Date of Award:
Doctor of Philosophy (PhD)
Animal, Dairy, and Veterinary Sciences
Dale L Barnard
Reverse genetics is a discipline that involves the use of genetic manipulation and modification to study an organism's altered phenotype. In this study, infectious recombinant viruses were rescued from altered cDNA clones encoding the antigenome of human parainfluenza virus type 3 and the resulting phenotypes were examined. In one clone, the gene for the enhanced green fluorescent protein was inserted into the virus antigenome to be expressed during viral replication, resulting in infected cells emitting green fluorescence. Viral titers, mRNA replication, and genomic replication for the virus expressing the enhanced green fluorescent protein were reduced when compared to the human parainfluenza virus type 3 wild-type strain. In addition, the sensitivity of the virus expressing the enhanced green fluorescent protein to antiviral compounds is increased when compared to the wild-type strain, which may lead to the identification of false positive antiviral compounds. An assay that measures the enhanced green fluorescent protein as a direct indicator of virus replication can be shortened to 3 days in duration and is a more robust assay compared to assays that measure cellular viability. In other clones, mutations were introduced into the phosphoprotein gene to eliminate the expression of the D domain of the PD protein in order to understand its function. The titers of two recombinant knockout viruses that are deficient in the expression of the D domain are reduced when compared to the wild-type strain in both MA-104 and A549 cells. In MA 104 cells, viral mRNA transcription and genomic replication of the two knockout viruses are reduced when compared to the wild-type strain. In A549 cells, cellular expression and secretion of antiviral cytokines infected with the two knockout viruses are either reduced or remain unchanged when compared to the wild-type strain. These results suggest that the D domain may play a role in viral RNA synthesis and not in counteracting the host cell's antiviral response. The results of these studies shed light on the influence an additional gene has on viral replication and possible functions of the D domain.
Roth, Jason Peter, "The Use of Reverse Genetics to Clone and Rescue Infectious, Recombinant Human Parainfluenza Type 3 Viruses" (2009). All Graduate Theses and Dissertations. Paper 467.
Copyright for this work is retained by the student.