Study On Gene Sequence Analysis And Biological Characterization Of H7N9 Subtype Influenza Virus From 2017 To 2019

Since the first patient infected with H7N9 was reported in March 2013,there have been five waves of H7N9 avian influenza in pandemics until 2020.Previous low pathogenic viruses in chickens mutated to highly in the last wave,and a total of human cases of H7N9 infection with deaths excedeed the previous outbreaks combined.It is very important to study the gene sequence and pathogenicity of the H7N9 influenza viruses because the viruses not only have caused huge economic losses to poultry industry but also have posed a public health concern to global.The occurrence of H7N9 outbreaks has been control when the H7N9 inactivated vaccine was introduced in poultry in the fall of 2017.In order to investigate the epidemic situation,genetic evolution and pathogenic characteristics of H7N9influenza viruses in poultry of China,in this study,samples from live poultry markets and poultry farms in some regions of China from 2017 to 2019 were isolated and identified,and 20 H7N9 subtype influenza viruses were selected for genomic sequence analysis and biological characteristics study.The whole genome of 20 H7N9 subtype avian influenza viruses was sequenced.Molecular characterization of 4 strains confirmed motif PEIPKGR↓GLF at its HA-cleavage site,which was the characteristic of low pathogenicity avian influenza viruses;however,some basic amino acids were inserted into the other 16 strains HA protein and confirmed motif PEVPKGKRTAR↓GLF,PEVPKRKRTAR↓GLF at its HA-cleavage site,which was the characteristic of highly avian influenza viruses.The analysis of the key amino acid sites had showed that G186V and Q226L double-site mutation in the HA protein in 3 viruses;R294K mutation in the NA protein in 2viruses;A588V mutation in the PB2 protein in 6 viruses;none of the E627K and D701N mutation in PB2 region that they favors avian adaptation and receptor-binding ability;all of them possessed the amantadine resistance mutation（S31N）in M2 region.Phylogenetic analysis of whole genome had showed that the branches of the H7N9 influenza viruses of HA and NA genes were divided into Yangtze River Delta and Pearl River Delta.The HA and NA genes of the 16 viruses were distributed in the branch of the Yangtze river delta;2 viruses（E112 and E128）were distributed in the branch of the pearl river delta;the HA gene of the 2 viruses（E65 and E656）was distributed in the branch of the Yangtze river delta but the NA gene was distributed in the branch of the pearl river delta.The internal genes of individual viruses were most close to human-origin H7N9 subtype influenza viruses and avian-origin H9N2subtype avian influenza viruses.Noteworthy,the prediction of potential glycosylation sites of HA and NA genes had increased one site in 2019,the mean rates of HA gene nucleotide substitutions and the mutation rates of first nucleotide,second nucleotide of each codon of the viruses during 2018-2019 were higher than the fifth epidemic.The standard antigen and standard serum（Re-1,Re-2）of the H7N9 subtype avian influenza vaccine were selected for the hemagglutination inhibition（HI）test.The results showed that Re-1 has a higher HI titer（5log2～9log2）for the isolates from2017,but a lower HI titer（3 log2～5log2）for the isolates from 2018-2019.Re-2 has a higher HI titer（6log2～9 log2）for the isolates from 2017-2019.Herein we selected four H7N9 subtype influenza viruses to study the pathogenicity in SPF chickens and BALB/c mice.Four-week-old SPF chickens and BALB/c mice were challenged intranasally with the viruses doses of 10⁶EID₅₀/200μL and 10⁶EID₅₀/50μL respectively.The results showed that the low pathogenicity avian influenza virus（E65）was not lethal to SPF chickens and the serum antibody turned positive after 14 days post-inoculation（dpi）;The highly avian influenza viruses（E664,F692,G375）were 100%lethal to SPF chickens,and the E664 and F692had stronger pathogenicity and horizontal transmissibility to SPF chickens than G375.Pathological tissue damage of heart,liver,spleen,lung and brain was observed in HE sections.E65 and G375 was not lethal to BALB/c mice but reduced the body weight gain transiently;E664 and F692 were 100%lethal to BALB/c mice and higher viruses titers were detected in the lungs and turbinates of dead mice.Moreover,the viruses titer could able to be detected in the lungs of the E65 and G375 groups of mice that did not die,which indicating that the lungs and turbinates were the main sites of viruses replication.Pathological tissue damage of lung,liver and brain was observed in HE section.