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. 2021 Mar:289:114038.
doi: 10.1016/j.jviromet.2020.114038. Epub 2020 Dec 7.

Expanding virus susceptibility spectrum of MDBK cells by expressing host receptors nectin 4 and TfR

Peiyu Han  1 , Xuanhao Zhang  2 , Siming Yang  2 , Xiaofeng Dai  3 , Qing Lv  4 , Dong Hua  4
Affiliations

Expanding virus susceptibility spectrum of MDBK cells by expressing host receptors nectin 4 and TfR

Peiyu Han et al. J Virol Methods. 2021 Mar.

Abstract

Cell-based vaccine manufacturing is a flexible and cost-effective approach for vaccine production which, however, requires cell adaptation to new vaccine strains. Generating one omnipotent or semi-omnipotent cell line feasible for the production of multiple viruses could help resolve this problem. We previously proposed virus Baltimore subtyping-based choice of receptors and a panel of minimally preferred receptors for the establishment of cells with a broad virus susceptibility spectrum. With the aim of establishing cells sensitive to viruses of livestocks including bovine, ovine and canine, we selected TfR and Nectin 4 from the minimally preferred receptor panel, and successfully sensitized the starting cell line MDBK to CPV and CDV infection. Our study is a preliminary validation of our previously identified associations between host receptor usage and virus Baltimore subtyping. Evidence from more viruses of the same Baltimore subtyping and more starting cell lines need to be used to consolidate our results.

Keywords: Host receptor; MDBK; Nectin 4; Susceptibility spectrum; TfR; Vaccine production.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Plasmid Design of pLVX-DogTfR-His and Agarose Gel after Double Endonuclease (BamHI and EcoRI) Cutting. (A) Plasmid design of pLVX-DogTfR-His. (B) Agarose gel after double endonuclease (BamHI and EcoRI) cutting.
Fig. 2
Fig. 2
Plasmid Design of pcDNA3.1 (+)-DogN4-Flag and Agarose Gel Imaging after Double Endonuclease (EcoRI and XbaI) Cutting. (A) Plasmid design of pcDNA3.1 (+)-DogN4-Flag. (B) Agarose gel imaging after double endonuclease (EcoRI and XbaI) cutting.
Fig. 3
Fig. 3
Comparisons on Transfection Efficiency among Different Transfection Approaches in MDBK. (A) Immunofluorescence imaging, and (B) transfection rates of calcium phosphate, lipofectamine 2000/3000, and electroporation mediated plasmid transfection in MDBK cells. (C) Cell survival rates after 7 days cultivation under different puromycin antibiotic concentrations.
Fig. 4
Fig. 4
Receptor Expression and Localization after Plasmid Transfection. (A) qPCR, (B) western blot, and (C) immunofluorescence imaging showing receptor expression and localization after plasmid transfection.
Fig. 5
Fig. 5
Virus Infection Detection in MDBK Cells Constructed with TfR. (A) Agarose gel, (B) immunofluorescence imaging, (C) TCID50 and (D) qPCR circle threshold showing CPV infection on MDBK-TfR cells.
Fig. 6
Fig. 6
Virus Infection Detection in MDBK Cells Constructed with Nectin 4. (A) Agarose gel, (B) immunofluorescence imaging, (C) TCID50 and (D) qPCR circle threshold showing CDV infection on MDBK-N4 cells.
Fig. 7
Fig. 7
Clonability and stability of MDBK-TfR and MDBK-N4. (A) Flow cytometry detecting TfR and Nectin 4 in MDBK-TfR and MDBK-N4 cells. (B) TfR and Nectin 4 expression during cell passaging.
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