scholarly journals Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2147
Author(s):  
Aspen M. Workman ◽  
Michael P. Heaton ◽  
Dennis A. Webster ◽  
Gregory P. Harhay ◽  
Theodore S. Kalbfleisch ◽  
...  
Keyword(s):  
Cell Line ◽  
Virus Entry ◽  
Mdbk Cell ◽  
Genomic Variation ◽  
Host Factors ◽  
Bovine Viral Diarrhea ◽  
Primary Host ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Endosomal Membranes

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.

 Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance - MDBK reads mapping to regions in genome  not shared with CRIB cell line v1

2021 ◽  
Author(s):  
aspen.workman not provided ◽  
mike.heaton not provided ◽  
Dennis A. Webster ◽  
Gregory P Harhay ◽  
Tim Smith ◽  
...  
Keyword(s):  
Cell Line ◽  
Bovine Viral Diarrhea Virus ◽  
Virus Entry ◽  
Mdbk Cell ◽  
Genomic Variation ◽  
Host Factors ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

Bovine viral diarrhea virus (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 genesPTPN12,GRID2, andRABGAP1L. 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 ofPTPN12,GRID2, orRABGAP1Lgene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

scholarly journals Complete Genome Sequence of Noncytopathic Bovine Viral Diarrhea Virus 1 Contaminating a High-Passage RK-13 Cell Line

2015 ◽  
Vol 3 (5) ◽  
Author(s):  
Bora Nam ◽  
Ganwu Li ◽  
Ying Zheng ◽  
Jianqiang Zhang ◽  
Kathleen M. Shuck ◽  
...  
Keyword(s):  
Cell Line ◽  
Genome Sequence ◽  
Bovine Viral Diarrhea Virus ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Bovine Viral Diarrhea ◽  
High Passage ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

A high-passage rabbit kidney RK-13 cell line (HP-RK-13[KY], originally derived from the ATCC CCL-37 cell line) used in certain laboratories worldwide is contaminated with noncytopathic bovine viral diarrhea virus (ncpBVDV). On complete genome sequence analysis, the virus strain was found to belong to BVDV group 1b.

scholarly journals Developing an Antiviral Drug Screening System for Anti-Bovine Viral Diarrhea Virus (BVDV) Therapies

2019 ◽  
Vol 69 (4) ◽  
pp. 1255
Author(s):  
A. Mokhtari ◽  
M. Mahzounieh ◽  
O. Madadgar ◽  
A. Ghalyanchi Langeroudi2
Keyword(s):  
Cell Line ◽  
Drug Screening ◽  
Bovine Viral Diarrhea Virus ◽  
Antiviral Drug ◽  
Bovine Viral Diarrhea ◽  
Screening System ◽  
Egfp Gene ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) is an economically important animal pathogen affecting cattle. Despite the use of vaccination, test and slaughter practices, BVD remains a serious problem of cattle breeding. This study was conducted in order to develop a cell line that expresses some of BVDV sub-replicons. BVDV-NADL NS3 and 5’UTR were cloned in pWPI-linker B lentiviral plasmid at the upstream of EGFP gene. Consequently, lentiviral vectors containing BVDV-NS3 and BVDV-5’UTR were produced by using the second-generation lentiviral packaging system. By these lentivectors, MDBK cells expressing BVDV-5’UTR and BVDV-NS3 partial fragments were prepared. The efficiency of the infection was evaluated by fluorescence microscopy, western blotting, and RT-PCR. The results indicated that the development of MDBK cell line expressing these transgenes provides a very sensitive antiviral drug screening system for anti-bovine viral diarrhea virus (BVDV) therapies.

scholarly journals Elimination of Non-cytopathic Bovine Viral Diarrhea Virus From the LFBK-αvβ6 Cell Line

2021 ◽  
Vol 8 ◽  
Author(s):  
Ashley R. Gray ◽  
Britta A. Wood ◽  
Elisabeth Henry ◽  
Donald P. King ◽  
Valérie Mioulet
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Analytical Sensitivity ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Mouth Disease Virus ◽  
Viral Diarrhea Virus

The LFBK-αvβ6 cell line is highly sensitive for the isolation of foot-and-mouth disease virus (FMDV) and porcinophilic vesicular viruses. However, LFBK-αvβ6 cells are contaminated with a non-cytopathic bovine viral diarrhea virus (BVDV), which complicates handling procedures in areas where other cell lines are maintained, as well downstream use of viral isolates. In this study, we used an aromatic cationic compound (DB772) to treat LFBK-αvβ6 cells using an approach that has been previously used to eliminate persistent BVDV from fetal fibroblast cell lines. After three cell passages with 4 μM DB772, BVDV could no longer be detected in unclarified cell suspensions using a pan-pestivirus real-time RT-PCR assay, and remained undetectable after treatment was stopped (nine passages) for an additional 28 passages. The analytical sensitivity of the DB772-treated LFBK-αvβ6 cultures (renamed WRL-LFBK-αvβ6) to titrations of FMDV and other vesicular virus isolates was comparable to untreated LFBK-αvβ6 cells. These new BVDV-free cells can be handled without the risk of cross-contaminating other cells lines or reagents, and used for routine diagnostics, in vivo studies and/or preparation of new vaccine strains.

Bovine viral diarrhea virus genomic variation within persistently infected cattle

2018 ◽  
Vol 58 ◽  
pp. 218-223 ◽  
Author(s):  
A. Chernick ◽  
A. Ambagala ◽  
K. Orsel ◽  
J.D. Wasmuth ◽  
G. van Marle ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Genomic Variation ◽  
Bovine Viral Diarrhea ◽  
Infected Cattle ◽  
Persistently Infected ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

scholarly journals Formation of bovine viral diarrhea virus E1–E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains

2008 ◽  
Vol 89 (9) ◽  
pp. 2114-2121 ◽  
Author(s):  
Saskia Ronecker ◽  
Gert Zimmer ◽  
Georg Herrler ◽  
Irene Greiser-Wilke ◽  
Beatrice Grummer
Keyword(s):  
Amino Acid ◽  
Bovine Viral Diarrhea Virus ◽  
Transmembrane Domain ◽  
Virus Entry ◽  
Transmembrane Domains ◽  
Site Directed Mutagenesis ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

The envelope of bovine viral diarrhea virus (BVDV) contains the glycoproteins Erns, E1 and E2. Complementation of a recombinant vesicular stomatitis virus (VSV) with BVDV glycoproteins resulted in infectious pseudotyped viruses. To elucidate the specific role of each of the single envelope glycoproteins during viral entry, pseudotypes were generated bearing the BVDV envelope proteins in different combinations. Pseudoviruses that contained E1 and E2 but not Erns were infectious, indicating that Erns is dispensable for virus entry. VSV/BVDV pseudotypes with chimeric proteins (the ectodomain of the BVDV glycoprotein and the transmembrane domain of the VSV-G protein) were not infectious. The fact that E1–E2 heterodimers were not detected if one of the proteins was chimeric indicated that the heterodimers are crucial for BVDV entry. It was shown by site-directed mutagenesis that the charged amino acids in the transmembrane domains of BVDV E1 (lysine and arginine) and the charged amino acid in the transmembrane domain of E2 (arginine) play a key role in heterodimer formation. Pseudoviruses bearing the mutation E2-R/A, where the charged amino acid was substituted by alanine, were not infectious, supporting the hypothesis that E1–E2 heterodimers are essential for BVDV entry.

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

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 859 ◽  
Author(s):  
Kevin P. Szillat ◽  
Susanne Koethe ◽  
Kerstin Wernike ◽  
Dirk Höper ◽  
Martin Beer
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Receptor Interaction ◽  
Bovine Viral Diarrhea ◽  
Viral Glycoprotein ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Dependent Cell ◽  
Viral Diarrhea Virus

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.

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