Effect of Heat Shock Treatment on the Virulence of Grass Carp Reovirus in Rare Minnow Gobiocypris rarus.

The mode and outcome of fish-virus interactions are influenced by many abiotic factors, among which water temperature is especially important in poikilothermic fish. Rare minnow Gobiocypris rarus is a eurythermal small cyprinid fish that is sensitive to infection with genotype II grass carp reovirus (GCRV). HSP70, a conservative and key player in heat shock response, is previously identified as an induced pro-viral factor during GCRV infection in vitro. Here, rare minnow was subjected to heat shock treatment (HST), 1 h treatment at 32 °C followed by reverting to a normal temperature of 24 °C, and subsequently challenged with GCRV-II at a dosage of 1 × LD50. The effect of HST on GCRV virulence in vivo was evaluated by calculating virus-associated mortality and viral load in both dead and survival fish. The results revealed that HST enhanced the mortality of rare minnow infected with GCRV; the fact that viral loads in the tissue samples of HST-treated fish were significantly higher than those in samples of the control group at 6, 8 d p.i. reflected a faster infection process due to HST. Quantitative gene expression analysis was further employed to show that the expression levels of Hsp70 in intestine and liver tissues from the HST group declined faster than muscle tissue after HST. HST W/O GCRV challenge upregulated proinflammatory cytokines such as MyD88 and Nf-κB, which was in consistence with the inflammation observed in histopathological analysis. This study shed light on the complexity of the interaction between fish abiotic and biotic stress response, which suggested that HST, an abiotic stress, could enhance the virulence of GCRV in Gobiocypris rarus that involved modulating the gene expression of host heat shock, as well as a pro-inflammatory response.

Genomic and Transcriptional Profiles of Kelch-like (klhl) Gene Family in Polyploid Carassius Complex.

Genome duplication supplies raw genetic materials and has been thought to be essential for evolutionary innovation and ecological adaptation. Here, we select Kelch-like (klhl) genes to study the evolution of the duplicated genes in the polyploid Carassius complex, including amphidiploid C. auratus and amphitriploid C. gibelio. Phylogenetic, chromosomal location and read coverage analyses indicate that most of Carassius klhl genes exhibit a 2:1 relationship with zebrafish orthologs and confirm two rounds of polyploidy, an allotetraploidy followed by an autotriploidy, occurred during Carassius evolution. The lineage-specific expansion and biased retention/loss of klhl genes are also found in Carassius. Transcriptome analyses across eight adult tissues and seven embryogenesis stages reveal varied expression dominance and divergence between the two species. The expression of klhls in response to Carassius herpesvirus 2 infection shows different expression changes corresponding to distinct herpesvirus resistances in three C. gibelio gynogenetic clones. Finally, we find that most C. gibelio klhl genes possess three alleles except eight genes that have lost one or two alleles due to genome rearrangement. The allele expression bias is prosperous for Cgklhl genes and varies during embryogenesis owning to the sequential expression manner of the alleles. The current study provides global insights into the genomic and transcriptional evolution of duplicated genes in a given superfamily resulting from multiple rounds of polyploidization.

Autophagy Delays Apoptotic Cell Death Induced by Siniperca chuatsi Rhabdovirus in Epithelioma Papulosum Cyprinid Cells.

Autophagy and apoptosis are two key cell fate determination pathways, which play vital roles in the interaction between viruses and host cells. Previous research had confirmed that one strain of fish rhabdoviruses, Siniperca chuatsi rhabdovirus (SCRV), could induce apoptosis and autophagy after infection. In the current study, we continued to analyze the interaction of autophagy and apoptosis in SCRV-infected EPC cell lines after treatment with different autophagy or apoptosis inhibitors. We found that SCRV infection could activate the mitochondrial apoptotic pathway by the detection of the activities of the caspase-3 and caspase-9 and by flow cytometry analysis in JC-1-stained cells, respectively. Furthermore, no significant autophagy-related factors were disturbed in SCRV-infected cell after apoptosis inhibitor Z-VAD-FMK treatment, while autophagy inducer rapamycin could obviously delay the occurrence of CPE and cell death. Meanwhile, rapamycin was able to reduce the proportion of apoptotic cells. Besides that, rapamycin could disturb the expression of p62 and LC3B-II, and the transcription level of SCRV nucleoprotein mRNA. The progeny virus titers did not show a big difference between the rapamycin treatment or without it. Collectively, our data preliminarily confirmed that SCRV-activated autophagy could delay apoptosis in EPC cells and may not affect virus production. Further study may need to focus on the crosstalk regulation and its roles on the SCRV infection.

Comparative Molecular Characterization of Novel and Known Piscine Toti-Like Viruses.

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5' or 3' ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3' end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3' end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters.

Establishment of a rare minnow (Gobiocypris rarus) model for evaluation of experimental vaccines against a disease induced by grass carp reovirus genotype II.

Vaccination is the most effective way to control the grass carp haemorrhagic disease (GCHD) with the primary pathogen grass carp reovirus genotype II (GCRV-II). However, due to the large difference in breeding conditions and unclear genetic background of grass carp, the results of the experiment were not reliable, which further hinders the effective prevention and control of GCHD. The rare minnow (Gobiocypris rarus) is highly sensitive to GCRV. Its small size, easy feeding, transparent egg membrane, and annual spawning are in line with the necessary conditions for an experimental aquatic animals culture object. In this study, immunogenicity and protective effects of attenuated and inactivated viruses for grass carp and rare minnow were evaluated in parallel. The expression of immune-related genes increased statistically significant after immunization. With the rise of specific serum antibody titers, the results of rare minnow and grass carp were consistent. In addition, there was no significant residue of adjuvant observed in both fish species injected with an adjuvanted and inactivated virus. Challenge of immunized grass carp and rare minnow with the isolate HuNan1307 resulted in protection rates of 95.8% and 92.6% for attenuated virus, 81.4% and 77.7% for inactivated virus, respectively, as well as the viral load changed consistently. The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCHD.

TBK1 regulates the induction of innate immune response against GCRV by phosphorylating IRF3 in rare minnow (Gobiocypris rarus).

Rare minnow (Gobiocypris rarus), a small cyprinid species that is highly sensitive to the grass carp reovirus (GCRV), is regarded as an ideal model to study the mechanisms of innate immunity in fish. In the present study, a TBK1 homologue from rare minnow (GrTBK1) was identified and its roles in defence against viral infection were investigated. Sequence analysis showed that GrTBK1 encoded a 727-amino acid peptide which shared 98% and 72% identity to the black carp (Mylopharyngodon piceus) and human (Homo sapiens) orthologues, respectively. The amino acid sequence analysis demonstrated that GrTBK1 contains a conserved Serine/Threonine protein kinases catalytic domain (S_TKc) at the N-terminus. Furthermore, cellular distribution proved that GrTBK1 was located in the cytoplasm region. Quantitative real-time PCR analysis revealed that GrTBK1 was ubiquitously expressed in all examined organs, but especially highly in liver. Temporal expression analysis in vivo showed that the expression levels of GrTBK1 were obviously up-regulated in response to GCRV infection. Meanwhile, qRT-PCR assay revealed that the levels of S7 RNA, an important segment of GCRV genome, were higher in the liver than in other tissues. This indicates that GrTBK1 might play a crucial role in responses to GCRV infection in fish. In addition, GrTBK1 activated several type I interferon (IFN) promoters and induced the expression of downstream type I IFN-stimulated genes (ISGs). Furthermore, GrTBK1 obviously phosphorylated the interferon regulatory factor 3 (IRF3). Furthermore, overexpression of GrTBK1 remarkably decreased the GCRV proliferation. In summary, we systematically characterized GrTBK1 and illustrated its role in the innate immune response to GCRV infections.

Molecular detection of a novel cyprinid herpesvirus in roach (Rutilus rutilus) and asp (Leuciscus aspius) showing typical signs of carp pox disease.

In the early spring of 2018, in Lake Balaton (Hungary), a roach (Rutilus rutilus) and an asp (Leuciscus aspius) were found in an fish trap at the outlet of the river Sió showing typical signs of the so-called carp pox disease, such as foci of epidermal hyperplasia on the head and the whole body surface, including the fins. Molecular tests revealed the presence of the DNA of an unknown fish herpesvirus. Three genes encoding the DNA-dependent DNA polymerase, major capsid protein and ATPase subunit of terminase were amplified and sequenced from the alloherpesviral genome. The gene sequences of the viruses obtained from the two different fish species shared 94.4% nucleotide sequence identity (98.1% amino acid sequence identity), suggesting that they belong to the same virus species. Phylogenetic analysis based on the DNA polymerase (and the concatenated sequences of the amplified genes, as well) implied that the detected virus belongs to the genus Cyprinivirus within the family Alloherpesviridae. The sequences of the novel alloherpesvirus diverge from those of the five cyprinivirus species described previously, so it putatively represents the sixth virus species in the genus.

MicroRNA expression profiling of caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection.

As a member of the genus Cyprinivirus in the family Alloherpesviridae, Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss in the aquaculture industry, mainly in C. auratus gibelio and goldfish. However, the molecular mechanisms underlying the pathogenicity of CyHV-2 remain elusive. In this study, high-throughput sequencing technology was employed to explore the miRNA expression profiles of C. auratus gibelio (GiCF) caudal fin cells in response to Cyprinid Herpesvirus-2 (CyHV-2) infection. A total of 631 novel miRNAs and 409 known miRNAs were identified. The expression levels of 7 miRNAs were found as significantly modulated (5 down-regulation and 2 up-regulation; P < 0.01, |logFC|>1, TPM>10) in CyHV-2 infected cells. 7 miRNA and their potential mRNA targets were validated by Real-time PCR (qRT-PCR), respectively. Targets prediction and functional analysis of these 7 miRNAs revealed significant enrichment for several signaling pathways, including PPAR, p53 and FoxO pathways. These studies provided more valuable basis for further study on the roles of miRNAs in CyHV-2 replication and pathogenesis.

Detection of cyprinid herpesvirus 2 by loop-mediated isothermal amplification in combination with a lateral flow dipstick.

We developed a convenient technique to detect Herpesviral haematopoietic necrosis attributed to cyprinid herpes virus 2 (CyHV-2), a serious disease of Crucian carp and goldfish related to high mortality. In the present study, we employed a lateral flow dipstick (LAMP-LFD) to present a loop-mediated isothermal amplification assay. The specificity was ascertained via other six viruses, and the sensitivity was compared using PCR method, which are the reaction conditions changes for the method improved. The results revealed that CyHV-2 performance was observable at 64 °C in a separated tube within 60 min, when the samples hybridized using an FITC-labeled probe. As the LAMP-LFD method's specificity was high, with its sensitivity identical to that of traditional PCR, the overall DNA collected revealed the lowest detection limit of 0.18 pg/µl from goldfish diseased by CyHV-2. In summary, the development of LAMP-LFD's method does not require expensive instruments, and it can be regarded as a fast, simple, and reliable method for CyHV-2 detection.

Virus susceptibility of a new cell line derived from the fin of black carp Mylopharyngodon piceus.

A continuous cell line MPF derived from the fin of black carp Mylopharyngodon piceus was established and characterised in this study. Mylopharyngodon piceus fin (MPF) cells were subcultured for more than 80 passages with high viability recovery after long-term storage. The karyotyping analysis revealed that MPF had a modal diploid chromosome number (2n = 48) and identical ribosomal RNA sequence with black carp. In addition, the expression of pluripotency-associated markers including nanog, oct4 and vasa, were detected in MPF. The transient transfection efficiency of MPF reached 23% with a fluorescent reporter by modified electroporation and stable expression of red fluorescent MPF was established by the baculovirus system, indicating that MPF is an ideal platform for studying gene functions in vitro. Lastly, cytopathic effects were also observed and RNA transcripts of a viral gene increased after infection by spring viremia of carp virus (SVCV), suggesting that MPF could be an alternative tool for investigating pathogen-host interactions in black carp. In conclusion, a fin cell line that is susceptible to SVCV was established as a potential adult stem-cell line, providing a suitable tool for future genetic analyses and pathogen-host studies in black carp.

Characterization of a Novel Megalocytivirus Isolated from European Chub (Squalius cephalus).

A novel virus from moribund European chub (Squalius cephalus) was isolated on epithelioma papulosum cyprini (EPC) cells. Transmission electron microscopic examination revealed abundant non-enveloped, hexagonal virus particles in the cytoplasm of infected EPC cells consistent with an iridovirus. Illumina MiSeq sequence data enabled the assembly and annotation of the full genome (128,216 bp encoding 108 open reading frames) of the suspected iridovirus. Maximum Likelihood phylogenetic analyses based on 25 iridovirus core genes supported the European chub iridovirus (ECIV) as being the sister species to the recently-discovered scale drop disease virus (SDDV), which together form the most basal megalocytivirus clade. Genetic analyses of the ECIV major capsid protein and ATPase genes revealed the greatest nucleotide identity to members of the genus Megalocytivirus including SDDV. These data support ECIV as a novel member within the genus Megalocytivirus. Experimental challenge studies are needed to fulfill River's postulates and determine whether ECIV induces the pathognomonic microscopic lesions (i.e., megalocytes with basophilic cytoplasmic inclusions) observed in megalocytivirus infections.

Molecular cloning, GTP recognition mechanism and tissue-specific expression profiling of myxovirus resistance (Mx) protein in Labeo rohita (Hamilton) after Poly I:C induction.

The myxovirus resistance (Mx) proteins belong to interferon-induced dynamin GTPase and play pivotal role in the inhibition of replication of numerous viruses. These antiviral proteins are released in usual or diseased condition to prevent the viral attack and to carry regular cellular activities like endocytosis and trafficking of nucleoproteins into the nucleus. The invasion of virus up-regulates the expression of Mx transcripts and double-stranded RNA mimic like polyinosinic polycytidyilic acid (Poly I:C). To understand the tissue-specific expression profiling and mechanism of GTP recognition of Mx protein from Labeo rohita (rohu), the full-length gene was cloned, sequenced and characterized through various Bioinformatics tools for the first time. The Mx cDNA was comprised of 2297 bp, and the open reading frame of 1938 bp encodes polypeptide of 631 amino acids. The coding sequence of Mx protein possess the signature motif of dynamin superfamily, LPRG(S/K)GIVTR, the tripartite guanosine-5/triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD) and the leucine zipper motifs at the C-terminal end, well conserved in all interferon-induced Mx protein in vertebrates. Western blotting confirmed the molecular weight of Mx protein to be 72 kDa. After the intraperitoneal challenge of L. rohita with a Poly I:C, up-regulation of Mx protein was observed in brain, spleen, liver, kidney, intestine, heart, muscle, and gill. Ontogeny study displayed pronounced expression of Mx protein in all stages of the developmental of Rohu after Poly I:C induction. However a persistent expression of Mx transcript was also observed in Rohu egg as well as milt without induction with Poly I:C. Higher expression of Mx gene was observed on 96 h where it was 6.4 folds higher than the control. The computational modelling of Mx protein portrayed the tripartite N-terminal G-domain that binds to GTP, the bundle-signaling element (BSE) which interconnects the G-domain to the elongated stalk domain and C-terminal helical stalk domain. In agreement with the experimental studies, a series of conserved residues viz., Gln52, Ser53, Ser54, Leu68, Pro69, Gly71, Gly73, Thr76, Asp151, Gly154, Thr220, Lys221, Val251, Cys253, Arg254, and Gly255 were computed to be indispensable for tight anchoring of GTP within binding cavity of G-domain. The binding free energy calculation study depicted that the van der Waals and electrostatic terms contributs significantly to molecular recognition of GTP. Collectively, our study provides mechanistic insights into the tissue-specific expression profiling and GTP binding mechanism of Mx protein from Labeo rohita, which is expected to drive further research on several cellular events including viral resistance and endocytosis in the near future.

Elucidation of mechanism for host response to VHSV infection at varying temperatures in vitro and in vivo through proteomic analysis.

Seasonal temperature has a major influence on the infectivity of pathogens and the host immune system. Viral hemorrhagic septicemia virus (VHSV) is one such pathogen that only causes the mortality of fish at low temperatures. This study aims to discover the host defense mechanism and pathway for resistance to VHSV at higher temperatures. We first observed the VHSV infection patterns at low and higher temperatures in fathead minnow (FHM) cells (20 °C and 28 °C) and zebrafish (15 °C and 25 °C). In comparison to the 20 °C infection, FHM cells infected at 28 °C showed decreased apoptosis, increased cell viability, and reduced VHSV N gene expression. In zebrafish, infection at 25 °C caused no mortality and significantly reduced the N gene copy number in comparison to infection at 15 °C. To explore the antiviral infection mechanisms induced by high temperature in vitro and in vivo, the changes in the proteomic profile were measured through UPLC-MSE analysis. ACADL, PTPN6, TLR1, F7, A2M, and GLI2 were selected as high temperature-specific biomarkers in the FHM cell proteome; and MYH9, HPX, ANTXR1, APOA1, HBZ, and MYH7 were selected in zebrafish. Increased immune response, anticoagulation effects, and the formation of lymphocytes from hematopoietic stem cells were analyzed as functions that were commonly induced by high temperature in vitro and in vivo. Among these biomarkers, GLI2 was predicted as an upstream regulator. When treated with GANT58, a GLI-specific inhibitor, cell viability was further reduced due to GLI2 inhibition during VHSV infection at varying temperatures in FHM cells, and the mortality in zebrafish was induced earlier at the low temperature. Overall, this study discovered a new mechanism for VHSV infection in vitro and in vivo that is regulated by GLI2 protein.

The antiviral mechanism of viperin and its splice variant in spring viremia of carp virus infected fathead minnow cells.

Viperin is known to play an important role in innate immune and its antiviral mechanisms are well demonstrated in mammals. Fish Viperin mediates antiviral activity against several viruses. However, little has been done to the underlying mechanism. Here, we discovered a novel Viperin splice variant named Viperin_sv1 from viral-infected FHM cells. Spring varimia of carp virus (SVCV) was able to increase the mRNA levels of both Viperin and Viperin_sv1, while poly(I:C) only has effect on Viperin. Viperin functions as an antiviral protein at 24 h post-SVCV infection, but the antiviral activity dramatically declined at late infection stages. However, Viperin_sv1 inhibited SVCV replication significantly at all the tested time. Viperin_sv1, but not Viperin can facilitate the production of type I IFN and IFN stimulate genes (ISGs) through activation of RIG-1, IRF3 and IRF7 signaling cascades. On the other hand, SVCV down-regulated Viperin_sv1 at the protein level through the proteasome pathway to keep itself away from the immune system monitoring. Taken together, these findings provide new insights into the regulation of Viperin from the posttranscriptional modification perspective and the role of splicing variant Viperin_sv1 in virus-host interaction.

Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model.

An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID50), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens.

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion-dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry.

Cyprinid viral diseases and vaccine development.

In the past decades, global freshwater fish production has been rapidly growing, while cyprinid takes the largest portion. Along with the rapid rise of novel forms of intensive aquaculture, increased global aquatic animal movement and various anthropogenic stress to aquatic ecosystems during the past century, freshwater fish farming industry encounter the emergence and breakout of many diseases, especially viral diseases. Because of the ability to safely and effectively prevent aquaculture diseases, vaccines have become the mainstream technology for prevention and control of aquatic diseases in the world. In this review, authors summarized six major cyprinid viral diseases, including koi herpesvirus disease (KHVD), spring viraemia of carp (SVC), grass carp hemorrhagic disease (GCHD), koi sleepy disease (KSD), carp pox disease (CPD) and herpesviral haematopoietic necrosis (HPHN). The present review described the characteristics of these diseases from epidemiology, pathology, etiology and diagnostics. Furthermore, the development of specific vaccines respective to these diseases is stated according to preparation methods and immunization approaches. It is hoped that the review could contribute to aquaculture in prevention and controlling of cyprinid viral diseases, and serve the healthy and sustainable development of aquaculture industry.

CRISPR/Cas9-mediated knockout of HIF-1α gene in epithelioma papulosum cyprini (EPC) cells inhibited apoptosis and viral hemorrhagic septicemia virus (VHSV) growth.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of HIF-1α and HIF-1ß, and its key role in the regulation of cellular responses to hypoxia has been well-demonstrated. The participation of HIF-1α in apoptosis has been reported in mammals, however, a little information is available on the role of HIF-1α in the progression of apoptosis in fish. In this study, to know the role of HIF-1α in the apoptosis of fish cells, we produced HIF-1α knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector, and a single cell clone showing a heterozygous insertion/deletion (indel) mutation (one nucleotide insertion and one nucleotide deletion in HIF-1α gene) was chosen for further experiments. To confirm the knockout of HIF-1α, cells were transfected with a hypoxia reporting vector containing hypoxic response elements (HREs). EPC cells transfected with the reporting plasmids showed significantly increased luminescence by exposure to cobalt chloride, a prolyl hydroxylases inhibitor. On the other hand, HIF-1α knockout EPC cells showed a non-responsiveness to a cobalt chloride exposure, suggesting that functional HIF-1α protein was not produced in the HIF-1α knockout EPC cells. Apoptosis progression induced by camptothecin and viral hemorrhagic septicemia virus (VHSV) infection was severely inhibited by HIF-1α knockout, and the replication of VHSV was significantly retarded in HIF-1α knockout EPC cells. These results suggest that HIF-1α in EPC cells acts as a pro-apoptotic factor in the progression of apoptosis triggered by a DNA damaging agent and rhabdoviral infection.

Application of a monoclonal antibody specific for the ORF92 capsid protein of Cyprinid herpesvirus 2.

Cyprinid herpesvirus 2 (CyHV-2) infection is a recently discovered viral disease of farmed silver crucian carp (Carassius auratus gibelio) in mainland China that has resulted in massive fish mortality and appears to be spreading cross the world. Various detection methods based on nucleic acids have been developed, but effective immunological diagnostic method has not been reported for CyHV-2. In this study, recombinant ORF92 protein was used as a capture antigen to identify cells and tissues infected with CyHV-2 by immunological methods. Firstly, the open reading frame of CyHV-2 ORF92 was cloned into the PGEX-4T-3 vector and expressed in Escherichia coli. Purified recombinant ORF92 protein was then used as an antigen to prepare monoclonal antibodies, and an efficient hybridoma cell line was selected by dot-blot assay. The resulting mAb-1B7 was applied to detect CyHV-2 in infected Ryukin goldfish fin (RyuF-2) cells and fish tissues by western blotting and immunofluorescence assays. The monoclonal antibody could specifically identify CyHV-2 in infected RyuF-2 cells and gill, kidney, and spleen tissues, suggesting it could be applied for CyHV-2 detection in crucian carp.

Involvement of interferon regulatory factor 3 from the barbel chub Squaliobarbus curriculus in the immune response against grass carp reovirus.

The barbel chub Squaliobarbus curriculus is an important commercial fish species in China, and has shown significant resistance to grass carp reovirus (GCRV). In this study, the cDNA sequence of interferon regulatory factors 3 (IRF3) from Squaliobarbus curriculus, designated as ScIRF3, was cloned, and its effect against GCRV was investigated. The full-length 1837 base pair (bp) cDNA of ScIRF3 contained a complete open reading frame of 1374 bp and encoded a putative polypeptide of 457 amino acid residues. The ScIRF3 protein contained conserved domains, including an N-terminal DNA-binding domain, a C-terminal IRF association domain, and a serine-rich domain. Phylogenetic analysis showed that ScIRF3 was closely clustered with IRF3s from Carassius auratus and Ctenopharyngodon idellus. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScIRF3 in Squaliobarbus curriculus were the highest in the spleen and lowest in the muscle. After GCRV infection, expression levels of both ScIRF3 and type I interferon (IFN) were initially up-regulated and subsequently down-regulated in the spleen and intestine. Correlation analysis showed that the expression level of type I IFN is significantly positively correlated with that of ScIRF3 (Pearson correlation coefficient: 0.883, P: 0.004) in the intestine. The expression level of type I IFN was also significantly up-regulated and the GCRV titer was significantly decreased (P < .05) in GCRV-infected ScIRF3-overexpressing Ctenopharyngodon idellus kidney cells. These results indicate that ScIRF3 may play a role in the type I IFN immune response against GCRV in Squaliobarbus curriculus and can also inhibit GCRV replication in Ctenopharyngodon idellus kidney cells.