Viral RNA of the 272 novel sequences was extracted from original biological samples (n = 261) and growth medium after passage in cell culture (n = 11). Reverse transcription and PCR assays targeting a 288 bp region of 5′UTR of BVDV were performed using previously described primers by Letellier et al. [31 (link)], with the exception of strains collected in Sicily, which were tested using primers by Vilcek et al. [32 (link)]. The samples collected in the Center and the South macroareas were also tested by primers for atypical Pestivirus [14 ]. For the BVDV-1 subtypes identified for the first time in Italy, a 428 bp region encoding autoprotease Npro was amplified using nested PCR, as previously described [20 (link), 29 (link)].
For each sample, the amplicons of the expected sizes were purified and sequenced using forward and reverse primers by cycle sequencing using a Big Dye Terminator version 1.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) and an ABI PRISM 3130 sequencing device or sent for outsource sequencing (Primm).
For each sample, the amplicons of the expected sizes were purified and sequenced using forward and reverse primers by cycle sequencing using a Big Dye Terminator version 1.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) and an ABI PRISM 3130 sequencing device or sent for outsource sequencing (Primm).
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