Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance

Authors: Workman, Aspen; Heaton, Michael - Mike; WEBSTER, DENNIS - Recombinetics, Inc; Harhay, Gregory; KALBFLEISCH, THEODORE - University Of Kentucky; Smith, Timothy - Tim; Falkenberg, Shollie; CARLSON, DANIEL - Recombinetics, Inc; SONSTEGARD, TAD - Acceligen Inc

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2021
Publication Date: 10/25/2021
Citation: Workman, A.M., Heaton, M.P., Webster, D.A., Harhay, G.P., Kalbfleisch, T.S., Smith, T.P.L., Falkenberg, S.M., Carlson, D.F., Sonstegard, T.S. 2021. Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance. Viruses. 13(11). Article 2147. https://doi.org/10.3390/v13112147.
DOI: https://doi.org/10.3390/v13112147

Interpretive Summary: Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle that causes significant morbidity and mortality, despite 50 years of vaccine availability. BVDV entry into susceptible host cells involves attachment of virions to cellular receptors, internalization, and fusion with host membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. Thus, to identify novel factors essential for BVDV entry and find potential therapeutic targets, we sequenced and compared the complete genomes of two commercially available bovine cell lines: Madin-Darby bovine kidney (MDBK) cells which are susceptible to BVDV infection and CRIB cells, an MDBK clone resistant to BVDV infection at the level of virus entry. Comparative genome analyses identified three large deletions in the BVDV-resistant CRIB cell line that were predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. CRISPR/Cas9 was then used to knockout these three genes, individually or in combination, in the parental MDBK cell line to evaluate the role of these genes in virus entry and replication. Infection studies showed that these three genes were not necessary for viral infection in MDBK cells. More complex analyses of smaller deletions, insertions, and loss-of-function mutations combined with global RNA expression data will be needed to reveal and rank additional candidate genes that may play a role in BVDV resistance. Ultimately, this approach is expected to identify cellular targets for novel intervention strategies to help alleviate the impact of this important viral pathogen of cattle.

Technical Abstract: Bovine viral diarrhea virus’s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors,internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.