Influenza viruses cause respiratory tract infections associated with substantial morbidity and mortality. Seasonal influenza epidemics affect between 5 and 15% of the world population, causing 3 to 5 million cases of severe disease and approximately 0.5 million deaths per year (54
). Influenza pandemics have also caused sporadic large-scale morbidity and mortality in the past century (15
). Type A influenza viruses are responsible for most of the influenza disease burden in human populations (46
). A novel H1N1 virus that emerged in 2009 caused an ongoing pandemic with excess morbidity and mortality (52
). The genome of influenza A viruses consists of eight negative-sense RNA molecules (35
) with highly conserved termini comprising the core promoter for transcription and replication (9
). Each viral RNA segment associated with nucleoprotein (NP) and RNA polymerase subunits (PB2, PB1, and PA) forming ribonucleoprotein (RNP) complexes.
Clinical and public health reference laboratories generally rely on embryonated chicken eggs or cell cultures of mammalian origin for isolation and propagation of influenza viruses (41
). However, culture of subtype H3N2 viruses from clinical specimens by inoculation into eggs is becoming increasingly problematic; currently very few specimens yield an isolate (34
). Viral isolation in cell cultures is handicapped by the relatively longer times required to obtain test results (5 to 7 days) and substantial requirements for specialized materials, equipment, and labor (42
), although culture systems such as R-Mix cells provide results faster (1 to 2 days) (2
). Cell lines expressing reporter genes inducible upon viral infection could mitigate this problem (23
). These reporter cells exploit the specificity of viral transcription factors for their target promoters in combination with the extreme sensitivity of reporter enzymes such as luciferase (31
). This approach expedites detection of specific viruses and amplifies the virus present in the clinical specimen, providing a live virus stock to be stored for further analyses. However, reporter cell lines have not been widely used for influenza virus, perhaps because the available HEK-293T reporter cells are not a favored substrate for virus isolation due to their susceptibility to the toxic effects of trypsin, which is required for the production of infectious influenza viruses in cultured cell lines (16
). MDCK cells have become a most widely used substrate for isolation of influenza viruses since they are known to be highly permissive for propagation of influenza viruses (47
) and resistant to the toxic effects of trypsin supplementation.
Some clinical virology laboratories continue to isolate influenza viruses in cultured cells, but the faster immunochromatographic or EIA (enzyme immunoassay)-based devices or PCR are most widely used for laboratory diagnosis of influenza (8
). This trend has become problematic for influenza surveillance programs because virus isolates are indispensable for monitoring antigenic drift, vaccine seed development, and drug sensitivity testing (12
). In addition, phenotypic analyses of viral isolates are critical to fully interpret results from vaccine and antiviral effectiveness clinical trials (3
). We report here on the development of a cell-based reporter system for influenza A virus using highly permissive MDCK cells expressing a luciferase-encoding amplicon controlled by canine RNA polymerase I (POL-I) promoter elements. This cell-based reporter system provides a sensitive method for the detection and isolation of influenza A viruses, and it is also useful for the screening of antiviral drugs or neutralizing antibody assays.