Identification Of Four Strains Of Genotype C DHAV And The Preliminary Study Of Interaction Mechanism Between Virus And Host

Duck hepatitis A virus(DHAV)which included three kinds of genotypes(type A?type B and type C)was a member of the picornavirus family.The DHAV-C was widespread in in domestic poultry,and hindered the development of duck industry at present.There are more incompletions in the research of genetic variation and pathogenicity mechanism of DHAV-C.For example,on the one hand,the highly variable amino acid regions were found in the major antigenic proteins of DHAV-C,which could change the antigenicity to affect the protection effects of conventional vaccines.However,there are rare studies on epidemiological investigation,genomic features and regulation of heredity and variation of the current epidemic DHAV-C strains,which should be reinforced and perfected.On the other hand,the interaction mechanism between DHAV-C and its host was still unknown,the role of key molecules in antiviral response of host animal was also not clear.Therefore,the confirmation of the kinds of PRRs related to the infected progress of DHAV-C and the finding of the antiviral molecules will be helpful to clarify the molecular mechanism of interaction between virus and susceptible animal.It also could provide references for the research on the novel antiviral agent.Taken together,the studies were conducted as follow:(1)The development of the RT-ii PCR methods for DHAV-A and DHAV-CInsulated isothermal PCR(ii PCR)was a novel method for amplifying DNA/RNA in vitro based on the principle of heating convection and fluorescent quantitative PCR.And it also was more sensitive,specific and convenient to fit for the on-site detection.Therefore,the primers and probes targeted to3C/3D gene and VP3 gene were designed to detect the DHAV-A and DHAV-C,respectively.The high specificities of these two novel methods were identified by 11 kinds of duck pathogens.And the lower limits of DHAV-A and DHAV-C of RT-ii PCR were:49.1 copies/?L?38.5 copies/?L,respectively.By2×2 contingency tables analyses,the coincidence rate between novel RT-ii PCR and referenced r RT-PCR were confirmed,which exceeded 97%in both two methods.However,the analytical sensitivity of ii PCR was higher than that of r RT-PCR.Therefore,these two new detection methods will be new and effective tools of on-site diagnosis and epidemiological investigation of DHAV.(2)Identification of 4 strains of DHAV-C and the analyses of genomes of DHAV-CThe positive rate of DHAV in the samples from three different Provinces were detected by previously established ii PCR.And the positive samples were processed and inoculated in duck embryos.The results showed that Four strains of DHAV-C(SWUNC1?SWUNC4)were isolated,and the ELD₅₀ were10^-4.5/0.2m L?10^-5.33/0.2m L?10^-5.33/0.2m L and 10^-7.65/0.2 m L,respectively.Then,these viruses were inoculated in DELC,but only SWUNC4 strains could infect primary cells and caused obvious CPE.The TCID₅₀ of SWUNC4 strains in DELC was 10^-5.34/0.2m L.The circular virions was observed by transmission electron microscopy.The sequencing and analyses of genomes of new isolates showed that full length genome of DHAV-C strains was 7778?7780nt,of which the percentage of homology was92.69%?99.1%compared to other registered sequences.The evolutionary tree based on VP1 gene and whole-genome of DHAV-C illustrated that all isolates belonged to G?subtype.SWUNC4 strain was classified in one branch,which is the same with South Korea strains but far away from SWUNC1?SWUNC3 strains.Furthermore,all new isolates in VP1 proteins deduced amino acid sequences were different from previously strains in Sichuan province.Especially for SWUNC4 strains has 21 mutation sites.Compared with 16 represented strains of Korea,Vietnam,china and conventional vaccine,3?7 unique mutation sites of 4 isolates were found.In addition,the antigenic indexes of four strains of DHAV-C had obviously changes to conventional vaccine strains(B63 and FS strains).A new predicted B cell epitope(101?111aa)was found in the VP1 gene of SWUNC4.It suggested that the mutation of DHAV-C isolates could cause the differentiation of antigenicity.(3)The study of interaction mechanism between DHAV-C and host in early stage of infectionThere are 211 molecules and 74 significantly enriched pathway,which related to the innate antiviral immune response were screened out by RNA-seq technology and bioinformatics analysis to the m RNA samples extracted from livers in early stage of DHAV-C infection.The results of q PCR verification were performed high consistency with transcriptome analysis in expression levels of key molecules and its changes.The enrichment analysis showed that the molecules related to RLRs pathway enriched at24h after infection,but not at 12h.It implied that the PAMPs of DHAV-C were insufficiently recognized by RLRs.Furthermore,q PCR detection confirmed that the m RNA levels of RIG-I and MDA5 had no changes in livers at 12h and 24h after inoculated by DHAV-C,but they expression levels obviously raised at 36h.However,these expression changes of RLRs coincided with that of IFN-?/IFN-?in early stage of DHAV-C infection.It demonstrated that DHAV-C could escape recognition of RLRs related to virus infection in early stage.Besides,IRF1 and IRF3 also might be very functional in antivirus immune response.The expression levels of IL8,IL10 and CCL19 might be related to liver lesions.All of these results were helpful to explore the interaction mechanism between DHAV-C and host in depth.(4)du IFITM1 inhibited the increase of DHAV-C titers in DELCinterferon induced transmembrane proteins(IFITMs)family were found in recently known as its antivirus efficiency.But whether the duck IFITMs(du IFITMs)will affect the proliferation of DHAV is still unknown.According to our studies of transcriptomic and q PCR,the expression of du IFITM1 was significant up-regulated at 24h and 36h in livers infected by DHAV-C.Therefore,it was presumed that du IFITM1was involved in the host antivirus response to virus.Then p EGFP-du IFITM1 recombinant was constructed and transferred in DELC,which was used to determinate the affection between du IFITM1 and DHAV-C.The results showed that viral loads were significant down-regulated in DELC with the high levels of du IFITM1(P<0.05).It demonstrated that du IFITM1 was a new effective molecule could inhibit the increase of DHAV-C.