Genotyping and Classification of Tunisian Strains of Avian Reovirus using RT-PCR and RFLP Analysis
Résumé
Since 1998, avian reovirus (ARV) infection has been detected in broiler and breeding chicken flocks in Tunisia. The genotype of avian reoviruses was established using simple and rapid approaches. Reverse transcription PCR (RT-PCR) on both sigma C (sigma C) and sigma B (sigma B)-encoding genes followed by restriction fragment length polymorphism (RFLP) analyses were used to better characterize Tunisian isolated strains. The RT-PCR amplified fragments of 738 and 540 bp for sigma C-and sigma B-encoding genes, respectively, of 15 ARV Tunisian strains. DNA fragments amplified from S1133 vaccine and isolated strains were digested with different restrictions enzymes. RFLP on the sigma C gene indicated that the field isolates and the S1133 vaccine strain have identical profiles when separately digested with TaqI, PstI, DdeI, and HincII. Considering the sigma B gene, RFLP profiles were identical with RsaI, BclI, DpnII, and NciI restriction enzymes for all the strains. However, using MseI and AciI enzymes, it was shown that all tested isolates could be clearly distinguished from the vaccine strain. ARV strains could be classified in groups with strong relatedness. Strain-typing based on cleavage site results are in agreement with ARV clustering based on nucleotide sequences of both the sigma C and sigma B genes. RT-PCR-RFLP provides a simple and a rapid approach for genotyping ARV isolates, especially when a large number of isolates are being studied. Additionally, this approach may also determine whether a new variant strain has been introduced into a flock or if a given virus strain is being spread from one flock to another.