![]() Mutations were introduced according to amino acid sequences observed in naturally occurring mutants with various amino acid substitutions. PSV1.0-A/Anhui HA was used as template to make the HA protein variants. KC853231) derived from A/Anhui were synthesized and separately cloned into the eukaryotic expression vector pSV1.0, generating 2 recombinant plasmids pSV1.0-A/Anhui HA and pSV1.0-A/Anhui NA. KC853228) and neuraminidase gene (NA GenBank accession no. The coding sequences of HA gene (GenBank accession no. Plasmid Construction and Site-Directed Mutagenesis All cells were grown at 37☌ in a humidified 5% CO 2 incubator. Madin-Darby canine kidney (MDCK) cells were grown in DMEM supplemented with 10% FBS, 2% HEPES, and 1% penicillin/streptomycin. CellsĢ93FT cells were cultured in growth medium (complete Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 2% HEPES, 1% nonessential amino acids and 1% penicillin/streptomycin) and maintained in DMEM with 1% FBS, 2% HEPES, 1% nonessential amino acids and 1% penicillin/streptomycin. We removed duplicate sequences from this data set, generating 902 influenza A(H7N9) virus HA protein sequences. All sequences were separately aligned with the reference by using Bioedit software in conjunction with manual editing. All previously published HA protein sequences of influenza A(H7N9) viruses isolated from human as of 30 September 2017 were downloaded from the National Center for Biotechnology Information and Global Initiative on Sharing All Influenza Data (GISAID) databases. The HA protein sequence derived from influenza A/Anhui/1/2013(H7N9) virus (hereafter, “A/Anhui”) was selected as a reference in this study. MATERIALS AND METHODS Determination of Naturally Occurring Single Amino Acid Substitutions of Influenza A(H7N9) Virus HA Protein In this study, we found some critical mutations that resulted in drastic changes in their reactions to neutralizing antibodies moreover, we demonstrated better protection against the dominant virus strains through DNA vaccination targeting these newly-emerged virus. Given the similar 3-dimensional structures in HA protein of all viruses analyzed so far, it would be of great interest to investigate the evolutionary pattern of the 5 antigenic sites in HA protein derived from influenza A(H7N9) viruses. Previous studies have identified 5 immune-dominant antigenic sites (AS referred to as A to E) in HA protein derived from H1 and H3. ![]() Clearly, mutations in the gene encoding HA protein could drastically alter the antigenicity of the virus, thereby rendering the vaccines less effective. As a result, HA protein is considered the most important component in current influenza vaccines. It is well established that HA protein is the antigenic target of vaccine-induced neutralizing antibodies. Antibodies against HA protein can neutralize the virus by directly blocking the initial attachment of the virus to the target cells. HA protein facilitates virus entry by binding sialic acid receptor on the host membrane through its receptor binding site (RBS), including the 190 helix and the 130, 150, and 220 loops. It is important to determine the evolution pattern of the hemagglutinin (HA) protein of this virus because it is the major viral glycoprotein mediating the adsorption and penetration of virus into the host cell. Since the first report of influenza A(H7N9) virus infections in humans, 5 epidemic waves have been observed, which had public health authorities around the world on high alert for the potential development of a human influenza pandemic. ( See the Editorial Commentary by Petrie and Lauring on pages 3–5.) H7N9, hemagglutinin, antigenic drift, amino acid substitutions, vaccine ![]()
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