Rapid identification of neuraminidase inhibitor resistance mutations in seasonal influenza virus A(H1N1), A(H1N1)2009, and A(H3N2) subtypes by melting point analysis

The high mutation rate of influenza virus, combined with the increasing worldwide use of influenza virus-specific drugs, allows the selection of viruses that are resistant to the currently available antiviral medications. Therefore, reliable tests for the rapid detection of drug-resistant influenza virus strains are required. We evaluated the use of a procedure involving real-time polymerase chain reaction (PCR) followed by melting point analysis (MPA) of hybrids formed between the PCR product and a specific oligonucleotide probe for the identification of point mutations in the influenza A virus neuraminidase gene (NA) that are associated with oseltamivir resistance [resulting in the amino acid change H275Y for seasonal and pandemic influenza A(H1N1) viruses and E119V for A(H3N2) viruses]. Therefore, 54 seasonal A(H1N1) (12 oseltamivir-resistant and 42 sensitive strains), 222 A(H1N1)2009 (5 resistant, 217 sensitive), and 51 A(H3N2) viruses (2 resistant, 49 sensitive) were tested by MPA, and the results were compared to those obtained by sequencing the NA gene. The results clearly indicate that the identification of drug resistance mutations by MPA is as accurate as sequencing, irrespective of whether MPA is performed using clinical material or the corresponding isolate. MPA enables a clear identification of mutations associated with antiviral resistance.