In vitro Transcription of Virus Genomic RNA

Synthesis of full-length DNA from the genomic RNAs of ACLSV, ASPV, and ASGV was done by subjecting total RNA samples to a reverse transcription reaction at 42°C for 60 min using the PrimeScript II RT enzyme and oligo dT primer from the PrimeScript^® High Fidelity RT-PCR Kit (TaKaRa) according to manufacturer protocols. Full-length DNAs of the three viruses were amplified by PrimeSTAR GXL DNA polymerase (TaKaRa) using primer pairs containing the T3 promoter sequence: ACLSV5’endT3(+) (5′-aattaaccctcactaaagtgatactgatacagtgtacact-3′) and ACLSV3’end(–) (5′-tttttttagtagtaaaatatttaaaagt-3′) for ACLSV, ASPV5′endT3(+) (5′-aattaaccctcactaaaggatacgcaaacaaactctg-3′ and ASPV3′end(−) (5′-ttttttgaaaatctagttaaaacaaaaataag-3′) for ASPV, and ASGV5′ endT3(+) (5′-aattaaccctcactaaaggaaatttaacaggcttaattt-3′) and ASGV3′end(−) (5′-ttttttttttttttttttttagagtggacaaactctagac-3′) for ASGV. PCR was performed as follows: 98°C for 30 s, 54°C for ACLSV, 59°C for ASPV, and 52°C for ASGV for 30 s, and 68°C for 10 min for 40 cycles. The PCR product was then purified using the MonoFas^® DNA Purification Kit II (GL Sciences Inc., Tokyo, Japan). Purified full-length genomic DNAs were then used as templates for the in vitro transcription of full-length genomic RNA of each virus using the MEGAscript^® Kit (Ambion, Thermo Fisher scientific, Tokyo, Japan). The reaction mixtures contained the full-length genomic cDNA of each virus, ATP, CTP, UTP (75 mM each), GTP (15 mM), Cap Analog (40 mM) Enzyme Mix, and RNasin^® Plus RNase Inhibitor (Promega, Madison, United States), and were incubated for 3.5 h at 37°C. The reaction mixtures were then treated with TURBO DNase (Thermo Fisher scientific) (2 U/μl) at 37°C for 15 min to decompose the template DNAs. RNA samples were then extracted using water-saturated phenol and chloroform, precipitated using ethanol, and suspended in SDW. RNA transcript concentrations were measured using NanoDrop, while RNA transcript quality was also checked using 1% agarose gel electrophoresis.