Research On The Function Of 3’ Untranslated Region And Interferon Signaling Pathway Of Duck Hepatitis A Virus

Duck viral hepatitis induced by Duck hepatitis A virus is an acute and fatal disease to young ducklings,characterized by liver necrosis,hemorrhage and high mortality.Natural infection of DHAV was associated with age,although it could bring a high mortality to young ducklings within 3 weeks old,no clinical signs could be found in infected adult duck.Additionally,there is no correlation between in vivo viral load and the age of the ducklings.It gives us the indication that DHAV could escape from the incomplete immune system of young ducklings to propagate and induce pathogenesis in host.As the only member of Avihepatovirus in Picornaviridae,DHAVs had been classified into three genotypes by phylogenetic analyses: the classical genotype 1(DHAV-1),the genotype only isolated in Taiwan(DHAV-2),and the genotype firstly identified in South Korea(DHAV-3).Based on the neutralization tests,DHAVs were divided into three serotypes corresponding to the three genotypes.Present study on DHAV is undergoing a stage from basic genetic analysis to functional research and interaction with host.According to computer algorithms and biochemical structure probing,some stable motifs had been proved to exist in the picornavirus 3’UTRs and involve in virus replication.An important cis-acting molecular genetic determinant for the replication is believed to reside in the 3’UTR where the stable motifs are presumably recognized by the replication complex to initiate the synthesis of the negative-strand RNA.Engineer mutation in the relevant structures would impair the viral replication.Those viewpoints had been confirmed in some later picornavirus’ research.However,endowed with the longest sequence among all the Picornaviridae viruses,little is known about structure and function of 3’UTRs of DHAV.Based on the established DNA-Launched infectious clone of DHAV-1(LY0802 strain)and DHAV-3(SD1201 strain),we constructed a series of mutant strains to investigate the key structure and function of 3’UTRs of DHAV.Besides,based on the RNA-seq of DHAV infecting DEF,we did some work on interferon signaling pathway and tried to explain pathogenic mechanism from the interaction between DHAV and organism immunity.(1)Research of DHAV 3’UTRs’ function on virus propagation and pathogenesis5 DHAV-1 strains,2 DHAV-2 strains and 5 DHAV-3 strains were selected from NCBI to do the Megalian analysis.Conservative sequences were found among the same genetype and two relatively conservative sequences were found between different genotypes.Using RNAfold online software,except poly(A),we found 7 Stem-loop structures in LY0802 3’UTR and 3 in SD1101 3’UTR.Besides,the conservative sequence α was located in 1L-C and 3L-B while β was located in 1L-FG and 3L-C.Based on the predicted result,we constructed a series of 3’UTR mutants and firstly used TEM to detect the loading effectiveness of DNA-launched infectious clone.Mutant 1Δ1-339 was found that it could still propagate in DEF and induce weak pathogenicity to host cells.It gave us the indication that 3’UTR was not necessary for DHAV’s propagation and pathogenesis.The following qRT-PCR、IFA test and ELISA were designed to verify this hypothesis from gene transcription and protein expression.Finally we got the conclusion that 3’UTR was important for DHAV’s propagation and pathogenesis but not necessary.To verify the key structure in DHAV 3’UTR,we established two models-transcription and infection and confirmed 1C/1G as the key Stem-loop structure for DHAV-1 and 3C for DHAV-3.Additionally,that 4033 and 415 top genes were found in RNA-seq of 1Δ1-314 and 1Δ1-339 infecting DEFs showed the poly(A)tail was not absolutely required but it did bring influence on viral replication and infectivity of DHAV-1.(2)RNA-seq based on DEF infected by DHAV-1This RNA-seq is based on the model of DEF cells infected by DHAV-1L.Finally,compared to uninfected group,we got 5828 differentially expressed genes in host transcriptome,with 2025 up-regulated and 3803 down-regulated genes.According to GO annotation,all the differentially expressed mRNAs could be classified into biological processes,cellular components and molecular functions.In KEGG annotation,they were significantly enriched in 35 signaling pathways and among which the signal transduction,endocrine system and immune system were the top three enriched signaling pathways.In pattern-recognition receptors signaling pathway-Toll-like receptor signaling pathway,RIG-I-like receptor signaling pathway and NOD-like receptor signaling pathway,IκB,IL-6 and IL-8 in infected DEFs were obviously up-regulated.Besides,P13 K,TRAF3 and TBK1 in Toll-like receptor signaling pathway,TRAF3,TBK1 and MEKK1 in RIG-like receptor signaling pathway,Erbin in NOD receptor signaling pathway were all obviously down-regulated.Up-regulation of IκB could inhibit the DNA-binding subunits of NF-kappa B to enter the nucleus,meanwhile down-regulation of TRAF3 and TBK1 could inhibit the activation of IKKε/TBK1.That the two signaling pathway working together to inhibit secretion of IFN-β indicated IFN-β gene might not transcript in infected DEF.(3)Interferon signaling pathway research in DHAV infecting DEFAfter infecting DEFs with equal DHAV,cells were collected in 4h,8h,12 h,16h,24 h,36h and 48 h and total RNA were used to detect interferon β transcription.No interferon β gene was detected.Then after transfecting poly(I:C)into infected DEF to be the positive control,ELISA and qRT-PCR were separately designed to detect the interferon protein in DEF culture medium and ISG expression in DEFs.Finally we got the conclusion that infection of DHAV-1L could not only inhibit dsRNA inducing IFN-β but also inhibit dsRNA inducing ISGs in host cell.