CD46 edited MDBK cells exhibit resistance to BVDV infection

Bovine viral diarrhea virus (BVDV) is a major pathogen in cattle, causing significant economic losses and frequently contaminating cell cultures through fetal calf serum, which can compromise biological products. Gene editing technologies, such as CRISPR/Cas9, offer promising tools for developing disease-resistant models. CD46, the primary cellular receptor for BVDV, has an incompletely understood role in viral infection. This study aimed to: (i) characterize and compare CD46 between bovine cell lines resistant (CRIB) and susceptible (MDBK); (ii) edit the BVDV-binding region of MDBK-CD46; and (iii) evaluate the susceptibility of CD46-edited MDBK cells to BVDV infection. CD46 was sequenced in BVDV-sensitive MDBK cells, BVDV-resistant CRIB cells, and bovine fibroblasts. CRISPR/Cas9 was used to delete exon 1 of CD46, which encodes the viral attachment platform. Two guide RNAs were cloned into px458 plasmids expressing GFP and co-transfected into MDBK cells. Genomic DNA was PCR-amplified, cloned, and sequenced. Results showed that CRIB cells express a wild-type CD46 protein, suggesting that CD46 is not responsible for their resistance to BVDV. This resistance is likely due to the truncated ADAM17 in CRIB cells, which may alter membrane composition and endocytic pathways, affecting viral entry. Three edited MDBK cell lines were generated: two with homozygous deletions (A and B), and one with a heteroallelic edit (C). The edited lines A and B exhibited over 90 % resistance to BVDV. These findings support the use of gene editing to generate BVDV-resistant models and to further explore alternative viral receptors and entry pathways.