title: Development of a novel influenza A antiviral assay.
abstract: Traditional methods used to monitor influenza infection typically require 2-5 days to perform, prompting a need for more rapid and quantitative methods for monitoring viral infection in 96-well formats. Such assays would find application in high-throughput screening for novel antiviral agents. A new method, based on branched DNA (bDNA) technology, is described for the specific detection of negative strand RNA of influenza A strains using a set of oligonucleotides designed for the A/PR/8/34 nucleoprotein (NP) transcript. By detecting the genomic strand, this signal amplification assay is appropriate for monitoring the kinetics of viral replication. Assay performance was monitored following infection of MDCK cells. The assay exhibited high reproducibility, good sensitivity over a range of multiplicity of infection and has a lower limit of detection of approximately 5 x 10 (5) RNA copies. Designed to quantitate the H1N1 strain A/PR/8/34, the assay also detects other influenza A subtypes, but not the evolutionarily more distant strain B/Yamagata/16/88. Validation as an antiviral assay was demonstrated with two influenza antivirals, zanamivir and rimantadine. The EC(50) values calculated following bDNA detection for zanamivir (265 nM) and rimantadine (9.4 microg/ml) in A/PR/8/34 infection correlate closely to data previously reported from visual CPE determinations, neutral red dye uptake and plaque assays, respectively. The advantages over the more time-consuming traditional assays suggest that the influenza bDNA assay is applicable to rapid screening of compound collections for antiviral activity.
authors.affiliation: Johnson & Johnson Pharmaceutical Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, USA.
authors.forename: Pamela C
authors.initials: PC
authors.lastname: Wagaman
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Leong
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Simmen
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D002460:Cell Line; D004285:Dogs; D006146:Guanidines; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology*; D008826:Microbial Sensitivity Tests; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism; D015345:Oligonucleotide Probes; D011714:Pyrans; D012367:RNA, Viral / Q000032:analysis; D012299:Rimantadine / Q000494:pharmacology*; D012794:Sialic Acids / Q000494:pharmacology*; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D053243:Zanamivir
chemical_list: D000998:Antiviral Agents; D006146:Guanidines; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D015345:Oligonucleotide Probes; D011714:Pyrans; D012367:RNA, Viral; D012794:Sialic Acids; D014758:Viral Core Proteins; D012299:Rimantadine; D053243:Zanamivir