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Largemouth bass (Micropterus salmoides) is an important freshwater-cultured species in China. Recently, a lethal and epidemic disease caused by Micropterus salmoides rhabdovirus (MSRV) results in huge economic losses to the largemouth bass industry. Current diagnostics for detecting MSRV are limited in sensitivity and speed and are inconvenient to be used for non-laboratory detection. In this study, three rapid and convenient detection assays of MSRV by recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD), targeting the conserved sequences of the MSRV-SS N gene, are described. With these RPA methods, the detection could achieve within 50 min at 38°C. Both methods of RPA-AGE and RPA-LFD could detect the viral DNA as low as 170 copies/μl of the MSRV standard plasmid and were 100-fold more sensitive than that in the method of routine PCR. Meanwhile, these RPA methods were highly specific for the detection of MSRV and can be feasibly applied to the diagnostic of MSRV infection. In brief, RPA-AGE, RPA-LFD and RT-RPA-LFD provide convenient, rapid, sensitive and reliable methods that could improve field diagnosis of MSRV with limited machine resources, and would enhance the production of largemouth bass.

Infections of Cyprinid herpesvirus 2 in goldfish and farmed crucian carp (Carassius auratus gibelio) are still an urgent problem worldwide. Detection and prevention are necessary for the control of haematopoietic necrosis disease caused by CyHV-2. Although many sensitive molecular diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV-2 are still important to CyHV-2 study. In this experiment, purified CyHV-2 was used as antigens to produce monoclonal antibodies (Mabs). Six Mabs bound to different proteins were selected by Dot-blot screening and Western-blot analysis, and no one had cross-reactivity with closely related koi herpesvirus. Among them, Mabs 2E1-B10, 1F5-A3 and 4C4-A7 belonged to IgG

Bcl2 family proteins play a critical role in cell death or survival. BAX, the death-promoting protein of bcl2 family, mediated mitochondrial pathway inducing cells' apoptosis in mammal. MiRNAs have been implicated as negative regulators down-regulating genes' expression after post-transcriptional level. At present, little is known about the regulatory mechanism of miRNA on the Bcl2 family proteins during CyHV-2 infection in silver crucian carp (Carassius auratus gibelio). In this study, the ccBAX (silver crucian carp BAX) gene was cloned and expressed, and polyclonal antibodies were raised in mouse against the purified ccBAX-GST fusion protein. The structure analysis indicated that ccBAX protein included four conserve domains (BH1, BH2, BH3 and transmembrane domains) and the expression of ccBAX protein occurred throughout the cells. Furthermore, two miRNAs (miR-124 and miRNA-29b) were identified to negatively regulate ccBAX gene expression in GiCF cell. miR-124 was found to suppress the expression of WT-ccBAX (wild type), but not the MT-ccBAX (mutant). Overall, the results demonstrated that the expression of the ccBAX gene was significantly down-regulated by miR-124 in silver crucian carp (Carassius auratus gibelio) during CyHV-2 infection.

(‐)‐Epigallocatechin gallate (EGCG), a catechin found in green tea, has been demonstrated to exhibit activity against grass carp reovirus (GCRV). In the current study, we found that EGCG is partially transformed in vivo into (‐)‐epicatechin gallate (ECG), which differs from EGCG only by the absence of a hydroxyl group, and exhibits similar pharmacokinetic behaviour to that of EGCG. ECG is also a major catechin in green tea, but little information on its antiviral activity is available. Therefore, we assessed whether ECG affects GCRV in vitro. We incubated grass carp (Ctenopharyngodon idellus) kidney (CIK) cells with ECG and GCRV‐JX01 at different concentrations, and typical cytopathogenic effect (CPE) values were observed for 5 and 10 µg/ml ECG. However, the CPE in 20 µg/ml ECG treatment group was low; no significant CPE was observed for 40 µg/ml ECG treatment; and a high ECG concentration (80 µg/ml) led to stress response in the CIK cells. Western blot results also revealed that ECG suppresses GCRV replication in CIK cells. Thus, the data indicate that ECG, as well as EGCG, exhibits potential as an antiviral agent for aquaculture.

Herpesviruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. In our previous studies, we found a new miRNA miR-KT-635 encoded by Cyprinid herpesvirus 2, which is predicted to target viral genes and cellular genes involved in innate immune signalling pathway and apoptosis. However, the function and target gene of miR-KT-635 are not proved. In this study, the regulating target gene of miR-KT-635 was proved as the viral gene ORF23 directly, the target point sequence on gene was verified and miR-KT-635 was identified to regulate the expression of ORF23 protein. According to the bioinformatics analysis, the tRNA domain and ribosome domain in the protein sequence of ORF23 were found to share high homology with R2i and P53R2i, which are related to the ribonucleotide reductase small subunit in the host (transform NTP to dNTP). Within expectations, silencing of viral ORF23 or transfecting miR-KT-635 mimics in Carassius auratus gibelio caudal fin cell line (GiCF) could suppress viral propagation significantly.

The freshwater crayfish Procambarus clarkii is native to North America and Mexico, and it was introduced to China in 1929. The production and consumption of P. clarkii in China are the highest worldwide, reaching 208.96 million tons in 2020. The white spot syndrome virus (WSSV) is a major pathogen that affects shrimp, crayfish, crabs and lobsters, and it has caused widespread loss to the P. clarkii industry. Epigallocatechin-3-gallate (EGCG), a small-molecule compound, has a multitude of biological functions and the ability to bind to the 37 kDa/67 kDa laminin receptor (LamR). EGCG has potential antiviral effects against WSSV. In this study, we evaluated the potential anti-WSSV applications of EGCG in P. clarkii. We demonstrated that various concentrations (10 μg/g·bw, 20 μg/g·bw and 40 μg/g·bw) of EGCG can suppress WSSV infection in P. clarkii. Histopathological examination revealed no characteristic pathological changes due to EGCG administration in P. clarkii tissues. Furthermore, pharmacokinetics studies of EGCG in P. clarkii revealed its rapid absorption (T

Cyprinid herpesvirus 2 (CyHV-2) is a viral pathogen worldwide and causing high mortality on goldfish and silver crucian carp (Carassius auratus gibelio). In order to establish a stable and sensitive immunological diagnostic approach, the recombinant ORF121 protein encoded by the CyHV-2 ORF121 gene, was selected as a capture antigen to identify cells and tissues infected with CyHV-2 by immunological methods in this study. Firstly, the open reading frame of CyHV-2 ORF121 was cloned into the PGEX-4T-3 vector and expressed in Escherichia coli. Purified recombinant ORF121 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-3D9 was applied to detect CyHV-2 in infected caudal fin of Carassius auratus gibelio (GiCF) cells and fish tissues by western blotting, immunofluorescence assays and immunohistological asays. The monoclonal antibody could specifically identify CyHV-2 in infected GiCF cells and the gills, the kidney and the spleen tissues, and it could attenuate CPE by CyHV-2 in vitro, suggesting it can be applied for CyHV-2 detection in the crucian carp and ORF121 may be a candidate vaccine against CyHV-2.

Genotype III grass carp reovirus (GCRV; representative strain, GCRV-104) belongs to the subfamily Spinareovirinae and encodes an outer-fiber protein, VP55, responsible for mediating the infection of target tissues by the virus and assisting the virus into cells. Fibulin-4/EFEMP2 protein was previously identified as a putative binding partner for VP55 in a yeast two-hybrid screening. Here, we have further characterized the association between Fibulin-4 and VP55 by using protein interaction assays. An intracellular co-localization assay showed that RFP-Fibulin-4 co-localized with GFP-VP55 in grass carp ovary (GCO) cells. Bacterially expressed GST-tagged Fibulin-4 was shown to associate with baculovirus-expressed His-tagged VP55 in a dot-blot overlay assay; moreover, baculovirus-expressed His-tagged VP55 was able to pull down GFP-Fibulin-4 expressed in the GCO cells. We performed real-time PCR and immunoblotting analysis and showed that endogenous Fibulin-4, although suppressed to a lower level in the late infection phase, is present throughout the infection course of GCRV-104 in CIK cells. In conclusion, our results indicate that grass carp Fibulin-4 interacts with VP55. The presence of Fibulin-4, a well-known secreted protein, during the infection course of GCRV-104 in grass carp cells implies its potential role during viral egression through interaction with VP55.

Neomycin belongs to the family of 2-deoxystreptamine-containing aminoglycoside antibiotics. It is widely used for bacterial infections, targeting most gram-negative and some gram-positive bacteria. Neomycin has also been reported to show antiviral activity. Here, we evaluated the toxicity of neomycin sulfate, and investigated its effect on non-specific immunity and viral infection in crucian carp (Carassius auratus gibelio). The safe concentration of neomycin sulfate for crucian carp was determined to be 102.9 mg/kg in vivo. In oxidative damage assays, neomycin sulfate increased superoxide dismutase and catalase activity and decreased malondialdehyde in the liver of crucian carp. In non-specific blood immune assays, the white blood cell count and complement 3 content significantly increased after neomycin sulfate treatment, while no significant changes were observed in antibacterial or lysozyme activity. In a challenge test, neomycin sulfate protected the crucian carp from cyprinid herpesvirus 2 (CyHV-2) infection and inhibited CyHV-2 replication. In cytotoxicity assays, low concentrations of neomycin sulfate had no cytotoxicity on cells from the fins of crucian carp. The results of the present study indicate that oral administration of neomycin sulfate reduced oxidative damage, enhanced immunity and provided protection against CyHV-2 in crucian carp. Keywords: Neomycin sulfate, Non-specific immunity, Oxidative damage, Crucian carp, Cyprinid herpesvirus 2

An acute cyprinid herpesvirus 2 (CyHV-2) infection of crucian carp (Carassius auratus gibelio) is characterized with hemorrhagic inflammation and high mortality. Berberine is a biologically active plant alkaloid with an anti-inflammation function involving the regulation of transcription factor NF-κB and its downstream inflammation-related genes in mammalian organisms. Our previous study had indicated that BBH (berberine chloride hydrate) inhibited gene expression and replication of CyHV-2, and might serve as a potential small molecular drug for controlling CyHV-2 infection in crucian carp. Here, NF-κB signaling in CyHV-2-infected crucian carp treated with BBH was investigated both in vitro and in vivo. Real-time RT-PCR and ELISA assays revealed that CyHV-2 could activate the NF-κB signaling resulting in up-regulation of its downstream pro-inflammatory gene expression including IL-6, IL-8, IL-1β and TNF-ɑ. BBH could attenuate NF-κB signaling and downregulate the expression of its downstream inflammatory cytokines in a dose-dependent manner both in vivo and in vitro. BBH not only dose-dependently inhibits viral load in CyHV-2-susceptible crucian carp cell lines in infection and inhibition assays, but also ameliorates inflammatory injury in CyHV-2-targeted organ in histopathology examinations. Thus, negative regulation of NF-κB by BBH should contribute to BBH-mediated protection of crucian carp from CyHV-2 infection via attenuating viral gene expression and host inflammatory response simultaneously.

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.

Fibulin-4 is not only involved in connective tissue development and elastic fiber formation, but also plays critical neoplastic roles in tumor growth by activating Wnt/β-Catenin signaling in human. Recently, Fibulin-4 was shown to associate with grass carp reovirus (GCRV) outer capsid proteins and might relate to viral hemorrhagic disease in grass carp Ctenopharyngodon idella. Here, we monitored the expression pattern of Fibulin-4 during the infection course of GCRV at both translational and transcriptional levels, and found that Fibulin-4 was significantly suppressed upon the viral challenge in grass cap GCO cells. Over expression of Fibulin-4 was achieved by transduction of pEGFP-Fibulin-4 plasmids into GCO cells, which was confirmed by both Western blot and Real time RT-PCR analysis. In GCO cells with over-expression of Fibulin-4, significantly increase of viral protein synthesis and progeny virus production was detected. Our study indicated that Fibulin-4 displayed pro-viral function and was inhibited during viral challenge. Thus, repression of Fibulin-4 expression seemed to be involved in anti-viral response in grass carp Ctenopharyngodon idella.

Crucian carp (Carassius auratus gibelio) is a popular food fish in Asia, and cyprinid herpesvirus 2 (CyHV-2) is the only known viral pathogen for crucian carp. Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms. In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge, a partial interferon transcript was identified to be significantly up-regulated in the kidney of infected fish, which was named as crucian carp IFNc (ccIFNc). The complete ORF of ccIFNc was further determined by RACE technique, which spanned over 546 bp and encoded a polypeptide containing 182 amino acids. Phylogenetic analysis revealed that ccIFNc clustered with known type I IFN genes from other aquatic organisms. Quantitative RT-PCR analysis demonstrated that ccIFNc was constitutively expressed in all investigated tissues with a comparably higher expression level in spleen, gill, kidney, and muscle. Following challenge with CyHV-2, the transcriptional levels of ccIFNc were dramatically up-regulated in all of the tested tissues, especially in the spleen and gill with increased folds of 436 and 158, respectively. The intramuscular (i.m.) injection of a eukaryotic expression plasmid encoding ccIFNc (pEGFP-cIFNc) resulted in increased ccIFNc expression and reduced the mortality after the CyHV-2 challenge significantly. In summary, our data suggested that the ccIFNc belongs to the type I interferon family with a potential role in countering CyHV-2 infection in crucian carp.

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.

Grass carp reovirus (GCRV) infection causes apoptosis in Ctenopharyngodon idella kidney cells (CIK). However, the cause of GCRV-induced apoptosis and its signaling pathways remain unknown. This study investigated the role of TNF-α-induced capase-8 pathways in mediating GCRV-induced apoptosis in the grass carp (Ctenopharyngodon idella). Recombinant TNF-α was expressed and purified from Escherichia. coli. The western blot assay indicated that TNF-α expression level in kidney and spleen was higher than that in liver. In apoptosis assay, recombinant TNF-α triggered significant apoptosis in CIK cells, which was characterized by increased mRNA levels of TNF-α, TRADD or caspase-8, and enhanced caspase-8 activity in CIK cells. To confirm the biological activity of TNF-α during GCRV infection, significant apoptosis in CIK cells was induced by GCRV correlating with enhanced caspase-8 activity, increased mRNA level of TNF-α, TRADD or caspase-8, increased protein level of TNF-α in CIK cells and cell supernatant, suggesting that TNF-α-induced capase-8 pathways might be involved in GCRV-triggered apoptosis. Furthermore, treatment with an anti-TNF-α polyclonal antibody significantly decreased the degree of apoptosis in infected CIK cells compared with cells treated with a control antibody, which confirmed that TNF-α was a key mediator involved in GCRV-induced apoptosis. Taken together, these results indicated that GCRV might trigger apoptosis via TNF-α induced capase-8 pathways in CIK cells.

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of herpesviral hematopoietic necrosis (HVHN). In order to identify the protein contained the genome of CyHV-2, virus particles were purified by sucrose density gradient in combination with ultracentrifugation and observed by transmission electron microscope. The viral proteins were separated by SDS-PAGE, and then identified by mass spectrometry identification. The anti-CyHV-2 polyclonal antibody prepared by immunizing mice with purified virus was subjected to Western blotting analysis and mass spectrometry identification to identify the major immunogenic proteins to mice. A large number of intact CyHV-2 virions were collected from the sucrose layer between 50% and 66%, and a small amount of viral envelops were damaged. The results of mass spectrometry identification showed that CyHV-2 containing 74 proteins, including 3 capsid proteins, 18 membrane proteins, 53 other proteins; Western blotting and mass spectrometry identification showed that there are eight major immunogenic proteins, namely pORF92, pORF115, pORF25, pORF57, pORF66, pORF72, pORF131 and pORF132. This study is the first to identify the proteins contained in CyHV-2 as well as the major immunogenic proteins, which will provide a more comprehensive theoretical and practical basis for the in-depth study of CyHV -2 virus.

Black spot disease causes significant economic losses in whiteleg shrimp Penaeus vannamei yet only limited information is available on Shewanella algae as a bacterial pathogen of this disease. In this study, a virulent strain, temporarily named SFH3, was isolated from diseased freshwater-cultured P. vannamei suffering from black spot disease, identified phenotypically and molecularly as S. algae. A phylogenetic tree was constructed to examine the taxonomic position and relatedness of isolate SFH3 to other S. algae isolates. When screened against a range of common veterinary antibiotics, isolate SFH3 is apparently susceptible to aminoglycosides and quinolones antibiotics in aquaculture. To the best of our knowledge, this is the first report of S. algae as a causal agent of black spot disease in live freshwater-cultured P. vannamei.

Ulcerative disease causes significant economic losses in largemouth bass Micropterus salmoides production. Yet information reported on Bacillus mycoides as a pathogen for largemouth bass is scarce. In this study, a virulent strain, temporarily named LYS1, was isolated from diseased M. salmoides suffering from ulcerative disease, identified phenotypically and molecularly as B. mycoides. A phylogenetic tree was constructed to examine isolate LYS1 and compare it to other known isolates. In addition, isolate LYS1 appears to be susceptible to aminoglycosides and quinolones drugs for veterinary use in aquaculture as seen when screened against a range of common antibiotics. To the best of our knowledge, this is the first report of B. mycoides as a pathogen of ulcerative disease in farmed largemouth bass

Acute hepatopancreatic necrosis disease (AHPND) of shrimps is an important disease, first appeared in China in 2009. Since then, AHPND has caused serious drops in shrimp production (up to 20 % worldwide). Although AHPND [originally termed as acute hepatopancreatic necrosis syndrome (AHPNS)] first appeared in 2009, it was not until 2013 that a laboratory infection model was devised and the causative agent identified as certain strains of Vibrio parahaemolyticus. AHPND has caused mortality from 40 to 100 % which usually occurs early (within approximately 35 days) after stocking shrimp fry in shrimp ponds; therefore, it was initially referred to as early mortality syndrome (EMS). Confusingly, other pathogens and environmental factors also cause EMS and are often attributed to AHPND by shrimp farmers. Frequently, farmers do not send samples for confirmatory tests requiring detection of the unique histopathology at the acute stage of disease (massive sloughing of hepatopancreatic epithelial cells without any accompanying signs of a pathogen). The gross signs presumptive of AHPND (lethargy, slow growth, empty stomach and midgut, and a pale to white, atrophied hepatopancreas) are insufficient for confirmatory diagnosis. Recently, molecular detection of AHPND bacteria using PCR has been developed, which has sped up diagnosis and increased research on the causative agent, alternative detection methods, and possible therapies. We hope that this review of research progress on AHPND will serve as a useful introduction for researchers who are currently unfamiliar with AHPND, but have backgrounds in bacterial virulence, detection, and epidemiology, and may be encouraged to participate in the research effort to reduce AHPND’s impact on shrimp cultivation.

Previous report demonstrated that grass carp reovirus (GCRV) infection resulted in unlinking cellular stress granule formation from aggregation of grass carp Ctenopharyngodon idella TIA1 (CiTIA1). Here, we provided evidence to show that CiTIA1 bound to synthesized ssRNA and dsRNA in vitro. Both GST-pull down assay and RNA immunoprecipitation analysis confirmed the association between GCRV-specific RNA and GST-tagged CiTIA1 in C. idella kidney (CIK) cells. Furthermore, CiTIA1 was shown to protect dsRNA of virus-origin from degradation in CIK cells through Northern blot analysis. Finally, transient overexpression of CiTIA1 enhanced the replication efficiency of GCRV in CIK cells. Taken together, our results suggested that cellular CiTIA1 might facilitate GCRV replication through sequestrating and protecting viral RNA from degradation., Highlights • CiTIA1 binds both ssRNA and dsRNA. • dsRNA is protected from degradation by CiTIA1. • Overexpression of CiTIA1 enhanced grass carp reovirus replication.

MicroRNAs (miRNAs) modulate many biological processes and can be up-or down-regulated in a variety of diseases states, including in bacterial infection. In this study, we characterized miRNAs associated with susceptibility or resistance to motile Aeromonas hydrophila in grass carp (Ctenopharyngodon idella), a commercially important farmed fish species in China. Using Illumina next-generation sequencing, we detected 185 miRNAs in A. hydrophila-susceptible (SGC) and -resistant grass carp (RGC) during immune activation. Twenty-one miRNAs were found to be differentially expressed between SGC and RGC, with 23.8% (5 out 21) exhibiting elevated expression in SGC. Further investigation of a selected five miRNAs indicated differences in the timing and tissue sources of expression. Target genes likely regulated by these miRNAs were identified using computational prediction combined with transcriptome expression data. MiRNA targets of differentially expressed mRNAs included those with functions important for bacterial resistance, such as tlr4 and nfil3-6. In addition, we demonstrated that nfil3-6 and tlr4 genes are direct targets of let-7i and cid-miRn-118. The present study suggested that the multiple miRNAs, displaying diverse kinetics and tissue sources on antibacterial immune processes.

Human tumor necrosis factor receptor-associated protein 1 (Trap1) is a mitochondrial protein identical to heat shock protein 75 (HSP75) that plays an important role in protecting cells from oxidative stress and apoptosis. In this study, grass carp (Ctenopharyngodon idella) tumor necrosis factor receptor-associated protein 1 (designated as CiTrap1) was identified through two-dimensional electrophoresis (2-DE) analysis and its pattern of expression was investigated in grass carp kidney (CIK) cells infected with grass carp reovirus (GCRV). The full length cDNA of CiTrap1 contained an opening reading frame of 2157 bp that encoded a peptide of 718 amino acids. Phylogenetic analyses indicated that the CiTrap1 shared 87% identity with its homologue from zebrafish (Danio rerio). The transcriptional level of CiTrap1 in CIK cells was upregulated post virus infection as well as poly (I: C) stimulation. Following virus infection, grass carp PTEN-induced putative kinase 1 (PINK1) and Sorcin, whose coding proteins interact with Trap1 in human, were simultaneously upregulated with CiTrap1. Typical characteristics of apoptosis were observed in CIK cells infected with GCRV by DAPI staining, DNA ladder electrophoresis, TUNEL assay and Annexin Ⅴ labeling. RNAi-mediated silencing of CiTrap1 in CIK cells resulted in the increased rate of virus-induced apoptotic cells. The results of this study suggest that CiTrap1 is involved in the host's innate immune response to viral infection possibly through protecting infected cells from apoptosis.

Bacteriosis has caused significant economic losses in farmed blunt snout bream Megalobrama amblycephala. Only scarce information is available on Acinetobacter johnsonii as a possible causal agent in M. amblycephala. In this study, a virulent strain temporarily named BY3, was isolated from diseased bream, identified phenotypically and molecularly as A. johnsonii. Phylogenetic trees of 16S rRNA and gyrB gene sequences were constructed to examine the taxonomic position of isolate BY3 and compare it to other known isolates. When screened against a range of common veterinary antibiotics, isolate BY3 has not shown resistance to florfenicol, neomycin, and sulfisoxazole antibiotics in aquaculture. To the best of our knowledge, this is the first report of A. johnsonii infection in farmed blunt snout bream.

Bacteriosis is a major economic problem affecting the farming industry of whiteleg shrimp Penaeus vannamei. Little information is available on Providencia rettgeri as a possible pathogen for P. vannamei. In this study, a virulent strain, here named S6, was isolated from diseased P. vannamei, identified phenotypically, and molecularly, as P. rettgeri. A phylogenetic tree was constructed to examine the taxonomic position and relatedness of isolate S6 to other P. rettgeri isolates. When screened against a range of common veterinary antibiotics, isolate S6 exhibited resistance to aminoglycosides and sulfonamides antibiotics in aquaculture. To the best of our knowledge, this is the first report of P. rettgeri as a causal agent of bacteriosis in freshwater farmed P. vannamei.

Bacteriosis has caused significant economic losses in farmed yellow catfish Pelteobagrus fulvidraco. Information is limited on Shewanella putrefaciens as a pathogen for yellow catfish. In this study, a virulent strain, temporarily named Y2, was isolated from diseased yellow catfish, identified phenotypically and molecularly as S. putrefaciens. A phylogenetic tree was constructed to examine isolate Y2 and compare it to other known isolates. In addition, isolate Y2 is apparently susceptible to aminoglycosides and tetracycline drugs for veterinary use in aquaculture as revealed when screened against a range of common antibiotics. To the best of our knowledge, this is the first report of S. putrefaciens as an emerging pathogen for cultured yellow catfish.

Enteritis has caused significant economic losses in farmed channel catfish Ictalurus punctatus. Yet information is limited on Enterobacter aerogenes as a potential causal agent for enteritis in channel catfish. In this study, a virulent strain, temporarily named HT2, was isolated from diseased channel catfish suffering from enteritis, identified phenotypically and molecularly as E. aerogenes. A phylogenetic tree was constructed to examine isolate HT2 and compare it to other known isolates. In addition, isolate HT2 is apparently susceptible to aminoglycosides and quinolones, drugs for veterinary use in aquaculture as seen when screened against a range of common antibiotics. To the best of our knowledge, this is the first report of E. aerogenes as a pathogen causing enteritis in farmed channel catfish.

Interleukin 11 (IL-11), Intelectin (ITLN) and Purine nucleoside phosphorylase 5a (PNP5a) play important roles in innate immunity. In a previous study to identify differentially expressed immune-related genes, suppression subtractive hybridization (SSH) assay was used to characterize differentially expressed genes in crucian carp (Carassius auratus gibelio) infected with Cyprinid herpesvirus 2 (CyHV-2) in which IL-11, ITLN and PNP5a were identified to be the three most significantly up-regulated genes (Xu et al., Archives of Virology, 2014, http://dx.doi.org/10.1007/s00705-014-2011-9). In this study, the complete open reading frames (ORF) of IL-11, ITLN and PNP5a genes were cloned and sequenced. The full-length cDNAs of the three genes contained an ORF of 597, 945 and 882 bp, encoding a polypeptide of 198, 314 and 293 amino acids, respectively. Phylogenetic analysis indicated that the three genes shared high homology to other bony fish species including Zebrafish. Interestingly, the ITLN gene of crucian carp lacked a 10 aa peptide that was found in the C-terminal of other fish species. A real-time RT-PCR assay was developed to quantitatively examine their tissue distribution. We found that IL-11, ITLN and PNP5a were expressed at low levels in all of the tissues examined. To monitor the response of these genes to CyHV- 2 or Aeromonas hydrophila (A. hydrophila) infection, we determined the expression level of IL-11, ITLN and PNP5a at different time points after infection in kidney. Significant up-regulation of IL-11, ITLN and PNP5a was only observed 72 h post-CyHV-2 injection (P < 0.01), whereas significant up-regulation was observed as early as 6 h after infection with A. hydrophila (P < 0.01). Our results demonstrated that host innate immune response to CyHV-2, at least in which IL-11, ITLN and PNP5a were involved, was slow in comparison to that induced by A. hydrophila. It suggested that CyHV-2 might suppress host innate response during early infection. The lack of a C-terminal peptide of crucian carp ITLN gene implied a possible functional difference of this gene during evolution, which merit further investigation.

Voltage-dependent anion channels (VDACs) located in the mitochondrial outer membrane are mitochondrial porins that play central roles in regulating cell life and death. In this present report, the VDAC protein 1 from grass carp Ctenopharyngodon idella (designated as CiVDAC1) was found to be upregulated by grass carp reovirus (GCRV) infection through two-dimensional gel electrophoresis and protein analysis of infected C. idella kidney (CIK) cells. The full-length cDNA of CiVDAC1 was 995 bp with an open reading frame (ORF) of 852 bp that encodes a putative 283-amino acid protein. Phylogenic analysis revealed that the complete ORF of CiVDAC1 demonstrated high identity with well characterized mammalian homologs. The deduced CiVDAC1 protein contains an α-helix at the amino terminal, 19 membrane-spanning β-strands, and one eukaryotic mitochondrial porin signature motif. Tissue tropism analysis indicated that CiVDAC1 is abundant in muscle, heart, skin, swim bladder, trunk kidney and spleen. Transcriptional expression profiles indicated that the CiVDAC1 gene was upregulated upon viral challenge in a manner similar to the Mx2 gene, which is a marker gene used to indicate activation of innate antiviral immunity. Similar expression patterns of the CiVDAC1 gene were observed in CIK cells stimulated with poly (I:C), as well as grass carp kidney tissue challenged with GCRV in vivo. CiVDAC1 silencing in CIK cells had no impact on progeny virus production, but over-expression of CiVDAC1 in vivo showed strongly protect against challenge with live virus. To interpret the role of other VDAC proteins in viral pathogenesis, CiVDAC2 was characterized and showed to respond positively to GCRV challenge, which suggested that CiVDAC2 might functionally complement CiVDAC1 in C. idella. The present data did demonstrate that CiVDAC1 might be mediated grass carp antiviral immune response.

Summary In order to control saprolegniosis in Prussian carp (Carassius gibelio (Bloch, 1782) eggs, it is important to screen herb extracts as potential anti-Saprolegnia drugs in Prussian carp hatcheries. For this purpose, an oomycete water mould (strain SC) isolated from Prussian carp [Carassius gibelio (Bloch, 1782)] eggs suffering from saprolegniosis was characterised morphologically as well as from ITS rDNA sequence data. Initially identified as a Saprolegnia sp. based on its morphological features, the constructed phylogenetic tree using the neighbour joining method further indicated that the SC strain was closely related to Saprolegnia australis R. F. Elliott 1968 strain VI05733 (GenBank accession no. HE798564), and which could form biofilm communities as virulence factors. In addition, aqueous extracts from forty Chinese herbs were screened as possible anti-Saprolegnia agents. Among them, a 1 g ml−1 extract from Radix sanguisorbae was the most efficacious anti-Saprolegnia agent, indicated by the minimum inhibitory concentration that was as low as 256 mg L−1. Relative survival of 73 and 88% was obtained against the SC strain in fish eggs at concentrations of 256 and 1280 mg L−1, respectively. This is the first known report of Saprolegnia australis R. F. Elliott 1968 infection in C. gibelio (Bloch, 1782) eggs involving the screening of R. sanguisorbae extracts as potential anti-Saprolegnia agents.

Tail rot disease is the cause of significant economic damage in freshwater farmed Murray cod Maccullochella peelii. Only scarce information is available on Aeromonas hydrophila as a possible causal agent for this disease. In this study, a virulent strain, temporarily named XY3, was isolated from diseased codfish suffering from tail rot disease, and identified as A. hydrophila through phylogenetic analysis and phenotypic characteristics. A. hydrophila possesses multiple virulence genes including aerA, ahpA, alt, ast and hlyA genes. In addition, it appears that isolate XY3 has developed multiple resistances to cephalosporin, chloromycetin, glycopeptides, macrolides, nitrofuran, and penicillin drugs, as well as to aminoglycosides, sulfonamides, and tetracyclines antibiotics for veterinary uses in aquaculture as revealed when screened against a range of common antibiotics. To the best of our knowledge, this is the first report of tail rot disease caused by A. hydrophila in freshwater farmed codfish.

Tail rot disease causes significant economic damage in freshwater farmed Japanese eel Anguilla japonica, yet information on Citrobacter freundii as a possible causal agent for this disease is scarce. In this study, a virulent strain, temporarily named MINA, was isolated from diseased A. japonica suffering from tail rot disease. It was identified through phylogenetic analysis and phenotypic characteristics and thus compared to other known isolates. Isolate MINA has developed multiple resistances to penicillin, quinolones and sulfonamide antibiotics as well as to amide alcohols, cephalosporin, glycopeptide and macrolide drugs used in aquaculture when screened against a range of common antibiotics. To the best of our knowledge, this is the first report of tail rot disease caused by C. freundii in freshwater farmed A. japonica.

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.

Whiteleg shrimp Penaeus vannamei is an important commercial shrimp species cultivated in China and many other countries worldwide. Bacteriosis is a major economic problem that inhibits the farming of this species in fresh water. White feces syndrome is an emerging epidemic in freshwater cultured P. vannamei and has caused significant economic damage. Only scarce information is available on Vibrio cholerae as a possible causal agent for this disease. In this study, a virulent strain BB31 was isolated from diseased P. vannamei suffering from white feces syndrome, and identified as a V. cholerae isolate through phylogenetic analysis and phenotypic characteristics. A phylogenetic tree was constructed to examine the relationship of isolate BB31 to other V. cholerae isolates. Three genes encoding hemolysin, outer membrane protein, and cholera toxin transcriptional activator were present in the BB31 isolate confirming its potential pathogenicity. In addition, isolate BB31 is known to have developed resistance to penicillin, sulfonamides and cephalosporin antibiotics. This was demonstrated when screened against a range of common antibiotics for aquaculture and veterinary use. To the best of our knowledge, this is the first report of white feces syndrome caused by V. cholerae in freshwater farmed P. vannamei.

Bacteriosis has become a major global economic problem in freshwater farmed whiteleg shrimp Penaeus vannamei. However, limited information is available on the incidence of Aeromonas schubertii infections in freshwater cultured P. vannamei. Red body disease, an epidemic frequently associated with P. vannamei freshwater farming, occurred in a P. vannamei farm in Shanghai China in June 2013. A pathogenic strain of A. schubertii (isolate HS1) was isolated from diseased freshwater cultured P. vannamei suffering from red body disease, and identified through phylogenetic analysis and phenotypic characteristics. A phylogenetic tree was constructed to examine the relatedness of isolate HS1 with other A. schubertii isolates. In addition, isolate HS1 showed no signs developing antibiotic resistance when screened against a range of common antibiotics used in aquaculture. To the best of our knowledge, this is the first report of A. schubertii infection in freshwater farmed P. vannamei.

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.

In the present study, a pathogenic strain, JL, was isolated from Pengze crucian carp (Carassius auratus) eggs suffering from saprolegniosis. It was initially determined as Saprolegnia sp. strain JL. Saprolegnia species have been implicated for significant fungal contamination, involving both living and dead fish and their eggs. A phylogenetic tree was constructed using the maximum parsimony method. The tree shows that the JL strain was closely related to Saprolegnia parasitica isolate SAP171, isolated from Salmo trutta suffering from saprolegniasis in Laukaa, Finland. The minimum inhibitory concentration (MIC) of 20 Chinese herbs was screened. Pseudolarix kaempferi Gord. (Pinaceae) was the most effective in inhibiting growth of the bacteria and was chosen for further trial. Significant protective efficacy of 52.63% and 73.68% was obtained against the JL strain in Pengze crucian carp eggs at P. kaempferi concentrations of 12.5 mg/ml and 25.0 mg/ml, respectively.