Abstract
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.
Highlights
Área de estudoO estudo foi realizado na Região Metropolinada de Fortaleza, no município de Maracanaú que apresenta como características geográficas uma altitude média de 48 m, latitude 3o 52’ 36” e longitude 38o 37’ 32”, com área absoluta de 105,70 km
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 authors alone are responsible for the content and writing of the paper
Summary
O estudo foi realizado na Região Metropolinada de Fortaleza, no município de Maracanaú que apresenta como características geográficas uma altitude média de 48 m, latitude 3o 52’ 36” e longitude 38o 37’ 32”, com área absoluta de 105,70 km. Os primers utilizados para amplificar o gene gag foram desenhados e confeccionados de acordo com Saltarelli et al [22]. Os primers externos GEX5 (5’-CAAG-CAGCAGGAGGGAGAAGCTG-3’) e GEX3 (5’-TCC ¬TACCCCCATAATTTGATCCAC-3’) foram utilizados na primeira amplificação, e os primers internos GIN5 (5’-GTTCCAGCAACTGCAAACAGTAGCAATG-3’) e GIN3 (5’-ACCTTTCTGCTTCTTCATTTAATTTCCC-3’) foram utilizados na segunda amplificação. Para a realização da Nested-PCR utilizando primers para a região LTR foram utilizados primers confeccionados de acordo com Ryan et al [21]. Os primers externos LTREX EFW (5′-ACTGTCAGGRCAGAGAACARATGCC-3′) e LTREX ERV (5′-CTCTCTTACCTTACTTCAGG-3′) foram utilizados na primeira amplificação, e os primers internos LTRIN IFW (5′-AAGTCATGTAKCAGCTGATGCTT-3′) e LTRIN IRV (5′-TTGCACGGAATTAGTAACG-3′) foram utilizados na segunda amplificação. Para testar o potencial de detecção dos primers descritos anteriormente foram utilizadas 58 amostras de DNA extraídas do lavado bronco-alveolar de ovinos abatidos na Região Metropolitana de Fortaleza, segundo protocolo de Martins et al [11]. As amostras extraídas foram armazenadas em eppendorffs® de 2 mL e conservadas a -20°C até subsequente uso
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