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Carassius auratus gibelio is susceptible to the herpesviral hematopoietic necrosis (HVHN) disease caused by cyprinid herpesvirus 2 (CyHV-2) infection during the breeding process. Nevertheless, the report on biological response of CyHV-2 with C. auratus gibelio was limited, especially in vitro. In this study, host gene expression profiling was mostly analyzed in caudal fin cells of Carassius auratus gibelio (GiCF) underlying CyHV-2 infection. Transcriptomics and proteomics were employed to study the differential expression gene and revealed the host genes involved in pathway during the CyHV-2 infection. Transcriptome analysis revealed that compared with the control group, there were 11 335 and 19 421 differentially expressed unigenes at 48 h and at 96 h, respectively. Furthermore, proteome analysis showed that there were a total of 9008 proteins, among which 169 proteins were differential expression in the 48 h group and 502 proteins in the 96 h group. Notably, 10 and 158 differentially co-expressed genes at mRNA and protein levels (cDEGs) were reliably quantified at 48 h and 96 h, respectively. Interestingly, significantly different expressed genes both in the transcriptome and the proteome were identified, including GNG7, Hsp90a, THBS1 and RRM2. The result suggested that PI3k-AKT pathway was activated, but the p53 signaling pathway was suppressed. The above result will lay the foundation for understanding the mechanisms of host defense virus invasion during CyHV-2 infection.

The eukaryotic proteasome is a large multi-subunit complex that plays an important role in a wide range of fundamental cellular functions by degrading un-needed or damaged proteins, which also can be inverted or manipulated by viruses to favor viral infection. In this study, we demonstrated that proteasome subunit beta-type 7 (PSMB7), a proteasome-constitutive protein that is important for proteasome assembly, interacts with grass carp reovirus (GCRV) capsid proteins. Yeast 2-hybrid assay indicates that capsid protein VP38 of genotype Ⅲ GCRV could bind PSMB7, and this mutual interaction was further confirmed by pull-down, co-immunoprecipitation and subcellular co-localization assays. Furthermore, VP38 homologous proteins, VP7 from genotype I and VP35 from genotype II GCRV, can also interact with host PSMB7 in similar protein-protein interaction assays. Finally, PSMB7 expression level remains stable during GCRV infection, while, psmb7 gene transcription was repressed upon GCRV challenge; interaction with PSMB7 doesn't result in protein degradation of either VP7 or VP38 during viral infection. Thus, the interaction between host PSMB7 and viral capsid protein might suggest that interfering with PSMB7-mediated proteasome assembly should be involved in efficient aquareovirus infection.

Grass carp Ctenopharyngodon idella Hsp70 has been identified to play a functional role in viral attachment of type III grass carp reovirus, GCRV-104. However, it remains to be clarified whether Hsc70, sharing 86% identity with Hsp70, plays a similar role during viral infection. In this study, grass carp Hsp70 was shown to be induced by GCRV-104 in different grass carp cell lines, whereas Hsc70 was expressed in a relatively constant level during the infection. The expression patterns of Hsc70 and Hsp70 were similar to their homologs in mammals. Notably, both inhibitor and over-expression assays indicated that Hsp70 was required for efficient viral replication. Thus, our study supported a novel pro-viral property of Hsp70 besides its reported role in the viral attachment. Results herein presented also suggested that the heat shock response of grass carp might be manipulated by aquareovirus to facilitate its replication in fish cells.

Mammalian EGF containing fibulin-like extracellular matrix protein 2 (Fibulin-4/EFEMP2), an extracellular matrix(ECM) protein and a member of the fibulin family, is involved in elastic fiber formation, connective tissue development and some human diseases. In a yeast-two hybrid screening of host proteins interacting with outer capsid protein of grass carp reovirus (GCRV), a grass carp homologue of Fibulin-4 (designated as GcFibulin-4) is suggested to hold the potential to bind VP7, VP56 and VP55, the outer capsid protein encoded by type I, II, III GCRV, respectively. GcFibulin-4 gene of grass carp was cloned and sequenced from the cDNA library constructed for the yeast two-hybrid screening. Full-length cDNA of GcFibulin-4 contains an open reading frame (ORF) of 1323 bp encoding a putative protein of 440 amino acids. Phylogenetic analysis of GcFibulin-4 indicated that it shared a high homology with zebra fish Fibulin-4 protein. Transcriptional distribution analysis of GcFibulin-4 in various tissues of healthy grass carp showed that GcFibulin-4 was highly expressed in muscle, moderately expressed in the intestine and brain, and slightly expressed in other examined tissues; the expression pattern is consistent with tissue tropism of GCRV resulting in hemorrhage symptom in the corresponding tissues. Our results suggested that Fibulin-4 might enable free GCRV particles, the pathogen for grass carp hemorrhagic disease, to target fish tissues more efficiently by interacting with viral outer capsid proteins.

Grass carp ( Ctenopharyngodon idella ) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. To better understand the molecular responses to GCRV infection, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization tandem mass spectroscopy were performed to investigate altered proteins in C. idella kidney (CIK) cells. Differentially expressed proteins in mock infected CIK cells and GCRV-infected CIK cells were compared. Twenty-three differentially expressed spots were identified (22 upregulated spots and 1 downregulated spot), which included cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism-associated proteins and ubiquitin proteasome pathway-associated proteins. Moreover, 10 of the corresponding genes of the differentially expressed proteins were quantified by real-time reverse transcription polymerase chain reaction to examine their transcriptional profiles. The T cell internal antigen 1 (TIA1) and Ras-GTPase-activating SH3-domain-binding protein1 (G3BP1) of the cellular stress granule pathway from grass carp C. idella (designated as Ci TIA1 and Ci G3BP1) were upregulated and downregulated during GCRV infection, respectively. The full-length cDNA of Ci TIA1 was 2753 bp, with an open reading frame (ORF) of 1155bp, which encodes a putative 385-amino acid protein. The 2271bp full-length cDNA of Ci G3BP1 comprised an ORF of 1455bp that encodes a putative 485-amino acid protein. Phylogenetic analysis revealed that the complete ORFs of Ci TIA1 and Ci G3BP1 were very similar to zebrafish and well-characterized mammalian homologs. The expressions of the cellular proteins Ci TIA1 and Ci G3BP1 in response to GCRV were validated by western blotting, which indicated that the GCRV should unlink TIA1 aggregation and stress granule formation. This study provides useful information on the proteomic and cellular stress granule pathway's responses to GCRV infection, which adds to our understanding of viral pathogenesis.

Myeloid-specific peroxidase (MPO), keratin 8 (KRT-8), and dual specificity phosphatase 1 (DUSP-1) are believed to play essential roles in innate immunity. Through suppression subtractive hybridization (SSH) analysis, we previously identified MPO, KRT-8, and DUSP-1 as the three genes that were the most significantly upregulated in crucian carp (Carassius auratus gibelio) that survived Cyprinid herpesvirus 2 (CyHV-2) infection. Here, we have further characterized these three genes and their response to pathogen challenge. The open reading frames (ORF) of MPO, KRT-8, and DUSP-1 were cloned by RACE technique and sequenced. The full-length cDNAs of the three genes contained ORFs of 2289, 1575 and 1083 bp respectively. The polypeptides from each ORF were projected to contain 762 (MPO), 524 (KRT-8), and 360 (DUSP-1) amino acids. Phylogenetic analysis showed that the three genes were most closely related to zebrafish. We found that MPO, KRT-8, and DUSP-1 were expressed at low levels in all of the tissues examined in healthy crucian carp. Quantitative real-time RT-PCR analysis indicated that MPO, KRT-8, and DUSP-1 mRNA expression was significantly upregulated within 72 h of CyHV-2 infection compared to mock infected controls. Maximum expression of MPO was detected at 24 hpi (2.71-fold, P < 0.05). While, 12 hpi (3.80-fold, P < 0.01) and 6 hpi (8.70-fold, P < 0.01) were the highest expression time points for KRT-8 and DUSP-1, respectively. In contrast, after Aeromonas hydrophila challenge, the transcripts of these three genes remained unchanged or slightly down-regulated. For the fish survived from viral infection, expression levels of MPO and KRT-8 were 2.72 fold and 2.47 fold higher than those of fish died from acute infection, and similar level of DUSP-1 was observed in samples of survived fish. These data suggested MPO, KRT-8 and DUSP-1 might be involved in the antiviral, but not antibacterial innate immune response in crucian carp. These findings also support the use of MPO and KRT-8 as immunological markers for a response to viral infection in crucian carp.

Relative quantification is the strategy of choice for processing real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) data in microRNA (miRNA) expression studies. Normalization of relative quantification data is performed by comparison to reference genes. In teleost species, such as grass carp ( Ctenopharyngodon idella ), the determination of reference miRNAs and the optimal numbers of these that should be used has not been widely studied. In the present study, the stability of seven miRNAs ( miR-126-3p , miR-101a , miR-451 , miR-22a , miR-146 , miR-142a-5p and miR-192 ) was investigated by RT-qPCR in different tissues and in different development stages of grass carp. Stability values were calculated with geNorm, NormFinder, BestKeeper and Delta CT algorithms. The results showed that tissue type is an important variability factor for miRNA expression stability. All seven miRNAs had good stability values and, therefore, could be used as reference miRNAs. When all tissues and developmental stages were considered, miR-101a was the most stable miRNA. When each tissue type was considered separately, the most stable miRNAs were 126-3p in blood and liver, 101a in the gills, 192 in the kidney, 451 in the intestine and 22a in the brain, head kidney, spleen, heart, muscle, skin and fin. 126-3p was the most stable reference miRNA gene during developmental stages 1–5, while 22a was the most stable during developmental stages 6–18. Overall, this study provides valuable information about the reference miRNAs that can be used to perform appropriate normalizations when undertaking relative quantification in RT-qPCR studies of grass carp.

Lipopolysaccharide-induced TNF-α factor (LITAF), which participates in innate immune response and regulates TNF-α transcription, has been identified and characterized in various organisms. In a study to screen interacting cellular proteins with grass carp reovirus using yeast two-hybrid system, a grass carp homologue of LITAF was identified to bind the NS26 protein encoded by the S11 genomic fragment of Grass carp reovirus (GCRV). In this study, grass carp LPS-induced TNF-α factor gene (designated as CiLITAF) was cloned and sequenced from the cDNA library constructed for the yeast two-hybrid screening. The CiLITAF cDNA contained an open reading frame (ORF) of 483 bp encoding a polypeptide of 161 amino acids with an estimated molecular mass of 17.0 kDa. In CIK cells infected with GCRV or treated with poly (I:C), transiently stimulated transcription of CiLITAF mRNA was noticed at 8 h post infection or poly (I:C) treatment. Grass carp TNF-α (CiTNFα) transcriptional level was also transiently induced to a high level following the stimulation of CiLITAF in these in vitro tests. In vivo analysis further showed that, significantly up-regulated transcriptional expression of both CiLITAF and CiTNFα were detected in the spleen tissue as early as 48 h post challenge with GCRV. This study thus characterized CiLITAF as an inducible gene responding to viral infection.