ◽  
...  
Keyword(s):  
Bovine Leukemia Virus ◽  
Viral Genome ◽  
Leukemia Virus ◽  
Biological Properties ◽  
Wild Type ◽  
Type Strains

Enzootic Bovine Leukosis and Bovine Leukemia Virus

1983 ◽  
pp. 231-245
Author(s):  
G. Marbaix ◽  
R. Kettmann ◽  
J. Deschamps ◽  
D. Couez ◽  
M. Mammerickx ◽  
...  
Keyword(s):  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Enzootic Bovine Leukosis ◽  
Bovine Leukosis

scholarly journals Mutant Tax Protein from Bovine Leukemia Virus with Enhanced Ability To Activate the Expression of c-fos

2002 ◽  
Vol 76 (5) ◽  
pp. 2557-2562 ◽  
Author(s):  
Shigeru Tajima ◽  
Yoko Aida
Keyword(s):  
Bovine Leukemia Virus ◽  
Mutational Analysis ◽  
Leukemia Virus ◽  
Upstream Sequence ◽  
Wild Type ◽  
Cell Leukemia Virus Type ◽  
Mutant Proteins ◽  
Tax Protein ◽  
Human T Cell ◽  
Carg Box

ABSTRACT Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. We previously identified several mutants of the BLV Tax protein with an ability to transactivate transcription via the BLV enhancer that is significantly greater than that of the wild-type Tax protein. Moreover, the mutant proteins also activated other viral enhancers, such as the enhancer of human T-cell leukemia virus type 1, which cannot be activated by wild-type BLV Tax. In this study, we demonstrated that the mutant proteins but not wild-type protein activate the upstream sequence of the human c-fos gene, which contains two major cis-acting elements, the CArG box and cyclic AMP-responsive element (CRE) motif. The mutant protein also strongly increased levels of endogenous c-fos mRNA in both human and bovine cell lines. On the other hand, the wild-type Tax protein has no activity to activate the expression of human c-fos, indicating that wild-type BLV Tax might discriminate between human and bovine c-fos promoter sequences. Deletion and point-mutational analysis of the cis-acting elements revealed that both the CArG box and the CRE motif were indispensable for the activation of c-fos by the mutant BLV Tax protein. Our results suggest that the mutant BLV Tax proteins might not only have the ability to enhance the production of virus particles but might also have increased ability to induce leukemia.

scholarly journals Function and Conformation of Wild-Type p53 Protein Are Influenced by Mutations in Bovine Leukemia Virus-Induced B-Cell Lymphosarcoma

Virology ◽  
1998 ◽  
Vol 243 (1) ◽  
pp. 235-246 ◽  
Author(s):  
Shigeru Tajima ◽  
Wen Zhong Zhuang ◽  
Mitsuo V. Kato ◽  
Kosuke Okada ◽  
Yoji Ikawa ◽  
...  
Keyword(s):  
B Cell ◽  
Bovine Leukemia Virus ◽  
P53 Protein ◽  
Leukemia Virus ◽  
Wild Type ◽  
Wild Type P53

scholarly journals Discordance between Bovine Leukemia Virus Tax Immortalization In Vitro and Oncogenicity In Vivo

2000 ◽  
Vol 74 (21) ◽  
pp. 9895-9902 ◽  
Author(s):  
Jean-Claude Twizere ◽  
Pierre Kerkhofs ◽  
Arsène Burny ◽  
Daniel Portetelle ◽  
Richard Kettmann ◽  
...  
Keyword(s):  
Viral Replication ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type Virus ◽  
Target Cells ◽  
Phosphorylation Sites ◽  
Primary Cells ◽  
Wild Type

ABSTRACT Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

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