The Evolution And Significance Of Viral Quasispecies Of Avian Influenza A (H7N9) Under The Selective Pressure Of Neuraminidase Inhibitor

Human infection with avian influenza is an acute respiratory infection caused by some subtypes of avian influenza virus that has crossed the species barrier.Since the mid and late 1990 s,human infection with avian influenza has occurred in a number of countries and regions.In March 2013,a previously unrecognized novel avian-origin influenza A(H7N9)virus associated with human deaths emerged in Shanghai and Anhui of China.Since the notifiation of human infection with H7N9 virus,the virus has caused five epidemic waves leading to a total of more than 1500 laboratory-confimed cases,with a high fatality rate.Furthermore,gene mutations have been found in H7N9 virus.On one hand,the H7N9 virus was recognized as easier to infect mammals and caused the largest number of human infections,more than other previously reported avian inflenza viruses did,on the other hand,the virus has evolved from LPAI(low pathogenic avian inflenza)virus to HPAI(highly pathogenic avian inflenza)virus.The emergence of clustered H7N9 cases suggests that the potential for the H7N9 virus to spread among the human population is still exist.H7N9 avian influenza has become a serious threat to human health and another challenge for human beings.H7N9 avian influenza virus belongs to the RNA virus.Because of the limited fidelity of the RNA dependent RNA polymerase,mutations are easily occured in H7N9 virus.The quasispecies of influenza virus are variant pedigrees comprised with the mixture of different but closely related viral genomes.Under the selective pressure(such as drug resistance,antigenicity,and pathogenicity,etc.),the quasispecies of influenza virus are dynamically changed which can affect many biological characteristics of the virus.With the selection of external pressure,the dominant influenza virus mutants will gradually be screened and become the dominant strain of influenza virus in the host.This research focuses on the effect of drug selection pressure on the evolution of H7N9 avian influenza virus quasispecies,and will serve as the foundation for clinical antiviral therapy.Currently,neuraminidase(NA)inhibitors such as oseltamivir and palaminivir are the major antiviral drugs used to treat patients with H7N9 avian influenza.The drug pressure of NA inhibitors is the main selection pressure of the H7N9 virus.With the use of NA inhibitors,the emergence of drug-resistant H7N9 virus variants directly affects the effect of clinical antiviral therapy.However,most of the reported resistant mutations of H7N9 virus were found in sporadic cases.In this study,using high-throughput sequencingtechnology,we investigated the quasispecies evolution pattern of H7N9 virus under the selective pressure of NA inhibitor(Peramivir),and pinpointed the effects of quasispecies evolution on the biological characteristics of H7N9 virus,such as drug resistance,antigenicity,and pathogenicity,etc.The main results we got are as follows:1.The quasispecies of H7N9 virus evolved under the selective pressure of NA inhibitor peramivir,leading to the formation of dominant species.The A/Nanjing/2/2013(H7N9)strain was used as the original strain and was passaged in MDCK cells for 3 times.Then the H7N9 virus was subjected to progressively increasing concentrations of peramivir.The results of the next generation sequencing showed that the presence of peramivir caused an increase in the number of SNVs that fixed during passaging.These loci were mainly located at HA and NA genes,suggesting the variants rapidly evolved and became the dominant species in the viral quasispecies.2.The variations of two key amino acid sites(I222T and H274Y)were found in the NA of H7N9 virus,and the withdrawal of drug pressure had no significant effect on the variation frequencies of I222 T and H274 Y.After twelve passages in the presence of peramivir,two mutations(I222T and H274Y)in the NA of H7N9 virus were identified.The frequencies of these two mutations increased rapidly after the emergence at the tenth passage.Further studies showed that the withdrawal of peramivir had no significant effect on the variation frequencies of I222 T and H274 Y,suggesting these two mutaitons might be neutral in the no drug environment.3.The sensitivity of NA-I222 T variant to oseltamivir carboxylate was decreased,while the NA-H274 Y variant showed resistance to both oseltamivir and peramivir.The results of NA inhibition assay showed that the sensitivity of NA-I222 T variant to oseltamivir carboxylate was significantly decreased(IC50: 3.02 n M,8.63-fold change),while the NA-H274 Y variant showed resistance to both oseltamivir and peramivir(IC50: 132.3 n M and 4.09 n M,respectively,426.77-fold change and 51.13-fold change,respectively).Consistent with the results of enzymatic reaction,oseltamivir carboxylate and peramivir both exhibited poor inhibitory effects against NA-H274 Y and NA-I222 T variants at cell level.However,the two variants(NA-H274 Y and NA-I222T)and control virus exhibited comparable sensitivity to T-705 and ribavirin.4.The proliferative ability of NA-H274 Y variant decreased at the early stage of infection which might be associated with the decline in the activity of the NA.The peak titer of NA-H274 Y variant and the control virus were comparable.To compare the proliferative property of variant and control H7N9 viruses in cell culture,we observed the replication kinetics of these viruses in MDCK cell.The results showed that although the proliferative ability of NA-H274 variant decreased at the early stage of infection,the peak titer of NA-H274 Y variant and the control virus were comparable after 48 h of infection.Then the kinetic analysis of NA activity was performed.We found that the NA-H274 Y variant showed lower activity(Vmax)and higher Km value than that of control.5.The H274 Y mutation had no obvious effect on the virulence of H7N9 virus,while the I222 T mutation enhanced the virulence of H7N9 virus to mice.Infection of mice with NA-I222 T variant resulted in more rapid weight loss and 100% lethality of mice,suggesting the virulence of NA-I222 T variant was increased as compared with control.Collectively,in this study,we systematically investigated the quasispecies evolution pattern of H7N9 virus under the selective pressure of NA inhibitor Peramivir,and pinpointed the effects of quasispecies evolution on the biological characteristics of H7N9 virus,such as drug resistance,antigenicity,and pathogenicity,etc.The findings of this study would probe into the variation trends of H7N9 virus at the molecular level,and provide guidance for the prevention and treatment of H7N9 avian influenza.