Cross-Species Transmission And Human Adaptive Mutation Of H7N9 Avian Influenza Virus

Since the spring of 2013,the H7N9 avian influenza virus(AIV)has broken through the species barrier to infect humans,with periodic outbreaks.Moreover,with the progress of the H7N9 avian influenza epidemic,the occurrence of clusters of human infections has also been continuously reported,posing a great threat to human health.The H7N9 AIV needs to continuously mutate to further adapt to the human body.The occurrence of clusters of human infections is a warning signal of the human adaptability of the virus.However,in China,the epidemiological characteristics,temporal and spatial distribution characteristics and clustering characteristics of H7N9 and other subtypes of AIVs in poultry and humans are still unclear.Therefore,research on the temporal and spatial distribution characteristics of H7N9 and other subtypes of AIVs outbreaks and the epidemiological characteristics of clustered epidemics is very important to ascertain the adaptation changes of H7N9 AIV infecting humans.Previous studies have shown that the PB2 protein of AIV is the molecular basis for the continuous adaptive mutation in humans.Although there have been reports of E627 K,D701N,K526 R and other adaptive mutation sites on the PB2 protein,it has been found that many H7N9 virus strains isolated from humans do not contain any reported adaptive mutation sites.The virus may use other unclear adaptive mutations in the PB2 protein to maintain virus replication in human cells.Therefore,further exploration and verification of potential adaptive sites on the PB2 protein are still needed.This study has three research purposes.First,clarify the epidemiological characteristics,temporal and spatial distribution characteristics,clustering characteristics,and host distribution characteristics of H7N9 and other subtypes of AIVs in poultry and humans in China,providing evidences for exploring the main epidemiological characteristics of human adaptability of H7N9 AIV after cross-species transmission.Second,explore and identify potential adaptive mutation sites in PB2 of H7N9 AIV.Third,verify the effects of potential adaptive mutation sites and the overlapping interactions with some reported mutation sites.According to the research purposes,this research is divided into three parts.In the first part of this study,we searched multiple public data sources related to avian influenza and adopted a systematic review method to analyze and excavate the epidemiological data of H7N9 and other subtypes of AIVs in poultry and humans,as well as the host distribution of the virus in China.Through epidemiological description,spatial autocorrelation analysis,spatio-temporal scanning and cluster analysis,explore the epidemiological characteristics,spatial autocorrelation and spatiotemporal gathering of H7N9 and other subtypes of avian influenza outbreaks in poultry and humans,and clarify host distribution characteristics.The results showed that human infection with H7N9 AIV has obvious regional gathering(Moran’s I = 0.5164,P = 0.001).High-high gathering areas are located in Zhejiang,Jiangsu,Anhui,Fujian and Jiangxi.Overally,the southeast coastal area is a hot spot and the central and northern parts of China are cold spots.The epidemic of human infection H7N9 virus is distributed in two spatiotemporal clusters,of which the first-level spatial and temporal aggregation area(RR = 5.60,LLR = 527.67,P< 0.001)is located in the southeast coastal area of China,and the gathering time is from January 1,2015 to December 31,2017.The secondary aggregation area includes 15 provinces in China(RR=2.10,LLR=34.84,P < 0.001),and the gathering time is from2017/1/1 to 2017/12 /31.Generally,there is a trend of spreading to the north and west of China;Guangxi is a high-high gathering area for poultry infected with H7N9 virus;the high incidence areas of other subtypes of AIVs are in southern China,such as Guangdong,Guangxi and Hunan;in addition,the host distribution of H7N9 subtype AIV is quite different from other subtypes,and it is mainly detected from humans(53.5%),chickens(31.5%)and the environment(12.3%).In the second part of this study,by downloading the PB2 protein sequences of the H7N9 AIV,based on the information of clusters of human infections obtained in the first part,to analyze the molecular evolution and mutation characteristics of the clustered epidemics of H7N9 avian influenza and combine with multi-factor regression model constructed by the PB2 protein sequences from different hosts and different subtypes to explore the potential adaptive mutation sites on the PB2 from two aspects.This study collected a total of 35 PB2 protein sequence information from clustered cases,and found that 34(34/35)strains isolate from clustered cases showed at least one reported adaptive mutation in the PB2 protein.In addition to reported adaptive mutation sites,single site mutations such as V292 I,K191E,N559 T,M676I/V,and combined mutations V511IM535L-M570I-I647 V occurred frequently;A total of 25565 PB2 protein sequences were downloaded,and analyzed by multivariate regression model and it was found that there were 5 adaptive sites related to human infection with H7N9 AIV,including K526 R,E627K and D701 N reported in the literature,and two new potential adaptative mutation sites V292 I and D740 A.In the third part of this study,based on the results of the second part,we further analyze the time trajectory of the mutation of key amino acid sites among different virus subtypes,and then use reverse genetics and cell biology techniques to verify the effects of these mutations.The study found that from 2013 to 2019,the proportion of PB2-V292 I in H7N9 AIV strains derived from poultry and humans has gradually increased,and it has a higher proportion in other subtypes such as H5N1 and H5N6.The D740 A mutation only exists in the H7N9 strains isolated in 2013.Further experimental studies have found that PB2-V292 I coupled with PB2-E627 K can increase viral polymerase activity and enhance virus transcription and replication,thereby enhancing the adaptation of PB2-E627 K.V292I alone does not promote the replication and transcription of the virus.the D740 A mutation on the PB2 will reduce the transcription and replication of the virus in mammalian cells.In summary,the main conclusions of this study are:1.The southeast coastal area of China is a high-risk area for human infection of H7N9 avian influenza virus.Compared with human infection with H7N9 virus,human infections with H5N1,H5N6 and H9N2 subtypes are relatively concentrated in the southern of China,such as Guangdong,Guangxi and Hunan.The distribution of hosts of H7N9 and other subtypes of AIVs is different.2.Almost all virus isolates derived from clusters of human infections of H7N9 virus have at least one reported adaptive mutation site in PB2 protein.Single site mutations such as V292 I and combined mutation V511I-M535L-M570I-I647 V occurred frequently.3.PB2-V292 I enhances the polymerase activity,the replication and transcription of H7N9 carring PB2-E627 K.PB2-V292 I coupled with E627 K mutation has a certain effect in the human adaptation process of human infection with H7N9 virus.