Complete Genomic Sequencing of Bagv Virus

Complete genomic sequencing of the virus genome was performed by bidirectional sequencing of RT-PCR fragments amplified. The virus strain was named BAGV/PT/2021. Seventeen primer sets were designed based on an alignment of all BAGV complete genomes available in GenBank database (January 2022) (Table 1). RT-PCR reactions were performed with the AgPath-ID™ One-Step RT-PCR Reagents kit (Applied Biosystems, Foster City, CA, USA). PCR products were purified using NZYGelpure purification kit (NZYTech, Lisbon, Portugal) and Sanger sequenced using the ABI Prism BigDye Terminator v3.1 Cycle sequencing kit on a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Whenever necessary for sequencing confirmation, individual RT-PCR products were sequenced after being cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.