High-resolution melting analysis for detection of nucleotide mutation markers in the polymerase-acidic (PA) gene of influenza virus that are associated with baloxavir marboxil resistance

The I38T substitution in the influenza virus polymerase-acidic (PA) subunit is a resistance marker of concern for treatment with the antiviral baloxavir marboxil (BXM). Thus, monitoring PA/I38T mutations is of clinical importance. Here, we developed three rapid and sensitive assays for the detection and monitoring of the PA/I38T mutation. In addition, we updated our previously developed methods to monitor the D197N mutation in the neuraminidase (NA) of influenza B virus, which is associated with resistance to oseltamivir. Real-time PCR high-resolution melting analysis (HRMA) was developed for the rapid detection of the PA/I38T and NA/D197N mutations using oligonucleotides with substitutions of interest and influenza viruses isolated in our laboratory. HRMA was subsequently performed on 94 clinical samples that were positive for A/H1N1pdm09, A/H3N2, and type-B influenza viruses and on viruses that were selected in vitro to grow in the presence of BXA (baloxavir acid, BXM active compound). The HRMAs were able to discriminate PA/I38 from the PA/I38T mutation and NA substitutions in synthetic oligonucleotides. However, the I38T mutation and NA mutations were not detected in any of our clinical samples, indicating the absence of these resistance markers in the circulating viruses examined. Only one out of 43 A/H3N2 clinical samples analyzed contained a virus with mutations associated with resistance to oseltamivir. All the HRMA results were confirmed by sequencing. Finally, HRMA was performed on A/H1N1pdm09 and A/H3N2 influenza viruses following BXA selection in vitro. The presence of the I38T mutation in the BXA-selected A/H3N2 variant, but not in the A/H1N1pdm09 variant, was identified by HRMA after 12 passages. Overall, these findings indicate that HRMA could be a powerful tool for rapidly monitoring BXM resistance in influenza viruses during seasonal circulation.