We adopted optimizations to amplify different segments of GFLV RNA2. The primers were the crucial part of such optimizations. Reagents were purchased from Fermentas (Lithuania). Reverse transcription was done by the use of oligo d(T)16 or a GFLV-specific primer. Initially, previously reported primers (Wetzel et al., 2001) were applied to give 810 bp fragment. New primers were designed after sequences of local isolates were determined to enhance efficiency of the PCRs. As such, GMPF1 and GMPR1 primers were designed for amplification of full length MP gene (1044 bp). Also, we designed GFLV-2048 and GFLV-3559 to amplify the virus full length CP gene. Amplification of the RNA2 was done with 5’-NC/M4 and GFLV2048F/3’NC primer pairs giving 2.2 and 1.65 Kbp segments of the GFLV RNA2, respectively, covering the partial 5’- non coding region, entire 2AHP and 2BMP, and the 2CCP with a partial segments from 3’- non-coding region (Nourinejhad-Zarghani et al., 2012). As a result of three independent surveys during 2003-2007, GFLV was detected by ELISA in collectively 84 out of 346 (24.3%) samples showing that nearly all sampled are as in the northwest were infected by GFLV. However, ELISA detected GFLV in a percentage of samples likely due to a relatively lower sensitivity of ELISA compared to that of RT-PCR. By the help of newly-designed primers different genomic segment of the virus were amplified, cloned and sequenced. By the primers GMPF1 and GMPR1, the full length MP gene (1044 bp) was amplified from 41 of the 86 ELISA-positive samples. Sequence analyses of seven PCR products (MP) revealed up to 17 and 8% divergence between the Iran isolates at NT and deduced AA sequence, respectively. A 1515 bp fragment was obtained for 16 out of 89 samples by the use of GFLV-2048 and GFLV-3559. CP fragment from eight isolates were cloned and the NT sequences determined. Alignment of previously reported GFLV strains/ isolates and ArMV-S showed that new isolates were GFLV.
