It is known that avian influenza penetrates into the host cell by binding with sialic acids, the terminal residues of oligosaccharides. Avian influenza A virus preferably recognises α(2,3)-linked sialic acid residues as a receptor for penetration whereas human influenza A virus preferably binds with α(2,6)-linked sialic acids. Prevention of transfer of sialic acids to sugar bond or removal of it could be a defensive strategy against viral infection. There are 6 known sialyltransferases (ST3Gal1-6) that transfer α(2,3)-linked sialic acid residues to sugar branches. Most avian influenza virus isolates bind strongly to a sugar chain containing Neu5Aca(2,3) residues. In our study, we have shown that knockout of sialyltransferases leads to inhibition of viral infection. To find the expressed sialyltransferases in respiratory and digestive tracts, we used RT-qPCR. Tissue samples were taken from 3 chickens of Haisex white cross. Expression of mRNA was measured by RT-qPCR in 3 repeats and serial dilutions. Data analysis was carried out using the 2−ΔΔCt method. The amount of total RNA was normalised using GAPDH mRNA. For CRISPR/Cas9 targeting sialyltransferases, 3 guide RNAs for each gene were designed. We confirmed knockout (KO) of ST3GAL1 and ST3GAL6 by T7E assay. To estimate sialylation level on the cell surface, we performed a lectin-binding assay. For the assay, cells were incubated with fluorescein isothiocyanate (FITC)-labelled Maackia amurensis lectins and then subjected to flow-cytometry analysis to quantify the percentage of α(2,3)-sialylated cells in DF1 knockout (KO) v. DF1 wild type (wt) cell line. To estimate resistance to viral infection, a hemagglutinin binding assay was done, using fluorescein isothiocyanate (FITC)-labelled HA1 from H5N1 (A/Vietnam/1203/2004). To quantify the percentage of agglutinated HA1 molecules, DF1 KO and DF1 wt cells were analysed by flow cytometry. We found that mainly ST3GAL4 and ST3GAL5 are expressed in the chicken intestine (3-fold and 20-fold less compared with GAPDH level, respectively; other STs were not detected), and mainly ST3GAL1 and ST3GAL6 are expressed in the chicken respiratory tract (5-fold and 1.2-fold more compared with GAPDH level respectively; other STs were not detected). The expression profile of α(2,3)-sialyltransferases in the DF1 chicken cell line showed the noticeable expression of ST3GAL1 and ST3GAL6 compared with others as has been shown for the respiratory tract (500- and 1000-fold less compared with GAPDH respectively; other STs were not detected). In this study, we adopted the CRISPR/Cas9 system to knock out ST3GAL1 and ST3GAL6 genes in the chicken DF1 cell line. We confirmed that knockout of the genes leads to extinction of α(2,3)-sialic residues from the cell surface (7% v. 100% for DF1 KO v. DF1 wt cell line). Finally, we showed that knockout of sialyltransferases in the DF1 cells increases resistance against influenza A infection (16% v. 100% for DF1 KO v. DF1 wt cell line). Thus, creation of transgenic poultry with tissue-specific knockout of the α(2,3) sialyltransferases might protect domestic birds against influenza virus and block possible transfer of avian flu to human population.
N-Acetylneuraminyllactosylceramide, GM3-NeuAc, a new influenza A virus receptor which mediates the adsorption-fusion process of viral infection. Binding specificity of influenza virus A/Aichi/2/68 (H3N2) to membrane-associated GM3 with different molecular species of sialic acid.
