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Herpesvirus infection usually relies on the interaction between viral protein and host protein to enhance replication of the enveloped virus. Fish Carassius auratus herpesvirus (CaHV) is highly pathogenic pathogen causing gill acute hemorrhages of crucian carp (Carassius auratus) and high moritality rates among those infected fish. The protein of CaHV (CaHV-138 L) containing two transmembrane (TM) domains and an immunoglobulin C-2 Type (IGc2) domain was predicted as a viral membrane protein. In this investigation, fluorescence observation showed that full-length CaHV-138 L mainly localized on the plasma membrane or around nuclear membrane of fish fathead minnow (FHM) cells in a punctate pattern. The TM domain deletion mutants of CaHV-138 L (ΔTM1, ΔTM2, and ΔTM1&ΔTM2) diffusely distributed in both the cytoplasm and the nucleus, mainly presented patchy fashion in the cytoplasm, and mainly presented both in the nucleus and in the cytoplasm, respectively. Obviously, the TM domain deletion mutants significantly affected CaHV-138 L subcellular localization. Meanwhile, colocalization assay showed that the full-length viral protein colocalized with mitochondria. Furthermore, the interaction between CaHV-138 L and host protein was identified by yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays. The host mitochondrial protein FoF1 ATP synthase (FoF1-ATPase) that interacts with this viral protein was screened. The data indicated that CaHV-138 L can target to mitochondrial protein FoF1-ATPase, which might provide energy for virus replication through mediating mitochondrial ATP synthesis. This study has provided valuable information for better understanding of the links of herpesvirus proteins with aquaculture animal proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Interferon (IFN) system plays a vital role in the first line of defense against viruses. In this study, we first identified multiple transcripts of 15 IFN system genes, including PRRs (TLR2, TLR3, RIG-I, and LGP2), PRR-mediated IFN signal pathway (MyD88, MITA, and MAVS), IFN regulatory factors (IRF1, IRF3, IRF7, and IRF9), IFNs (IFNφ1 and IFNφ3), and ISGs (Mx and viperin), and one transcript of TLR9 in de novo transcriptome assembly data of gibel carp head-kidney. Multiple nucleotide alignments and phylogenetic analysis of common region showed that the transcripts of every of the 15 IFN system genes were classified into two homologs with distinctly divergent sequences, indicating that hexaploid gibel carp may be an allopolyploid. During Carassius auratus herpesvirus (CaHV) infection, gibel carp resistant clone H significantly suppressed CaHV replication with markedly less viral loads than those in highly susceptible clone A + and moderately resistant clone F. Then, qPCR analyses were performed to reveal their differential and dynamic expression changes during CaHV infection in head kidney, spleen and liver among three gibel carp gynogenetic clones. Through qPCR and hierarchical clustering analysis, 8 genes, such as RIG-Is, LGP2s, IRF1-B, IRF3s, IRF7s, IRF9-B, Mxs, and viperins, were identified as candidate resistant-related genes. They remarkably increased their expression in immune tissues of three clones after CaHV infection. Significantly, the up-regulation folds of these genes in clone A + , F and H were related to their resistance ability to CaHV, progressively increasing from susceptible clone to resistant clone at 1 dpi. The positive correlation to the resistance ability suggested that resistant clone H immediately triggered stronger IFN response. IFNφ3 showed a different dynamic change and was sharply induced in moderately resistant clone F at 3 dpi. The other 5 IFN system genes (TLR2, TLR3, TLR9, MyD88, and MITA) maintained a low expression level after CaHV challenge. Interestingly, the A or B copies/homologs of almost these IFN system genes exhibited differential transcript abundance in immune tissue after CaHV challenge, suggesting A or B homologs might occur dominant or biased expression of homeologs during gibel carp evolution. These data provide candidate resistant-related genes for disease-resistance breeding of gibel carp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Diverse immunoglobulin (Ig) domain-containing protein (DICP) family is a novel bony fish-specific multi-gene family encoding diversified immune receptors. However, their function and the implication of binding partners remain unknown. In this study, we first identified 28 DICPs from three gibel carp gynogenetic clones and revealed their high variability and clone-specific feature. After crucian carp herpesvirus (CaHV) infection, these DICPs were significantly upregulated in head kidney, kidney and spleen. The up-regulation folds in clone A + , F and H were related to the susceptibility to CaHV, progressively increasing from resistant clone to susceptible clone. Overexpression of gibel carp DICPs inhibited interferon (IFN) and viperin promoter-driven luciferase activity. The additions of E. coli extracts and lipid A significantly enhanced the inhibition effect. In addition, gibel carp DICPs can interact with SHP-1 and SHP-2. These findings suggest that gible carp DICPs, as inhibitory receptors, might specifically recognize lipid A, and then interact with SHP-1 and SHP-2 to inhibit the induction of IFN and ISGs., (Copyright © 2018. Published by Elsevier Ltd.)

The freshwater cyanobacterial virus (cyanophage) A-4L, a podovirus, can infect the model cyanobacterium Anabaena sp. strain PCC 7120 resulting in a high burst size and forming concentric plaques on its lawns. The complete genome sequence of A-4L was determined by the combination of high-throughput sequencing, terminal transferase-mediated polymerase chain reaction and restriction mapping. It contains 41,750 bp with 810 bp direct terminal repeats and 38 potential open reading frames. As compared with other cyanobacterial podoviruses in diverse ecosystems, the A-4L has the longest terminal repeat and shares similar genome organizations with freshwater members. Furthermore, phylogenetic analysis based on concatenated sequences of eight core proteins indicated that freshwater cyanobacterial podoviruses were clustered together and distinct from marine counterparts, suggesting a clear divergence in the cyanobacterial podovirus lineage between freshwater and marine ecosystems. Our findings uncover the unique genome structure of A-4L which contains long direct terminal repeats, and create the first model system to address knowledge gaps in understanding cyanobacterial virus-host interactions at the molecular level., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A ranavirus-induced thymus cDNA library was constructed from Chinese giant salamander, the largest extant amphibian species. Among the 137 putative immune-related genes derived from this library, these molecules received particular focus: immunoglobulin heavy chains (IgM, IgD, and IgY), IFN-inducible protein 6 (IFI6), and T cell receptor beta chain (TCRβ). Several unusual features were uncovered: IgD displays a structure pattern distinct from those described for other amphibians by having only four constant domains plus a hinge region. A unique IgY form (IgY(ΔFc)), previously undescribed in amphibians, is present in serum. Alternative splicing is observed to generate IgH diversification. IFI6 is newly-identified in amphibians, which occurs in two forms divergent in subcelluar distribution and antiviral activity. TCRβ immunoscope profile follows the typical vertebrate pattern, implying a polyclonal T cell repertoire. Collectively, the pioneering survey of ranavirus-induced thymus cDNA library from Chinese giant salamander reveals immune components and characteristics in this primitive amphibian., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

An Ussuri catfish Pseudobagrus ussuriensis skin (UCS) cell line was developed and subcultured for more than 60 passages. UCS cells consisted of mostly epithelial-like cells and multiplied well in TC199 medium supplemented with 10% fetal bovine serum at 25°C. Chromosome analysis revealed that most UCS cells had a normal diploid karyotype with 2n=52. UCS cells showed differential cytopathic effects (CPEs) after inoculation of spring viremia of carp virus (SVCV, a negative-strand RNA virus), grass carp reovirus (GCRV, a multi-segmented double-stranded RNA virus) and Rana grylio virus (RGV, a large double-stranded DNA virus), and were indicative of high sensitivities to these three aquatic animal viruses by a virus titration study. The CPE caused by SVCV appeared as rounded and granular cells, grape-like clusters and small lytic plaques. Characteristic CPE containing plaque-like syncytia was induced by GCRV. RGV-infected cells produced typical CPE characterized by cells shrinkage and aggregation, formation of clear plaques and cell sheet detachment. Furthermore, significant fluorescent signals were observed after UCS cells were transfected with green fluorescent protein reporter plasmids, and the development of CPE induced by a recombinant RGV, ΔTK-RGV, in UCS cells was illustrated using a combination of light and fluorescence microscopy. The data from this study suggested that UCS cell line can potentially serve as a useful tool for the comparison study of different aquatic animal viruses and the isolation of some newly emerging viruses in Ussuri catfish farming., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, a recombinant RGV (i53R-RGV-lacIO) containing the inducible lac repressor/operator system was constructed. i53R-RGV-lacIO was a conditional lethal mutant in which the expression of envelope protein 53R was regulated by IPTG. i53R-RGV-lacIO shared characteristics similar to RGV in the presence of IPTG. However, the expression level of 53R, the ability of plaques formation, and the virus titers were strongly reduced in the absence of IPTG. Electron microscopy showed that the number of progeny virus produced by i53R-RGV-lacIO was remarkably reduced without IPTG. Furthermore, over-expression of 53R in vitro could increase titers of i53R-RGV-lacIO in the absence of IPTG. Therefore, the current data suggested that the lac repressor/operator system could regulate gene expression in the recombinant iridovirus. Our study was thought to be the first report of the system in aquatic virus., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In the present study, Rana grylio virus (RGV, an iridovirus) thymidine kinase (TK) gene and viral envelope protein 53R gene were chosen as targets for foreign gene insertion. ΔTK-RGV and Δ53R-RGV, two recombinant RGV, expressing enhanced green fluorescence protein (EGFP) were constructed and analyzed in Epithelioma papulosum cyprinid (EPC) cells. The EGFP gene which fused to the virus major capsid protein (MCP) promoter p50 was inserted into TK and 53R gene loci of RGV, respectively. Cells infected with these two recombinant viruses not only displayed plaques, but also emitted strong green fluorescence under fluorescence microscope, providing a simple method for selection and purification of recombinant viruses. ΔTK-RGV was purified by seven successive rounds of plaque isolation and could be stably propagated in EPC cells. All of the plaques produced by the purified recombinant virus emitted green fluorescence. However, Δ53R-RGV was hard to be purified even through twenty rounds of plaque isolation. The purified recombinant virus ΔTK-RGV was verified by PCR analysis and Western blotting. These results showed EGFP was expressed in ΔTK-RGV infected cells. Furthermore, one-step growth curves and electron microscopy revealed that infection with recombinant ΔTK-RGV and wild-type RGV are similar. Therefore, RGV was demonstrated could be as a viral vector for foreign gene expression in fish cells., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA(7)) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (ORF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fold up-regulation of the beta-defensin within first 12h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair (bp) sequence (+26 to -73) and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappaB) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappaB and Sp1.

The complete genome of mandarin fish Siniperca chuatsi rhabdovirus (SCRV) was cloned and sequenced. It comprises 11,545 nucleotides and contains five genes encoding the nucleoprotein N, the phosphoprotein P, the matrix protein M, the glycoprotein G, and the RNA-dependent RNA polymerase protein L. At the 3' and 5' termini of SCRV genome, leader and trailer sequences show inverse complementarity. The N, P, M and G proteins share the highest sequence identities (ranging from 14.8 to 41.5%) with the respective proteins of rhabdovirus 903/87, the L protein has the highest identity with those of vesiculoviruses, especially with Chandipura virus (44.7%). Phylogenetic analysis of L proteins showed that SCRV clustered with spring vireamia of carp virus (SVCV) and was most closely related to viruses in the genus Vesiculovirus. In addition, an overlapping open reading frame (ORF) predicted to encode a protein similar to vesicular stomatitis virus C protein is present within the P gene of SCRV. Furthermore, an unoverlapping small ORF downstream of M ORF within M gene is predicted (tentatively called orf4). Therefore, the genomic organization of SCRV can be proposed as 3' leader-N-P/C-M-(orf4)-G-L-trailer 5'. Orf4 transcription or translation products could not be detected by northern or Western blot, respectively, though one similar mRNA band to M mRNA was found. This is the first report on one small unoverlapping ORF in M gene of a fish rhabdovirus.

Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish, but little is known about the relationship between its gene structure and nuclear localization of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. auratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poly I:C, or CAB IFN-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NLS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids ("95 KDKSINK 101" and "75 KTWKANFR 82"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K75, K78, R82, K95, and K101) and one non-conserved basic amino acid (K97) are present in this NLS from CaIRF-1. This observation suggests that K97 of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic amino acids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1.

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system.

The heme-regulated initiation factor 2alpha kinase (HRI) is acknowledged to play an important role in translational shutoff in reticulocytes in response to various cellular stresses. In this study, we report its homologous cDNA cloning and characterization from cultured flounder embryonic cells (FEC) after treatment with UV-inactivated grass carp haemorrhagic virus (GCHV). The full-length cDNA of Paralichthys olivaceus HRI homologue (PoHRI) has 2391 bp and encodes a protein of 651 amino acids. The putative PoHRI protein exhibits high identity with all members of eIF2alpha kinase family. It contains 12 catalytic subdomains located within the C-terminus of all Ser/Thr protein kinases, a unique kinase insertion of 136 amino acids between subdomains IV and V, and a relatively conserved N-terminal domain (NTD). Upon heat shock, virus infection or Poly I:C treatment, PoHRI mRNA and protein are significantly upregulated in FEC cells but show different expression patterns in response to different stresses. In healthy flounders, PoHRI displays a wide tissue distribution at both the mRNA and protein levels. These results indicate that PoHRI is a ubiquitous eIF2alpha kinase and might play an important role in translational control over nonheme producing FEC cells under different stresses.