To obtain the U
S1 sequence from strains F, KOS, OD4, CJ394, TFT401, CJ311, 970, and 994, the U
S1 gene was amplified from purified viral genomic DNA and then directly sequenced. To amplify the U
S1 gene, we performed a PCR reaction consisting of 2.5 μg viral DNA, 1× reaction buffer, 1× enhancer buffer, 1 mM MgSO
4, 4 mM dNTP, 0.1 μg each primer, and 1 μL Pfx high-fidelity polymerase (Invitrogen Inc., Carlsbad, CA) in a total volume of 50 μL The cycling conditions were 1 cycle of 94°C for 5 minutes, 30 cycles of 94°C for 15 seconds, 50°C for 30 seconds, 68°C for 2 minutes, and a final cycle of 58°C for 7 minutes. The U
S1 gene extends between genome nucleotides 132,363 and 133,906. The forward primer annealed at nucleotide 132,363 (5′-TTTTGCACGGGTAAGCAC-3′), and the reverse primer annealed at nucleotide 133,992 (5′-CCGACTTCCTCACATCTGCT-3′). The PCR products were directly sequenced using multiple primers (
Table 2). The sequencing mixture consisted of 200 μg amplification product, 1× reaction buffer, 0.1 μg primer, and 2 μL dye terminator (Big Dye, v3.1; Applied Biosystems, Foster City, CA) in a total volume of 20 μL. The cycling conditions were 1 cycle of 95°C for 3 minutes, 45 cycles of 95°C for 20 seconds, 45°C for 30 seconds, and 60°C for 4 minutes followed by a final cycle of 72°C for 7 minutes. Sequences were determined at the University of Wisconsin-Madison Biotechnology Center sequencing facility. To reconcile discrepancies in the sequence, chromatograms were visually inspected or sequencing reactions were repeated. The constructs were sequenced and found to match the sequences of the original strains.