Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus

Carrozza, Maria Luisa; Mazzei, Maurizio; Bandecchi, Patrizia; Fraisier, Christophe; Pérez, Marta; Suzan‐Monti, Marie; Andrés, D. de; Amorena, B.; Rosati, Sergio; Andrésdóttir, Valgerður; Luján, Lluís; Pépin, Michel; Blacklaws, Barbara; Tolari, Francesco; Harkiss, G. D.

doi:10.1016/j.jviromet.2010.01.013

Cited by 11 publications

(10 citation statements)

References 37 publications

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“…A maioria dos estudos realizados para detecção de LVPR é realizada utilizando primers desenhados para o gene gag, uma vez que este apresenta sequências conservadas em diferentes amostras de LVPR [24]. Dos animais testados, apenas o animal P1 foi positivo [7,16]. No entanto, para detecção mais eficiente do MVV em amostras de ovinos, muitos trabalhos tem demonstrado que a escolha de primers para a região LTR é mais acertada [2,8,26], uma vez que esses primers apresentam uma melhor capacidade de detecção do MVV mesmo quando se tem uma ampla variedade de cepas circulantes do vírus [20].…”

Section: Discussionunclassified

Detection of Maedi-Visna Virus from Sheep Bronchoalveolar Lavage by Nested PCR Evaluation of Different Primers Pairs

Marinho

Martins

Souza

et al. 2016

Acta Scientiae. Vet.

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Background: Small ruminant lentiviruses (SRLV) are characterized by a high degree of genetic variability related to your replication process, resulting in several strains in different geographic regions. The Polymerase Chain Reaction (PCR) is very successful in the detection of proviral DNA of SRLV, however, due to the high variability of the lentivirus genome, the efficiency and sensibility of PCR depends mainly on the specificity of the primers designed and the choice of the amplified target viral region. The aim of this study was to compare detection of Maedi Visna Virus (MVV) from bronco alveolar lavage samples of sheep by Nested PCR using primers for the gag and LTR genes.Materials, Methods & Results: Samples of sheep bronchoalveolar lavage (n = 58) from slaughterhouse in the Metropolitan Region of Fortaleza were previously tested by nested PCR using primers for region gag. Thereafter, these samples were tested by nested PCR with primers designed for the LTR region. Both tests were conducted using thermocycler (Biocycler®) under the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing of primers at 56°C for 1 min and extension of DNA at 72°C for 45 s with a final extension at 72 for 7 min. The first and second round were performed under the same conditions. Every amplification was performed using a positive control MVV-K1514 and water RNA/DNA free with a negative control. After the amplification, the PCR products were separated by agarose gel electrophoresis at 1% stained with ethidium bromide in TBE buffer. The tests revelead only 1 sample (P1) was detected exclusively for the primer of gag gene, while 8 samples were positive only for the test performed with primers of the LTR region, 5 samples were positive for both sets of primers tested and 30 samples were negative for all tests. The test with the LTR gene demonstrated 37.93% (22/58) positives of Maedi Visna in the samples studied.Discussion: In recent years, with advances in molecular biology techniques, some PCR protocols have been developed for the diagnosis of SRLV. However, these viruses exhibit a high instability and mutation rate becomes very difficult to use the same primers in different geographic regions. In this study, comparing the MVV detection capability by nested PCR with differents primer sets was possible to demonstrate that primers LTR gene were more efficient in detecting positive animals when compared with the primers designed for the gag region. In all tests, only the animal (P1) was positive for the nested PCR performed with the primers for the gag gene, not being detected by the LTR gene. Some studies suggest success in the detection of MVV using primers for the gag gene. However, for more efficient detection of MVV in sheep samples, many studies have shown that the choice of primers for the LTR region is more accurate, since these primers have better MVV detection capability even when it has a large range of circulating virus strains. it is known that the genetic diversity of SRLV generate difficulties in carrying out molecular tests, since the molecular diagnostic tests depend on factors such as the percentage of identity of nucleotides of the viral populations circulating in the herds and the sequences used for testing. In this study it is possible to conclude that the effective control of lentiviruses diagnostic methods should be chosen properly in order to be applied in disease control programs.

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Section: Discussionunclassified

Detection of Maedi-Visna Virus from Sheep Bronchoalveolar Lavage by Nested PCR Evaluation of Different Primers Pairs

Marinho

Martins

Souza

et al. 2016

Acta Scientiae. Vet.

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“…The PCR and the early detection of amplicons have been combined in this technique [206,207]. The rtPCR has been useful for detection and quantification of viral nucleic acids in different cells or tissues, although its use in SRLV routinary diagnosis is not widespread [207,208,209]. Primers designed in almost all SRLV genetic regions [205] have yielded reliable and fast results.…”

Section: In Vitro Diagnosismentioning

confidence: 99%

Small Ruminant Lentiviruses: Genetic Variability, Tropism and Diagnosis

Ramírez

Reina

Amorena

et al. 2013

Viruses

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126

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Small ruminant lentiviruses (SRLV) cause a multisystemic chronic disease affecting animal production and welfare. SRLV infections are spread across the world with the exception of Iceland. Success in controlling SRLV spread depends largely on the use of appropriate diagnostic tools, but the existence of a high genetic/antigenic variability among these viruses, the fluctuant levels of antibody against them and the low viral loads found in infected individuals hamper the diagnostic efficacy. SRLV have a marked in vivo tropism towards the monocyte/macrophage lineage and attempts have been made to identify the genome regions involved in tropism, with two main candidates, the LTR and env gene, since LTR contains primer binding sites for viral replication and the env-encoded protein (SU ENV), which mediates the binding of the virus to the host’s cell and has hypervariable regions to escape the humoral immune response. Once inside the host cell, innate immunity may interfere with SRLV replication, but the virus develops counteraction mechanisms to escape, multiply and survive, creating a quasi-species and undergoing compartmentalization events. So far, the mechanisms of organ tropism involved in the development of different disease forms (neurological, arthritic, pulmonary and mammary) are unknown, but different alternatives are proposed. This is an overview of the current state of knowledge on SRLV genetic variability and its implications in tropism as well as in the development of alternative diagnostic assays.

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“…The first is by direct measurement of proviral concentration [73,79,80,81,82] in peripheral blood, specific tissues, or both [83]. Such measurements have the advantage of reflecting viral replication.…”

Section: Development Of Genetic Marker Tests For Small Ruminant Lementioning

confidence: 99%

Expanding Possibilities for Intervention against Small Ruminant Lentiviruses through Genetic Marker-Assisted Selective Breeding

White

Knowles

2013

Viruses

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Small ruminant lentiviruses include members that infect sheep (ovine lentivirus [OvLV]; also known as ovine progressive pneumonia virus/maedi-visna virus) and goats (caprine arthritis encephalitis virus [CAEV]). Breed differences in seroprevalence and proviral concentration of OvLV had suggested a strong genetic component in susceptibility to infection by OvLV in sheep. A genetic marker test for susceptibility to OvLV has been developed recently based on the TMEM154 gene with validation data from over 2,800 sheep representing nine cohorts. While no single genotype has been shown to have complete resistance to OvLV, consistent association in thousands of sheep from multiple breeds and management conditions highlight a new strategy for intervention by selective breeding. This genetic marker-assisted selection (MAS) has the potential to be a useful addition to existing viral control measures. Further, the discovery of multiple additional genomic regions associated with susceptibility to or control of OvLV suggests that additional genetic marker tests may be developed to extend the reach of MAS in the future. This review will cover the strengths and limitations of existing data from host genetics as an intervention and outline additional questions for future genetic research in sheep, goats, small ruminant lentiviruses, and their host-pathogen interactions.

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Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus

Cited by 11 publications

References 37 publications

Detection of Maedi-Visna Virus from Sheep Bronchoalveolar Lavage by Nested PCR Evaluation of Different Primers Pairs

Detection of Maedi-Visna Virus from Sheep Bronchoalveolar Lavage by Nested PCR Evaluation of Different Primers Pairs

Small Ruminant Lentiviruses: Genetic Variability, Tropism and Diagnosis

Expanding Possibilities for Intervention against Small Ruminant Lentiviruses through Genetic Marker-Assisted Selective Breeding

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