Effects Of PA-X And PB1-F2 On The Pathogenicity Of H1N1 Swine Influenza Virus

Influenza A virus(IAV)is one of the most important zoonotic viruses.IAV can cause infection in multiple hosts including humans,birds,pigs,dogs and horses.IAV makes a great threat to public health and causes sever economy losses to agriculture.For example,the 2009 pandemic H1N1 influenza virus(pdm09)was a triple reassortment virus whose genome was originated from swine,human and avian influenza viruses.It is believed that the genome reassortment between influenza viruses could improve their global pandemic potential.PA-X and PB1-F2 proteins function to modify the host innate immune response.Both PA-X and PB1-F2 were truncated after transspecies transmission and had significant effects on the virulence and transmission of virus,consequently cause great attention.To better understand the comprehensive effect of PA-X and PB1-F2 proteins on the biology of pdm09 H1N1 swine influenza,a WT strain(WT?PAX-41/F2-11)and three mutated recombinant viruses(pdm09?PAX?41/F2?91,pdm09?PAX?61/F2?11,pdm09?PAX?61/F2?91)were rescued by reverse genetic system and site-direct mutagenesis method based on backbone of a H1N1 swine influenza virus,which express truncated PA-X and PB1-F2 protein.The effects of PA-X and PB1-F2 on the replication and virulence of H1N1 swine influenza virus were evaluated in both cells and mice.Results showed that two signal-mutated strains(pdm09?PAX-61/F2-11 and pdm09?PAX-41/F2-91)had reduced the early replication ability in MDCK cells but enhanced early replication ability in PK-15 cells.However,the double mutated strain(pdm09?PAX-61/F2-91),had significantly enhanced the virus early replication ability in both MDCK and PK-15 cells,indicating that PA-X and PB1-F2 proteins are hostspecific and have certain synergistic effects.The vivo test in mice showed that two single mutations enhanced the pathogenicity and lethality of the virus in mice,promoted the replication ability of the virus on the early infection stage in mice lung,and induced higher levels of inflammatory cytokine expression,caused more severe pathological changes in mice lung.The double mutant strain showed a synergistic effect.These results demonstrated that both PA-X and PB1-F2 protein no only effect foundation characters of H1N1 swine influenza virus in vitro,but also modified the virulence of swine influenza virus in vivo,consequently discovered distinct functions of truncated and full-length of both PA-X and PB1-F2 protein in swine influenza virus.Furthermore,the PB1-F2 protein,truncated or not,could not change the virulence of influenza virus obviously when accompany with truncated PA-X.However,PB1-F2 could cause the significant effect to virulence of virus if the expression of PA-X protein is full-length.A swine influenza virus expressing both full-length PA-X and PB1-F2 protein could significant enhance virulence and replication in early infection stage but suppressed the maintenance of viral titer in vivo compare with other mutated and WT strain.This study is the first to manipulate PA-X and PB1-F2 simultaneously,also offer basic information to explain the interaction between PA-X and PB1-F2 protein.