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Title: Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

Author
item Roth, Jason
item LI, JOSEPH - Utah State University
item BARNARD, DALE - Utah State University

Submitted to: Current Protocols in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2010
Publication Date: 5/1/2010
Citation: Roth, J.P., Li, J.K., Barnard, D.L. 2010. Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP). Curr. Protoc. Microbiol. 17;15F.1.1-15F.1.22.

Interpretive Summary: The ability to clone a virus has lead to new ways of studying its growth cycle and disease and can also assist in the discovery of treatments against the virus. With recent advancements in technology, it is now possible to clone a virus by converting its RNA genome into a DNA plasmid, which this method describes for the Human parainfluenza type 3 virus. This method also details the insertion of the gene encoding a green fluorescent protein, which glows green under UV light, into the viral DNA plasmid. The final step of this method describes the procedure to rescue or convert the DNA plasmid back into an infectious RNA virus. An infectious virus that glows green is a very useful tool in research that can be used to track the location and growth of the virus in living tissue. In addition, the amount of green fluorescence can be measured and is directly proportional to virus growth, which is very useful when testing antiviral drugs and vaccines that should reduce virus growth.

Technical 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 enhanced green fluorescent protein (EGFP) gene into the human parainfluenza virus type 3 (HPIV-3) antigenome and rescuing a recombinant, infectious virus is explained. The first step in this process includes the generation of a cDNA clone copied from 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.