Location: Animal Health GenomicsTitle: Resistance to bovine viral diarrhea virus (BVDV) infection in the bovine kidney CRIB cell line is not due to PTPN12, GRID2, or RABGAP1L mutations
|Heaton, Michael - Mike|
|WEBSTER, DENNIS - Recombinetics, Inc|
|KALBFLEISCH, THEODORE - University Of Kentucky|
|Smith, Timothy - Tim|
|CARLSON, DANIEL - Recombinetics, Inc|
|SONSTEGARD, TAD - Recombinetics, Inc|
Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/16/2020
Publication Date: 5/20/2020
Citation: Workman, A.M., Heaton, M.P., Webster, D.A., Kalbfleisch, T.S., Smith, T.P., Carlson, D.F., Sonstegard, T.S. 2020. Resistance to bovine viral diarrhea virus (BVDV) infection in the bovine kidney CRIB cell line is not due to PTPN12, GRID2, or RABGAP1L mutations [abstract]. In: Proceedings of American Society for Virology Meeting, June 13-17, 2020, Fort Collins, Colorado. Poster No. P37-1.
Technical Abstract: Bovine viral diarrhea virus (BVDV; family Flaviviridae, genus Pestivirus) entry into bovine cells is a multistep process involving attachment of virions to cellular receptors, internalization, and pH-dependent membrane fusion with endosomal membranes. CD46 is the main cellular receptor for BVDV; however, the complete spectrum of host factors required for virus entry is unknown. A commercially available bovine kidney cell line (CRIB) resistant to BVDV was previously obtained by cloning Madin-Darby bovine kidney (MDBK) cells that survived infection with a cytolytic strain of BVDV. Early characterization of CRIB cells uncovered a defect in entry of BVDV and other related pestiviruses despite expressing CD46 on the cell surface. The goal of this study was to identify and evaluate any major genomic changes that could be responsible for the resistant phenotype of CRIB cells. Since propagation of cell lines frequently results in chromosomal deletions and rearrangements that might affect viral susceptibility, we first characterized the parent and resistant cell lines for homozygous deletions by whole-genome sequencing (WGS). To that end, short-read WGS from MDBK (48 GB) and CRIB (62 GB) cell lines were generated and aligned to the bovine reference assembly UMD3.1 yielding approximately 15- and 19-fold coverage, respectively. A search for large, homozygous deleted regions identified three compound deletions in the CRIB cell line that were predicted to disrupt the expression or function of the genes PTPN12, GRID2, and RABGAP1L. CRISPR/Cas9-mediated knockout of these genes individually or in combination in MDBK cells did not significantly impact virus growth kinetics in multistep growth-curve experiments. Thus, the genetic changes that led to the development of the resistant phenotype remain to be determined. A more comprehensive list of CRIB cells genomic differences and transcriptional changes are needed for future investigations to gain a better understanding of host factors required for pestivirus entry.