| Highly pathogenic H5 subtype avian influenza virus(AIV)is widely prevalent with a variety of hosts and frequent variations.It is easy to be recombined with other genotype viruses,which can lead to virulence,host tropism and protective antigen changes.As a natural host and an important vector of avian influenza virus,waterfowl closely tracks the variation trend of H5 avian influenza wild strain in waterfowl,which is of great significance to actively prevent H5 avian influenza and maintain public health and safety.From February 2015 to December 2018,1107 samples of poultry tissues and808 samples of cotton swabs were collected from the Pearl River Delta region.Among which a total of 110 H5N6 subtype AIV HA and NA genes were identified and obtained.According to the nucleotide analysis and phylogenetic analysis,it is suggested that the HA genes of 55 newly indentifed strains between 2017 and 2018 were classified into a new clade termed as clade2.3.4.4f branch.Meanwhile,HA genes of54 strains and 1 strain were clustered into clade2.3.4.4d branch and clade2.3.4.4e branch,respectively.The amino acide analysis indicated that,the cleavage site of these 110 newly identified strains were showing highly pathogenic strain characteristics.S137 A,T160A,D1818 N,D1818S,A188 E,Q196K,Q226 L,S227R,G228 S amino acid mutations could be found in the newly found strains which may enhence ?2,6-sialic acid receptors binding ability.The potential glycosylation sites at the70-72,135-137,140-142,209-211 site were also carried by the HA proteins,and the 140-142 glycosylation sites could be found in all strains located in the clade2.3.4.4f branches.Amino acid deletions in the stem of NA were observed in all16 seletced strains.According to NA gene sequencing,nucleotide sequence similarity and genetic evolution analysis,the results showed that the NA gene of the above 110 strains belonged to N6 serotype,and the amino acid deletion in the stemof NA was found in all the tested strains.The six inner genes segements of 16 selected strains were further analyzed.The PB2 genes of 16 strains were clusted into ST339-like lineage.The other five genes,known as PB1,PA,NP,M,NS genes were all clusted into the H5-like lineage.M2 and NA protein show no amino acid mutations relating to the drug resistance.The analysis showed that the similarity between NP gene of 16 strains virus and A/duck/Guangdong/A9/2016(H9N2)strain isolated from duck population in 2016 was 97.7% ~ 99.6% respectively which suggested that such NP gene commonly exists in different serotypes of avian influenza viruses in the Pearl River Delta region.In this experiment,a representative test strain A/Goose/Guangdong/1807-6/2018(H5N6)(GS/1807-6)with 140-142 new glycosylation sites was selected from the clade2.3.4.4f branch of waterfowl.The vaccine strain A/chicken/Guizhou/4/2013(H5N1)(Re-8)was used as control.Construction of prokaryotic expression system of HA protein and optimization of expression conditions.The purified HA protein can be deteced by Western Blot with anti-His tag and anti-H5 HA protein antibodies.The results showed that the HA(GS/1807-6)protein and the HA protein of the vaccine strain Re-8 were successfully expressed.It provides a basis for the downstream test of H5 subtype AIV HA protein in the laboratory. |
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