Mechanism Of Neurotropism And Pathogenicity Of H9N2 And H10N7 Subtype Avian Influenza Viruses In Mice

The research on the mechanism of avian influenza viruses(AIV)crossing host barrier and transmitting to mammals is very important.Clinically,except for human respiratory system,AIV can also infect central nervous system(CNS)directly,inducing acute encephalopathy combined with high mortality.However,most of the researches focus on the mechanism of infection and pathogenicity of AIV in mammals.There are only few researches studying on the neurotropism of AIV in mammals.Therefore,it is important to investigate the neurovirulence and adaptation mechanism of AIV in mammals.H9N2 are circulating in poultry worldwide,which had increased adaptation ability in mammals recently.H10N7 AIVs are endemic in multiple avian species.It can also break through the host barrier and transmit to mammals and even humans.Many cases of human infected with H10N7 AIV have been reported.Whether H9N2 and H10N7 viruses have neurovirulence and high pathogenicity after adapted in mammals and what are the key factors affecting these biological characteristics are still largely unknown.In the present study,we passaged an H9N2 A/chicken/Shandong/16/05(SD16)and an H10N7 A/mallard/Beijing/27/2011(BJ27)AIV in Balb/c mice by lung to lung to mimic the natural selection of AIV in mammals.After 13 passages and 9 passages respectively,the mouse-adapted H9N2 and mouse-adapted H10N7 viruses which showed increased pathogenicity with high mortality and neurovirulence in mice were generated.They are named as SD16-MA and BJ27-MA after three times plaque purification in MDCK cells.Sequence analysis of the mouse-adapted viruses indicated that eight amino acid substitutions including PB2 M147L,PB2 V250G,PB2 E627K,PB1 Y657H,HA L226Q,HA R211K,M1 R210K and NS1 L214F and five amino acid mutations including PB2 E158G,PB2 M631L,HA G218E,NA K110E and NA S453I were appeared in SD16-MA and BJ27-MA viruses respectively.Among them,H9N2-MA and H10N7-MA all had adapted mutations on PB2 and HA protein.Structure analyses indicated that PB2 E627K is adjacent to PB2 M631L.They are all located in PB2-PB1 and PB2-NP interaction region and play important roles in increasing polymerase activity of AIV in mammalian cells.HA G218E and HA L226Q are all located in 220-loop region and determine the sialic acid receptor binding affinity.It indicated that mutations in receptor binding region and polymerase protein are important for the adaptation of AIV in mammals.To further determine the viral factors that are important for the viral neurovirulence,a series of recombinant viruses were generated by reverse genetics under wild type SD16 and BJ27 viruses background.SD16 recombinant viruses included rSD16-PB2,rSD16-PB1,rSD16-HA,rSD16-M1,rSD16-NS1 and rSD16-MA.BJ27 recombinant viruses included rBJ27-PB2,rBJ27-HA,rBJ27-NA,rBJ27-PB2/HA,rBJ27-PB2/NA,rBJ27-HA/NA and rBJ27-MA.We found that the adapted mutations in PB2 and HA were important for the neurovirulence of H9N2 influenza virus in mice,and the combination of adapted PB2 and NA confered the neurovirulence of H10N7 influenza virus in mice.Then we deeply explored the invasion route into mice brain of SD16-MA.A group of fifteen mice were inoculated with rSD16-MA and three of them were euthanized per day from 1 dpi to 5 dpi.Tissues including nasal mucosa,olfactory bulb,olfactory nerves ganglion,trigeminal nerves ganglion,vagal nerves ganglion,cerebrum and cerebellum were collected for detecting the viral antigen-positive cells by immunohistochemical method.The result showed that with time going on,rSD16-MA viral antigen was presented in nasal mucosa,olfactory bulb,olfactory nerve ganglion,trigeminal nerve ganglion,cerebrum and cerebellum with time going on.The virus could not be detected in blood at 3dpi and 5dpi,which indicated that SD16-MA could only use olfactory nerve and trigeminal nerve to enter into mice CNS,but not hematogenous route.Previous studies indicated that Fragile X mental retardation protein(FMRP)is implicated in intracellular RNA transport and regulation of translation at synapses.It targeted human influenza virus RNA synthesis machinery and facilitated virus replication.Based on the distribution and function of FMRP,we speculated that FMRP might play an important role of AIV in infection of CNS.In our research,the function of FMRP in neurovirulence of influenza virus was proved by using WT and FMRP-/-mice.The virus could not be detected in FMRP-/-mice brain infected with rSD16-MA virus.The role of FMRP in promoting influenza virus replication ability in neuron cells was also determinated in vitro.Compared with WT murine primary cortical neuron cells,rSD16-MA infection and replication ability were sharply decreased in FMRP deficient cells,which further verified the important roles of FMRP in the infection and replication ability of influenza virus in neuron cells.Further studies showed that enhanced polymerase activity was conferred by adapted mutations on PB2,which facilitated the viral NP expression in N2a cells.The interaction between NP and FMRP contributed to increased transcription ability of AIV in neuron cells.In this study,we identified the interacted protein in mammalian neuron cells essential for neurovirulence of AIV in mice.SD16-MA and BJ27-MA are all showed increased pathogenicity in mice after adaptation.The machenism of adaptation of H9N2 AIV in mammals has been studies before.In this study,we deeply investigated the molecular adaptation mechanism of H10N7 influenza viruses in mammals.To pinpoint the key factors of the increased pathogenicity of BJ27-MA,three single segment recombination viruses and five point mutant viruses were generated with the rBJ27 backbone.And then,we compared the viral pathogenicities in mice,replication abilities in vivo and in vitro,polymerase activities and NA enzymatic activities of these viruses.We found that PB2 M631L,PB2 E158G and NA K110E were responsible for the increased virulence of H10N7 virus in mammals by increasing polymerase activity and NA enzymatic activity.Collectively,we deeply explored the molecular mechanism of the neurovirulence and increased pathogenicity of mouse adapted H9N2 and H10N7 AIVs in mice.Adapted mutations on PB2,HA or/and NA were important for the neurovirulence of H9N2 and H10N7 AIV.FMRP played dominant roles in the process of H9N2-MA using olfactory nerve and trigeminal nerve ways in invading into mice brain.Therefore,our study revealed the theoretical basis for the adaptation of avian influenza virus in mammals and provided better strategies for the control and prevention of influenza virus nerovirulence.