Substitution of I222L-E119V in neuraminidase from highly pathogenic avian influenza H7N9 virus exhibited synergistic resistance effect to oseltamivir in mice

Jing Tang,Rongbao Gao,Dayan Wang,Weijuan Huang,Qiongqiong Fang,Liqi Liu,Shuxia Zhang,Zhaomin Feng,Cuiling Xu,Jia Liu,Xiyan Li

doi:10.1038/s41598-021-95771-4

Abstract

That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC50 fold change of oseltamivir to HPAI H7N9 with NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than the E119V mutant virus. HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log10 TCID50/ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research.

Highlights

Human infection with highly pathogenic avian influenza (HPAI) H7N9 virus appeared at the end of 2016 and re-emerged at 2 0191–3
We want to test if the two framework mutations (E119V and I222L) of NA could coexist in HPAI H7N9 virus, and study its effect on viral fitness and drug resistance effect in mammalian cells and mice
When influenza viruses possess drug resistant mutations, their replication level in cells or their pathogenicity to animals may be reduced to varying degrees, especially when mutations occur in catalytic residues that directly contact the sialic acid (SA)[19,27]

Summary

Introduction

Human infection with highly pathogenic avian influenza (HPAI) H7N9 virus appeared at the end of 2016 and re-emerged at 2 0191–3. Amino acid mutation at the key site of neuraminidase (NA) may lead to reduced susceptibility of influenza virus to NAIs and weaken the therapeutic effect[17]. When the conserved amino acids in the NA active site of influenza virus mutated, Scientific Reports | (2021) 11:16293. Our previous research found that the rate of strains isolated from human with reduced susceptibility to NAIs in HPAI H7N9 virus was high[23]. HPAI H7N9 viruses with reduced susceptibility to neuraminidase inhibitors (NA R292K, E119V or H274Y) showed good replication capacity in mammalian c ells[24]. NA 119V or D substitutions in HPAI H7N9 showed reduced susceptibility to oseltamivir and zanamivir r espectively[24]. We want to test if the two framework mutations (E119V and I222L) of NA could coexist in HPAI H7N9 virus, and study its effect on viral fitness and drug resistance effect in mammalian cells and mice

Methods

Results

Conclusion