Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility

Abstract

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model

“…Furthermore, a recombinant I38T-variant A/H1N1pdm09 virus demonstrated similar fitness characteristics in ferrets to the patient-derived A/H1N1pdm09 isolate, suggesting that transmission of the variant by respiratory droplets/aerosols was restricted to a greater extent than transmission by direct contact. Our study revealed that populations of I38T-variants are outcompeted by WT viruses in both A/H1N1pdm09 and A/H3N2; however, pure populations of I38T-variant viruses were still able to be transmitted in ferrets in accordance with previous studies (Imai et al, 2019; Jones et al, 2020).…”

“…In existing studies investigating the fitness of PA/I38T-substituted influenza viruses in animal models, variant influenza viruses of recombinant and patient-derived origins have been capable of robust viral replication in mice, hamsters and ferrets (Checkmahomed et al, 2019; Chesnokov et al, 2019; Imai et al, 2019; Jones et al, 2020). Ferrets represent the gold standard transmission model for evaluating the fitness of influenza viruses (Belser, Eckert, Tumpey, & Maines, 2016); of the available reports, patient-derived and recombinant I38T viruses have previously been shown to transmit from experimentally inoculated DFs to naïve recipients by direct (Jones et al, 2020) and airborne contact (Imai et al, 2019; Jones et al, 2020) routes. In our study, patient-derived A/H3N2 and A/H1N1pdm09 I38T-variants, when evaluated as pure viral populations, could sustain a chain of three sequential generations of direct contact transmission.…”

Comprehensive assessment of amino acid substitutions in the trimeric RNA polymerase complex of influenza A virus detected in clinical trials of baloxavir marboxil

“…However, these substitutions were unable to restore the growth impairment of the I38T-substituted viruses. Given that I38-substituted viruses show different patterns in terms of replicative capacity dependent on the type/subtype or isolated year, 11,19,26,28…”

Detection of baloxavir resistant influenza A viruses using next generation sequencing and pyrosequencing methods

“…However, virus specimen from a baloxavir-treated patient was reported to contain I38K as a mixture (I38 T/K/I) in Japan during 2018–19 season ( Takashita et al, 2019a ). Recently Jones and colleagues have shown that introduction of polar, positively charged lysine (K) at PA-38 reduced the polymerase activity by 50% compared to wildtype (I38) in minireplicon assay ( Jones et al, 2020 ). However, they apparently did not investigate the effect of this amino acid substitution on virus properties.…”

“…Virological surveillance conducted in Japan during 2018–19 season detected viruses carrying PA-38 substitutions with frequency of 2.3% in A(H1N1)pdm09 and 8.0% in A(H3N2) viruses, while no I38-substituted influenza B virus was reported ( https://www.niid.go.jp/niid/images/flu/resistance/20191227/dr18-19e20191227-1.pdf ). In ferret transmissibility experiments, influenza A and B viruses carrying I38T and I38M were shown to efficiently transmit to naïve ferrets by direct contact and airborne routes ( Imai et al, 2020 ; Jones et al, 2020 ). Moreover, limited human-to-human transmission of I38T-carrying influenza A viruses have been documented, raising public health concerns ( Imai et al, 2020 ; Takashita et al, 2019a ).…”