Briefly, modified BVDV-1 and BVDV-2 RNA were transcribed in vitro from complete clones of cDNA pXYKE-B-NdN (corresponding to the modified BVDV-2) and pKANE99C (corresponding to the modified BVDV-1). Later on, the BVDV strains were propagated in MDBK-B2 cells lineage and the viral RNA was extracted to establish the cDNA standard library and to its extension by polymerase (RT-PCR) chain reaction. The target living virus, modified and attenuated, were obtained by cDNA elaboration after in vitro transcription and RNA transfection of MDBK cells.

Sequencing of master virus seeds (MVS) to modified BVDVs were used to inoculation in MDBKB2 cells and, after 48 hours of incubation, the supernatant material was removed and cells were cleansed and lysed. Total RNA was extracted from cells, examined for integrity and later converted into cDNA for sequencing. Results of sequencing the established MVS confirmed the presence of introduced deletions in Npro and Erns. When compared, the MVS sequences and original isolated KE9 and NY93 showed the presence of just minor differences in nucleotides that were determined to have no influence over the attenuated phenotype.

PCR data obtained for MVS and WVS (MVS + 5) of strains in vaccines of modified BVDV have also confirmed the presence of the introduced Npro deletion. The in vitro data were also supported by PCR data obtained in BVDV (virus isolation method) positive samples which showed that the introduced Npro deletion remained stable for several in vitro passages. Jointly, the data show that BVDV genomes containing the inserted deletions are genetically stable in vitro and in vivo.