Preliminary Study On The Mouse-Adapted Mechanism Of H1N1 Subtype Canine Influenza Virus

Canine influenza（CI）is a canine respiratory infection caused by canine influenza virus（CIV）.Currently,as the susceptible host of influenza virus,dogs can naturally be infected by a variety of influenza viruses with different hosts and subtypes.As the companion animals of human,there is a close relationship between pet dogs and humans.Once CIV acquired the ability to adapt to new hosts through constant reassortment and mutation,it will pose a potential threat to human health.The novel reassortment swine-origin H1N1 subtype CIVs were isolated for the first time in the previous study in this laboratory,and some of CIVs can preferentially bind to human receptors and have a certain transmission capacity.However,the cross-host adapted mechanism of those viruses is still unclear.Therefore,this study intends to establish a mouse-lung adapted model for H1N1 subtype CIV,and to elucidate the molecular mechanisms that may affect host adaptation of H1N1 subtype CIV by exploring the effect of adaptive mutation sites on viral pathogenicity.（1）The H1N1 subtype CIV（A/canine/Guangxi/LZ57/2015,LZ57）isolated from our lab is selected through 13 passages in the lung of mice,the adaptative model was established successfully,one mouse-adapted virus is obtained by plaque purification,named as LZ57-MA.Through the experimental evaluation of mice,it was found that compared with the wild-type virus（LZ57-WT）,the lung virus titer of mice infected with the LZ57-MA increased significantly（P<0.001）,and the highest could reach 7.39 Log₁₀PFU/m L.The survival rate of the mice is only 0%after 4 days post infection.Comparative analysis of the whole genome sequence between LZ57-MA and LZ57-WT found that a total of7 amino acid mutation sites appeared on the PB2,PA,HA,NS1 and NEP proteins of the LZ57-MA,respectively PB2-E578D,PA-T97I,HA-N198D,HA-A227E,NS1-A53D,NS1-R154K and NEP-R42K.（2）We generate 4 reassortant viruses by reverse genetic containing one gene from LZ57-MA in the background,named as r LZ57-MA（PB2）,r LZ57-MA（PA）,r LZ57-MA（HA）and r LZ57-MA（NS）.The differences of replication of recombinant viruses are compared by multi-step growth curve,results showed that the viral titers possessing PB2,PA and HA gene mutations in each time period are higher than of r LZ57-WT.Among these viruses,single gene replacement virus（r LZ57-MA（PB2））owns the highest titer,which could reach to 8.77 Log₁₀PFU/m L at 36hpi.In contrast,the viral titer of r LZ57-WT only reached to 7.15 Log₁₀PFU/m L,suggesting that the virus possessing PB2mutation can increase the ability to replicate in MDCK cells.The pathogenic differences between different recombinant viruses are evaluated by mouse study,and the results showed that the PB2 gene mutation in r LZ57-WT dramatically increased the virulence(MLD₅₀ 3.75 versus>6.0).Because there is a Glu-to-Asp substitution at position 578（PB2-E578D）,suggesting that this substitution could be the potential determinants of enhanced virulence.The polymerase activity caused by the substitution of PB2（E578D）and PA（T97I）is verified by dual-luciferase reporter assay,and the results revealed that both of them could increase polymerase activity,especially PA gene（T97I）from adapted strain could significantly increase polymerase activity by at least 2 times（P<0.05）.In conclusion,the mouse-adapted model of H1N1 CIV was established successfully.It is confirmed initially that the PB2 gene（E578D）has a certain effect on the pathogenicity of CIV.It need further study to understand whether PB2 and other genes synergistically enhance the pathogenicity of the virus.The results of this study reveal the potential molecular markers of CIV,which is responsible for the differences of pathogenicity.It will lay the foundation for further studying the mechanism of adaptation to new mammals.