| Canine influenza virus(CIV)is the influenza A virus that can cause clinical symptoms such as fever,sneeze and runny nose in dogs.It is often followed by other viral and bacterial diseases that can cause respiratory failure and death.In China,CIV is mainly derived from avian H3N2 subtype influenza virus,which has been able to spread steadily in the canine population after a long period of adaptive evolution.Although census information is patchy,the global canine population has been estimated to be ~700 million.If we consider that dogs are used as companion and working animals,it is feasible to think that they can become—like pigs—another “mixing vessel” species in which avian,human,and canine viruses can reassort and originate new viruses with pandemic potential.Therefore,the research on CIV is of great significance to public health and human health.Tetherin(BST2/CD317/HM1.24)has emerged as a key host cell defense molecule,inhibiting the release and spread of diverse enveloped virions from infected cells.This paper first studied the ability and mechanism of tetherin limiting CIV.The gene was amplified by RT-PCR from beagle peripheral blood lymphocytes,and then the recombinant eukaryotic expression plasmid(p3×FLAG-CMV-10)was established.The biological features of tetherin protein including properties,secondary structure and transmembrane domain were analyzed by online software such as ProtParam,Protscale,SOPMA,TMHMM,TargetP,SignalP,MotifScan and Interproscan.The EditSeq software analysis showed that the 567 nucleotides of canine tetherin coding sequence(CDS)encode 188 amino acids whose molecular weight 20626.84 Daltons.The amino acid sequence homology are 42%,42%,60.4%,54%,30.5%,41.9%,37.3%,42.6%,42.2% and 58.3% respectively when compared with human,chimpanzee,domestic cat,domestic ferret,domestic guinea pig,horse,house mouse,pig,rhesus monkey and tiger.Canine tetherin has the following characteristics: unstable hydrophilic protein,no signal peptide,one transmembrane domain,a type ? transmembrane protein,have glycosylation and phosphorylation Sites.Then,the analysis of tetherin's tissue expression profile found that compared to other tissues and organs,tetherin was particularly abundant in the immune organs.The tetherin gene sequence contains the sequence of interferon response elements,which can be regulated and expressed by the canine IFN-?.The mRNA expression level of tetherin can be significantly improved when CIV infection hosts.CCK-8 detection proved tetherin has the ability to effectively help cells resist the damage caused by CIV infection.In the experiment of whether tetherin affects CIV in cell proliferation ability,we also found that overexpression of tetherin can inhibit the replication of CIV,and interfering with tetherin gene will enhance the proliferation ability of CIV in cells.In order to explore the mechanism of tetherin inhibiting CIV,We used BiFC and Co-IP to identify proteins in viruses that interact with tetherin.We found that both M1 and M2 of CIV can interact with tetherin,and tetherin can be dose-dependent in the degradation of M2 proteins.Because of M1 and M2 proteins are important in CIV replication assembly and budding,we believe that tetherin,in addition to being able to bind virus particles on the cell surface through its own unique topological structure as previously studied,prevents its release,also can pass the virus encoded protein degradation leads to lack of its function to suppress viral replication.MicroRNAs,a class of noncoding RNAs 18 to 23 nucleotides(nt)in length,play critical roles in a wide variety of biological processes.The objective of this study was to examine differences in microRNA expression profiles derived from the lungs of beagle dogs infected with the avian-origin H3N2 canine influenza virus(CIV)or the highly pathogenic avian influenza(HPAI)H5N1 virus(canine-origin isolation strain).After dogs were infected with H3N2 or H5N1,microRNA expression in the lungs was assessed using a deep-sequencing approach.To identify the roles of microRNAs in viral pathogenicity and the host immune response,microRNA target genes were predicted,and their functions were analyzed using bioinformatics software.A total of 229 microRNAs were upregulated in the H5N1 infection group compared with those in the H3N2 infection group,and 166 microRNAs were downregulated.MicroRNA target genes in the H5N1 group were more significantly involved in metabolic pathways,such as glycerolipid metabolism and glycerophospholipid metabolism,than those in the H3N2 group.The inhibition of metabolic pathways may lead to appetite loss,weight loss and weakened immunity.Moreover,miR-485,miR-144,miR-133 b,miR-4859-5p,miR-6902-3p,miR-7638,miR-1307-3p and miR-1346 were significantly altered microRNAs that potentially led to the inhibition of innate immune pathways and the heightened pathogenicity of H5N1 compared with that of H3N2 in dogs.In addition,we found a significant reduction in miR-182 targeting the Tetherin gene when the dog infected with CIV.It indicates that the organism can regulate the expression level of tetherin through miRNA,promote the ability of antiviral response,and inhibit the replication of virus.In summary,as the first study of the canine tetherin protein,this paper confirmed that the natural immune factor tetherin plays an important role in CIV infection,suggesting tetherin's application prospect in the antiviral target drug.Through systematic analysis the expression of miRNA in the lungs of dogs infected with H3N2 and H5N1,revealing the important role of miRNA in the pathogenesis of CIV and regulating natural immune response,deepens our understanding of the complex relationships among microRNAs,the influenza virus-mediated immune response and immune injury in dogs. |
PDF Full Text Request