Molecular Mechanism Of H9N2 Subtype Avian Influenza Virus To Increase Transmitting In Chickens By Preference Of ?-2,6 Sialic Acid Receptor

H9N2 Avian Influenza Virus(AIV)is one of the most common pathogens to the poultry in China which causing huge economic losses.At present,the majority of H9N2 AIV isolated in nature tends to bind to human sialic acid receptor(named as Sia ?-2,6-Gal).However,recent studies have demonstrated that avian sialic acid receptor(named as Sia ?-2,3-Gal)mainly existed in chicken tracheal cells.Thus,what is the function of the H9N2 AIV binding to human sialic acid receptor? What is its molecular mechanism? These questions are not clear.Thus,we performed some experiments as below in this study.First,we investigated the role of the key amino acids that decided to binding to Sia ?-2,6-Gal involving in H9N2 AIV transmitting in chickens.According to previous results,we chose two H9N2 AIVs with different binding preference,A2093 virus(HA-226 L,mainly binding to Sia ?-2,6-Gal)and F98 virus(HA-226 Q,mainly binding to Sia ?-2,3-Gal).These viruses and their mutants were rescued by reverse genetic system,named as rA2093,rA2093-HA-L226Q(mainly binding to Sia ?-2,3-Gal,rF and rF-HA-Q226L(mainly binding to Sia ?-2,6-Gal,and were used to perform chicken transmission experiment.The result indicated that only rA2093 were detected in the oropharynx swabs from the contacted group.It suggested that HA-Q226 L could efficiently improve the transmission ability of rA2093 in chickens.Conversely,HA-Q226 L obviously increased the replication ability of rF in the oropharynx swabs from the infected group.However,neither rF or rF-HA-Q226 L were detected from the contacted group.In a further study,HA-Q226 L obviously increased the virus titers of rA2093 and rF in the tracheas from either the infected group or the contacted group.In a word,we found that HA-Q226 L could significantly increase the virus replication ability of H9N2 AIV in the up respiratory tract,and then played an important role in the transmission among chickens.Next,we studied the mechanism of enhancing H9N2 AIV replicating and transmission abilities in chickens by HA-Q226 L from 3 aspects.1)Receptor binding process: HA-Q226 L downregulated abilities of rA2093 and rF binding to chicken tracheal cells.2)Virus releasing process: HA-Q226 L made virus like particles of rA2093 and rF easier to release from cells.3)Heat stability and Acid stability: HA-Q226 L could increase the heat stability and acid stability of rA2093 and rF more stable.These results demonstrated that HA-Q226 L could make progeny viruses of H9N2 AIVs easier to release from cells,and make the viruses more stable in the environment.Both of these were benefit for H9N2 AIV to replicate and transmit among chickens.In a summary,this study confirmed that HA-Q226 L could cause the receptor binding preference of H9N2 AIV from avian Sia ?-2,3-Gal to human Sia ?-2,6-Gal,and played an important role to enhance the virus transmission ability among chickens.The molecular mechanism was that HA-Q226 L improve H9N2 AIV releasing from cells and then was benefit for virus replicating.Moreover,HA-Q226 L enhanced the heat stability and acid stability of H9N2 AIV contributing to virus better adapting in the environment.