Effect Of Deletion Of Amino Acids At The C-terminal Of NS1 Protein Of H1N1 Canine Influenza Virus On Its Pathogenicity

The non-structural protein NS1 encoded by the eighth fragment of influenza A virus has a regulatory function on its virulence and replication.Many studies have confirmed that NS1 protein can antagonize host cell interferon（IFN）and plays an important role in enhancing viral replication.Conversely,the replication ability of viruses truncated NS1 protein will be significantly reduced due to IFN-mediated antiviral responses.In our previous study,there is one strain with a 136-amino-acid deletion at the NS1C-terminus（named CIV-LZ57ΔNS）was isolated and purified from pet dogs.In this study,stains with the deletion/truncation/mutant at the NS1 C-terminus and full-length NS1 protein will be compared for studying the differences of their biological characteristics in terms of growth curves and replication kinetics,mouse study and plaque phenotypes,which will explore the key domains or sites located in the NS1 C-terminus influenced on viral virulence and pathogenicity.It will provide theoretical basis for the development of modified live virus vaccines（MLV）.To investigate whether the a 136-amino acids deletion in the C-terminal NS1 protein of H1N1 subtype canine influenza virus（CIV）will affect the pathogenicity in mice.In this study,the strains with deletion of NS1 gene（r LZ57ΔNS）or complete NS1 gene（r LZ57c NS）are rescued.BALB/c mice are infected with a dose of 10⁶ PFU/50μL for comparing their pathogenicity.Results that both r LZ57ΔNS and r LZ57c NS could infect BALB/c mice without adaptation.The body weight of mice in r LZ57c NS group decreases continuously at 3 d.p.i.,and the survival rate is 0%,while r LZ57ΔNS group does not cause significant weight loss,and the survival rate is 100%.Both of them could replicate in the lungs and turbinates of mice.Compared with r LZ57c NS group,the viral titer in the lungs of r LZ57ΔNS group is lower,which only reached to 10^3.89 PFU/m L.The results show that the deletion of NS1 protein could reduce the pathogenicity to mice to some extent.The truncated and complete NS1 protein are identified by Western Blot and expressed at the expected size of 12k Da and 26k Da,respectively,indicating that the truncated expression of NS1 protein is resulted from frameshift mutation.In order to understand which functional domains plays a role in attenuating the virus,in this study,the truncated/mutant viruses（r LZ57del NS73,r LZ57del NS99,r LZ57del NS113,r LZ57del NS126）are rescued successfully by reverse genetics in the background of r LZ57c NS.Results show that there are differences of replication in MDCK.Among these strains,the viral titer of r LZ57del NS99 is significantly lower than that of r LZ57c NS and other truncated strains,which is only 10^6.67 PFU/m L at 48 h.p.i,comparing with other strains.In addition,the average plaque diameter of r LZ57del NS126 on MDCK cells is significantly greater than that of the parent strain r LZ57c NS（P<0.05）,but the replication level of r LZ57del NS126 in MDCK cells is not significantly increased,and the plaque diameter of r LZ57mut NS84 mutated with C-terminal PBM sequence is significantly higher than that of r LZ57ΔNS（P<0.01）.Pathogenicity test in mice showed that without Ed region,the pathogenicity of r LZ57del NS73 was significantly reduced,there were no clinical signs and sustained weight loss in mice,and only 10^4.14 and 10^2.73 PFU/m L were replicated in the lungs and turbinates.r LZ57del NS99 and r LZ57mut NS84,enhanced the pathogenicity of the virus to mice and the survival rates were 33%and 0%,respectively.The replication titers in respiratory tract exceeded r LZ57c NS and reached 10^5.3 and 10^5.14PFU/m L,respectively.The results show that the deletion of different functional domains in ED of NS1 protein could cause the changes of the replication level in different degrees.In summary,the differences of NS1 C-terminal strain and non-deletion strain in pathogenicity are evaluated by mouse study.Although both of them could infect mice without adaptation,the results showed that the a136-amino-acids deletion of NS1 protein could reduce the pathogenicity for mice.In order to determine the domain causing the differences in pathogenicity,five different truncated/mutant strains（del NS73,del NS99,del NS113,del NS126 and mut NS84）are successfully rescued by reverse genetics.The results showed that the in vitro replication of del NS99 was relatively weak within 48 hours,but in the mouse pathogenicity test,del NS99and PBM mutant Mut84 were more pathogenicity to mice,and the clinical signs were obvious,which could result in higher lethal rate and respiratory replication titer than other strains,the results showed that ED domain and PBM domain had some effect on the pathogenicity of mice,while del NS73without full-length ED decreased the pathogenicity of mice,and DELNS113and del NS126 did not show similar pathogenicity to del NS99.Whether it is due to its interaction with host Cytokines,or whether it is related to up-regulation of antiviral factors,further research is needed.