Molecular Mechanism Of Transmissibility Of H9N2 Subtype Influenza Virus In Guinea Pig Model

H9N2 subtype AIV is popularly circulating in poultry and caused several human infection cases. Serological surveillance for H9N2 virus exposure in humans suggests that 2.3%-13.7% of workers exposed to poultry in China have detectable antibodies against the H9 influenza hemagglutinin protein. Human infected by H9N2 subtype AIV show a typical human-like illness that can easily go undetected, providing the viruses a greater opportunity to adapt to humans. To more important, parts of H9N2 subtype AIV have possessed the ability to bind to human-like receptor. In this study, a strain H9N2 subtype AIV was selected for adaptive passaging in mice and guinea pigs. And the virulence and transmissibility of mouse-adapted virus and guinea pig-adapted virus were evaluated. Further, we preliminary studied the molecular mechanisms for these adapted virus.Recent studies have documented that H5N1 subtype AIV and H7N1 subtype AIV could obtain droplet transmissibility in ferret by passages. Here, we used a guinea pig model to examine whether serial passages results in adaptive viral changes that confer a transmissible phenotype to a wild-type H9N2 virus. After nine serial passages of an H9N2 virus through guinea pigs, productive transmission by direct contact occurred in 2/3 guinea pig pairs. The efficiency of transmission by direct contact increased following the fifteenth passage and occurred in 3/3 guinea pig pairs. Three amino acid substitutions, HA1-Q227 P, HA2-D46 E, and NP-E434 K, were sufficient for efficient transmission via direct contact in guinea pigs(2/3 pairs). The two HA amino acid substitutions enhanced receptor binding to α2,3-linked sialic acid receptors. Additionally, the HA2-D46 E substitution increased virus thermostability whereas the NP-E434 K mutation enhanced viral RNA polymerase activity in vitro. Besides above three mutations, PB2-D195 N substitution also has a synergistic effect on transmission of SD2 virus in guinea pigs.We have proved that SD2 virus could obtain direct transmissibility in guinea pig model by passaging in guinea pigs. To compare the effect of host differences on adaptive evolution of influenza virus, we also adapted SD2 virus in mice by lung to lung. The mouse-adapted virus acquired high virulence in mice by only five passages and obtained three mutations. HA-N313D/N496 S substitutions could increase the ability to bind to α2,6-linked sialic acid receptors. Moreover, PB2-E627 K substitution significantly increases viral polymerase activity in mammalian cells. However, unlike guinea pig-adapted virus, the increase of human-like receptor and viral polymerase activity did not confer a transmissible phenotype to the mouse-adapted virus in guinea pigs.The activity of polymerase is critical for replication ability and transmissibility of influenza virus in hosts.We have found that PB2-D195 N substitution also particially contributed to the transmission of H9N2 subtype AIV among guinea pigs. According Influenza Data of Genbank, a PB2-195 N residue is present in several subtype influenza virus, including H5N1 viruse, H6N1 AIV, H7 subtype and H9N2 subtype AIV, particularly few H5N1 virus possessed both PB2-195 N and PB2-627 K. Therefore, we explored the effect of the two mutations on transmissibility and virulence of SD2 virus. Our results showed that PB2-D195 N and PB2-E627 K contributed to direct transmission of the H9N2 subtype AIV in guinea pigs and expand viral tropism in mice with virus replication in brain tissue of mouse.Our studies demonstrated that influenza virus could acquire a serial amino acid mutation with different phynotype of transmissibility and virulence via adaption in different mammalian modles. The results indicated that the evolution of influenza virus was also determined by the interaction between the virus and different hosts. Three adaptive mutations(HA1-Q227 P, HA2-D46 E and NP-E434K) could conferred direct transmissibility to H9N2 subtype AIV in guinea pigs. While the PB2-627 K residue, which is the important virulent molecular marker, together with PB2-D195 N contributed to direct transmission of H9N2 subtype AIV. Furthermore, receptor binding ability and viral polymerase activity decided the transmissibility of influenza virus. Our study could enrich the knowledge of H9N2 subtype AIV and are important for a better preparation for influenza pandemic.