Gabrielle Rosemblit Martins

Duplex nested-PCR for detection of small ruminant lentiviruses

Small ruminant lentiviruses (SRLV) have high genetic variability which results in different viral strains around the world. This create a challenge to design sensible primers for molecular diagnosis in different regions. This work proposes a protocol of duplex nested-PCR for the precise diagnosis of SRLV. The technique was designed and tested with the control strains CAEV Co and MVV 1514. Then, field strains were submitted to the same protocol of duplex nested-PCR. Blood samples of sheep and goats were tested with AGID and nested PCR with specific primers for pol, gag and LTR. The AGID results showed low detection capacity of positive animals, while the nested PCR demonstrated a greater capacity of virus detection. Results demonstrated that LTR-PCR was more efficient in detecting positive sheep samples, whereas gag-PCR allowed a good detection of samples of positive goats and positive sheep. In addition, pol-PCR was more efficient with goat samples than for sheep. Duplex nested PCR performed with standard virus samples and field strains demonstrated that the technique is more efficient for the detection of multiple pro-viral DNA sequences. This study demonstrated a successful duplex nested PCR assay allowing a more accurate diagnosis of SRLV.

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

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.

Goat umbilical cord cells are permissive to small ruminant lentivirus infection in vitro

Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.