Team:Jiangnan/Design

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Broad Spectrum

The key for a cell line to be sensitive to the infection of a broad spectrum of viruses is to have the corresponding receptors expressed on the cell surface. We established an association between virus Baltimore subtyping and cell surface receptors mediating virus infection, and found that given the current sensitivity spectrum of our chassis cell line MDBK, only Nectin4 and TfR need to be artificially expressed on cell surface to make it sensitive to, in principle, all commonly encountered animal viruses. The sequence design is as below, where the sequences encoding each receptor are inserted into the designed cassette,respectively, and the proteins are expressed after cell transfection.

Suspension Culture

Through comparing two sets (CHO and BHK-21) of transcriptome of suspended and adherent cells with the same origin, we obtained a list of candidate genes responsible for cell suspension. Following computational modeling of these genes, we identified the target gene “PABPC 1” and conducted wet lab experiments to verify our assumption. The gene regulatory network is constructed as below.

High Titer

We conducted two sets of work to increase virus titer.

First, we constructed the computational network and identified the key gene (IRF7) responsible for virus multiplication, which is shown as below.

Second, we established an approach, namely cold atmospheric plasma, with the device manufactured to further increase virus titre. Plasma is the fourth fundamental state of matter, and a cocktail of ions, free electrons, reactive species, etc. Plasma may increase virus titre by increasing the permeability of cell membrane or stimulating cell receptors mediating virus internalization.

Reference

1. Talekar A, Moscona APorotto M. Measles virus fusion machinery activated by sialic acid binding globular domain [J]. J Virol, 2013, 87(24): 13619-13627.
2. Delpeut S, Noyce R SRichardson C D. The Tumor-Associated Marker, PVRL4 (Nectin-4), is the Epithelial Receptor for Morbilliviruses [J]. Viruses, 2014, 6(6): 2268-2286.
3. Lofling J, Lyi S M, Parrish C R, et al. Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic acid N-glycolyl neuraminic acid [J]. Virology, 2013, 440(1): 89-96.
4. Vicentemanzanares M, Choi C KHorwitz A R. Integrins in cell migration - the actin connection [J]. Journal of Cell Science, 2009, 122(Pt 2): 199-206.
5. Forzan M, Marsh MRoy P. Bluetongue Virus Entry into Cells [J]. J Virol, 2007, 81(9): 4819-4827.
6. Schneider-Schaulies J, Meulen VSchneider-Schaulies S. Measles virus interactions with cellular receptors: Consequences for viral pathogenesis [J]. J NeuroVirol, 2001, 7(5): 391-399.
7. Von Messling V, Oezguen N, Zheng Q, et al. Nearby clusters of hemagglutinin residues sustain SLAM-dependent canine distemper virus entry in peripheral blood mononuclear cells [J]. J Virol, 2005, 79(9): 5857-5862.
8. Pratakpiriya W, Seki F, Otsuki N, et al. Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence [J]. J Virol, 2012, 86(18): 10207-10210.