Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA

Eltahir, Yassir Mohammed; Dovas, Chrysostomos Ι.; Papanastassopoulou, Maria; Koumbati, Maria; Giadinis, Nektarios D.; Verghese-Nikolakaki, S.; Koptopoulos, G.

doi:10.1016/j.jviromet.2006.03.010

Cited by 38 publications

(36 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Over the last decade several PCR assays for SRLV have been developed and published (Zanoni et al, 1992;Rosati et al, 1995;Wagter et al, 1998;Celer et al, 2000;Extramiana et al, 2002;Carrozza et al, 2003;Kuźmak et al, 2003;Álvarez et al, 2006;Eltahir et al, 2006;Gil et al, 2006;Leginagoikoa et al, 2006). The use of PCR in routine settings, however, is still limited because of the low sensitivity attained so far and the difficulties arising from the notorious genomic heterogeneity of the SRLV since lentiviruses are among the most rapidly evolving genomes (Blacklaws et al, 2004;Angelopoulou et al, 2005;De Andrés et al, 2005;Reina et al, 2006), which severely complicates the design of generally applicable oligonucleotides for priming and probing.…”

Section: Introductionmentioning

confidence: 99%

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction

Brinkhof

Maanen

Wigger

et al. 2008

Journal of Virological Methods

View full text Add to dashboard Cite

Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated.Priming oligonucleotides were designed on the highly conserved 5 untranslated leader-gag region while those on the long terminal repeat (LTR) assay were derived from literature. DNA was extracted from the buffycoat interlayer of centrifuged blood samples. Real-time PCR was performed by means of LightCycler technology (Roche Applied Science) using melting temperature analysis (SYBR Green I) for detection. Results were compared with those of serology using samples from Dutch sheep and goat flocks with known SRLV statuses, with sequential samples from a natural transmission experiment and samples from different regions in Norway, France, Spain and Italy.Real-time PCR testing, especially the application of oligonucleotides for priming the leader-gag region appeared promising in detecting SRLV specific proviral DNA in blood samples from both sheep and goats.

show abstract

Section: Introductionmentioning

confidence: 99%

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction

Brinkhof

Maanen

Wigger

et al. 2008

Journal of Virological Methods

View full text Add to dashboard Cite

show abstract

“…OPPV provirus is found in cells of the macrophage and monocyte lineage without causing cellular ablation (13,14). Specifically, OPPV provirus has been detected in many cell types, including lung type I and II pneumocytes, interstitial and alveolar macrophages, endothelial cells, fibroblasts, colostrum cells, cumulus cells, peripheral blood mononuclear cells, and peripheral blood leukocytes (PBL) (2,4,6,8,11,17,18,27). Plasma viremias are thought to be low in OPPV-infected sheep (3); therefore, measuring OPPV provirus loads may be the best way to quantitate virus in vivo.…”

mentioning

confidence: 99%

Development and Validation of an Ovine Progressive Pneumonia Virus Quantitative PCR

Herrmann‐Hoesing

White

Lewis

et al. 2007

Clin Vaccine Immunol

View full text Add to dashboard Cite

Ovine progressive pneumonia virus (OPPV) infects at least one sheep in 81% of U.S. sheep flocks, as determined by serology, and can cause viral mastitis, arthritis, dyspnea, and cachexia. Diagnostic tests that quantify OPPV proviral load in peripheral blood leukocytes (PBL) provide an additional method for identification of infected sheep and may help to further understanding of the pathogenesis of OPPV-induced disease. In this study, we compared a new OPPV real-time quantitative PCR (qPCR) assay specific for the transmembrane region of the envelope gene (tm) with a competitive inhibition enzyme-linked immunosorbent assay (cELISA) using 396 PBL samples and sera from Idaho sheep. The OPPV qPCR had a positive concordance of 96.2% ؎ 2.3% and a negative concordance of 97.7% ؎ 2.5% compared to the cELISA, with a kappa value of 0.93, indicating excellent agreement between the two tests. In addition, the presence of tm in the three OPPV qPCR-positive and cELISA-negative sheep and in 15 sheep with different OPPV proviral loads was confirmed by cloning and sequencing. These data indicate that the OPPV qPCR may be used as a supplemental diagnostic tool for OPPV infection and for measurement of viral load in PBLs of infected sheep.Ovine progressive pneumonia virus (OPPV) is also referred to as maedi-visna virus (MVV) or ovine lentivirus (OvLV) and is a member of the family Retroviridae. As with other lentiviruses, infection with OPPV is defined as successful integration of virus into the sheep genome, and this integrated virus is called provirus. OPPV provirus is found in cells of the macrophage and monocyte lineage without causing cellular ablation (13,14). Specifically, OPPV provirus has been detected in many cell types, including lung type I and II pneumocytes, interstitial and alveolar macrophages, endothelial cells, fibroblasts, colostrum cells, cumulus cells, peripheral blood mononuclear cells, and peripheral blood leukocytes (PBL) (2,4,6,8,11,17,18,27). Plasma viremias are thought to be low in OPPV-infected sheep (3); therefore, measuring OPPV provirus loads may be the best way to quantitate virus in vivo.Determination of the severity of OPPV-induced clinical disease has been primarily based upon postmortem pathological assessment of lung tissue, since sheep with lung lesions typically manifest one or more of various clinical signs, such as dyspnea, mastitis, arthritis, cachexia, and central nervous system disorders, prior to euthanasia (5). Subsequent reanalysis of the data from an earlier pathogenesis study showed that the presence of lung lesions (mild, moderate, or severe) correlates significantly with the presence of provirus in alveolar macrophages using PCR specific for ltr and pol (Fisher's exact test, two-sided P value ϭ 0.0003) (4). In addition, sheep with lung lesions had Ͼ10-fold more pol copies in their alveolar macrophages, determined by a quantitative PCR (qPCR), than sheep without lung lesions (27). Currently, it is unknown whether OPPV provirus load in PBL can be used as predictor of clinical diseas...

show abstract

“…although this infection is very common in Greek flocks (Eltahir et al 2006), it is not considered very common in the Centro-african countries and usually when it is found it has been introduced by European breeds (Mekonnen et al 2010). However, the findings of the present study confirm the opinion that this condition is not common in Centroafrican small ruminant breeds.…”

Section: Discussionmentioning

confidence: 99%

“…Detection of the proviral SrLV DNa was performed using DNa extracts from separated peripheral blood mononuclear cells (pBMCs) and nested-pCr (Eltahir et al 2006) modified by using 0.5 units platinum® Taq DNa polymerase (Invitrogen, The Netherlands) per 20 μl assay.…”

Section: Methodsmentioning

confidence: 99%

Assessment of some health parameters in West African Pygmy Goats and Cameroon Dwarf Sheep of a Zoo in Greece

Tahas¹,

Giadinis

Kritsepi‐Konstantinou

et al. 2017

J Hellenic Vet Med Soc

View full text Add to dashboard Cite

Ten A frican pygmy goats and five Cameroon sheep from the Attica Zoological Park in Greece were examined for some health parameters including a thorough clinical examination, haematology, serum biochemistry, serological and P CR examination for small ruminant lentivirus, milk bacteriology for common pathogens of clinical or subclinical mastitis and faecal parasitology. These were compared with the existing literature for these two exotic breeds of small ruminants. Haematologic and biochemistry values are compared to existing literature for the pygmy goats whereas this is the first report of such values in Cameroon sheep. A ll animals proved clinically healthy and were free of common pathogens found in small ruminants in Greece as well as for intestinal and respiratory parasites

show abstract

Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA

Cited by 38 publications

References 35 publications

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction

Development and Validation of an Ovine Progressive Pneumonia Virus Quantitative PCR

Assessment of some health parameters in West African Pygmy Goats and Cameroon Dwarf Sheep of a Zoo in Greece

Contact Info

Product

Resources

About