The Genetic Evolution Of H7N9 Subtype Avian Influenza Virus In Chicken Flocks In China And The Effect Mechanism Of PA-X Protein On The Infection Of Highly/Low Pathogenic Strains

Since the first report of the H7N9 subtype avian influenza virus(AIV)in 2013,five epidemic waves have been formed in Chinese population.1567 cases of H7N9 infection have been confirmed in laboratory by September 5th,2018,with a fatality rate of nearly 40%.Particularly,the fifth epidemic wave during 2016 to 2017 caused the largest number of human infections and widely distributed in China.During this period,the highly pathogenic(HP)H7N9 AIV was firstly isolated.Compared with the Asian HP H5N1 virus which has been endemic inChina for more than 20 years,the H7N9 AIV has resulted far more human cases in just 5 years.In addition,the coexisting of HP and low pathogenic(LP)strains poses a significant threat to the poultry industry and public health.Therefore,it is necessary to study the genetic evolution and biological characteristics of H7N9 AIV in China and to understand the future epidemic trends.Since H7N9 infections in human were mainly derived from exposure to poultry,the monitoring and prevention of H7N9 AIV in poultry is the most effective way to reduce and control human infections.Therefore,the establishment of rapid detection methods and the preparation of poultry vaccines with good immunogenicity are necessitated.Meanwhile,studying the pathogenic mechanism of H7N9 AIV on poultry can provide a scientific support for more effective prevention and control strategies.Concurrence of HP and LP strains to poultry in the same H7N9 subtype provides a good virus model for investigating the effect of the newly discovered PA-X protein which has been poorly studied in H7N9 AIVs.Therefore,exploring the effect of PA-X on H7N9 pathogenicity and host antiviral immunity will reveal the key role of PA-X protein in newly-emerged influenza virus and benefit the research of the pathogenic mechanism of H7N9 virus.Based on the epidemiological surveillance of H7N9 AIV in chicken flocks in part regions of China,the genetic evolution of H7N9 AIVs has been summarized from 2013 to 2017,and the biological characteristics of some HP H7N9 AIVs have been determined.Given the current prevalence of H7N9,we successfully constructed a vaccine candidate RGW with good immunogenicity and established a RRT-PCR detection method based on Taqman-MGB probe with high sensitivity for differentiated diagnosis of HP and LP H7N9 viruses.Besides,we have investigated the effect and mechanism of PA-X on regulating the replication and pathogenicity of H7N9 AIV.1.Genetic evolution and biological characterization of H7N9 avian influenza viruses in chicken flocks in China since 2013Based on our etiological surveillance of avian influenza in chicken farms and live poultry markets in part regions of China since 2013,the epidemiology and genetic evolution of 116 isolates of H7N9 AIV were analyzed.Our study showed that avian origin H7N9 virus has been local prevalent in China while the time of epidemic peak and region of H7N9 in poultry were similar to those of the human-origin H7N9,further confirming that human H7N9 was highly homologous to avian H7N9 virus.Phylogenetically,HA and NA genes of H7N9 avian isolates were both clustered chronologically during the surveillance period.However,the HP H7N9 strains isolated during the fifth epidemic wave(October 2016-March 2017)clustered independently in the clade of the 2015 isolates and diverged from the 2016-2017 branch,implying that these HP variants might evolved from the 2015 viruses directly.Generally,the evolution of internal genes is relatively conservative,with most H7N9 strains derived from the H9N2 virus of predominant genotype S that containing the PB2 and M genes of the G1-like lineage and the other internal genes of the F98-like lineage.However noteworthy,the PB2 gene was divided into two distinct evolutionary branches and the M gene of F98-like lineage appeared in few strains isolated in 2015,suggesting that H7N9 viruses may still undergo gene reassortment during evolution.Analysis of key amino acids showed that during the fifth epidemic wave,we isolated 7 HPAIVs with 4-amino-acid insertion at the cleavage site and there were two motif patterns:PEVPKGKRTAR↓GLF and PEVPKRKRTAR↓GLF.G186V and Q226L mutation at the receptor binding site of HA indicated that most of the H7N9 AIVs had already acquired the dual receptor binding property for human-type and avian-type receptors.The proportion of R140K mutation in HA antigenic epitope A increased year by year,suggesting that H7N9 AIVs may have had antigenic drift during the evolution process.Meanwhile,the amino acid substitutions of PB2-K526R,PB1-I368V,PA-K356R,NS1-P42S became increasingly established in the H7N9 isolates as year went by in this study,suggesting that the avian H7N9 viruses have acquired the potential to reproduce and gain virulence in mice.These viruses will pose a huge threat to public health once they acquired the adaption in mammals.The biological characterization of HP H7N9 and animal experiments showed that HP-H7N9 was highly pathogenic in chickens,but varied in pathogenicity in mice.GD15 strain showed both higher virulence and stronger replication than GD4 strain in mice and chickens,whether of which is related to the different cleavage site patterns required further study.Furthermore,animal infection and transmission experiments suggest that the transmission mode of H7N9 AIV was limited to contact transmission,but the transmission efficiency was extremely high.2.Construction of vaccine candidate strain for highly pathogenic H7N9 avian influenza virus and establishment of RRT-PCR detection method based on Taqman-MGB probeBased on the characteristics of epidemiology and genetic evolution of H7N9 virus circulating in China,we constructed a well-designed RGW vaccine strain with good immunogenicity and established a MGB probe RRT-PCR detection method for diagnosis of H7N9 with different virulence in order to effectively prevent and control the spread of H7N9 AIV in poultry.We successfully constructed a recombinant attenuated vaccine strain RGW using mutagenesis and reverse genetics strategies.The HA(deletion of 4 amino acids KRTA at cleavage site)and NA genes were derived from the HP H7N9 virus GD15 and the six internal gene segments were derived from H9N2 vaccine strain WJ57 of the S genotype.The mutagenesis strategy not only ensured the attenuation of the virulence of HP strain,but also not reducing its immunogenicity,which provide the possibility to prevent poultry from the infection of both of HP H7N9 and LP H7N9 viruses.RGW vaccine strain showed the same characteristics of good reproductive and immunogenicity with a high HA titer of 11 log2 after 10 passages in eggs,and maintained a high HI titer of 8log2 four months after immunization in chicken.In addition,it can not only produced high titers of HI antibodies against the HP H7N9 AIV,but also provided good crossreaction with the current epidemic LP H7N9 AIV with a cross-protection titer ≥ 91og2.Furthermore,the candidate vaccine strain RGW provided 100%clinical protection(death protection)of chickens challenged with HP H7N9 viruses,and prevented vaccinated chickens from virus shedding(80%～100%),which could effectively control the spread and circulationof H7N9 virus in the chicken flocks and thus reduce the human cases by contacting with poultry.At present,HP H7N9 strains and LP H7N9 strains are prevalent at the same time in poultry.It is important to establish a quick detection method for both of HP H7N9 and LP H7N9 in one reaction.Therefore,we established a dual channel RRT-PCR method based on Taqman-MGB probe technology to distinguish HP and LP H7N9 viruses.The Taqman-MGB probe had higher sensitivity than the common RRT-PCR method,and this was the first time using the MGB probe for the detection of H7N9 viruses.This method with probes designed on the base of the different motifs of the cleavage site in HP and LP H7N9 viruses can efficiently detect HP-H7N9 or LP-H7N9 under dual channel conditions with a sensitivity of 50 copies and achieved the effect of differentiation.Compared with the traditional chicken embryo isolation and RT-PCR method,our method was more sensitive with a total coincidence rate 92.7%.The clinical operation was easy and fast avoiding possible cross-contamination.3.Investigation of the effect and mechanism of PA-X protein on highly/low pathogenic H7N9 virusesIn order to explore the role of PA-X protein in the infection of highly/low pathogenic H7N9 viruses,we have successfully constructed the mutated highly pathogenic viruses GD15-PAX3,GD15-PAX5 and the mutated low pathogenic viruses r15-PAX3 and r15-PAX5 with decreased expression of PA-X by genetically modifying the ribosomal frameshift region of PA gene using the highly pathogenic strain A/chicken/Guangdong/GD15/2016(GD15)and the low pathogenic strain r15,which was attenuated at the cleavage site of GD15.Our study systematically compared the differences between the wildtype and mutated strains in the course of infection.Compared to the wildtype GD15 strain,the mutated viruses GD15-PAX3 and GD15-PAX5 became more virulent to SPF chicken embryos when using highly pathogenic strains as the backbone.The viral replication level increased in MDCK and DF-1 cells and SPF chickens,and the polymerase activity also increased in mammalian and avian cells.Besides,the apoptosis-inducing ability of GD15-PAX3 also enhanced.However,transcriptomics analysis of chicken lungs showed GD15-PAX3 induced a lower level of host innate immune response than GD15,mainly in that the amount of significantly differentially expressed genes induced by GD15-PAX3 was smaller than that of the GD15.Besides,the response levels of GD15-PAX3-stimulated inflammatory cytokines such as IL-6,IL-18,INF-P were also significantly lower than that of GD15.Therefore,when using highly pathogenic strains as backbones,PA-X can significantly inhibit viral replication and virus-induced apoptosis,and enhance the host’s innate immune response.However,when using low pathogenic strains as backbones,most of the results were contrary to those of the highly pathogenic strains.Compared with the primary virus r15,the modified virus r15-PAX3 had lower replication level in vivo and in vitro,with a significant decrease of the polymerase activity in mammalian and avian cells and a decrease in the ability to induce apoptosis.Meanwhile,transcriptomics analysis of chicken lungs showed that the amount of significantly differentially expressed genes induced by r15-PAX3 during host’s immune response was lower than that of r15,but the expression levels of important inflammatory cytokines such as IL-6,IL-18,TNF-a were significantly increased.Therefore,when using low pathogenic strains as backbones,PA-X can significantly enhance the virus replication and virus-induced apoptosis,and inhibit the host’s innate immune response ability.Therefore,our study found for the first time that PA-X protein played a role in regulating replication and virulence in HP/LP H7N9 virus,and that H7N9 virus PA-X protein could not induce significant "Host shutoff" activity in chicken lungs.We infered that the ability of PA-X protein to inhibit host protein expression may be host-or subtype-specific.Meanwhile,our results also indicate that PA-X may regulate the replication and pathogenicity of high/low pathogenic H7N9 virus by regulating the response level of inflammatory cytokines and varying degrees of virus-induced apoptosis,furthering our understanding of the intricate pathogenesis of HP/LP H7N9 viruses.