Effects Of The PA-X And PB1-F2 Proteins On The Virulence Of The Human H1N1 And Canine H3N2 Influenza Virus

Influenza A virus is an important zoonotic pathogen,which genome consists of eight single-stranded negative-strand RNA segments,and can infect human,horses,pigs,dogs,birds and other animals.Influenza A virus makes a great threat to the public health and causes huge losses to agricultural economy.Furthermore,the cross-species transmission of influenza viruses increased the damage to public health.To date,all strains of influenza virus that has caused pandemics in the human population came from the cross-species transmission of influenza viruses.For example,the genome of 1918 Spanish flu virus was originated from avian influenza virus,while the genome of 2009 human H1N1 pandemic influenza(pdm09)virus was derived from the reassortment of influenza viruses from human,swine,and avian.Canine influenza virus can lead to upper respiratory tract infections,accompanied by coughing,fever and inhibiting host immune response in dogs.The dog served as one of the companion animals and always has a close relationshiop with people and that may increase the possibility that the canine influenza virus infecting human.It is not a long time that the canine influenza virus transmitted and circulated in the dogs,furthermore,the canine influenza only has few recombination or rearrangement with other influenza viruses during the circulation in dogs.Therefore,the evolutionary history of canine influenza is relatively clear compare with other influenza virus.Thus,the canine influenza is considered to be an ideal model for studying the factors related with the cross-species infection of influenza virus.In recent years,with the further research related with influenza viruses,the mutations related with cross-species infection and the interaction relationship between viruses with the host innate immune response has got more and more attention.The PA-X and PB1-F2 are non-structural proteins encoded by the genome of influenza virus.It has been confirmed by many studies that both PA-X and PB1-F2 proteins have functions of regulating the viral replication efficient,pathogenicity and the host innate immune response.Moreover,both proteins undergo a structure change during the cross-species infection of influenza virus.Thus,they may be factors related with the cross-species infection of influenza virus.Nowadays,there have been some reports about the PA-X and PB1-F2 proteins of the human 2009 H1N1 pandemic strains.But there are no similar studies targeting canine influenza yet.Therefore,to understand the effects of PA-X and PB1-F2 proteins on the virulence of influenza virus,we made a further research on the effects of PA-X and PB1-F2 of pdm09.Based on this,the effects of PA-X and PB1-F2 proteins from canine H3N2 influenza strains were characterized.The 2009 H1N1 pandemic influenza was spread to the whole world within one year and continued to circulate among human and swine.It is the latest global influenza outbreak.The pdm09 viruses harbored both truncated 232 aa PA-X and 11 aa PB1-F2 proteins.In our study,based on the pdm09 strain A/Guangdong/1057/2010(GD1057),we constructed a reverse genetic system and rescued four recombinant viruses expressing different combinations of PA-X and PB1-F2 proteins,and named as WT＿PAX＿41/F2＿11,Pdm09＿PAX＿61/F2＿11,Pdm09＿PAX＿41/F2＿90 and Pdm09＿PAX＿61/F2＿90.To investigate the effects of PA-X and PB1-F2 on the virulence of pdm09 virus,we have compared the replication efficiency,the polymerase activity and the host-shutoff activity in cells,and the effects of virus pathogenicity and immune response in mice of each reconstructed virus respectively.The results show that when we simultaneously prolonged the PA-X and PB1-F2 proteins of the pdm09 viruses,the replication efficiency of virus was significantly improved.With the dual luciferase assay and western blot assay,the results showed that the expression of both full-length PA-X and PB1-F2 protein increased the expression level of viral RNP protein,thereby increased the replication efficiency of the virus.Furthermore,the results demonstrated that the reconstructed virus which expressing full-length PA-X and PB1-F2 proteins lead to the most pathogenic to mice on the early infection stage and caused more damage to the lung of mice.Our study further proved that PA-X and PB1-F2 proteins have combined effects on regulating the host immune response,and the expression pattern of PB1-F2 protein can affect the host-shutoff activity of PA-X protein.In summary,we have studied,for the first time,the combined effects of PA-X and PB1-F2 proteins on the pdm09 viruses.Canine H3N2 influenza virus was discovered in 2008,and widely circulated in North America and Asia in 2015.It has infected more than 1,000 dogs and therefore became one of the major pathogens of dogs.The canine H3N2 strain which used in our study is harboring a truncated 232 aa PA-X protein and a full length 90 aa PB1-F2 protein.In this study,seven reconstructed viruses expressing different PA-X proteins and PB1-F2 proteins patterns were rescued based on the reverse genetic system of canine influenza A/canine/Guangdong/02/2011(GD02)strains.We have compared the viral replication efficiency,the polymerase activity in cells and the pathogenicity in mice of each reconstructed virus.The results showed that single prolonged or deleted PA-X of canine influenza increased the polymerase activity of the virus,thereby increased the replication efficiency of virus on MDCK cells and increased the viral pathogenicity in mice.After then,our reserch truncated or removed the PB1-F2 protein of canine influenza and the results show that the replication efficiency of both reconstructed viruses was increased,but only the virus lacking PB1-F2 harbored a higher polymerase activity in cells and pathogenicity in mice.When canine influenza expressed both full-length PA-X protein and PB1-F2 protein,a significant increased of the viral polymerase activity was observed,and this result was consistent with the results of the pdm09 strain,demonstrated that the full-length PA-X and PB1-F2 has combined effect to increase the polymerase activity of the virus.Finally,the different combination of PA-X proteins and PB1-F2 proteins lead to different effects on the viral replication efficiency,the polymerase activity in cells and pathogenicity in mice.This indicated that the two proteins have combined effects when regulating viral virulence.Our research has constructed the reverse genetic systems for human 2009 pandemic H1N1 influenza strain GD1057 and canine H3N2 influenza strain GD02.Our research has studied the effects of PA-X and PB1-F2 proteins on the viral replication efficiency,the polymerase activity in cells and the pathogenic in mice.Our study targets the effects of different PA-X and PB1-F2 protein of canine H3N2 influenza virus on the virus virulence.This study is important for understanding the virulence alters of influenza viruses and the effects on host immune response,provide a theoretical basis for the research,control and prevention of influenza virus.