Grass Carp Ovary Research Articles

Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages

Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.

Vaccine Molecule Design Based on Phage Display and Computational Modeling against Rhabdovirus.

Rhabdoviruses with rich species lead a variety of high lethality and rapid transmission diseases to plants and animals around the globe. Vaccination is one of the most effective approaches to prevent and control virus disease. However, the key antigenic epitopes of glycoprotein being used for vaccine development are unclear. In this study, fish-derived Abs are employed for a Micropterus salmoides rhabdovirus (MSRV) vaccine design by phage display and bioinformatics analysis. We constructed an anti-MSRV phage Ab library to screen Abs for glycoprotein segment 2 (G2) (G129-266). Four M13-phage-displayed Abs (Ab-5, Ab-7, Ab-8 and Ab-30) exhibited strong specificity to target Ag, and Ab-7 had the highest affinity with MSRV. Ab-7 (300 μg/ml) significantly increased grass carp ovary cell viability to 83.40% and significantly decreased the titer of MSRV. Molecular docking results showed that the key region of Ag-Ab interaction was located in 10ESQEFTTLTSH20 of G2. G2Ser11 and G2Gln12 were replaced with alanine, respectively, and molecular docking results showed that the Ag-Ab was nonbinding (ΔG > 0). Then, the peptide vaccine KLH-G210-20 was immunized to M. salmoides via i.p. injection. ELISA result showed that the serum Ab potency level increased significantly (p < 0.01). More importantly, the challenge test demonstrated that the peptide vaccine elicited robust protection against MSRV invasion, and the relative percentage survival reached 62.07%. Overall, this study proposed an approach for screening key epitope by combining phage display technology and bioinformatics tools to provide a reliable theoretical reference for the prevention and control of viral diseases.

Identification and molecular characterization of Rab7 form Yellow River Carp Cyprinus carpio

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. In this study, full-length cDNA sequences of Rab7 were cloned from Yellow River Carp Cyprinus carpio, which were designated as CcRab7, and its function during pathogen infection was also investigated. The results showed that the full-length cDNA of CcRab7 was composed of 1511 bp, and open reading frame is 618 bp encoding 205 amino acids with 23.10 kDa. The deduced amino acid sequence of CcRab7 shared high similarity to that of Pimephales promelas (99.02%). In this paper, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression levels in 11 tissues and organs of healthy Yellow River Carp, and the tissue distribution of CcRab7 as well as its defense mechanism against Spring viremia of carp virus (SVCV) and Aeromonas hydrophila in Yellow River Carp C. carpio was investigated. The results revealed that the mRNA transcripts of CcRab7 were widely detected in various tissues of C. carpio, with the highest expression levels in head kidney and blood, indicating that CcRab7 was involved in the innate immune process. Yellow River Carp was immunized with A. hydrophila and SVCV respectively, and the expression changes of CcRab7 were detected in gill, spleen, liver and skin of Yellow River Carp. The mRNA transcripts of CcRab7 in the main organs (gill, and liver, skin and spleen) were significantly affected by SVCV and A. hydrophila infection. The results showed that the CcRab7 expression levels was obviously up-regulated at different time points. The eukaryotic recombinant plasmids of CcRab7 and pEGFP-N3 transfected into Grass carp ovary cell lines (GCO) cells for subcellular localization. The results provide a basis for further investigation into the immunological role of CcRab7 in Yellow River Carp.

Microcystin-LR-induced autophagy regulates oxidative stress, inflammation, and apoptosis in grass carp ovary cells in vitro

MC-LR is one of the cyanotoxins produced by fresh water cyanobacteria. Previous studies showed that autophagy played an important role in MC-LR-induced reproduction toxicity. However, information on the toxicological mechanism is limited. In this study, MC-LR could induce autophagy and apoptosis in GCO cells in vitro. In GCO cells that had been exposed to MC-LR, the inhibitor of 3-MA effectively decreased cell viability and damaged cell ultrastructure. Oxidative stress was significantly increased in the 3-MA + MC-LR group, accompanied by significantly increased MDA content and decreased CAT activity and GST, SOD1, GPx, and GR expression levels (P < 0.05). Inflammation was more serious in the 3-MA + MC-LR group than that of MC-LR group, which was evidenced by increasing expression levels of TNFα, IL11, MyD88, TNFR1, TRAF2, JNK, CCL4, and CCL20 (P < 0.05). Interestingly, the significant decrease of Caspase-9, Caspase-7, and Bax expression and significant increase of Bcl-2 and Bcl-2/Bax ratio in 3-MA + MC-LR group compared to MC-LR group, suggesting that extent of apoptosis were reduced. Taken together, these results indicated that MC-LR induced autophagy and apoptosis in GCO cells, however, the inhibition of autophagy decreased the extent of apoptosis, induced more serious oxidative stress and inflammation, which eventually induced cell death. Our findings provided some information for exploring the toxicity of MC-LR, however, the role of autophagy require further study in vivo.

A therapeutic agent of ursolic acid demonstrates potential application in aquaculture

Micropterus salmoides rhabdovirus (MSRV) has a high mortality rate and causes huge economic losses to the aquaculture industry. In this study, we identified that ursolic acid (UA) had antiviral efficacy against MSRV in vitro and in vivo. The results showed that UA inhibited MSRV replication in grass carp ovary (GCO) cells with a half-maximal inhibitory concentration (IC50) of 5.55 μM, reduced viral titers and decreased cytopathic effects (CPE). Mechanistically, UA does not directly damage viral particles. On the other hand, UA inhibits MSRV replication by altering viral binding and release. Furthermore, pre- and post-treatment assays revealed that UA had preventive and therapeutic effects. For in vivo studies, UA could enhance the survival rate of MSRV-infected largemouth bass. Similarly, UA reduced the viral load of MSRV in the heart, spleen and brain at 3, 5 and 7 d post-infection. In conclusion, UA is an effective inhibitor of rhabdovirus in aquaculture.

Hsp90 Regulates GCRV-II Proliferation by Interacting with VP35 as Its Receptor and Chaperone.

The frequent outbreak of grass carp hemorrhagic disease caused by grass carp reovirus (GCRV), especially the mainly prevalent type II GCRV (GCRV-II), has seriously affected the grass carp culture in China. However, its pathogenic mechanism is still far from clear. In this study, the GCRV-II outer capsid protein VP35 was used as bait to capture interacting partners from Ctenopharyngon idellus kidney (CIK) cells, and heat shock protein 90 (Hsp90) was selected and confirmed interacting with VP35 through the C-terminal domain of Hsp90. Knockdown of Hsp90 or inhibition of Hsp90 activity suppressed GCRV-II proliferation, demonstrating that Hsp90 is an essential factor for GCRV-II proliferation. The confocal microscopy and flow cytometry showed that Hsp90 localized at both membrane and cytoplasm of CIK cells. The entry of GCRV-II into CIK cells was efficiently blocked by incubating the cells with Hsp90 antibody or by pretreating the virus with recombinant Hsp90 protein. Whereas overexpression of Hsp90 in CIK cells, grass carp ovary (GCO) cells, or 293T cells promoted GCRV-II entry, indicating that the membrane Hsp90 functions as a receptor of GCRV-II. Furthermore, Hsp90 interacted with clathrin and mediated GCRV-II entry into CIK cells through clathrin endocytosis pathway. In addition, we found that the cytoplasmic Hsp90 acted as a chaperone of VP35 because inhibition of Hsp90 activity enhanced VP35 polyubiquitination and degraded VP35 through the proteasome pathway. Collectively, our data suggest that Hsp90 functions both as a receptor for GCRV-II entry and a chaperone for the maturation of GCRV-II VP35, thus ensuring efficient proliferation of GCRV-II. IMPORTANCE Identification of viral receptors has always been the research hot spot in virus research field as receptor functions at the first stage of viral infection, which can be designed as efficient antiviral drug targets. GCRV-II, the causative agent of the grass carp epidemic hemorrhagic disease, has caused tremendous losses in grass carp culture in China. To date, the receptor of GCRV-II remains unknown. This study focused on identifying cellular receptor interacting with the GCRV-II outer capsid protein VP35, studying the effects of their interaction on GCRV-II proliferation, and revealing the underlying mechanisms. We demonstrated that Hsp90 acts both as a receptor of GCRV-II by interacting with VP35 and as a chaperone for the maturation of VP35, thus ensuring efficient proliferation of GCRV-II. Our data provide important insights into the role of Hsp90 in GCRV-II life cycle, which will help understand the mechanism of reovirus infection.

Functional and expression analysis reveals the involvement of integrin αvβ3 in antiviral immunity of grass carp (Ctenopharyngodon idella)

Integrins are α-β heterodimeric cell receptors that can bind the protein components of pathogens, and play crucial roles in mammalian immune responses, but the immune functions mediated by integrins remains largely unknown in teleost fish. In this study, an integrin αvβ3 (GCαvβ3) originally assembled by αv (GCαv) and β3 (GCβ3) subunits, was identified from a teleost fish grass carp Ctenopharyngodon idella. The pairwise alignment analyses showed that the amino acid sequences of GCαv and GCβ3 shared high similarity (75.2–95.1%) and identity (58.6–90.7%) with their homologs from other vertebrates. Both GCαv and GCβ3 harbored the conserved protein domains and motifs, and were clustered in fish branch of the phylogenetic tree containing the counterparts from various vertebrates. Co-immunoprecipitation displayed that GCβ3 could interact with the grass carp reovirus (GCRV) outer capsid protein VP5. Two incubation experiments revealed that the interaction of GCRV or VP5 proteins with GCβ3 could induce the expressions of type I interferons (IFNs) including IFN2 and IFN3 in grass carp ovary cell line. The functional analysis demonstrated that GCαvβ3 served as a receptor of viral protein components to be involved in antiviral immunity as human integrin αvβ3 did. In addition, both GCαv and GCβ3 were significantly upregulated in various tissues of grass carp after GCRV infection. This study might provide fundamental basis for understanding the molecular characteristics and immune functions of GCαvβ3, and offer a new insight into the antiviral immune mechanism specific to the integrins in grass carp.

Synthesized Magnolol Derivatives Improve Anti-Micropterus salmoides Rhabdovirus (MSRV) Activity In Vivo.

Micropterus salmoides rhabdovirus (MSRV) is a primary viral pathogen in largemouth bass aquaculture, which leads to tremendous economic losses yearly. Currently, there are no approved drugs for the treatment and control of this virus. Our previous studies screened the herb Magnolia officinalis from many traditional Chinese medicines, and we isolated and identified magnolol as its main active compound against multiple rhabdoviruses, including MSRV. On the basis of the structure–activity relationship and pharmacophore model of magnolol, two new magnolol derivatives, namely, hydrogenated magnolol and 2,2′-dimethoxy-magnolol, were designed and synthesized. Their anti-MSRV activities were systematically investigated both in vitro and in vivo. By comparing the half-maximal inhibitory concentration (IC50), it was found that hydrogenated magnolol possessed a higher anti-MSRV activity than magnolol and 2,2′-dimethoxy-magnolol, with an IC50 of 13.37 μM. Furthermore, hydrogenated magnolol exhibited a protective effect on the grass carp ovary (GCO) cell line by reducing the cytopathic effect induced by MSRV. Further studies revealed that hydrogenated magnolol did not directly impact virions or interfere with MSRV adsorption. It worked within the 6–8 h of the phase of virus replication. In vivo treatment of MSRV infection with magnolol and hydrogenated magnolol showed that they significantly improved the survival rate by 44.6% and 62.7%, respectively, compared to MSRV-infected groups. The viral load measured by the expression of viral glycoprotein in the organs including the liver, spleen, and kidney also significantly decreased when fish were intraperitoneally injected at a dose of 20 mg/kg. Altogether, the structural optimization of magnolol via hydrogenation of the propylene groups increased its anti-MSRV activity both in vitro and in vivo. These results may provide a valuable reference for anti-MSRV drug discovery and development in aquaculture.

Non-pathogenic grass carp reovirus infection leads to both apoptosis and autophagy in a grass carp cell line

A novel GCRV strain isolated from healthy grass carp was named as grass carp reovirus - HH196 (GCRV-HH196), and its infection mechanism remains unclear. In this study, the grass carp ovary cell line (GCO cells) was used to investigate the cell death involved in GCRV-HH196 infection. The results showed that DNA damage, cells volume reduction and cytoplasm shrinkage happened during GCRV-HH196 infection. The mRNA expression levels of pro-apoptotic genes were up-regulated during infection. Two initiators of apoptosis, caspase 8 and caspase 9, and the executioner of apoptosis, caspase 3, were all significantly activated in GCRV-HH196-infected cells. Flow cytometry analysis showed that the number of apoptotic cells in infected cells was significantly higher than that in control cells as the infection progress. Meanwhile, autophagy was also involved in the regulation of GCRV - HH196 infection. We observed that LC3 puncta existed in cytoplasm in GCRV-HH196-infected cells. Furthermore, the protein level of LC3-Ⅱ and Beclin-1 increased, while that of p-Akt decreased in GCRV-HH196-infected cells. These results demonstrated that GCRV-HH196 may regulate apoptosis and autophagy for the virus proliferation and spread, which set a foundation for further research on the interaction between GCRV-HH196 and host.

Evaluation on the antiviral effect of natural product arctigenin against Micropterus salmoides rhabdovirus (MSRV) in vitro and in vivo

Micropterus salmoides rhabdovirus (MSRV) is a fish viral pathogen that causes severe disease and huge economic losses in the largemouth bass (M. salmoides) farming industry in aquaculture. Currently, there is no treatment strategy for MSRV. Thus, anti-MSRV drugs are urgently needed. In this study, it was found that the ethanol extract of Arctium lappa L. had good anti-MSRV activity, and the inhibition rate of G and N protein expression of MSRV was >95%. Further studies showed that arctigenin, a natural component of A. lappa L., could inhibit MSRV infection in a dose-dependent mode on grass carp ovary (GCO) cells. In addition, arctigenin could effectively inhibit the cytopathic changes caused by virus and significantly reduce the titer of virus. The pretreatment and post-treatment experiments showed that arctigenin had preventive and therapeutic effects on MSRV infection. TOA and TOR studies indicated that arctigenin may play an antiviral role in the early stage of virus replication. Interestingly, we found that arctigenin treatment significantly inhibited mitochondrial damage in GCO cells by preserving mitochondrial function and maintaining mitochondrial membrane potential levels (ΔΨm). More importantly, arctigenin was also effective against MSRV infection in largemouth bass. The survival rate of infected fish was increased by 16% and 20% respectively by adding 0.5% and 1% arctigenin in the diet. Additionally, the virus titer of the largemouth bass in the arctigenin treatment group was significantly decreased. In summary, arctigenin is confirmed to be a natural product with the excellent anti-MSRV activity, and its application in the treatment of MSRV is promising in aquaculture.

Quinoline, with the active site of 8-hydroxyl, efficiently inhibits Micropterus salmoides rhabdovirus (MSRV) infection in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is an significant pathogen that causes high mortality and related economic losses in bass aquaculture. There is no effective or approved therapy to date. In this study, we evaluated the anti-MSRV effects of 22 quinoline derivatives in grass carp ovary (GCO) cells. Among these compounds, 8-hydroxyquinoline exhibited valid inhibition in decreasing MSRV nucleoprotein gene expression levels of 99.3% with a half-maximal inhibitory concentrations (IC50 ) value of 4.66μM at 48h. Moreover, 8-hydroxyquinoline significantly enhanced a protective effect in GCO cells by reducing the cytopathic effect (CPE). By comparing the anti-MSRV activity of 22 quinoline derivatives, we found that 8-hydroxyquinoline possessed the efficient active site of 8-hydroxyl and inhibited MSRV infection in vitro. For in vivo studies, 8-hydroxyquinoline via intraperitoneal injection exhibited an antiviral effect in MSRV-infected largemouth bass by substantially enhancing the survival rate by 15.0%. Importantly, the viral loads in the infected largemouth bass notably reduced in the spleen on the third days post-infection. Overall, 8-hydroxyquinoline was considered to be an efficient agent against MSRV in aquaculture.

Discrepant dose responses of bisphenol A on oxidative stress and DNA methylation in grass carp ovary cells

Bisphenol A (BPA), is a common contaminant in diverse environmental compartments and its endocrine disruptive effect on living organisms has been widely reported. Further works are still required to facilitate the research on cytotoxicity and genotoxicity. In the present study, grass carp ovary (GCO) cells were used to investigate cellular oxidative stress and genomic DNA methylation under BPA exposure. Results showed that BPA exposure for 48 h arrested cell proliferation and viability. The oxidative stress was distinctly enhanced with increased reactive oxygen species (ROS), malondialdehyde level, and oxidation of reduced glutathione (GSH) in 30 μM BPA group. Furthermore, the global 5-methylcytosine (5 mC) level was elevated and showed inverted U-shaped responses to the BPA doses. Besides, one-carbon metabolism and de novo GSH synthesis were disrupted at 30 μM BPA. Current data suggested that low dose of BPA exposure could exhibit hormesis in recycling circular biosynthesis of GSH and scavenging ROS to create a relatively reductive intracellular environment, and up-regulate transcripts of methyltransferases that increased the 5 mC level in GCO cells. While high dose of BPA distinctly induced oxidative stress, elevated de novo GSH synthesis, and then attenuated transmethylation activity and decreased 5 mC level. Current study highlighted the discrepant dose responses of BPA in fish ovary cells that facilitated the understanding of pleiotropic consequences in organisms.

Grass carp (Ctenopharyngodon idella) NRF2 alleviates the oxidative stress and enhances cell viability through upregulating the expression of HO-1.

As a member of the Cap 'n' Collar (CNC) family, NRF2 contains a basic leucine zipper (bZip) and can regulate the downstream target gene heme oxygenase 1 (HO-1) in response to oxidative stress. In the present study, a grass carp (Ctenopharyngodon idella) NRF2 ORF was cloned and identified. The largest ORF (1782 bp) encodes a polypeptide of 593 amino acids. The deduced amino acid sequence of grass carp NRF2 (CiNRF2) contains a well-conserved DNA-binding domain (BRLZ domain). Phylogenetic tree analysis revealed that CiNRF2 has a closer evolutionary relationship with other fish counterparts. After CIK (C. idellus kidney) cells were persistently stimulated with tunicamycin (TM), CiNRF2 was significantly upregulated from 12 to 36 h. Then, the expression was dropped at 48 h post-infection. Additionally, when TM or TG (thapsigargin) stimulated CIK cells, overexpression of CiNRF2 in cells downregulated the expression of Bip mRNA, a marker protein of oxidative stress, suggesting that fish NRF2 can alleviate the oxidative stress level induced by TM or TG. To study the protective mechanism of fish NRF2, the DNA sequences of CiNRF2 and CiATF4 (grass carp ATF4) were separately sub-cloned into the expression vectors pEGFP and pCMV-Flag for co-immunoprecipitation and GST pull-down assays. These assays showed that CiNRF2 can combine with CiATF4 through its Neh1 domain. Meanwhile, we cloned grass carp HO-1 promoter sequence and constructed the recombinant plasmid of pGL3-HO-1. Soon afterwards, pGL3-HO-1 was co-transfected into grass carp ovary (CO) cells with pcDNA3.1-CiNRF2 or pcDNA3.1-CiATF4, respectively. The results showed that the luciferase activity of pGL3-HO-1 in the overexpressed CiNRF2 plus CiATF4 cells was significantly increased, along with the increase of cell viability (~ 133%). However, when HO-1 was knocked down in cells, CiNRF2 was unable to perform its function. These results demonstrated that CiNRF2 was effective in protecting grass carp against the oxidative stress induced by TM and increasing cell viability by upregulating HO-1 expression.

Sequence and expression analysis of the cytoplasmic pattern recognition receptor melanoma differentiation-associated gene 5 from the barbel chub Squaliobarbus curriculus

MDA5 is a cytoplasmic viral double-stranded RNA recognition receptor that plays a pivotal role in the aquatic animal innate immune system. To decipher the role of MDA5 of Squaliobarbus curriculus (ScMDA5) in the immune response, full-length cDNA of ScMDA5 was cloned using the RACE technology, mRNA and protein expression levels of ScMDA5 signalling pathway members in response to stimulation were detected and effects of overexpression of ScMDA5 on the immune response were investigated. ScMDA5 comprises 3597 bp and is composed of an open reading frame (2958 nucleotides long) that translates into a putative peptide of 985 amino acid residues. ScMDA5 possesses two N-terminal caspase-recruiting domains, DEAD-like helicases superfamily, helicase superfamily C-terminal and RIG-I_C-RD domains, and differences in these domains among species were mainly observed with respect to their length and location. ScMDA5 was closely clustered with those of Carassius auratus, Ctenopharyngodon idellus and Mylopharyngodon piceus. ScMDA5 transcripts were most abundant in the spleen and the lowest in the liver. Expression levels of ScMDA5 in healthy tissues were significantly correlated with those of ScIRF3, ScIRF7 and ScIFN. Besides, mRNA expression levels of ScIRF3 were significantly correlated with those of ScIRF7 (0.956, P < 0.01). Expression level changes, including downregulation, upregulation and initial upregulation followed by downregulation, were found in ScMDA5 signalling pathway molecules in tissues after grass carp reovirus infection. Protein levels of ScMDA5 were the highest in the liver and the lowest in the spleen in detected healthy tissues. Overexpression of ScMDA5 led to significantly enhanced CiIRF7 and CiMx transcription in grass carp ovary cells (P < 0.05). The results of this study helped to clarify the role of ScMDA5 in the immune reaction against grass carp reovirus and provided fundamental information for fish breeding to achieve strong resistance to infection.

Isolation and characterization of a novel strain (YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system.

As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.

Characterization of the interaction between outer-fiber protein VP55 of genotype III grass carp reovirus and Fibulin-4 of grass carp

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.

In vitro antiviral efficacy of moroxydine hydrochloride and ribavirin against grass carp reovirus and giant salamander iridovirus.

Moroxydine hydrochloride (Mor) and ribavirin (Rib) have been reported to exhibit multi-antiviral activities against DNA and RNA viruses, but their antiviral activities and pharmacologies have seldom been studied in aquaculture. This paper has selected 3 aquatic viruses including a double-stranded RNA virus (grass carp reovirus, GCRV), a single-stranded RNA virus (spring viraemia of carp virus, SVCV) and a DNA virus (giant salamander iridovirus, GSIV) for antiviral testing. The results showed that Mor and Rib can effectively control the infection of GCRV and GSIV in respective host cells. Further study was undertaken to explore the antivirus efficiencies and pharmacological mechanisms of Mor and Rib on GCRV and GSIV in vitro. Briefly, compounds showed over 50% protective effects at 15.9 µg ml-1 except for the group of GSIV-infected epithelioma papulosum cyprinid (EPC) cells treated with Mor. Moreover, Mor and Rib blocked the virus-induced cytopathic effects and apoptosis in host cells to keep the normal cellular structure. The expression of VP1 (GCRV) and major capsid protein (MCP; GSIV) gene was also significantly inhibited in the virus-infected cells when treated with Mor and Rib. Cytotoxicity assay verified the 2 compounds had no toxic effects on grass carp ovary (GCO) cells and EPC cells at ≤96 µg ml-1. In conclusion, these results indicated that exposing GCRV-infected GCO cells and GSIV-infected EPC cells to Mor and Rib could elicit significant antiviral responses, and the 2 compounds have been shown to be promising agents for viral control in the aquaculture industry.

Spring viraemia of carp virus enters grass carp ovary cells via clathrin-mediated endocytosis and macropinocytosis.

Spring viraemia of carp virus (SVCV) is the causative pathogen of the outbreaks of an acute haemorrhagic and contagious viraemia responsible for the significant mortality in several cyprinid species. However, the endocytic pathway(s) and their regulatory molecules have not been characterized for SVCV. Here, using a combination of specific pharmacological inhibitors, transmission electron microscopy, immunofluorescence microscopy and real-time quantitative PCR, we found that SVCV entered grass carp ovary cells via clathrin-mediated endocytosis and macropinocytosis in a low-pH-dependent manner. We also discovered that dynamin II, actin microfilaments and microtubules were essential for SVCV internalization. Moreover, we found that the P21-activated kinase 1 inhibitor IPA-3 and the protein kinase C inhibitor rottlerin could block SVCV cell entry and replication, while phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 could promote SVCV infection. Results presented in this study provide helpful insight into revealing the initial steps of SVCV infection, and they may facilitate the development of therapeutic interventions.

Effects of L-carnitine against H2O2-induced oxidative stress in grass carp ovary cells (Ctenopharyngodon idellus).

This study was designed in vitro to investigate the effects of L-carnitine against H2O2-induced oxidative stress in a grass carp (Ctenopharyngodon idellus) ovary cell line (GCO). GCO cells were pre-treated with different concentrations of L-carnitine, followed by incubation with 2.5mM H2O2 for 1h to induce oxidative damage. The results indicated that adding L-carnitine at concentrations of 0.01-1mM into the medium for 12h significantly increased cell viability. Pre-treatment with L-carnitine at concentrations of 0.1-5mM for 12h significantly inhibited 2.5mM H2O2-induced cell viability loss. The significant decreases in the level of reactive oxygen species and cell apoptosis were observed in 0.5mM L-carnitine group compared to the H2O2 group. Malondialdehyde values of all of the L-carnitine groups were significantly lower than those of the H2O2 group, while total glutathione levels of all of the L-carnitine groups were significantly higher than of the H2O2 group. The activity of antioxidant enzymes, such as total superoxide dismutase (0.1 and 0.5mM L-carnitine), catalase (0.5mM L-carnitine) and γ-glutamyl cysteine synthetase (0.5 and 1mM L-carnitine), was significantly increased. In addition, pre-treatment of L-carnitine in GCO cells exposed to 2.5mM H2O2 significantly increased the mRNA expression of copper, zinc superoxide dismutase, catalase (0.5mM L-carnitine), glutamate cysteine ligase catalytic subunit (0.1-1mM) and glutathione peroxidase (0.1mM L-carnitine). In conclusion, L-carnitine promotes GCO cell growth and improves antioxidant function, it plays a protective role against oxidative stress induced by H2O2 in GCO cells, and the appropriate supplemental amount of L-carnitine is 0.1-1mM.

Characterization and virus susceptibility of a skin cell line from red-spotted grouper (Epinephelus akaara)

A red-spotted grouper Epinephelus akaara skin (RGS) cell line was established and characterized. RGS cells had a normal diploid chromosome number of 2n = 48, the morphology of which was fibroblastic-like in 3 days and epithelial-like over 5 after 16 passages. The cells multiplied well in Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum at 25°C. Susceptibilities of RGS and grass carp ovary (GCO) cells to two viruses were tested, and the results showed that the titer of an iridovirus Rana grylio virus (RGV) in RGS cells was 10³·⁵ TCID₅₀ ml⁻¹, which was much higher than a rhabdovirus spring viremia of carp virus (SVCV) in the cells (10⁰·⁵ TCID₅₀ ml⁻¹). The titers of RGV and SVCV in GCO were 10⁶·⁰ TCID₅₀ ml⁻¹ and 10⁸·⁰ TCID₅₀ ml⁻¹, respectively, which were higher than those in RGS cells. The data may imply that RGS cells could be selectively resistible to some viruses during infection. RT-PCR analysis of RGV-infected RGS cells showed that RGV could replicate in RGS cells. Further study of virus replications in RGS cells was conducted by electron microscopy and immunofluorescence microscopy has shown that virus particles scattered in the cytoplasm and virus protein appeared in both the cytoplasm and nucleus. The results suggested that RGS cells could be used as a potential in vitro model to study the cutaneous barrier function against virus infection.