Abstract
The production and use of pseudotyped viral particles are widely established for many viruses, and applications in the fields of serology and vaccine development are manifold. Viral pseudotypes have proven to be powerful tools to study the effects of viral evolution on serological outcomes, viral tropism and immunogenicity studies. Pseudotyped viruses are chimeric constructs in which the outer (surface) glycoprotein(s) of one virus is combined with the replication-defective viral “core” of another virus. Pseudotypes allow for accurate, sequence-directed, sensitive antibody neutralisation assays and antiviral screening to be conducted within a low biosecurity facility and offer a safe and efficient alternative to wildtype virus use.The protocol outlined here represents a rapid and reliable method for the generation of high-titre pseudotype viral particles with the MERS-CoV spike protein on a lentiviral core, and is adapted from previously published protocols. This protocol is optimised for transfection in a 100mm Petri dish with 7ml of supernatant harvested, however it can be readily scaled to different production volumes.This protocol has a number of advantages including:•Use of readily available reagents.•Consistent, high virus titres.•Rapid generation of novel glycoproteins for research into strain variation.
Highlights
Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Chatham Maritime, Kent, United Kingdom GRAPHICAL ABSTRACT
Optional step: Additional media may be added to cells to allow a second harvest 18–24 h later by adding further DMEM/10% FBS/1% P/S
Note: A control pseudotype virus may be created by following the steps outlined above but leaving out the pCAGGS-MERS-CoV spike construct
Summary
Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Chatham Maritime, Kent, United Kingdom GRAPHICAL ABSTRACT. 4. Add the following plasmids (0.9:1:1.5 envelope:core:vector ratio) for transfection to tube 1: a. 5. Add 200 ml Opti-MEM1 to the plasmid DNA mix (tube 1). While transfection mix is incubating, the culture media on the 293T/17 cells should be removed and 7 ml of fresh DMEM/10% FBS/1% P/S added.
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