Genetic Evolution Analysis And Biological Characteristics Evaluation Of Wild Animals-origin H5Nx Avian Influenza Virus

In recent years,the outbreak of the H5Nx AIVs has brought serious threats to the poultry industry and human health.Furthermore,the H5Nx influenza virus has rapidly evolved.Constant appearance of new subtypes through mutations and reassortments brought new challenges to the prevention and control of influenza.Wild animals play an important role in the evolution,spread and transmission of the H5Nx influenza virus.Wild birds,which serve as the reservoir pool of avian influenza virus,provide excellent opportunities for the reassortment of viruses from different regions due to their large-scale,long-distance migration habits on a global scale.Understanding and mastering the pattern of epidemic and the genetic evolution and biological characteristics of H5Nx avian influenza virus in wild birds has important implications for the epidemiology of influenza and public health.Moreover,the lethal H5N1 avian influenza virus that spreads across species to wild mammals such as viverrids,tigers and lions threaten the biodiversity of wild mammals seriously.To monitor the prevalence,analyze the genetic evolution,assess the pathogenicity and infectivity of wild-animals origin H5Nx influenza viruses are critical to evaluate the risk of cross-species transmission.In this study,we conducted active early warning monitoring and emergency outbreak detection clarified the circulation of H5Nx avian influenza virus in wild animals.Phylogenetic analysis of wild-animals origin H5Nx influenza viruses were performed by genome sequencing and multiple sequence aligning.Amino acid mutation site analysis were conducted to reveal the genetic evolution.We carried out replication kinetics tests in vitro and in vivo to evaluate the pathogenicity and infectivity of H5Nx avian influenza virus from different wild animal species.In order to evaluate the transmission of H5Nx influenza virus in mammals,experiments that test receptor binding ability and transmission ability in guinea pig have been carried out.In the first place,we conducted active warning monitoring and emergency outbreak detection of wild birds and wild mammals.We collected 9082 samples including throat swabs,anal swabs and feces from different wild animals'species in migratory routes,stopover sites and wintering areas.For active warning monitoring,we detected and isolated low-pathogenic avian influenza virus such as H3N8?six strains?,H4N6?twelve strains?,H9N2?eight strains?,H6N5?six strains?,H7N8?14 strains?,H10N4?one strain?,H10N7?four strains?and H12N5?one strain?but no H5Nx influenza virus was isolated in wild animals.For the detection of emergency outbreaks,we isolated clade 2.3.4.4 H5N6 and H5N8 avian influenza viruses from dead wild birds and clade 2.3.2.1c H5N1 avian influenza viruses from dead tigers and lions.Furthermore,we performed phylogenetic analysis on the H5Nx isolates,identified their genetic evolution,and analyzed the key amino acid sites that determined their biological characteristics.It was found that the reassortment of H5Nx avian influenza viruses was complex.Amino acid mutations that carry enhanced mammalian pathogenicity were found on wild-animals origin H5Nx influenza virus belonging to different clades.Based on the above studies,we infected wild-mammals origin H5N1 and wild-bird origin H5Nx avian influenza virus to MDCK cells or BALB/c mice to study the replication ability in vivo and in vitro,respectively.The tissue tropism,LD₅₀ and the pathogenicity of H5Nx isolates were measured as well.We further evaluate the receptor binding capacity by red blood cell and transmission in guinea pig.The experimental results showed that the clade 2.3.2.1c H5N1 avian influenza virus were equipped with stronger replication ability both in vitro and in vivo,and had extensive tissue tropism and high pathogenicity to mice.Clade 2.3.4.4 wild-birds origin H5 subtype avian influenza virus can directly infected and replicated in the respiratory system without previous adaptation.Among them,wild-bird origin H5N8 showed low pathogenicity to mice,and wild-bird origin H5N6 assumed moderate pathogenicity to mice.Clade2.3.4.4 wild-birds origin H5Nx influenza virus had lower ability to replicate in vivo and in vitro compared to the wild mammalian source clade 2.3.2.1c H5N1 avian influenza virus and showed limited tissue tropism.Clade 2.3.4.4 wild-bird origin H5 isolates retains avian-like receptor binding capacity and cannot transmute between guinea pigs.In summary,our research has deepened the understanding of the H5Nx influenza virus in wild animals.The evaluation of the biological characteristics and analysis of genetic evolution of the wild animal-derived H5Nx flu virus provided reference to biosafety risk assessment of HPAIV H5Nx influenza virus.