The MicroRNA And Transcriptomic Profile Of GF-1 And SSN-1 Cells Infected With Red-spotted Grouper Nervous Necrosis Virus

Nervous necrosis virus(NNV),also known as betanodavirus,is a member of the Nodaviridae family.NNV infection can cause serious fish disease,named viral nervous necrosis(VNN)or viral encephalopathy and retinopathy(VER).NNV was initially isolated from diseased marine fish and has been reported in more than 120 different cultured and wild-ranged fish species,and caused huge economical losses in aquaculture industry.Recently,there is increasing evidence that NNV has also been epidemic in freshwater fish.Experimental infections also showed that some freshwater fish were susceptible to NNV.Although extensive efforts have been made to develop therapeutic and prophylactic regiments against the viral infection,the molecular mechanisms underlying the pathogenicity of NNV are still poorly understood.Analysis of the microRNA and transcriptomic profilings of cells infected with red-spotted grouper nervous necrosis virus(RGNNV)will reveal the global host-RGNNV interactions.The obtained data will facilitate the studies of the replication and pathogenesis of RGNNV,which will shed a new light on the development of effective strategies for the prevention of VER.To date,several fish cell lines have been shown to be susceptible to NNV.However,due to the lack of susceptible cell lines derived from fish nervous tissue,the neurotrophy of NNV remains enigmatic.Therefore,it is highly expected to establish an NNV-susceptible cell line derived from fish nervous tissue.In this study,we firstly investigated the microRNA(miRNA)expression profiles of grouper fin(GF-1)cells infected with RGNNV at 3 and 24 hours post of infection(poi)using deep RNA sequencing technique;In addition,the transcriptomic profiles of GF-1 cells and striped snakehead fish(Channa striatus)cells(SSN-1)infected with RGNNV at the same time point were investigated by solexa deep sequencing and de novo assembly,respectively.Taken the advantage of the established Chinese perch brain(CPB)cell line derived from the brain tissue of Mandarin fish(Siniperca chuatsi),we further investigated the susceptibility of CPB cell as well as Mandarin fish to RGNNV.The establishment of the fish brain originated CPB cell line,which was susceptible to NNV infection,will facilitate the study of the replication and pathogenicity of NNV in the future.The obtained results were as follows:1.Through HiSeq 2500 sequencing,a total of 220 miRNAs were identified by aligning the small RNA sequences with the known miRNAs of zebrafish in miRbase 21.0 database,and 18 novel miRNAs were predicted using miRDeep2 software.Compared with the mock-infected GF-1 cells,51 and 16 differentially expressed miRNAs(DE-miRNAs)were identified in GF-1 cells infected with RGNNV at 3 and 24 hours,respectively.Six randomly selected DE-mi RNAs were measured by quantitative reverse transcription polymerase chain reaction(qRT-PCR),similar expression patterns of the detected DE-miRNAs were observed by both qRT-PCR and HiSeq 2500.Six novel miRNAs were randomly selected to validate their expressions using RT-PCR.The target genes of the DE-miRNAs covered a wide range of functions,including regulation of transcription,oxidation-reduction process,proteolysis,apoptotic and immune responses,et al.The effects of four DE-miRNAs including miR-1,miR-30 b,miR-150,and mi R-184 on RGNNV replication were evaluated,and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV.2.Through HiSeq 2500 sequencing,a total of 127,259,258 clean reads were obtained and they were assembled into 111,860 unigenes with a mean size of 624 bp.35,390 unigenes(31.64%)were annotated via blastx searches against with seven protein databases.Compared with the mock-infected GF-1 cells,226 and 117 differentially expressed genes(DEGs)were identified in GF-1 cells infected with RGNNV at 3 and 24 hours,respectively.Further analysis on the apoptosis pathway suggested that RGNNV infection might induce apoptosis of GF-1 cells via Drp1 and Bax-meditated mitochondrial pathway.Five DEGs were randomly selected to validate their expressions using qRT-PCR,the results showed that their expression profiles were consistent with those obtained by HiSeq 2500 sequencing;The activity of caspase-3,-8,-9 were assayed by using luminescence detection,the results showed that RGNNV infection can significantly enhance the activities of Caspase-3,-8 and-9 in GF-1 cells.3.Through HiSeq 2000 sequencing,a total of 253,338,544 clean reads were obtained and they were assembled into 111,860 unigenes with a mean size of 829 bp.18,974 unigenes(20.32%)were annotated via blastx searches against with four protein databases.Compared with the mock-infected SSN-1 cells,2,640 and 3,211 DEGs were identified in SSN-1 cells infected with RGNNV at 3 and 24 hours,respectively.Further analysis on the apoptosis pathway suggested that RGNNV infection might induce apoptosis of SSN-1 cells via Endonuclease G(EndoG)-associated mitochondrial pathway.Seven DEGs were randomly selected to validate their expressions using qRT-PCR,the results showed that their expression profiles were consistent with those obtained by HiSeq 2000 sequencing;Annexin V-FITC/PI double staining and DAPI staining was used to measure the cellular apoptosis of SSN-1 cells upon RGNNV infection or EndoG over-expression,the results showed that RGNNV infection or over-expression of EndoG could induce apoptosis in SSN-1 cells.4.The apoptosis pathway induced by RGNNV infection in GF-1 cells and SSN-1 cells was compared using the transcriptomic profiles of GF-1 and SSN-1 cells infected with RGNNV.The expression of the genes in extrinsic apoptosis pathway was not significantly altered in both two cells.In the intrinsic apoptosis pathway,the expression level of Bax and Drp1 in GF-1 cells,and EndoG in SSN-1 cells were all significantly up-regulated upon RGNNV infection.The three proteins were involved in mitochondrial-mediated apoptosis.In addition,in the Endoplasmic reticulum(ER)stress response,the expression level of Bip was significantly up-regulated in both GF-1 and SSN-1 cells upon RGNNV infection,and eventually leading to mitochondrial mediated apoptosis,this phenomenon was also observed in the transcriptome of NNV infected grouper kidney(GK)cells.Therefore,we hypothesized that RGNNV infection may induce apoptosis through mitochondria-ER mediated apoptosis pathway,the detailed mechanism needs to be investigated in the future.5.CPB cell that was derived from brain tissues of a freshwater Mandarin fish was chosen to evaluate its susceptibility to RGNNV.The results of qRT-PCR showed that RGNNV replicated well in CPB cells.Annexin V-FITC/PI double staining and DAPI staining were used to confirm that RGNNV infection could induce apoptosis in CPB cells.Moreover,the susceptibility of Mandarin fish to RGNNV was also evaluated.Abnormal swimming of the Mandarin fish was observed after RGNNV was injected into the fish through eyeball.The cellular vacuolation was also observed in brain tissues of RGNNV-infected Mandarin fish by Hematoxylin-eosin(HE)staining.A lot of ~30 nm particles closely arranged in the inclusion body were observed in cytoplasm of the brain cells of RGNNV-infected Mandarin fish under electronic microscope(EM),and the mitochondria of brain cells from RGNNV-infected fish were swollen compared with those from mock-infected fish.Whether this phenomenon was associated with the loss of mitochondrial membrane potential(MMP)needs further study.The established RGNNV susceptive CPB cell line will pave a new way for the study of the pathogensis of RGNNV.