Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease PCR using a 3′ minor groove binder fluorogenic probe

Lew, Ala E.; Bock, R.E.; Molloy, J.B.; Minchin, Catherine M.; Robinson, Susan; Steer, Penelope A.

doi:10.1016/j.jviromet.2003.09.029

Cited by 41 publications

(36 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…To correct for differences in DNA concentrations and amplification efficiencies between samples, the 18S ribosomal DNA was quantified in parallel as described in ref. 24. Under these conditions, the sensitivity of the technique was below one viral copy detected in 100 ng of DNA.…”

Section: Ex Vivo Short-term Cultures Of Peripheral Blood Mononuclear mentioning

confidence: 90%

“…One hundred nanograms of genomic DNA were used for real-time PCR amplification of BLV proviral sequences essentially as described in ref. 24. A standard curve was generated after amplification of defined proviral copy numbers (from 1 to 10 7 of plasmid pBLV344) diluted in 100 ng of control genomic DNA.…”

Section: Ex Vivo Short-term Cultures Of Peripheral Blood Mononuclear mentioning

confidence: 99%

See 1 more Smart Citation

Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo

Achachi

Florins

Gillet

et al. 2005

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

View full text Add to dashboard Cite

show abstract

Section: Ex Vivo Short-term Cultures Of Peripheral Blood Mononuclear mentioning

confidence: 90%

Section: Ex Vivo Short-term Cultures Of Peripheral Blood Mononuclear mentioning

confidence: 99%

Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo

Achachi

Florins

Gillet

et al. 2005

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

View full text Add to dashboard Cite

show abstract

“…In contrast, R3-G4 mutant proviruses were nonpathogenic in 19 animals but not in sheep 245. In this animal, the proviral loads, as determined by real-time PCR (18), increased sharply 6 years postinoculation, reaching almost 6,000 viral genome copies per mm 3 of blood (Fig. 1A).…”

mentioning

confidence: 84%

Even Attenuated Bovine Leukemia Virus Proviruses Can Be Pathogenic in Sheep

Florins

Gillet

Boxus

et al. 2007

J Virol

View full text Add to dashboard Cite

Based on a reverse genetics approach, we previously reported that bovine leukemia virus (BLV) mutants harboring deletions in the accessory R3 and G4 genes persist at very low proviral loads and are unable to induce leukemia or lymphoma in sheep, indicating that these R3 and G4 gene sequences are required for pathogenesis. We now show that lymphoma can occur, albeit infrequently (1 case of 20) and after extended periods of latency (7 years). Direct sequencing and reinfection experiments demonstrated that lymphomagenesis was not due to the reversion of the mutant to the wild type. Similar observations with another type of attenuated mutant impaired in the transmembrane protein (TM) YXXL signaling motifs were made. We conclude that the R3 and G4 genes and the TM YXXL motifs are not strictly required for pathogenesis but that their integrity contributes to disease frequency and latency.Viral persistence results from a dynamic interplay between the host immune defense and the replicative capacity of the virus. Bovine leukemia virus (BLV) replication occurs via two main processes: the infection of new cellular targets and the mitosis of the host cell (recently reviewed in references 7 and 11). Although these two mechanisms can theoretically occur simultaneously, the former operates mainly in the early stages of infection, i.e., the seroconversion period. In contrast, viral spread by mitotic cell division accounts for most of the proviral loads during the asymptomatic and leukemic phases (20). Amazingly, the leukemic clone that generates the tumor originates from a cell that was infected soon after seroconversion. It seems, therefore, that the premalignant cell clone and/or its progeny persist throughout the long latency period. However, the molecular mechanisms that govern viral persistence and spread are still largely unknown.In an attempt to define the viral determinants required for transformation, we first established a model system based on the infection of sheep with a cloned BLV provirus (36). Among a series of proviruses, the 344 strain recapitulated all aspects associated with a lymphoproliferative disease followed by the onset of leukemia, lymphoma, or lymphosarcoma. In order to discover the role of viral genes in the leukemogenesis process (which includes all events leading to leukemia, lymphoma, or lymphosarcoma), we used reverse genetics by introducing directed deletions, insertions, and point mutations into the 344 proviral clone (35). By this approach, we previously demonstrated that the R3 and G4 accessory genes are dispensable for infection but are required for the maintenance of high proviral loads (34). Furthermore, the G4 protein, but not R3, exhibits transforming potential in primary rat embryo fibroblasts when coexpressed with the Ha-ras oncogene (15). Indeed, the G4-and Ha-ras-double-positive cells form transformed foci in cell cultures and induce tumors in nude mice. Mechanistically, G4

show abstract

“…Real-time RT-PCR was done using the Taqman MGB method (24). The primers and probe were designed using Primer Express software (version 2.0; PE Applied Biosystems).…”

Section: Translational Relevancementioning

confidence: 99%

Molecular Diagnosis of Sentinel Lymph Node Metastases in Cervical Cancer Using Squamous Cell Carcinoma Antigen

Yuan

Xiaoming

Jia³

et al. 2008

Clinical Cancer Research

View full text Add to dashboard Cite

Purpose: To clarify the prognostic value of molecular diagnosis of SLN metastases in cervical cancer using SCCA. Experimental Design: All SLNs and primary tumors, part of non-SLNs, were harvested from 36 patients with cervical cancer. Expression levels of SCCA, cytokeratin 19 (CK19), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in 178 samples (29 primary tumors, 5 histologic positive nodes, 60 histologic negative SLNs, 69 non-SLNs, and 15 normal nodes) were assessed by quantitative reverse transcription-PCR assay. The quantitative value of SCCA or CK19 mRNA was described as each value relative to GAPDH mRNA. The cutoff value was set at the upper limit of the quantitative value of nodes from noncancer patients, and those above this value constituted the molecular metastasis group. Results: The SCCA mRNA expression values were more than 1 Â10 3 in 28 primary tumors and all histologic positive nodes, and its expression levels in SLNs were higher than in non-SLNs. SLNs from patients with adverse prognostic features had higher SCCA mRNA expression levels. Four histologic negative SLNs were diagnosed molecular metastases based on SCCA mRNA. Two cases with histologically uninvolved pelvic nodes recurred. Survival analysis indicates that molecular lymphatic metastasis based on elevated SCCA mRNA level is the best predictor of recurrence. However, CK19 is not a suitable marker due to its low specificity and relative higher baseline expression in normal nodes. Conclusions: SCCA mRNA levels for molecular diagnosis of SLN metastases in cervical cancer more accurately identifies patients at risk for recurrence than the routine histology does.

show abstract

Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease PCR using a 3′ minor groove binder fluorogenic probe

Cited by 41 publications

References 35 publications

Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo

Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo

Even Attenuated Bovine Leukemia Virus Proviruses Can Be Pathogenic in Sheep

Molecular Diagnosis of Sentinel Lymph Node Metastases in Cervical Cancer Using Squamous Cell Carcinoma Antigen

Contact Info

Product

Resources

About