Assay for Inhibitors of Influenza NS1 Protein Function: Influenza virus replication assay in MDCK cells infected with virus A/PR/8/34
Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill more ..
BioActive Compounds: 23
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH084878-01A1
Assay Submitter (PI): Daniel Engel
Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus.
We have miniaturized and optimized a cell based assay in which NS1 from influenza A is expressed in the budding yeast S. cerevisiae. NS1 is a multi-functional protein that counters the host innate immune response and facilitates viral versus cellular gene expression. Expression of NS1 causes a pronounced slow growth phenotype in yeast due to its intrinsic molecular activities. Small molecules that suppress the slow growth phenotype can be identified by a straightforward growth recovery assay using optical density (OD) as the measurement. The same yeast strain not expressing NS1 was used as positive control in the yeast growth recovery assay. The present experiment attempted to confirm the activity of compounds observed in the primary screen.
This assay confirms the activity of compounds from the primary screen by measuring their affect on viral replication.
Protocol details are given in Basu et al. J. Vir. 83(4):1881-1891 2009. Titers of influenza virus stocks were determined by 50% tissue culture infective dose (TCID50) analysis on MDCK cells, maintained in Iscove medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. All media and sera were from Invitrogen. The viruses were propagated in 10-day-old embryonated chicken eggs at 37C. Confluent cell monolayers were infected at a multiplicity of infection (MOI) of 0.1 for 48 h in the presence or absence of 5uM compound. Compounds were added at the beginning of infection and were present throughout the infection. After 48 h virus titers were determined by standard TCID50 analysis.
Vehicle control gave a TCID50 of 8.5. Compounds that reduced TCID50 by 1 unit or greater were considered active and given a score of 80. Compounds with less activity were considered inactive and given a score of 0.
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** Test Concentration.
Data Table (Concise)