Detection of neuraminidase inhibitors-resistant influenza virus through genetic and phenotypic analysis in Korea

Kim Heui Man, Lee Namjoo, Kim Mi-Seon, Kang Chun, Chung Yoon-Seok
Division of Viral Diseases, Center for Laboratory Control of Infectious Diseases, KCDC

The applicable anti-influenza drugs were classified into neuraminidase inhibitors (Oseltamivir, Zanamivir, Peramivir, and Lanaminivir), ion channel blockers (Rimantadine and Amantadine) and a cap-dependent endonuclease (Baloxzvir), according to their respective mechanisms. In South Korea, Oseltamivir, Zanamivir, and Peramivir are mainly prescribed for patients exhibiting influenza-like illness and reports of antiviral drug-resistant A(H1N1)pdm09 viruses are rare. The Korea Centers for Diseases Control and Prevention operates the Korea Influenza and Respiratory Viruses Surveillance System (KINRESS) to monitor epidemic features of influenza viruses and analyze viral characteristics, such as anti-drug resistance. First, the neuraminidase (NA) gene of type A or B influenza was analyzed to screen for resistance to neuraminidase inhibitors (NAIs). H275Y was found to be a major mutation that inhibited NAIs application, especially Oseltamivir, against A(H1N1)pdm09 infection. After confirming the presence of mutations in NA function, NA activity of isolated viruses was calculated via a fluorescence-based assay using 4-(methylumbelliferyl)-N-acetylneuraminic acid (MUNANA) substrate. Neuramindase inhibition assay was conducted by calculating NA activity of NAIs-treated viruses. KINRESS aims to monitor patients with influenza-like illness who visit clinics (primary hospitals). NAIs-resistant virus was not detected during KINRESS surveillance for the past five seasons. However, recently A(H1N1)pdm09 viruses harboring the H275Y mutation in NA were detected in two hematological malignancy patients infected with influenza. The patients exhibited prolonged respiratory symptoms, with released viruses in the respiratory tract in spite of Oseltamivir and Peramivir application. The results of neuraminidase inhibition assay indicated that the viruses were resistant to Oseltamivir and Peramivir, but not Zanamivir. Therefore, it is essential to monitor influenza-like illness in both inpatients and outpatients, including those who are immunocompromised.

Keywords: Influenza, Drug Resistance, Neuraminidase, Oseltamivir, Zanamivir, Influenza A Virus, H1N1 Subtype, Antiviral Agents