Molecular Mechanism Of H6 Subtype Avian Influenza Virus Receptor Binding Specificity

The haemagglutinin of Influenza viruses which could recognize the host cell surface receptors,is a major determinant for host tropism of influenza viruses.The switch of the receptor binding preference of HA may enable influenza virus cross-species transmission.The avian influenza viruses obtaining human upper respiratory tract receptors(?-2,6SA)avidity may cause interspecies transmission to human,and then stably transmit between humans to cause pandemics.A/TaiWan/2/2013(H6N1)preferentially recognizes ?-2,6SA receptor and can infect people.Studies have shown that P186 L,E190V and G228 S enable A/TaiWan/2/2013(H6N1)to bind to?-(17),6SA preferentially.Although the H6 subtype avian influenza virus with priority binding to?-(17),6SA was not isolated in China,34% of the viruses had acquired the ability to bind to ?-(17),6SA receptor and some of which could transmit efficiently between guinea pigs by contact.Sequence alignment analysis indicates that A/Chicken/GuangDong/S1312/2010(H6N2)(hereinafter referred to as GD/S1312)and A/Chicken/HuNan/S3003/2009(H6N6)(hereinafter referred to as HuN / S3003)only has five amino acid differences in the receptor binding domain.GD/S1312 had a combination of?-(17),3SA and ?-(17),6SA receptors,while HuN/S3003 only has the ability to combine to ?-2,3SA.In order to study which amino acids make H6 subtype avian influenza viruses recognize ?-(17),6SA receptor and to assess the possibility that if the H6 subtype avian influenza viruses in China could obtain the ability to exclusively binding to ?-2,6SA through amino acid mutations in HA protein by natural evolution.By using reverse genetics technology and glycan test method,we rescued 10 mutant viruses and detected the receptor binding specificity of these 10 rescued viruses.In the rescued mutant viruses receptor binding specificity test,we found that 128 T,131N,133 E played an important role in the process of the rGD / S1312 recognizing human ?-(17),6SA,thus 128 T,131N,133 E may enable H6 subtype avian influenza viruses to bind to ?-2,6SA.We also found that Q226 L and G228 S mutations as well as Q226L/G228 S combined mutations could make rGD/S1312 preferentially bind to ?-2,6SA,and Q226 L mutation can change the receptor binding preference of rGD/S1312 from both ?-2,3SA and?-2,6SA to only ?-2,6SA.We conducted replication and transmission studies using guinea pigs in order to evaluate the respiratory droplet transmission ability of rGD/S1312 after it obtains the ability of specifically binding to ?-2,6SA.The results showed that Q226 L substitution and Q226L/G228 S combined substitutions could make rGD/S1312 transmissible by respiratory droplet between guinea pigs inefficiently.In conclusion,H6 subtype avian influenza viruses pose a clear threat to human health because Q226 L substitution and Q226L/G228 S combined substitutions could lead H6 subtype avian influenza viruses to preferentially recognizing ?-2,6SA.Although the transmission efficiency of rGD/S1312-Q226L/G228 S and rGD/S1312-Q226 L was poor,it may obtain high efficiency to transmit between mammals by accumulating gene mutations or gene combination.The study clarifies the mechanism of the H6 subtype avian influenza virus receptor binding specificity,which is of great significance to the early warning of another influenza pandemic.