Fitness of influenza A and B viruses with reduced susceptibility to baloxavir: A mini-review.

Neuraminidase inhibitors (NAIs), that currently include oseltamivir (Tamiflu® ), zanamivir (Relenza® ), peramivir (Rapivab® ) and laninamivir (Inavir® ), constitute an important class of antivirals recommended against seasonal influenza A and B infections. NAIs target the surface NA protein whose sialidase activity is responsible for virion release from infected cells. Because of their pivotal role in the transcription/translation process, the polymerase acidic (PA) and polymerase basic 1 and 2 (PB1 and PB2, respectively) internal proteins also constitute targets of interest for the development of additional anti-influenza agents. Baloxavir marboxil (BXM), an inhibitor of the cap-dependent endonuclease activity of the influenza PA protein, was approved in the United States and Japan in 2018. Baloxavir acid (BXA), the active compound of BXM, demonstrated a potent in vitro activity against different types/subtypes of influenza viruses including seasonal influenza A/B strains as well as avian influenza A viruses with a pandemic potential. A single oral dose of BXM provided virological and clinical benefits that were respectively superior or equal to those displayed by the standard (5 days, twice daily) oseltamivir regimen. Nevertheless, BXM-resistant variants have emerged at relatively high rates in BXM-treated children and adults. Consequently, there is a need to study the fitness (virulence and transmissibility) characteristics of mutants with a high potential to emerge as such variants can compromise the clinical usefulness of BXM. The purpose of this manuscript is to review the fitness properties of influenza A and B isolates harbouring mutations of reduced susceptibility to BXA.

Viral Fitness of Baloxavir-Resistant Recombinant Influenza B/Victoria- and B/Yamagata-like Viruses Harboring the I38T PA Change, In Vitro, Ex Vivo and in Guinea Pigs.

Seasonal influenza A and B viruses may cause severe infections requiring therapeutic interventions. Baloxavir, the latest antiviral drug approved against those infections, targets the endonuclease activity encoded by the polymerase acidic (PA) protein. While appearing effective at cessation of viral shedding, baloxavir demonstrated a low barrier of resistance. Herein, we aimed to assess the impact of PA-I38T substitution, a major marker of baloxavir-resistance, on the fitness of contemporary influenza B viruses. Recombinant wild-type (WT) influenza B/Phuket/2073/13 (B/Yamagata/16/88-like) and B/Washington/02/19 (B/Victoria/2/87-like) viruses and their respective PA-I38T mutants were used to evaluate replication kinetics in vitro, using A549 and Calu3 cells, and ex vivo, using nasal human airway epithelium (HAE) cells. Infectivity was also assessed in guinea pigs. In the B/Washington/02/19 background, there were no major differences between the recombinant WT virus and its I38T mutant when viral replication kinetics were evaluated in human lung cell lines and HAE as well as in nasal washes of experimentally infected guinea pigs. By contrast, the I38T mutation moderately impacted the B/Phuket/2073/13 viral fitness. In conclusion, contemporary influenza B viruses that may acquire baloxavir-resistance through the PA-I38T substitution could retain a significant level of fitness, highlighting the importance of monitoring the emergence of such variant.

Generation and Characterization of Drug-Resistant Influenza B Viruses Selected In Vitro with Baloxavir Acid.

Baloxavir marboxil (BXM) is an antiviral drug that targets the endonuclease of the influenza polymerase acidic (PA) protein. Antiviral resistance, mainly mediated by the I38T PA substitution, readily occurs in both A(H1N1) and A(H3N2) viruses following a single dose of BXM. Influenza B resistance to BXM remains poorly documented. We aimed to generate baloxavir-resistant contemporary influenza B/Yamagata/16/1988- and B/Victoria/2/1987-like viruses by in vitro passages under baloxavir acid (BXA) pressure to identify resistance mutations and to characterize the fitness of drug-resistant variants. Influenza B/Phuket/3073/2013 recombinant virus (rg-PKT13, a B/Yamagata/16/1988-like virus) and B/Quebec/MCV-11/2019 (MCV19, a B/Victoria/2/1987-like isolate) were passaged in ST6GalI-MDCK cells in the presence of increasing concentrations of BXA. At defined passages, viral RNA was extracted for sequencing the PA gene. The I38T PA substitution was selected in MCV19 after six passages in presence of BXA whereas no PA change was detected in rg-PKT13. The I38T substitution increased the BXA IC50 value by 13.7-fold in the MCV19 background and resulted in reduced viral titers compared to the wild type (WT) at early time points in ST6GalI-MDCK and at all time-points in human epithelial cells. By contrast, the I38T substitution had no impact on MCV19 polymerase activity, and this mutation was genetically stable over four passages. In conclusion, our results show a similar pathway of resistance to BXA in influenza B viruses highlighting the major role of the I38T PA substitution and suggest that I38T may differently impact the fitness of influenza variants depending on the viral type, subtype, or lineage.