Primary cells from a CD46-edited bovine heifer have reduced BVDV susceptibility despite viral adaptation to heparan sulfate

Authors: KRUEGER, ALEXANDRIA - Orise Fellow; VANDER LEY, BRIAN - University Of Nebraska; Heaton, Michael; SONSTEGARD, TAD - Acceligen Inc; Workman, Aspen

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2025
Publication Date: 4/28/2025
Citation: Krueger, A.C., Vander Ley, B.L., Heaton, M.P., Sonstegard, T.S., Workman, A.M. 2025. Primary cells from a CD46-edited bovine heifer have reduced BVDV susceptibility despite viral adaptation to heparan sulfate. Viruses. 17. Article 634. https://doi.org/10.3390/v17050634.
DOI: https://doi.org/10.3390/v17050634

Interpretive Summary: Gene editing offers a promising approach for breeding livestock that are resistant to disease. Editing technology was recently used to produce the first calf with genetic resistance to bovine viral diarrhea virus (BVDV), a major pathogen responsible for respiratory, gastrointestinal, and reproductive diseases that cost the cattle industry approximately $1 billion annually. However, pathogens can evolve rapidly compared to their host, which raises concerns that viruses may eventually circumvent the genetic barriers created by gene editing. To investigate this in the laboratory, BVDV evolution was studied by repeatedly growing the virus on gene-edited cells with alterations in the host receptor that facilitates viral entry. Whole genome sequencing was used to track the genetic changes in the virus genome as it adapted to overcome the genetic resistance. In this laboratory model, BVDV did not adapt to use the gene-edited virus receptor. Instead, the virus evolved to use an alternative receptor to infect cells. These adapted viruses were then used in an attempt to infect multiple cell types isolated from the gene-edited calf. Notably, white blood cells from the edited calf remained largely resistant to infection with the adapted viruses. This suggests that the viral adaptations observed in a laboratory model may not reflect the range of evolutionary dynamics that occur in a living animal. Thus, gene editing in cattle may lead to reduced susceptibility to BVDV, even when the virus has adapted to use alternative entry mechanisms.

Technical Abstract: A precision genome edit in the bovine CD46 gene (A82LPTFS87) dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned heifer. However, pathogen evolution threatens the long-term efficacy of such interventions. Here, our aim is two-fold: first, to determine whether BVDV can adapt in vitro to use the edited CD46 receptor to infect Madin–Darby bovine kidney (MDBK) cells, and second, to evaluate the ex vivo infectivity of culture-adapted viruses in cells from the CD46-edited heifer. Serial passage of BVDV on CD46-edited MDBK cells selected for virus variants capable of CD46-independent infection. Virus genome sequencing revealed mutations in the viral ERNS gene predicted to enhance HS-mediated entry. HS adaptation was confirmed by inhibiting virus infection with heparin or Heparinase I/III treatment. A naturally occurring HS-adapted field isolate from a persistently infected calf showed similar results. However, when tested on primary cells from the CD46-edited heifer, HS-adapted viruses showed reduced infectivity in skin fibroblasts, monocytes, and lymphocytes in a manner that correlated with HS expression. Thus, although BVDV can adapt to use HS as an alternative entry receptor, HS adaptation does not overcome the protection conferred by the CD46 edit in all relevant cell types.