Insertion of a GFP Reporter Gene in Influenza Virus

Insertion of a GFP Reporter Gene in Influenza Virus

Insertion of a GFP Reporter Gene in Influenza Virus

ABSTRACT

The incorporation of a fluorescent reporter gene into a replication-competent influenza A
virus (IAV) hasmade it possible to trace IAVinfection in vivo. This protocol describes the
process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using
the established reverse genetics system. The strategy begins with the reorganization of
segment eight of the IAV genome, during which the open reading frames of nonstructural
protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP
fusion to the NS1 protein. The NS1, GFP, and NEP open reading frames (ORF) are then
cloned into the IAV rescue system backbone. Upon construction of the GFP-encoding
segment eight rescue plasmid, recombinant NS1-GFP influenza virus can be rescued
via co-transfection with the remaining seven rescue plasmids. The generated NS1-GFP
IAV can subsequently be used to visualize infected cells, both in vitro and in vivo.
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