KEYWORDS:
Influenza virus, antiviral, baloxavir, susceptibility, replicative capacity
INTRODUCTION
Influenza is an acute infectious disease caused by the influenza virus and the worldwide epidemics each year result in approximately 3-5 million seriously ill cases and approximately 290,000-650,000 deaths.1 Antiviral treatment is recommended for the management of influenza infections, particularly in high risk individuals such as elderly and immunocompromised persons. Neuraminidase inhibitors (NAIs: oseltamivir, zanamivir, peramivir) are widely used as the current treatment for influenza,2 while adamantanes, M2 ion channel inhibitors, are no longer used due to widespread resistance in circulating influenza viruses.3 However, A(H1N1) viruses developed oseltamivir resistance in the 2007-09 influenza seasons,4,5,6 emphasizing the need for antivirals with a novel mechanism of action.
Baloxavir marboxil (BXM) became available for the treatment of uncomplicated influenza in otherwise healthy and high-risk patients in a number of countries, following its approval in Japan and the United States in 2018.7,8. Baloxavir acid (BXA), the active form of BXM, selectively and potently blocks a catalytic center of cap-dependent endonuclease (CEN) located in the polymerase acid (PA) protein of the influenza polymerase complex, which consists of PA, polymerase basic 1 (PB1) and PB2 subunits.9,10 The CEN is highly conserved across all types of influenza viruses11 and plays an essential role in the transcription, protein synthesis, and viral genome replication,12 and therefore BXA displays broad-spectrum activity against influenza A, B, C, and D viruses.13,14 In clinical trials, single-dose BXM treatment was superior to placebo in relieving influenza symptoms and additionally, superior to both Oseltamivir and placebo in reducing the viral load.7,8 However, amino acid (AA) substitutions at position I38 (T/M/F) in the PA subunit have been identified as the most common treatment-emergent substitutions associated with reduced susceptibility to BXA.10,15 Influenza surveillance studies conducted in Japan during the 2018-19 influenza season confirmed treatment-emergence of PA/I38T and PA/I38M variants in A(H3N2)-infected subjects.16,17 A(H1N1) and A(H3N2) viruses harboring PA/I38T substitution were detected in some few subjects without prior BXM-treatment, suggesting the possibility of human-to-human transmission of the variant viruses.17,18 In addition to the I38 substitutions, E23K/G, A37T and E199G substitutions were identified in the PA subunit that affect BXA susceptibility by less than 10-fold.10,19 20 Therefore, consecutive monitoring of variant viruses with reduced BXA susceptibility is required to identify new potential genetic markers for the purpose of influenza surveillance.
It has been well demonstrated that mutations in NA conferring resistance to NAIs can negatively impact the viral replicative capacity, but additional HA mutations can also compensate these fitness cost.21 K229R in the PB1 subunit of influenza A viruses confers resistance to the viral RNA polymerase inhibitor favipiravir, and the fitness cost caused by this mutation can be compensated by a P653L substitution in PA that restores the fitness while maintaining favipiravir resistance.22 Therefore, AA substitutions located at distal position from drug-binding sites may impact drug sensitivity or compensate impaired fitness.
Here, we report phenotypic analyses of AA substitutions in PA, PB1, and PB2 subunits which were detected in clinical trials and influenza surveillance. This additional information on BXA susceptibility and replicative capacity of viruses with these substitutions will further support influenza surveillance.