Analysis Of The Key Amino Acid Of PB2 Protein Affecting The Adaptability Of H10N8 Avian Influenza Virus In Mice And Chickens

H10N8 avian influenza virus?AIV?had caused two fatal human infections among three infections and recently in Northern Europe,avian origin H10N7 influenza outbreak among seals,demonstrated that H10 viruses can transmit between mammals and cause death in humans effectively.As the six internal segments of human infection H7N9 and H10N8 were from H9N2 AIV,we called these three types of virus as H9N2 original influenza virus.Previous study has revealed that the PB2 of H9N2 viruses had changed regularly,which were mainly focused on 292?I?V?,389?R?K?,598?T?M?,648?L?V?and 676?T?M?.We found that the sites of 292,389,588,598,648,and 676 had an obvious evolutionary turning point around 2012.Our previous research has revealed that except for E627 K and A588 V,the residue of I292 V,L648V,and T676 M of H10N8 AIV were correlated with mammalian adaptability.To verify whether these residues evolution towards to the adaption to mammals and avian simultaneously or not,a series of experiments were carried out.By using the reverse genetic technology,we rescued 11 strains,including r JX102,r JX102-PB2-I292 V,r JX102-PB2-R389 K,r JX102-PB2-A588 V,r JX102-PB2-T598 M,r JX102-PB2-E627 K,r JX102-PB2-L648 V,r JX102-PB2-T676 M,r JX102-PB2-D701 N,r JX102-PB2-F5?I292V,R389 K,T598M,L648 V and T676M?and r JX102-PB2-F6?I292V,R389 K,A588V,T598 M,L648V and T676M?,respectively.Experiments were carried out in vitro and in vivo.A mini-genome assay was performed in human HET293 T and avian DF-1 cells to evaluate the influence of mutation on polymerase activity.In HET293 T,the polymerase activity of r JX102-PB2-E627 K and r JX102-PB2-D701 N was much higher than r JX102-PB2.Besides,r JX102-PB2-F6 was 9 times and r JX102-PB2-A588 V was 6 times as high as r JX102.In addition,there was significant difference in polymerase activity between r JX102-PB2-I292 V,r JX102-PB2-T598 M,r JX102-PB2-T676 M,r JX102-PB2-F5 and r JX102.In DF1,r JX102-PB2-E627 K,r JX102-PB2-L648 V,r JX102-PB2-T676 M,r JX102-PB2-D701 N,r JX102-PB2-F5 and r JX102-PB2-F6 were extremely significant and was 3⁴ times higher than r JX102.r JX102-PB2-A588 V and r JX102-PB2-T598 M were almost 2 times higher than r JX102.r JX102-PB2-I292 V and r JX102-PB2-R389 K were about 1.5 times higher than r JX102.In A549 cells,except r JX102-PB2-R389 K and r JX102-PB2-T598 M exhibited low replication ability,all strains showed high titers than r JX102 in different time points or no significant differences.Among these strains,r JX102-PB2-A588 V,r JX102-PB2-E627 K,r JX102-PB2-D701 N and r JX102-PB2-F6,had a high virus titer than r JX102 in almost all the timepoints basically.However,better replication ability of r JX102-PB2-I292 V was displayed in the early stage?12h?while r JX102-PB2-F5 mainly appeared in the late stage?36h and 48h?.In CEF,the replication ability can be classified into two categories,that the unchanged and the increased in varying degrees.Concretely,r JX102-PB2-R389 KK,r JX102-PB2-L648 V,r JX102-PB2-T676 M,and r JX102-PB2-F5 were consistent with r JX102 at the timepoint detected.The rest of the variants displayed different enhancements at different time points.Among these,r JX102-PB2-I292 V,r JX102-PB2-T598 M,and r JX102-PB2-F5 had higher virus titers in the early?12h?and late?48h?stages,while the virus titers of r JX102-PB2-A588 V,r JX102-PB2-E627 K,r JX102-PB2-D701 N,and r JX102-PB2-F6 were significantly higher than that of r JX102 at all time points we have tested.BALB/c mice were infected with 10⁶EID₅₀/50?L virus and showed diverse body weight changes during the observation period: r JX102-PB2-T676 M with no apparent discrepancies with the control.Weight loss of the groups infected with r JX102-PB2-I292 V,r JX102-PB2-R389 K,r JX102-PB2-T598 M,and r JX102-PB2-F5 was lower than r JX102 and all mice were survived during the observation period.Virus titer was detected neither in brain nor nasal turbinate and was similar to that of r JX102 in lung.r JX102-PB2-A588 V,r JX102-PB2-L648 V caused 14.7% and 15.9% body weight loss and the survival rate of these two rates were 70% and 50%.All mice were died in r JX102-PB2-E627 K,r JX102-PB2-D701 N or r JX102-PB2-F6 group within 8 days.The virus titers of these five strains in lung were higher than r JX102.r JX102,r JX102-PB2-A588 V,r JX102-PB2-L648 V were chosen in the chickens' assay.Virus was detected in the vertebrate trachea.Among the infection groups,virus can be detected in 2/3 chickens and in the contact groups,both r JX102-PB2-A588 V and r JX102-PB2-F6 were higher than the r JX102 and r JX102-PB2-L648 V,for the former was 2/3 and the latter was 1/3.Both in infection and contact groups,r JX102-PB2-A588 V and r JX102-PB2-F6 had much high virus titer than r JX102.On the 4th day and the 6th day,the virus titer of throat swaps of r JX102-PB2-A588 V and r JX102-PB2-F6 was much higher than r JX102.The results of cloacal swabs of r JX102-PB2-A588 V and r JX102-PB2-F6 group can be detected in the early stage harbor virus for a long time.As shown in the results above,r JX102-PB2-A588 V and r JX102-PB2-F6,had a higher polymerase activity and the replication ability in both mammalian and avian cells,and replication well on both mice and chickens,indicating that r JX102-PB2-A588 V and r JX102-PB2-F6 can adapt to both mammals and avian.These results strongly suggest that it is particularly important to strengthen the prevention and control of H10N8 influenza virus.