The Mechanism Of Mammalian Adapation Of The Novel H10N8 Avian Influenza Virus

As one of the important zoonotic pathogens, avian influenza virus(AIV) can infect mammals and birds, usually cause yearly seasonal epidemics or occasional global pandemics in humans. Currently, there are virus strains from 16 HA subtypes and nine NA subtypes circulating in birds. Normaly, AIV cannot infect human beacause of its low affinity to the sialic acid receptor of human. However, virus reassortment and adaptive mutations contribute to the evolution of influenza virus and exhibit a serious threat to breeding industry and human public health security.After the first human infection with H7N9 occurred in east China, the first human infection with an avian influenza A(H10N8) virus was confirmed in Jiangxi Province On 17 December 2013.The patient died after nine days in hospital and so far, there are three people had infected with H10N8 virus,two of them died. Stduies had confiremed that the H10N8 virus retains a strong preference for avian-type receptors. However, the biological characteristics of this H10N8 virus and the molecular mechanism of its pathogenicity remain unclear.In this study, we characterized two H10N8 isolates, a human-origin strain A/JiangxiDonghu/346-1/2013(JX346) and an avian-origin strain, A/chicken/Jiangxi/ 102/ 2013(JX102).The results indicated that JX346 exhibited a higher replicative ability than JX102 in different cells. We evaluated the pathogenicity of these two viruses in a mouse model. JX346 showed high pathogenicity(MLD50=102.25EID50), the mice all died in 5 days post challenge(dpi) in the dose of 106EID50, while JX102 was nonpathogenic(MLD50> 106EID50). Then we used reverse genetics to exchange the genes of JX346 and JX102. The results of the mice experiments showed that PB2 gene of JX346 could enhance the virulence of JX102, infected mice all died within 5 dpi. Conversely, the virulence of the reassortant bearing the PB2 gene from JX102 in the JX346 background was significant attenuated. The JX346-102PB2 inoculated mice experienced just a slightly body weight loss and were recovery in 8 days. These results demonstrated that PB2 gene was the key determinant for the pathogenicity of the H10N8 virus.To identify amino acids in PB2 that contribute to the high virulence of JX346 in mice, we compared the PB2 sequences of JX346 and JX102 and found that there were eleven amino acid differences. PB2 627 K, a well-characterized mammalian adaptation marker was present in JX346.The results of the mutagenesis analysis demonstrated that lysine at position 627 of PB2 is not the only key virulence determinant of JX346 in mice. JX346-PB2 627 E still showed high pathogenicity in mice(MLD50=103.5EID50). Further studies indicated that other residues such as 87 E, 292 V, 340 K, 588 V, 648 V, 676 M were also responsible for the pathogenicity of the human H10N8 virus.Next, we focus on the residue 588 V, which located in the 627-domain of PB2 and found that compared with the parent viruses, the avian-origin H10N8, H7N9 and H9N2 influenza virus, harboring PB2-588 V exhibited higher polymerase activity, more efficient virus replication in mammal and avian cells and higher virulence in mice. Importantly, analyses of the PB2 sequences obtained from the database showed that avian H9N2 or human H7N9 influenza viruses bearing PB2-588 V had the tendency to be increase. Above all, we demonstrated that the substitution A588 V may be a new strategy of mammalian adaptions for the avian influenza virus.Furthermore, we explored the molecular mechanism of the mammalian adaptation of the residue 588 in the JX102 background. Some similar properties of aliphatic amino acids(T, I, L, S, G)were introduced into the positon 588 of JX102 PB2. The results showed that 588V、588T、588I and 588 L significantly enhanced the polymerase activity and replicative ability of JX102 in mammalian cells, while 588 S and 588 G showed no effects. Results of mice experiments showed that viruses bearing 588 V, 588 T, 588 I and 588 L exhibited high virulence and induced serve inflammatory reaction and pathological inflammatory injury in lungs. The inflammatory cytokines such as IL-6, TNF-α and IP-10 were high expressed and the lungs displayed acute inflammatory cell infiltration and hemorrhage. In the end, we explored the mechanism of residue 588 in PB2. The results showed that 588 V, 588 T, 588 I and 588 L exhibited a lower polymerase activity and replicative ability in importin-α1 silenced cells. But these results could not be found in other importin-α(α3, α4, α5, α7) silenced cells. These results demonstrated that residue 588 of PB2 promotes the mammalian adaptation of avian influenza virus was regulated by importin-α1.In conclusion, this study determined the biological characteristics of the novel H10N8 avian influenza virus and showed that human JX346 virus exhibited high lethality and the residues 87 E, 292 V, 340 K, 588 V, 627 K, 648 V and 676 M were responsible for the high pathogencity. Moreover, we found the residue in 588 of PB2 related to the mammalian adaptation of H10N8, H7N9 and H9N2 avian influenza viruses. And this promotion was regulated by the host cell protein, importin-α1, which provided reference for the treatment and prevention of avian influenza viruses.