The Application Of NGS In The Detection Of AIV And The Characterization Of H5 Subtype HPAIVs Isolated From Wild Birds In China From 2015 To 2017

Outbreaks of the H5 subtype highly pathogenic avian influenza(HPAI)have become more frequent globally in recent years with diverse circulating NA subtypes.Such viruses are also isolated from poultry and wild birds in China,mainly belonging to clade 2.3.4.4 and clade 2.3.2.There are so many subtypes and co-infections of AIV with multiple subtypes,posing a great challenge to the diagnosis of this disease.Next-generation sequencing(NGS)is economic and efficient.Moreover,it does not depend on known sequences and can obtain all 8 segments’ sequences of AIV just with one reaction,which are incomparable by sanger sequencing.As the natural reservoir for AIV,wild birds play a crucial role in the evolution and cross-species transmission,increasing the probability of the emergence of new viruses through gene recombination.To seek a more efficient method,explore the application of NSG in the detection of AIV,and understand the biological characteristics of H5 subtype HPAIVs isolated from wild birds in China in recent years,in this study,whole genomes of two AIV samples were amplified by AIV universal genomic amplification primers as NGS templates,which then were sequenced by Illumina and bioinformatically analyzed,including sequencing depth and the prediction of AIV subtypes.At the same time,systematical researches of H5 subtype HPAIVs causing outbreaks in wild birds in China from 2015 to 2017 were did,including whole genome sequencing and analysis of HA cleavage sites,potential glycosylation sites,receptor binding sites,amino acids deletion in NA stalk,amino acid sites in internal genes associated with enhanced pathogenicity of AIV to mice,homology,phylogenic evolution and genotypes.Besides,the pathogenecity in mammalian of some representatives was also evaluated in mice model.As a result,NGS templates of AIV were successfully prepared by a single PCR reaction;the M and NS genes were sequenced most deeply;the sample 1 was the co-infection of H5N2 and H9N2,and H9N2 was the dominant strain.The sample 2 was H5N8,of which genomic sequences were obtained completely.All HPAIVs contained the polybasic amino acid motif(-R/KRRKR*G-)at HA cleavage sites,which was the typical characteristic of HPAIV.Except for the highly conserved glycosylation sites,such as 26(NNS)、27(NST)、39(NVT)、181(NNT)、302(NSS)、499(NGT)、558(NGS),viruses from clade 2.3.2.1c possessed 156(NSS),isolates from clade2.3.2.1d contained 170(NDT)and 135(NSS)was found in SW/HuN/4/2016(H5N6)and WG/HuN/1/2017(H5N6)that were from clade 2.3.4.4.The receptor-binding sites at 226-228(H3 numbering)of all viruses exhibited avian-like receptor(α-2,3-SA)preference;there were amino acids deletions in N1 stalk and N6 stalk,whereas no deletion in N2 stalk and N8 stalk;some important mutations responding for enhanced pathogenicity of AIVs to mice,such as M1(30D,215A)and NS1(42S)were observed in all isolates and PB2(627K)was found in GBH Gull/QH/9/2015(H5N1)and PS/HuN/2/2016(H5N6);Amino acids and nucleotides homology rates of HA,NA and M were similar respectively,which of other 5 genes were significantly different however;The results of phylogenic analysis showed that HA genes belonged to clade2.3.2.1c,clade2.3.2.1d and clade2.3.4.4 respectively,the N1 gene from clade2.3.2.1c and clade2.3.2.1d evolved into 2 groups,N2,N6 and N8 genes were in the Eurasian lineage and internal genes evolved into multiple groups respectively.Moreover,most of the viruses derived from AIV with the same subtype,while some were multi-subtype recombinants;25 viruses formed 10 genotypes,indicating that the H5 subtype HPAIVs isolated from wild birds in China from 2015 to 2017 displayed genetic diversity.The mice test suggested that all representative strains in this study could infect mice without pre-adaptation and replicate effectively in the breathing system.But there were differencs in the pathogenicity.This study indicated that when NGS was used to detect co-infections of AIV with multiple subtypes,there were stricter requirements in the construction of libraries and the selection of splicing softwares,providing references for the detection of AIV in the laboratory by NGS and highlighted that comprehensive and active surveillance on wild birds should be implemented,laying the foundation for the prevention and control of H5 subtype HPAI.