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.
Highlights
Bovine viral diarrhea virus (BVDV; family Flaviviridae, genus pestivirus) is one of the most widespread and economically important viral infections in cattle [1] with average direct losses approaching $175–200/head USD [2]
Propagation of cell lines frequently results in chromosomal deletions and rearrangements that represent potential candidates for the loss of viral susceptibility observed in the CRIB cell line
Our first analysis focused on identifying homozygous deleted regions present in the CRIB cell line compared to the Madin-Darby bovine kidney (MDBK) parent line since these could potentially cause the altered phenotype
Summary
Bovine viral diarrhea virus (BVDV; family Flaviviridae, genus pestivirus) is one of the most widespread and economically important viral infections in cattle [1] with average direct losses approaching $175–200/head USD [2]. Acute infection results in systemic spread that is associated with respiratory and gastrointestinal diseases, immunosuppression, and reproductive failure [3,4]. BVDV can cross the placenta and infect the developing fetus resulting in abortion, malformations, or the birth of immunotolerant and persistently infected (PI) calves [5]. PI calves often have increased morbidity and mortality and are the most important sources of virus spread in the population [6]. Effective disease control relies on a combination of vaccination, identification and removal of PI calves, and implementation of biosecurity protocols [7].
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