Arvia Rosaria, Rocca Arianna, Casciato Benedetta, Stincarelli Maria Alfreda, Giannecchini Simone
Department Experimental and Clinical Medicine, University of Florence, Florence, Italy.
Dipartimento di Medicina Sperimentale e Clinica, Università di Firenze, Viale Morgagni 48, I-50134, Firenze, Italy.
Arch Virol. 2025 Jan 6;170(2):29. doi: 10.1007/s00705-024-06214-0.
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.
流感病毒聚合酶酸性（PA）亚基中的I38T替换是抗病毒药物巴洛沙韦酯（BXM）治疗中一个值得关注的耐药标志物。因此，监测PA/I38T突变具有临床重要性。在此，我们开发了三种快速灵敏的检测方法来检测和监测PA/I38T突变。此外，我们更新了之前开发的监测乙型流感病毒神经氨酸酶（NA）中D197N突变的方法，该突变与对奥司他韦的耐药性有关。利用带有感兴趣替换位点的寡核苷酸和我们实验室分离的流感病毒，开发了实时PCR高分辨率熔解分析（HRMA）用于快速检测PA/I38T和NA/D197N突变。随后，对94份A/H1N1pdm09、A/H3N2和乙型流感病毒阳性的临床样本以及在体外选择在巴洛沙韦酸（BXA，BXM的活性化合物）存在下生长的病毒进行了HRMA。HRMA能够区分合成寡核苷酸中的PA/I38与PA/I38T突变以及NA替换。然而，在我们的任何临床样本中均未检测到I38T突变和NA突变，这表明在所检测的循环病毒中不存在这些耐药标志物。在分析的43份A/H3N2临床样本中，只有1份样本中的病毒带有与对奥司他韦耐药相关的突变。所有HRMA结果均通过测序得到证实。最后，在体外进行BXA选择后，对A/H1N1pdm09和A/H3N2流感病毒进行了HRMA。经过12代培养后，HRMA鉴定出在BXA选择的A/H3N2变体中存在I38T突变，而在A/H1N1pdm09变体中不存在。总体而言，这些发现表明HRMA可能是在季节性流行期间快速监测流感病毒对BXM耐药性的有力工具。
