Genetic Analysis And Transmission Mechanisms Among Mammals Of Migratory Bird-origin Avian Influenza Virus

Influenza virus is currently one of the most concerned zoonotic pathogens.It is widely distributed in the natural environment,poultry and migratory birds.It can infect poultry and a variety of mammals including human beings and has an important impact on national security and industrial economy.Influenza virus can cause an influenza pandemic,seasonal influenza and pandemics and zoonotic influenza.Migratory birds are the reservoir of avian influenza viruses such as H3N8,H9N2 and H5N8,and these viruses continue to be circulating in migratory birds in China.In recent years,these viruses appear new biological phenotypes of genetic characteristics.Some viruses not only cause asymptomatic infection in waterfowls,sometimes can be lethal.Some migratory bird-derived viruses can be transmitted from waterfowl to land fowl and poultryderived H9N2 and H5N8 infect humans.The prevalence,evolution,and public health risks assessment of these virus subtypes in Chinese migratory birds need to be addressed urgently.This study starts with monitoring the epidemic dynamics of avian influenza viruses in migratory birds in eastern China.We analyze the intercontinental spread of avian influenza viruses between Asia and North America and assess the genetic variation of avian influenza viruses in natural hosts.Then we aim to provide data on avian influenza virus spreads between waterfowl and landfowl.Next,we assess the risk of mammalian infection by AI viruses from their natural reservoirs in animal models such as guinea pigs and ferrets.Lastly,we identify molecular features in the transmission and host adaptation of the virus to mammals.Part 1: Molecular epidemiology investigation of avian influenza in migratory birds in eastern china.First of all,we carry out the monitoring of transcontinental transmission of avian influenza virus and the new reassortant strains in migratory bird.In the context of global migratory bird migration,we select key migration locations such as the East Dongting Lake,Bohai Bay Wetlands,Inner Mongolia Tumuji,Shanghai Chongming Dongtan along the Atlantic-American migratory bird migration route in eastern China to carry out active monitoring of avian influenza virus.Over the 4-year period between 2016 and 2019,35,604 samples from 96 different bird species were collected and 88 strains of the virus were isolated.The prevalence of avian influenza in migratory birds was was about 0.247%.Twenty-three HA/NA subtypes of influenza A virus,including 12 different HA subtypes and 9 NA subtypes were isolated.H3 and H6 subtypes were the dominant epidemic strains,but the HPAIV H5N6 subtype was also widely distributed in migratory birds in2016.Virological and serological data show that the H9N2 avian influenza virus is widely prevalent in migratory birds.H9N2 avian influenza virus can infect a variety of wild birds including small owls,kestrels,goshawks and pheasants.In small multi-host ecosystems,the H9N2 virus can be effectively transmitted within and across species.Antigenicity analysis showed that the current poultry H5 vaccine strain had reduced protection against 2020 HPAIV H5N8 virus due to antigenic drift.The clade 2.3.4.4b HPAIV H5N8 viruses might pose a serious threat to poultry industry in China because of reduced efficacy of the H5 vaccine agaist 2020 HPAIV H5N8.Therefore,monitoring and preventive measures should be further strengthened.Part 2: The evolutionary genetics of avian influenza virus from migratory bird.We isolate and sequence the viruses from birds and performed genetic analysis.The evolution rate and most recent common ancestor(t MRCA)of each gene segment of avian influenza virus in the natural host are calculated by Bayesian Markov chain Monte Carlo(MCMC).Focusing on the intercontinental spread of avian influenza virus,we reconstruct the evolutionary dynamics of influenza based on a set of virus sequences which have been isolated at different points in time using BEAST.The aim is to estimate the ancestral locations of the virus using a Bayesian discrete phylogeographic approach to identify possible routes of introduction of the virus.Based on the above method,we can analysis of the role of migratory birds in the spread within continents and intercontinental.Genetic variation analysis revealed that there was rich genetic diversity in AIVs gene fragments of migratory bird-origin AIV in eastern China.Epidemiological investigation showed that the H3N8 subtype avian influenza virus was widely prevalent in migratory birds,and with rich genotypes.We classified eight H3N8 viruses into seven different genotypes based on genomic diversity.There was gene flow among species and different subtypes within an ecosystem.It was found that genetic material intercontinental exchange in H13 and H3N8 subtypes.The HPAI H5N8 virus detected in wild birds in China in autumn-winter of 2020 shows the highest identity with clade 2.3.4.4b HPAI A(H5N8)strains that circulating in Europe in a similar period,but different from the first semester of 2020 in Europe.The HPAI H5N8 virus in China and Europe originate from a single genetic group that firstly appeared in Iraq in May 2020 then subsequently in Kazakhstan and Russia in September.This cluster of HPAI H5N8 shares a common ancestor with viruses last detected in Egypt since 2017.Wild waterbirds(whooper swans)are implicated in the spread of HPAI H5N8 viruses along flyways from breeding grounds in Kazakhstan to wintering sites in China.The movements of some waterbird species(tundra swans)along the autumn-winter migration routes furtherly triggered a wider distribution of H5N8 from Northern China to Southern China.Part 3: Public health risk assessment of avian influenza virus in migratory birds.Then we evaluated the virulence and transmissibility of the novel reassortant strain in non-natural hosts.Receptor binding preference test are performed on representive wild bird-origin viruses.The public healthe risk of 2020 H5N8 HPAI,H9N2 and H3N8 viruses from wild birds are evaluated on animal model such as mice,guinea pigs and ferrets.The potential risk of avian influenza virus human-human transmission is analyzed based on the evaluation results.The H5N8 avian influenza viruses remain at very low risk of zoonotic transmission based on the results of avian-like receptor binding preference and poor ability of transmission in the guinea pig.However,it is worth noting that recently isolated HPAI H5N8 viruses in 2020 displayed moderate virulence in mice.Antigenicity analysis showed that all the H5N8 virus cross-reacted poorly with Re-11 antisera,which means the vaccine used in poultry in China could not provide efficient protection once the H5N8 virus spill into poultry from wild birds.Considering the ability of the H5N8 virus to rapidly spread among avian species,and generate novel strains by reassortment,it is necessary to carry out continuous closely monitoring in birds and risk assessment in mammals.The H9N2 isolates could directly infect mice without adaptation but were restricted to replicate in the respiratory system.Glycan binding preference analyses suggested that the H9N2 isolates have acquired a binding affinity for the human-like receptor.Notably,results from transimission experiment in guinea pigs and ferrets demonstrated the wild birds-derived H9N2 influenza virus exhibits efficient transmission phenotypes in mammalian models via respiratory droplets.Six of eight H3N8 viruses isolated naturally from wild birds have acquired the ability to bind to the humantype receptor.However,the affinity for ?-2,6-linked SAs was lower than that for ?-2,3-linked SAs.Experiments on guinea pigs demonstrated that three viruses transmitted efficiently to directcontact guinea pigs without prior adaptation.Notably,one virus transmitted efficiently via respiratory droplets in guinea pigs but not in ferrets.Part 4: Research on the molecular mechanism of transmission among mammals of H3N8 migratory bird-origin avian influenza virus.Finally,using the H3N8 avian influenza strain with distinct transmission ability but the similar genetic background,combined with reverse genetics,the key molecular markers determining the transmission of H3N8 in mammals were discovered.These results can provide a theoretical basis for monitoring and early warning of an influenza pandemic caused by the migratory bird-origin avian influenza virus.We further found that the PB1 S524 G mutation conferred T222 virus airborne transmissibility between ferrets.We also determined that the 524 G mutant increased viral pathogenicity slightly in mice compared with the WT(wild type).Based on these results,we elucidated the potential human health threat and molecular basis of mammalian transmissibility of H3N8 influenza viruses.We emphasized the need for continued surveillance of the H3N8 influenza viruses circulating in birds.The above results suggest that control of influenza viruses,continuous global surveillance,as well as understanding the mechanism of interspecies transmission and molecular determinants through which the emerging avian influenza viruses can acquire the ability to transmit to humans and other mammals are important keys in evaluating their potential risk to public health.