The Transcriptomic Analysis Of Mandarin Fish Brain Cells Infected With Infectious Spleen And Kidney Necrosis Virus And Its Replication

Mandarin fish(Siniperca chuatsi)is one of the most important cultured fish species in China.Recently,with the rapid expansion as well as higher density of the Mandarin fish culture,diseases have become the major hinders for the sustainable culture of the fish.Among them,infectious spleen and kidney necrosis virus(ISKNV)is a major pathogen,which has caused infectious spleen and kidney necrosis(ISKN)of Mandarin fish.ISKNV is a species of the genus Megalocytivirus that belongs to the family Iridoviridae.ISKNV is an enveloped virus with a double-stranded DNA genome of 111,362 bp,which encodes124 potential open reading frames(ORF).Due to the large viral genome and substantial viral proteins,it is reasonable to believe that the host-ISKNV interactions are extremely intricate and remain enigmatic.The analysis of the transcriptomic profilings of cells infected with ISKNV will reveal the global host-ISKNV interactions.The obtained data of transcriptome will facilitate the studies of the replication and pathogenesis of the ISKNV,which will shed a new light on the development of effective strategies for the prevention of ISKN.In this study,RNA-Seq(Illumina)technique was used for analyze the transcriptomic profiles of Mandarin fish brain cells(CPB)at progressive time points after the infection of ISKNV.The CPB cell transcriptomic database was established by de novo assembly using Trinity software.The functional annotation and pathway analysis were performed by sequence alignment against four protein databases,including nr,Swiss Prot,COG and KEGG.The differentially expressed genes(DEGs)related to ISKNV infection at progressive time points were identified.Twelve of DEGs were randomly chosen for quantitative real time PCR(q RT-PCR)verification.Based on the transcriptomic profilings,retinoic acid inducing gene-I like receptors(RLRs)pathway and apoptosis pathway in CPB cells after the infection of ISKNV were further analyzed.Further more,glutamine metabolic pathway and host cyclophilin A involved in the viral replication were also investigated.The results were as follows.1.A total of 96,206,040 clean reads were obtained by RNA-Seq.These data were assembled into 66,787 unigenes with a mean size of 927 bp.Blast searches for all unigenes were performed against the above mentioned four protein databases.Among them,33,362 unigenes(49.95%)were annotated.A total of 80,208 unigenes were mapped to 48 GO terms,and 25,943 unigenes were classed into 25 COG classifications.KEGG pathway analysis showed that there were 16,364 unigenes were annotated into 240 pathways.Blast searches revealed that 33,022 unigenes had been predicted CDS.Using the EST scan software to predict the CDS of the rest unigenes,the results showed that1,189 unigenes might be new protein CDS.2.In the samples harvested at 24 or 72 h post of infection(poi),a total of 10,834 or7,584 genes were differentially expressed in ISKNV-infected CPB cells in comparison with the mock,respectively.KEGG annotation revealed that 12 pathways were involved in immune responses and inflammation,and there were 516 or 449 DEGs at 24 or 72 h poi,respectively.Twelve DEGs were measured by q RT-PCR,similar expression patterns of the detected genes were observed by both q RT-PCR and RNA-Seq,indicating that the data of the RNA-Seq were reliable.3.The analysis of RLRs pathway showed that there were 10 DEGs in CPB cells after infection with ISKNV.At the early time point of ISKNV infection,virus inhibited the activation of NF-κB via over-expression of the IKKB-α and IKKB-β and lower-expression of interleukin-1 receptor-associated kinase 4(IRAK4).4.The analysis of apoptosis pathway showed that there were 11 DEGs in CPB cells after infection with ISKNV.ISKNV infection could induce apoptosis mainly via tumor necrosis factor(TNF)mediated death receptor pathway.Meanwhile,PI3 Ks related cellular signaling pathways were also activated during ISKNV infection.5.Some genes related to the glutamine metabolism have been changed significantly basing on DEGs analysis.Depriving ISKNV-infected CPB cells of exogenous glutamine led to a substantial decrease in infectious virion production in the supernatant,while the vitality of the cells cultured in glutamine deprived medium was not significantly different within 72 h,indicating that glutamine was essential for the efficient multiplication of ISKNV in CPB cells.Viral yield in glutamine-deprived cells could be rescued by the addition of multiple tricarboxylic acid(TCA)cycle intermediates.Thus,ISKNV infection induced a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle.Further more,the m RNA expressions of some ISKNV genes were decreased in glutamine-deprived cells,suggesting that the transcription step of the ISKNV was regulated by glutamine,resulting in the decrease of the viral protein synthesis and virion production.6.It has been shown that host protein cyclophilin(Cyp A)played a key role in the replication of several viruses.Based on the Mandarin fish Cyp A unigene in the CPB transcriptomic database,primers were designed and the full ORF of Mandarin fish Cyp A(SC-Cyp A)was obtained.The ORF of SC-Cyp A encoded a polypeptide of 164 amino acids with calculated molecular weight of 17.59 k Da.The deduced amino acid sequences of the SC-Cyp A shared highly conserved structures with Cyp As from the other species,indicating that SC-Cyp A should be a new member of the Cyp A family.The m RNA expression of SC-Cyp A was measured in ISKNV-infected CPB cell and non-infected cell at progressive time points.The results showed that the m RNA level of SC-Cyp A in the infected cells was decreased firstly,then increased,and eventually dropped to the basal level when it was compared with that in non-infected cells.Cyclosporin A(Cs A)is a specific inhibitor of Cyp A.ISKNV replication was inhibited by the addition of Cs A in a manner of dose dependent.Cs A could effectively inhibit the replication of ISKNV at different time points after ISKNV infection,and the strongest inhibitory effect was observed at 72 h poi.The addition of Cs A could inhibit the m RNA expressions of IL1-β,IL8,IL18 and ISG15 in CPB cells after the infection of ISKNV.By contrast,it induced the m RNA expression of TNF-α.