Drug Susceptibility Profile Of Human Infection With Highly Pathogenic Avian Influenza H7N9 Virus

Human infection with highly pathogenic avian influenza(HPAI)H7N9 virus appeared in the fifth wave of H7N9 epidemic at the end of 2016 and re-emerged at 2019 year.As the mutant of H7N9,HPAI H7N9 virus is characterized by the insertion of polybasic amino acids at the cleavage of its hemagglutinin(HA)protein.Although the mortality of HPAI H7N9 virus is similar to that of its low pathogenic(to birds)counterpart in human laboratory confirmed cases,the disease progresses of HPAI H7N9 is faster and with more severe clinical symptoms.As of April 27,2020,HPAI H7N9 virus has caused 33 laboratory confirmed cases,16 of which diedNeuraminidase inhibitors(NAIs)played a critical role in the clinical treatment of HPAI H7N9 infection.However,due to genetic variation of influenza virus,monitoring of its antiviral drug resistance is of great importance for guiding clinical treatment.In this study,a systematic and comprehensive drug susceptibility profile of HPAI H7N9 isolates covering 30 cases was studied for the first time.1.Characterization of drug susceptibility of human infection with highly pathogenic avian influenza H7N9 virus to NAIs and its drug resistance sites Firstly,thirty HPAI H7N9 isolates in mainland China from 2016-2019 year were analyzed with neuraminidase inhibition test(NAI test).Totally,13%(4/30)HPAI H7N9 isolates exhibited more than 10-fold elevated IC50 to oseltamivir or zanamivir.In all,23%(7/30)of the isolates had drug-resistant related mutations in NA which are R292K,E119V and H274Y by the next generation sequencing Recombinant viruses in the homogeneous genetic background of A/Guangdong/17SF006/2017(H7N9)were generated to examine the effect of NA amino acid substitutions on NAIs(oseltamivir or zanamivir)susceptibility and viral replication efficiency in vitro.All of the three substitutions conferred either reduced or highly reduced susceptibility to oseltamivir or zanamivir.All tested NAI-resistant viruses can replicate effectively in MDCK cells.NA R292K substitution of HPAI H7N9 resulted in a slight decrease of viral replication(P<0.05),while NA E119V or NA H274Y of the virus did not affect viral replication in MDCK cells(except for NA H274Y at 96h,P<0.05).The replication level of HPAI H7N9 virus bearing NA E119D substitution was similar to that of its sensitive strain at the early stage of replication,which conferred highly reduced susceptibility to zanamivir.2.The generation of resistance mutation sites and its potential compensated mutation of HPAI H7N9 virus:The proportion of HPAI H7N9 resistant strains was significantly higher than that of seasonal influenza virus and LPAI H7N9 virus.To study the generation of these mutations,HPAI H7N9 reassortment virus was prepared and passaged in the presence of oseltamivir or zanamivir for 10 passages.The oseltamivir induced R292K or E119V substitution in NA of the reassortment virus in vitro basically simulated that of HPAI H7N9 cases treated with NAIs,which indicated that the high frequency of resistant strain in HPAI H7N9 isolates is related to the use of NAIs.In addition,zanamivir induced an E119G substitution in NA of the reassortment virus.No amino acid mutations were identified in the internal genes of the recombinant virus,but R238I,A146E,G151E and G234T substitutions,which appeared in the presence of NAIs,were concentrated in the HA1 region of HA crystal structure.Considering that our first study above found that the drug resistance mutation of HPAI H7N9 virus did not significantly reduce its replication ability,the occurrence of HA mutation may compensate for the reduction of replication ability caused by NA mutation.3.The study of synergistic resistance sites to NAIs of influenza virus using a mouse model:Although NA I222L of HPAI H7N9 virus did not lead to drug resistance itself;it could have combinatorial resistant effect when it coexisted with NA E119V.The IC50 fold change of oseltamivir to HPAI H7N9 bearing 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 6log10TCID50/ml in MDCK cells.H7N9/PR8 virus bearing NA222L-1 19V 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.In conclusion,the proportion of strains with reduced susceptibility to NAIs in HPAI H7N9 viruses was high.Substitution of R292K,E119V or H274Y in NA induced by the use of NAIs during treatment leads HPAI H7N9 viruses to reduced susceptibility to NAIs.The replication ability of HPAI H7N9 with reduced susceptibility to NAIs(including synergistic resistance)in mammalian cells and pathogenicity(of H7N9/PR8)in mice were not significantly impaired.Therefore,it is necessary to strengthen drug resistance surveillance and new drug research.