Esterase Activity Sites Identification For Hef Glycoprotein Of Influenza C Virus & The Pathogenic Of Influenza C Virus And H10n8 Influenza Virus For Swine

Influenza is an important zoonotic disease caused by influenza virus,which have a wide range of hosts,including human and many kind animals.There are many reports that new type influenza virus interspecies transmission and infect human in recent years,espercially the newly emerged H7N9,H10N8 and H5N6 influenza virus,which have pose huge challenges for public health.Influenza virus can be divided into four different types so far,including A,B,C and D types.Because pig have a special receptor distribution characteristic on respiratory tract,and it is considered to playing as a “mixer” for influenza virus transmission,and plays an important role in the ecology for influenza virus.Thus,it is very important to monitor influenza virus in swine herds.We have studied the pathogenic of influenza C virus(Influenza C Virus,ICV)and H10N8 influenza virus for swine,and indentified the esterase activity sites for HEF glycoprotein of ICV.In addition,we also studied the protein expressed level in the lung of mice that infected with H10N8 influenza virus.The results in our study may lay the foundation for the pathogenic mechanism research for ICV and H10N8 influenza virus.Firstly,we tried to identify the esterase activity sites for HEF glycoprotein of ICV.When three amino acids(Ser71,Asp366 and His369)on HEF glycoprotein mutation into Ala,respectively,we found the replication ability of mutation virus have been reduced significantly.Then we tried to promote the release of mutation virus from cell surface by treatment with neuraminidase,and the results showed virus titer have been improved significantly for mutation virus after treatment with neuraminidase,while the wild type virus have no obviously influence.Finally,substrate pNPA was used to determine the esterase activity for three mutation viruses and wild type virus,and we found the esterase activity of three mutation viruses have been decreased significantly compared with wild type virus.And the the esterase activity have been decreased about 95.2%,88.0% and 97.6% when Ser71,Asp366 and His369 mutation into Ala,respectively.These results indicate that Ser71,Asp366 and His369 on HEF glycoprotein of ICV is critical for its esterase activity,and may be the catalytic triad of HEF esterase.In order to reveal the role of swine plays on the ecology of ICV,we have conducted epidemiological investigation for ICV in swine herds in south China for the first time,and further investigate the pathogenic of ICV for swine and the transmission of ICV in swine herds.Serological investigation results shows 54 serum samples were positive for ICV by hemagglutination inhibition assay(HI)in a total of 2050 serum samples collected form south China during 2016 to 2017,and the positive rate is 2.63% for ICV.The HI titer is distribution between 1:40 to 1:160,and the geometric mean titer(GMT)is 58.04(95% CI: 54.17 to 65.83),neutralizing antibody also can be detected by mircoserum neutralization assay(MN).These results indicate that there have ICV infection in swine herds in south China,and there also have co-infection between ICV and IAV.The results of pathogenic experiment of ICV for siwne indicate swine can be infected with ICV and cause clinical signs,virus can be detected from nasal swab.To our surprise,ICV can replicate and cause pathological changes in trachea,but can not replicate in lung,this may because the most suitable temperature for ICV replicate is 33?.Swine infected with ICV have seroconverted at 7 days post infection,and the HI titer can be reached to 1:160 at 14 days post infection.Howerver,ICV can not transmission by directly contacted between swine.These results indicate swine may be another reservoir for ICV apart from human.During the monitor for influenza virus in swine herds,antibody specific to H10N8 influenza virus have been detected,and the result have been confirmed by MN assay,which indicate there have nature infection of H10N8 influenza virus in swine herds.However,the pathogenic of H10N8 influenza virus to swine have never been reported.In this study,we reported the pathogenic of avian-origin(GD-E1)and human-origin H10N8(JX346)influenza virus to swine as well as the transmission ability.The results indicate swine infected with JX346 can be caused clinical signs and virus can be detected from nasal swabs in all infected swine at 3 days post infection.In addition,virus can replicate in trachea and lung,and cause serious pathological changes in lung,virus antigen also can be detected by immunohistochemistry(IHC)in lung.Although virus can not be deceted from nasal swabs in contact group,the serum from one pig have seroconverted,which indicate JX346 can be transmission between swine by contat,but the efficiency for transmission is low.GD-E1 have limited ability to infect swine,no virus can be detected from nasal swabs as well as in trachea and lung.However,pigs infected with GD-E1 have seroconverted at 14 days post infection,and GD-E1 can not transmission between swine.These results indicate epidemiology monitor for H10N8 influenza virus shoud be conducted in swine,which will helpful for the prevention and control of H10N8 influenza virus.Previous research have reported the pathogenic for mice between avian-origin and human-origin was significantly difference,and we also found GD-E1 and JX346 have difference pathogenic for mice.In order to know about the molecular mechanism for this difference pathogenic,we compared the protein expressed level in the lung of mice that infected with GD-E1 and JX346 using proteomics methods.Compared with GD-E1 group,a total of 836 differentally expressed proteins(DEPs)was identified in JX346 infection group,569 DEPs were up-regulated and 267 EDPs were down-regulated.These DEPs were significantly enrichment in signaling pathway that related with immune response,disease and metabolism.The signaling pathway related with immune response that significantly enrichment by DEPs including antigen processing and presentation,phagosome,complement and coagulation cascades,NOD-like receptor signaling pathway,natural killer cell mediated cytotoxicity,NF-?B and TNF signaling pathway.The differentally expressed level of some pivotal proteins that participate in these signaling pathway may be the reason that caused difference pathogenic for mice.These DEPs include ?-2m,MHCI and MHCII in antigen processing and presentation signaling pathway;C3 and C4 in complement and coagulation cascades;STAT1,STAT2,OAS2,IFI4,STING,IFIH1,OASL1,RSAD2,IFIT1 and IFIT3 that related to cytokines produce and exert its biological functions;Casp8,Bid and MCL1 that related to apoptosis.The expression level for 5 DEPs have confirmed by Western-Blot and 11 genes for DEPs have also confirmed by real-time qPCR,which have the same results with proteomics.These DEPs identified in this study will lay the foundation for the research of pathogenic mechanism,interaction between virus and host proteins,as well as drug targets selection for H10N8 influenza virus.