Abstract
The ability to rescue an infectious, recombinant RNA virus from a cDNA clone has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting the enhanced green fluorescent protein (EGFP) gene into the human parainfluenza virus type 3 (HPIV-3) antigenome and rescuing a recombinant, infectious virus is described. The first step in this process includes the generation of a cDNA clone copied from viral RNA isolated from an HPIV-3 wild-type infection. Next, the EGFP gene is inserted into the viral genome so that it is expressed independently during virus replication. Third, the viral support genes that are responsible for viral replication are cloned into T7 expression plasmids. Finally, an infectious, rHPIV3-EGFP virus is rescued from the cDNA clone with assistance from the viral support genes. Ultimately, cells infected with the rHPIV3-EGFP virus will emit green fluorescence that can be photographed and quantitated.