Discrimination of Influenza A(H1N1)pdm09 virus with H275Y mutation Conferring Resistance to Oseltamivir by Duplex Real-time RT-PCR
Oseltamivir, a selective neuraminidase enzyme inhibitor, is one of the most important drugs for the treatment and prophylaxis of influenza. The oseltamivir resistance of seasonal H1N1 and A(H1N1)pdm09 influenza virus was mainly caused by a histidine (H) to tyrosine (Y) mutation at residue 275 of the neuraminidase (based on the N1 numbering). It is essential to develop a specific molecular method for the identification of H275Y mutation conferring resistance to oseltamivir. Here, a duplex real-time RT-PCR assay, using FAM and HEX dye labeled probes, was established the genotyping method to simultaneously determine the gene sequence coding for H or Y in the 275 site. The discriminative capacity of H275Y using this method was assayed by testing the armored RNA containing wild-typed or mutated target. Specificity was determined using the viral RNA from other influenza viruses. The method was as sensitive as the real-time method in detection of influenza A recommended by WHO. A total number of 923 A(H1N1)pdm09 positive samples collected from May, 2009 to Dec, 2018 were used for the oseltamivir resistance virus screening and one sample with mixed wildtype and mutated viruses was detected, the genotype ratio of oseltamivir resistant and sensitive viruses was 3.5:1.
Keywords - Pandemic Influenza A (H1N1); Oseltamivir Resistance; H275Y; Real-Time RT-PCR