Cloning And Functional Study Of Antiviral TRIM And IFN Genes In Fish

The tripartite motifs(TRIMs)constitute a large family of proteins containing a Really Interesting New Gene(RING)domain,one or two B-box domain and Coiled-coil region followed by different C-terminal domains.TRIM proteins play multiple roles in various cellular processes,including cell growth,differentiation,apoptosis and antiviral immunity.Fish novel large multigene TRIM genes(finTRIM/ftr)appear only in teleost and play a vital role in antiviral responses.In the present study,phylogenetic analysis revealed the existence of different subsets of novel fish TRIM 14 genes(finTRIM14/ftr14),ftr51,ftr67,ftr72,ftr82,ftr83,and ftr99 in grass carp(Ctenopharyngodon idella),suggesting lineage-specific diversification events.Therefore,the number of finTRIM genes varies greatly among species.The ftr genes in grass carp,which are closely related to zebrafish and possess various evolutionary branches,have evolved faster than human TRIMs.The predicted protein domains were almost identical RING zinc finger domains,with the exception of ftr72,the B-box domain(excluding ftr67,ftr82,ftr83),and the B30.2 domain,which evolved under positive selection(with the exception of ftr67,and ftr72).The genes were predominantly expressed in the spleen,gill and head kidney.These findings indicate that the ftr genes in grass carp are involved diverse cellular processes,including innate immune responses.In zebrafish(Danio rerio),the functions of the finTRIM(ftr)family are unclear.In the present study,we investigated the expression pattern of ftr12,ftr51,ftr67,ftr82,ftr83,and ftr84 in zebrafish for the first time.The results showed that ftr12,ftr67,and ftr84 are maternally expressed in the oocyte and highly expressed at the early stage(0 ~ 4 hpf)of embryo(P < 0.05),suggesting their involvement in the embryonic innate defense system.The ftr82 gene was highly expressed at 8 hpf(P < 0.05),which implied that the embryos could synthesize their own immunity-related mRNAs.However,ftr51 and ftr83 were highest at 8 hpf,and might mediate embryonic development.The expression levels of ftr12,ftr51,and ftr67 were highest in the gill,intestines,and liver,respectively.Ftr82,ftr83,and ftr84 were predominantly expressed in the kidney,suggesting that these finTRIMs might play roles in both immunity and non-immunity-related tissue compartments.Zebrafish embryonicfibroblast(ZF4)cells were infected with Grass carp reovirus(GCRV)and Spring viremia of carp virus(SVCV).During GCRV infection,the expression of ftr12 was significantly upregulated from 12 h to 24 h;and ftr51 and ftr67 increased from 3 h to12 h.The expressions of ftr82,ftr83,and ftr84 were only upregulated at 12 h,12 h,and 24 h,respectively.All of these genes were significantly downregulated at 48 h(P< 0.05).Challenge with SVCV upregulated the expressions of ftr12 and ftr51 at 12 h and 48 h(P < 0.05),respectively,and ftr67 reached its highest expression level at 3 h.ftr82 showed only a slight upregulation at 6 h and 48 h,and ftr83 and ftr84 were consecutively increased,reaching their highest levels at 12 h(P < 0.05).Meanwhile,ftr67 and ftr83 were significantly downregulated at 48 h(P < 0.05).Our research demonstrated that ftr12,ftr51,ftr67,ftr82,ftr83,and ftr84 probably have important roles in innate immune responses and in non-immunity-related tissues.Interferon(IFN)plays vital roles in the antivirus immune response.However,the Dabry's sturgeon IFN sequences were not available.Hence,the present study aimed to characterize comprehensively transcriptome profile of the head kidney from Dabry's sturgeon infected with Aeromonas hydrophila using Illumina platform.Over42 million high-quality reads were obtained and de novo assembled into a final set of195240 unique transcript fragments(unigenes),with an average length of 564 bp.Approximately 41702 unigenes were annotated in the NR NCBI database.The results provide valuable references for studies in sturgeons that lack complete genomic sequences,and could also be helpful for the analyzing evolution among cartilaginous and teleost fish.The AdIFN?(Acipenser dabryanus IFN gamma),AdIFNe1 ? AdIFNe2 and AdIFNe3 were obtained from RNA-Sequencing analysis of spleen tissues and searched using a query set of available sequences in the NCBI non-redundant database.Similar to other teleost IFN gene,the AdIFN? possesses 4 exons and 3introns,and AdIFNe1,AdIFNe1 were both have 5 exons and 4 introns.The AdIFNe1?AdIFNe2 and AdIFNe3 were highly expressed at the early stage of embryo development,and then decreased,suggesting that there was maternal expression to protect embryo from stimulation outside.AdIFN? was no markedly changes during the embryo development.AdIFN?,AdIFNe1 ? AdIFNe2 and AdIFNe3 wereconstitutively expressed in different tissues,revealing that IFN is the basic component of organization to maintain normal activity.The primary spleen cells were induced by poly I:C(polyinosinic-polycytidylic acid)and LPS(lipopolysaccharides,from Escherichia coli 055:B5).After poly I:C administration,AdIFNe1,AdIFNe2 and IFNe3 could be stimulated by poly I:C,and AdIFNe3 was upregulated from 6 h to 24 h,AdIFNe1 and IFNe2 were upregulated from 6 h to 48 h,and subsequently decreased.AdIFN? can also be induced by poly I:C,and upregulaed from 12 h to 72 h,and the fold change was lower than that of type I IFN.It illustrated that AdIFN? also has the antiviral effect,and its ability of antivirus is weaker than that of I type IFN.LPS could also induce the expression of AdIFNe1,AdIFNe2 and IFNe3.Compared with AdIFN? induced by LPS,the expression changes of AdIFNe1,AdIFNe2 and IFNe3 were shorter and weaker against bacteria.The results showed that AdIFN?,AdIFNe1,AdIFNe2 and IFNe3 had both antiviral and anti bacterial capabilities,but type I IFN had stronger antiviral ability than type II IFN,and type II IFN had stronger antibacterial activity than type I IFN.