Inheritance of a Single Edited CD46 Allele Is Associated with Reduced Ex Vivo Susceptibility to Bovine Viral Diarrhea Virus

Abstract

Bovine viral diarrhea virus (BVDV) remains an economically important pathogen of cattle despite widespread vaccination. A homozygous CD46-edited Gir heifer (Ginger) was previously shown to have significantly reduced susceptibility to BVDV. The edited allele contains an in-frame six amino acid substitution within the virus-binding domain of the BVDV entry receptor CD46, replacing residues G82QVLAL with A82LPTFS. Here, we investigated whether reduced BVDV susceptibility is maintained when the edited allele is inherited in the heterozygous state. Ginger was artificially inseminated with semen from an unedited Gir bull and produced a healthy heterozygous CD46-edited bull calf (Giraldo). Whole-genome sequencing confirmed the inheritance and structural integrity of Giraldo’s edited allele. Compared with Ginger, Giraldo exhibited similarly reduced ex vivo BVDV susceptibility across primary fibroblasts, lymphocytes, and monocytes, despite inheriting a wild-type CD46 allele from the sire. Allele-specific CD46 RNA expression analysis demonstrated expression of both the edited and wild-type CD46 alleles. Thus, the reduced-susceptibility phenotype was not attributable to transcriptional silencing of the wild-type allele. Lentiviral complementation studies in CD46-knockout Madin-Darby bovine kidney (MDBK) cells further demonstrated that this wild-type CD46 allele was competent to support BVDV infection when expressed independently. Together, these findings indicate that the CD46 A82LPTFS allele can confer reduced BVDV susceptibility in the heterozygous state despite expression of a functional wild-type CD46 allele. This result suggests the potential to more rapidly disseminate reduced BVDV susceptibility through conventional breeding using homozygous CD46-edited sires. Importance Bovine viral diarrhea virus (BVDV) continues to cause substantial economic losses to the cattle industry despite the availability of vaccines. We previously developed cattle homozygous for a precisely edited version of the BVDV entry receptor gene CD46 that significantly reduces susceptibility to BVDV infection. Here, we show that this reduced-susceptibility phenotype is maintained when only one copy of the edited CD46 gene is inherited, despite the presence of a functional unedited copy of the gene. This finding suggests that the edited allele can reduce BVDV susceptibility in heterozygous animals, making it feasible to disseminate this trait through conventional breeding. More broadly, this study demonstrates the potential for precise editing of host genes exploited by viruses to generate heritable disease resistance, offering a practical strategy to improve livestock health and resilience.