Regulation Of Apoptosis By Host MiRNA Targeting TRAIL During Anguillid Herpesvirus Infection

Eels(Anguilla spp.)are one of the important aquaculture fishes in China.As the expansion of the aquaculture scales of eels,the diseases of eels occur frequently,and the Anguillid herpesvirus disease could cause huge losses.The pathogenicity of Anguillid herpesvirus isolated from different hosts or regions is quite different,and the diseases associate with high risks for latent infection,while the mechanism of it is unknown now.Numerous studies have shown that the host can regulate the apoptosis by miRNA targeted the apoptosis related genes under virus infection.The death receptors apoptotic pathway mediated by TRAIL is very important in host antiviral immunity.It is speculated that the latent infection of Ang HV-1 in the host is related to the regulation of apoptosis by the host miRNA under virus infection.In order to reveal the reasons of latent infection in eels,we investigated the roles of miRNA in the apoptosis regulation and screen potential drug targets for prevention and control of Anguillid herpesvirus 1 infection,and the following studies were carried out.First,Ang HV-1-FC was isolated from diseased A.rostrata and identified as Anguillid herpesvirus 1.EES was used as sensitive cell line for Ang HV-1-FC proliferation and enrichment.The pathogenicity of Ang HV-1-FC to healthy American eels was investigated through artificially infection.Meanwhile,the genome of Ang HV-1-FC was sequenced and performed comparative analysis with the reported genomes from other isolates of Ang HV-1 and the phylogenetic analysis was conducted.Second,absolute quantitative PCR was established for counting the virus numbers.The apoptotic phenotype changes of EES were detected after virus infection.RNA-seq was used to explore the underlying mechanism of apoptosis caused by virus infection with Illumina Nova Seq 6 000 system and the data mining was performed on12 mRNA libraries and 12 miRNA libraries at different time points(0 h,24 h,48 h and 72 h)after virus infection.Finally,we screened the miRNAs targeting TRAIL from mRNA and miRNA transcriptome association analysis,and verified that aan-miR-181b-3p could target TRAIL in apoptosis regulation by dual-luciferase reporter assay.In order to screen potential drug targets for prevention and control of Anguillid herpesvirus 1 infection,the miR-181b-3p mimics,miR-181b-3p inhibitors,p CMV-TRAIL and si RNA of TRAIL were transfected into the EES with or without virus infection and the functions of them were further investigated,respectively.The results showed as follows:1,Ang HV-1-FC could infect A.rostrata with the clinical symptoms as same as those in A.anguilla and A.japonica.The Ang HV-1 infection of EES was characterized by the formation of syncytium,and a large number of viral nucleocapsids with a morphology of concentric circle could be detected by TEM.Ang HV-1-FC caused the same symptoms as the naturally diseased eels through experimental infection.During the infected period,although all eels in the experimental group got sick,there was a phenomenon of clinical symptoms hysteresis,for the virus was latent with a low copy number and the eels appeared normal in early infection,suggesting that eels could be latently infected by the virus.The genome sequencing results showed that Ang HV-1-FC was similar to the reported Ang HV-1 isolates,and the average nucleotide identity was up to99% between Ang HV-1-FC and the others reported.However,phylogenetic analysis showed that there were significant genetic differences among them.2,The apoptotic phenotype was detected by Annexin V apoptosis kit,and we found Ang HV-1-FC could inhibit apoptosis of EES.The mRNA expression profiles showed that compared with 0 h post infection,the others exhibited a lot of differentially expressed genes(DEGs),with 2626,8 982,and 13 847 DEGs occurring at 24 h,48 h,and 72 h post infection,respectively.These DEGs were first enriched in apoptotic signaling pathway,p53 signaling pathway,TNF signaling pathway,NF-κB signaling pathway,which were involved in the initiation of death receptors apoptotic pathway,activation of mitochondrial apoptotic pathway,and inhibition of apoptosis by survival factors.Then,they were enriched in proteasome related to caspase protease activation and oxidative phosphorylation related to energy metabolism.Finally,they were enriched in DNA replication,cell cycle regulation and necrotic apoptosis.DEGs in the apoptotic pathway were screened,and apoptosis as well as the related protease activities were consistent throughout the process.Pro-apoptotic related factors such as TRAIL,FADD,Caspase 8,and Bax down-regulated,while anti-apoptotic related factors such as Bcl-2 and Gadd45 were continuously up-regulated,indicating that apoptosis might be temporarily activated after viral infection,then the virus subsequently regulated key apoptosis-related factors and activates NF-κB signaling pathways to inhibit apoptosis and promote viral replication.At last,cells activated caspase-independent necrotic apoptotic pathways as antiviral immune response.In the results of miRNA RNA-seq,926 mature miRNAs were identified,in which 396 were known miRNAs and 530 were novel miRNAs,but the expression levels of the latter were low.50 DE miRNAs with TPM expression greater than10 were screened as well as the target genes were predicted.The results showed that these miRNAs could target 4 329 genes,involving 558 passways,such as cell cycle regulation,p53 signaling pathway,MAPK signaling pathway and cytoskeletal regulation.Nine target genes related to apoptosis regulated by miRNA were chosen,and the interactions between miRNA and mRNA in apoptotic pathways were predicted.3,The miRNAs targeting TRAIL were screened from transcriptome association analysis,and verified that aan-miR-181b-3p could target TRAIL in apoptosis regulation.The results of function research of aan-miR-181b-3p and TRAIL showed that mimics transfection could inhibit apoptosis regardless of virus infection.It could also promote the virus proliferation.The inhibitor transfection could slightly promote apoptosis without virus infection.Although it played a limited role,it could slightly inhibit viral replication.The p CMV-TRAIL transfection could promote apoptosis,while the apoptotic effect was weakened on virus infection,and the virus replication was inhibited to a certain extent.The si RNA transfection had no significant effect on apoptosis as virus infection.In a word,Ang HV-1-FC was a new isolate from American eels,and it could cause Anguillid herpesvirus disease which might lead to huge economic losses in China.Ang HV-1 infection could inhibit EES apoptosis,and the miRNA of host could play an important role in the complex regulation of apoptosis.It was speculated that the inhibited apoptosis may be related to the latent infection of Ang HV-1-1FC.It was screened and verified that aan-miR-181b-3p could target TRAIL,and we found aan-miR-181b-3p could inhibit apoptosis and promote virus proliferation,while TRAIL could promote apoptosis and inhibit viral replication.The above targets could be used to screen potential drugs for prevention and control of Anguillid herpesvirus 1 infection.