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A recombinant, infectious human parainfluenza virus type 3 expressing the enhanced green fluorescent protein for use in high-throughput antiviral assays.

ABSTRACT: The ability to rescue an infectious, recombinant, negative-stranded, RNA virus from a complementary DNA (cDNA) clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted into the human parainfluenza virus type 3 (HPIV-3) antigenome and a recombinant, infectious virus was rescued. Maximum EGFP expression levels, measured by fluorescence, were seen at day 3. Comparison of a 3-day, viral expressed EGFP fluorescence assay to a 7-day, neutral red assay, based on complete cell destruction in virus infected MA-104 cells, yielded Z'-factor values of 0.83 and 0.70, respectively. A 3-day, endpoint EGFP-based antiviral assay and a 7-day, endpoint neutral red based antiviral assay were run in parallel to establish antiviral sensitivity profiles of 23 compounds based on selective index (SI) values. Using an SI threshold of 10, the EGFP-based antiviral assay had a sensitivity of 100% and a specificity of 54%. Thus, the use of an EGFP-based antiviral assay for testing potential antiviral compounds against HPIV-3 in a high-throughput format may be justified.

SUBMITTER: Roth JP

PROVIDER: S-EPMC2701465 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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A recombinant, infectious human parainfluenza virus type 3 expressing the enhanced green fluorescent protein for use in high-throughput antiviral assays.

Roth Jason P JP Li Joseph K-K JK Smee Donald F DF Morrey John D JD Barnard Dale L DL

Antiviral research 20090202 1

The ability to rescue an infectious, recombinant, negative-stranded, RNA virus from a complementary DNA (cDNA) clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted into the human parainfluenza virus type 3 (HPIV-3) antigenome and a recombinant, infectious virus was rescued. Maximum EGFP expression levels, measured by fluorescence, were seen at day 3. Comparison of a ...[more]

PMID: 19189850

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Project description:Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

Dataset Information

A recombinant, infectious human parainfluenza virus type 3 expressing the enhanced green fluorescent protein for use in high-throughput antiviral assays.

Publications

A recombinant, infectious human parainfluenza virus type 3 expressing the enhanced green fluorescent protein for use in high-throughput antiviral assays.

Similar Datasets

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