Reduced susceptibility to heparan sulfate-adapted BVDV in primary cells from a CD46-edited bovine heifer

Authors: KRUEGER, ALEXANDRIA - Oak Ridge Institute For Science And Education (ORISE); VANDER LEY, BRIAN - University Of Nebraska; Heaton, Michael; Workman, Aspen

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 10/17/2024
Publication Date: 1/2/2025
Citation: Krueger, A.C., Vander Ley, B., Heaton, M.P., Workman, A.M. 2025. Reduced susceptibility to heparan sulfate-adapted BVDV in primary cells from a CD46-edited bovine heifer [abstract]. Research Workers in Animal Diseases Conference Proceedings, January 18-21, 2025, Chicago, Illinois. p. 70.

Interpretive Summary:

Technical Abstract: Objective: Bovine viral diarrhea virus (BVDV) uses heparan sulfate (HS) and the protein receptor CD46 to bind and enter bovine cells. A CD46 gene-edited heifer was recently reported to have significantly reduced susceptibility to BVDV. A fundamental question is whether BVDV can adapt to circumvent the infection restriction imposed by the CD46 gene-edit. Here, our aims were two-fold: 1) determine whether BVDV can adapt in vitro to utilize the edited CD46 protein receptor to initiate infection, and 2) assess the ability of in vitro adapted BVDV isolates to infect primary cells ex vivo from the CD46 gene-edited heifer. Methods: BVDV isolates were serially cultured 11 passages on CD46 gene-edited Madin-Darby bovine kidney (MDBK) cells, and changes in viral infectivity in edited cells was quantified by flow cytometry. Viral adaptation to HS was investigated by pre-incubating adapted BVDV isolates with heparin, a HS analog, or by treating cells with Heparinase I/III to remove cell surface HS. Flow cytometry was also used to compare infection efficiency between unadapted and in vitro adapted virus pairs in primary cells from the CD46 gene-edited heifer and an unedited control. Results: Adapted BVDV isolates used a HS dependent entry mechanism to infect CD46 gene-edited MDBK cells with efficiency comparable to the unedited MDBK cells. Thus, BVDV did not adapt in vitro to use the edited CD46 protein for infection. Importantly, infection by HS adapted BVDV was reduced in primary skin fibroblasts from the CD46 gene-edited heifer and monocytes and lymphocytes remained highly resistant compared to the unedited control in ex vivo experiments. Conclusions: Ex vivo studies indicate that primary cells expressing the edited CD46 protein receptor maintain varying levels of resistance to in vitro adapted BVDV in a manner that correlates with HS expression. Additional research is needed to determine whether HS adapted viruses would be naturally selected for in CD46 gene-edited cattle and, if so, whether they would have similar tropism, pathogenesis and disease outcomes in vivo.