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Article

A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line—A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV)

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany
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Viruses 2020, 12(8), 859; https://doi.org/10.3390/v12080859
Received: 29 June 2020 / Revised: 31 July 2020 / Accepted: 2 August 2020 / Published: 6 August 2020
(This article belongs to the Special Issue Bovine Viral Diarrhea Virus and Related Pestiviruses)
Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin–Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus–receptor interaction. View Full-Text
Keywords: bovine viral diarrhea virus (BVDV); pestivirus; escape mutant; CD46; ERNS; adaptation; CRISPR; knockout; MDBK; cell entry bovine viral diarrhea virus (BVDV); pestivirus; escape mutant; CD46; ERNS; adaptation; CRISPR; knockout; MDBK; cell entry
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MDPI and ACS Style

Szillat, K.P.; Koethe, S.; Wernike, K.; Höper, D.; Beer, M. A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line—A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV). Viruses 2020, 12, 859. https://doi.org/10.3390/v12080859

AMA Style

Szillat KP, Koethe S, Wernike K, Höper D, Beer M. A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line—A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV). Viruses. 2020; 12(8):859. https://doi.org/10.3390/v12080859

Chicago/Turabian Style

Szillat, Kevin P., Susanne Koethe, Kerstin Wernike, Dirk Höper, and Martin Beer. 2020. "A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line—A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV)" Viruses 12, no. 8: 859. https://doi.org/10.3390/v12080859

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