title: An optimised method for the production of MERS-CoV spike expressing viral pseudotypes.
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 100 mm Petri dish with 7 ml 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.
authors.affiliation: Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Chatham Maritime, Kent, United Kingdom.
authors.forename: K
authors.initials: K
authors.lastname: Grehan
authors.affiliation: Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Chatham Maritime, Kent, United Kingdom.
authors.forename: F
authors.initials: F
authors.lastname: Ferrara
authors.affiliation: Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Chatham Maritime, Kent, United Kingdom.
authors.forename: N
authors.initials: N
authors.lastname: Temperton
keywords: Lentiviral pseudotype; MERS coronavirus; Virus neutralisation