Mutations in the Neuraminidase Gene of the Epidemic Influenza Virus Strain Isolated in the 2012-2013 Season and Improvement of a PCR Assay for the Detection of Influenza Virus

In 2011, we developed a real-time RT-PCR method to rapidly and sensitively detect three subtypes of influenza A virus [H1N1, H3N2, and influenza (H1N1) 2009] and influenza B virus (the conventional PCR method). This method was useful during the 2011-2012 epidemic season. However, epidemic influenza A virus strain H3N2 in the 2012-2013 season was undetectable by this method, possibly due to mutation in the neuraminidase (NA) gene of epidemic influenza A virus strain H3N2. Therefore, we improved the method by using the hemagglutinin (HA) gene instead of the NA gene as the target for the detection of influenza A virus strain H3N2. In addition, this improved PCR method also included a PCR detection system for the matrix (M) gene, well conserved and common to all influenza A virus strains. As a result, influenza A virus strain H3N2, which was undetectable by the conventional PCR method, was positive by the improved PCR method. Testing of specimens from 219 influenza-like illness patients during the 2012-2013 season by the influenza antigen immunochromatographic assay and conventional and improved PCR methods showed influenza virus A-positive rates of 24.2, 1.8, and 28.3%, respectively. All influenza A virus strains were positive for the M gene (in 62 [28.3%] of the 219 patients). These results suggest that the improved PCR method can determine the presence or absence of influenza A virus infection, even if a mutation in the HA or NA gene occurs in the future.