Identification Of Duck Mx Gene Transcripts And Expression Pattern Analysis Of Mx Gene Induced By Duck Hepatitis A Virus-1/-3 In Duck Embryos

Duck Mx protein is one of the antiviral molecules of the interferon mediated natural immune response in waterfowl.There are relatively few studies on the transcripts and expression patterns of duck Mx genes,while DHAV-1/DHAV-3 induces changes in the transcript levels of type I and type III interferons and their downstream Mx gene,the expression patterns of Mx protein and viral load in duck embryos remain unclear.In this study,the transcripts of Shao duck Mx gene were examined by Northern blot from DEF cells infected by DHAV-1,and two Shao duck Mx transcripts of different sizes were obtained.The 5’ end sequence of the duck Mx gene was amplified by 5’ RACE and spliced with the CDS sequence of the duck Mx gene already cloned by our group to get the full length 2241 bp.Sequence analysis showed that it contained three potential coding regions,with the length of 2241 bp,2166 bp and 2109 bp,encoding three different molecular weight of duck Mx proteins of 747 aa,721 aa and 702 aa.Then the transcripts were cloned by RT-PCR and to the sequence matched with the spliced sequences,which confirmed the 2241 bp transcript of the duck Mx gene was true,and it was named duck Mx-2241.Then,construct recombinant eukaryotic expression plasmids for three coding regions of the duck Mx gene and transfect HEK-293 T cells to obtain transient expression products.The 2109 bp CDS of duck Mx expressed an Mx protein of approximately 79 k Da,and2166 bp CDS expressed two Mx proteins of approximately 79 k Da and 82 k Da,finally,the 2241 bp CDS expressed three Mx proteins,which have approximately molecular weight of 79 k Da,82 k Da and 85 k Da.At the same time,natural Mx protein from DEF cells infected with DHAV-1 can also express 85 k Da,82 k Da and 79 k Da Mx protein.The presence of the Mx-2241 transcript and its encoding of three duck Mx proteins of varying sizes were further confirmed from the expression level.On the other hand,103ELD50 DHAV-1 and DHAV-3 virus were injected into 10-day-old SPF duck embryos and total RNA was extracted at 12 h,24 h,48 h and 72 h post infection.RT-q PCR was performed to detect the transcript levels of Mx,IFNα,IFNβ and IFNλ genes in six kinds of tissues,including heart,liver,brain,muscle,stomach and intestine.At the same time,the viral load of each tissue was measured using the established DHAV-1 and DHAV-3 absolute quantitative RT-q PCR method,and the expression of Mx protein in ducks was detected by Western blot.The results showed that both DHAV-1 and DHAV-3 could induce the transcription of interferon and Mx genes after infection,and the trend of both changes were consistent.Between them,DHAV-1stimulated higher transcription levels of IFN and Mx genes than DHAV-3,demonstrating a stronger innate immune response;in addition,the upregulation of Mx gene transcript levels activated by DHAV-1 infection in heart,brain and stomach tissues was mainly mediated by type III interferon,while in liver,muscle and intestinal tissues Mx gene upregulation was mediated by both type I and type III interferon;the upregulation of Mx transcript levels in heart,liver,brain and muscle tissues of duck embryos after DHAV-3 infection was mainly mediated by type I interferon The upregulation of Mx transcript levels in heart,liver,brain and muscle tissues after DHAV-3infection was mainly mediated by type I interferons,while in stomach and intestine tissues it was induced by both type I and type III interferons.From 12 to 72 h after DHAV-1 and DHAV-3infection,the viral load in all tissues showed a gradual increase or first increase and then decrease.Western blot showed that the expression of duck Mx proteins of different molecular weight were detected in all tissues,and the relative expression trends were generally consistent with the changes in Mx transcript levels.In this study,we obtained a new duck Mx gene transcript and characterized its expression in vivo and in vitro,and preliminarily investigated the expression patterns of Mx and interferon genes in the DHAV-1 and DHAV-3 activated interferon pathways,laying the foundation for studying the differences in antiviral activity of different duck Mx transcripts.