Study On The Genetic Variation Of Chicken H9N2Influenza Viruses

Vaccination is the effective means to prevent avian influenza. It is important to enhance thedevelopment of the poultry industry in China. The frequent and widely use and even abuse ofthe commercial vaccines in poultry drive the antigenic variation of influenza viruses. As themost predominant subtype in China, H9N2virus not only infects humans, but also frequentlyprovides internal genes for other subtypes. Given the epidemic potential and public healththreat, it emphasizes the necessity for monitoring genetic variation and biologicalcharacteristics of H9N2viruses to lay the foundations for developing influenza vaccine andantiviral drugs.In this study, the genetic variations and biological characteristics of four H9N2representatives were analyzed from the immunized chicken flocks with commercial SD96vaccine in Jilin, China during March to December,2012. Phylogenetic analysis showed thatH9N2viruses include two distinctive lineages: North American lineage (Ty66-like) andEurasian lineage. The Eurasian lineage can be further classified into four sublineages:Y439-like, KR323-like, G1-like and Y280-like. In the Y280-like sublineage prevalent inChina, there appears a novel clade (designated as JL12-like clade) including BJ94-like,SH98-like, G9-like, SD98-like and Y280-like clades. Moreover, it found that JL12-likeviruses are the predominant donors of the internal genes of H7N9viruses. Phylogenetically,JL12-like viruses were far away from those vaccine strains, indicating an antigenic differencebetween them.For the four new isolates, there were at least3-4variations at the antigenic sites comparedto the SD96vaccine strain. The cross-HI test demonstrated a definite difference inantigenicity between different clades in the Y280-like sublineage. There was relatively lowantigenic difference between JL12-like and Y280-like clades (R≥35.4), compared withG9-like, BJ94-like, SD98-like, or SH98-like clades (R≤25.0%). All these indicated that the antigenicity of JL12-like clade is changing or has changed and it is necessary to update thevaccine strain.The solid-phase binding assay showed that four isolates preferred SAα2,6to SAα2,3receptors, indicating that they acquired the potential for human infection. The dynamicssimulation showed that there was a stronger free binding-energy between four H9N2HAsand SAα2,6than SAα2,3, which was almost identical with the results of the solid-phasebinding assay. In addition, the amino acid at position226of HA may not bind to receptors,demonstrating that this site is not enough to change the receptor-binding preference. However,the Q/L substitution results in the change of binding sites and influences the contour betweenHA and receptors.With the increase of human infection with AIVs, antiviral drugs still represent the first lineof defense. Here, the susceptibility to Zanamivir of four isolates was detected. The NAenzymatic inhibition assay showed an obviously lower IC50value for DH104than those forDH102, DH108, or DH109. The molecular modeling demonstrated that the H264N mutationinduced lower binding affinity and the loss of hydrogen bonding between276E at the enzymeactive sites and Zanamivir. Therefore, the additional glycosylation chain makes DH104resistant to Zanamivir.Taken together, the commercial vaccines used in China cannot keep pace with theevolution of H9N2viruses. This virus not only plays a donor role in providing the internalgenes to other subtypes of AIV or seasonal influenza viruses, but also acquires the potentialfor human infection and the susceptibility to anti-influenza inhibitors. All these underscorethe necessity to reassess the significance of H9N2virus in veterinary and zoonosis, to payclose attention to its epidemic dynamics, and to enhance viral surveillance and prevention inorder to protect the health of animals and humans.