Location: Animal Health GenomicsTitle: Impact of CD46 knockout on bovine viral diarrhea virus (BVDV) replication in Madin-Darby bovine kidney cells
|WEBSTER, DENNIS - Recombinetics, Inc|
|CARLSON, DAN - Recombinetics, Inc|
|Heaton, Michael - Mike|
|SONSTEGARD, TAD - Recombinetics, Inc|
Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/2/2019
Publication Date: 7/31/2019
Citation: Workman, A.M., Webster, D.A., Carlson, D.F., Heaton, M.P., Falkenberg, S.M., Neill, J.D., Sonstegard, T.S. 2019. Impact of CD46 knockout on bovine viral diarrhea virus (BVDV) replication in Madin-Darby bovine kidney cells [abstract]. In: Proceedings of American Society for Virology Meeting, July 20-24, 2019, Minneapolis, Minnesota. Poster No. P17-16.
Technical Abstract: Bovine viral diarrhea virus (BVDV; family Flaviviridae, genus Pestivirus) causes subclinical to severe acute disease in cattle. CD46 is considered to be the dominant receptor for BVDV on Madin-Darby bovine kidney (MDBK) cells, leading to virus entry. In the present study, we evaluate the impact of CD46 disruption on virus binding and susceptibility to BVDV infection. MDBK CD46 knockout clones were generated using CRISPR-Cas9 technology to create a homozygous deletion encompassing the entire CD46 coding region. CD46 knockout clones were then assayed for their ability to support infection with BVDV strains and clinical samples belonging to both genotypes (1 and 2) and biotypes (cytopathic and non-cytopathic) of the virus. Cells were infected with BVDV at a low multiplicity of infection and viral genome copy number was monitored by RT-qPCR for 96 hours. At 24 hours post infection, the BVDV genome copy number was significantly reduced for all strains tested compared to the same strain grown in the parental MDBK cells. However, after 24 hours post infection, substantial variation in the efficiency of viral infection was observed. At 96 hours, the BVDV genome copy number in CD46 knockout cells ranged from 1% to 100% of the genome copy number for the same strain grown in the parental MDBK cells. It was recently shown that after the initiation of an infection cycle, BVDV can spread in a CD46-independent manner by direct cell-to-cell transmission in MDBK cells. Thus, we hypothesize that the difference in BVDV genome copy number observed at 96 hours post infection may be due to strain differences in the ability of the virus to spread via this mechanism. Viral genome evolution in CD46 knockout cells is also being monitored by next generation sequencing to determine whether the virus is able to adapt to more efficiently infect CD46 knockout cells.