Unveiling the emerging role of curcumin to alleviate ochratoxin A-induced muscle toxicity in grass carp(Ctenopharyngodon idella):in vitro and in vivo studies

Background Ochratoxin A(OTA),a globally abundant and extremely hazardous pollutant,is a significant source of contamination in aquafeeds and is responsible for severe food pollution.The developmental toxicity of OTA and the potential relieving strategy of natural products remain unclear.This study screened the substance curcumin(Cur),which had the best effect in alleviating OTA inhibition of myoblast proliferation,from 96 natural products and investigated its effect and mechanism in reducing OTA myotoxicity in vivo and in vitro.Methods A total of 720 healthy juvenile grass carp,with an initial average body weight of 11.06±0.05 g,were randomly assigned into 4 groups:the control group(without OTA and Cur),1.2 mg/kg OTA group,400 mg/kg Cur group,and 1.2 mg/kg OTA+400 mg/kg Cur group.Each treatment consisted of 3 replicates(180 fish)for 60 d.Results Firstly,we cultured,purified,and identified myoblasts using the tissue block culture method.Through preliminary screening and re-screening of 96 substances,we examined cell proliferation-related indicators such as cell viability and ultimately found that Cur had the best effect.Secondly,Cur could alleviate OTA-inhibited myoblast differentiation and myofibrillar development-related proteins(Myo G and MYHC)in vivo and in vitro and improve the growth performance of grass carp.Then,Cur could also promote the expression of OTA-inhibited protein synthesis-related proteins(S6K1 and TOR),which was related to the activation of the AKT/TOR signaling pathway.Finally,Cur could downregulate the expression of OTA-enhanced protein degradation-related genes(murf1,foxo3a,and ub),which was related to the inhibition of the Fox O3a signaling pathway.Conclusions In summary,our data demonstrated the effectiveness of Cur in alleviating OTA myotoxicity in vivo and in vitro.This study confirms the rapidity,feasibility,and effectiveness of establishing a natural product screening method targeting myoblasts to alleviate fungal toxin toxicity.

Mannan oligosaccharides alleviate oxidative injury in the head kidney and spleen in grass carp(Ctenopharyngodon idella)via the Nrf2 signaling pathway after Aeromonas hydrophila infection

Background Mannan oligosaccharides(MOS)are recommended as aquaculture additives owing to their excellent antioxidant properties.In the present study,we examined the effects of dietary MOS on the head kidney and spleen of grass carp(Ctenopharyngodon idella)with Aeromonas hydrophila infection.Methods A total of 540 grass carp were used for the study.They were administered six gradient dosages of the MOS diet(0,200,400,600,800,and 1,000 mg/kg)for 60 d.Subsequently,we performed a 14-day Aeromonas hydrophila challenge experiment.The antioxidant capacity of the head kidney and spleen were examined using spectrophotometry,DNA fragmentation,qRT-PCR,and Western blotting.Results After infection with Aeromonas hydrophila,400-600 mg/kg MOS supplementation decreased the levels of reactive oxygen species,protein carbonyl,and malonaldehyde and increased the levels of anti-superoxide anion,antihydroxyl radical,and glutathione in the head kidney and spleen of grass carp.The activities of copper-zinc superoxide dismutase,manganese superoxide dismutase,catalase,glutathione S-transferase,glutathione reductase,and glutathione peroxidase were also enhanced by supplementation with 400-600 mg/kg MOS.Furthermore,the expression of most antioxidant enzymes and their corresponding genes increased significantly with supplementation of 200-800 mg/kg MOS.mRNA and protein levels of nuclear factor erythroid 2-related factor 2 also increased following supplementation with 400-600 mg/kg MOS.In addition,supplementation with 400-600 mg/kg MOS reduced excessive apoptosis by inhibiting the death receptor pathway and mitochondrial pathway processes.Conclusions Based on the quadratic regression analysis of the above biomarkers(reactive oxygen species,malondialdehyde,and protein carbonyl)of oxidative damage in the head kidney and spleen of on-growing grass carp,the recommended MOS supplementation is 575.21,557.58,531.86,597.35,570.16,and 553.80 mg/kg,respectively.Collectively,MOS supplementation could alleviate oxidative injury in the head kidney and spleen of grass carp infected with Aeromonas hydrophila.

Dietary protein levels changed the hardness of muscle by acting on muscle fiber growth and the metabolism of collagen in sub-adult grass carp(Ctenopharyngodon idella)

Background:Nutrient regulation has been proven to be an effective way to improve the flesh quality in fish.As a necessary nutrient for fish growth,protein accounts for the highest proportion in the fish diet and is expensive.Although our team found that the effect of protein on the muscle hardness of grass carp was probably related to an increased collagen content,the mechanism for this effect has not been deeply explored.Moreover,few studies have explored the protein requirements of sub-adult grass crap(Ctenopharyngodon idella).Therefore,the effects of different dietary protein levels on the growth performance,nutritional value,muscle hardness,muscle fiber growth,collagen metabolism and related molecule expression in grass carp were investigated.Methods:A total of 450 healthy grass carp(721.16±1.98 g)were selected and assigned randomly to six experimen-tal groups with three replicates each(n=25/replicate),and were fed six diets with 15.91%,19.39%,22.10%,25.59%,28.53%and 31.42%protein for 60 d.Results:Appropriate levels of dietary protein increased the feed intake,percentage weight gain,specific growth rate,body composition,unsaturated fatty acid content in muscle,partial free amino acid content in muscle,and muscle hardness of grass carp.These protein levels also increased the muscle fiber density,the frequency of new muscle fibers,the contents of collagen and IGF-1,and the enzyme activities of prolyl 4-hydroxylases and lysyloxidase,and decreased the activity of matrix metalloproteinase-2.At the molecular level,the optimal dietary protein increased col-lagen type Iα1(Colα1),Colα2,PI3K,Akt,S6K1,La ribonucleoprotein domain family member 6a(LARP6a),TGF-β1,Smad2,Smad4,Smad3,tissue inhibitor of metalloproteinase-2,MyoD,Myf5,MyoG and MyHC relative mRNA levels.The levels of the myostatin-1 and myostatin-2 genes were downregulated,and the protein expression levels of p-Smad2,Smad2,Smad4,p-Akt,Akt,LARP6 and Smad3 were increased.Conclusions:The appropriate levels of dietary protein promoted the growth of sub-adult grass carp and improved muscle hardness by promoting the growth of muscle fibers,improving collagen synthesis and depressing collagen degradation.In addition,the dietary protein requirements of sub-adult grass carp were 26.21%and 24.85%according to the quadratic regression analysis of growth performance(SGR)and the muscle hardness(collagen content),respectively.

Implications of vitamin D for flesh quality of grass carp(Ctenopharyngodon idella):antioxidant ability,nutritional value,sensory quality,and myofiber characteristics

Background Muscle represents a unique and complex system with many components and comprises the major edible part of animals.Vitamin D is a critical nutrient for animals and is known to enhance calcium absorption and immune response.In recent years,dietary vitamin D supplementation in livestock has received increased attention due to biological responses including improving shear force in mammalian meat.However,the vitamin D acquisition and myofiber development processes in fish differ from those in mammals,and the effect of vitamin D on fish flesh quality is poorly understood.Here,the influence of dietary vitamin D on fillet quality,antioxidant ability,and myofiber development was examined in grass carp(Ctenopharyngodon idella).Methods A total of 540 healthy grass carp,with an initial average body weight of 257.24±0.63 g,were allotted in 6 experimental groups with 3 replicates each,and respectively fed corresponding diets with 15.2,364.3,782.5,1,167.9,1,573.8,and 1,980.1 IU/kg vitamin D for 70 d.Results Supplementation with 1,167.9 IU/kg vitamin D significantly improved nutritional value and sensory quality of fillets,enhancing crude protein,free amino acid,lipid,and collagen contents;maintaining an ideal pH;and reduc-ing lactate content,shear force,and cooking loss relative to respective values in the control(15.2 IU/kg)group.Average myofiber diameter and the frequency of myofibers>50μm in diameter increased under supplementation with 782.5–1,167.9 IU/kg vitamin D.Levels of oxidative damage biomarkers decreased,and the expression of antioxi-dant enzymes and nuclear factor erythroid 2-related factor 2 signaling molecules was upregulated in the 1,167.9 IU/kg vitamin D treatment compared to respective values in the control group.Furthermore,vitamin D supplementation activated cell differentiation by enhancing the expression of myogenic regulatory factors and myocyte enhancer factors compared to that in the control group.In addition,supplementation with 1,167.9 IU/kg vitamin D improved protein deposition associated with protein synthesis molecule(target of rapamycin)signaling and vitamin D receptor paralogs,along with inhibition of protein degradation(forkhead box protein 1)signaling.Conclusions Overall,the results demonstrated that vitamin D strengthened antioxidant ability and myofiber devel-opment,thereby enhancing nutritional value and sensory quality of fish flesh.These findings suggest that dietary vitamin D supplementation is conducive to the production of nutrient-rich,high quality aquaculture products.

Comparative Microsatellite Analysis of Grass Carp Genomes of Two Gynogenetic Groups and the Xiangjiang River Group

The genomes of three groups of grass carp, namely the Xiangjiang River grass carp group （Xiangjiang group）, a one-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-1 group）, and a two-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-2 group）, were comparatively analyzed with microsatellite markers. Genetic polymorphism had been observed in the Xiangjiang group and most of the examined loci had more than two alleles. But the degree of genetic diversity was not very high. Although all the examined genetic loci in the analyzed individuals were in homozygous state, the genotypes of different individuals of the group were not identical in the meio-gynogenetic-1 group. In the meio-gynogenetic-2 group, not only the examined genetic loci of each individual were homozygous but also the genotypes of all the analyzed individuals of the group were the same. These results suggested that the examined meio-gynogenetic-2 group is a homozygous group and homozygous clone could be produced by continuous artificial induction of gynogenesis for two generations. It was found that the polymorphism existed not only at the allele level but also at the locus level; many alleles of the microsatellite loci and some of the microsatellite loci had been lost during the process of artificial gynogenesis. Therefore, both protection of the diversity of natural grass carp resource and selection of homozygous traits with desired economic genotypes are very important aspects for grass carp breeding.

Comparative Analysis on Growth and Morphology in Yangtze River ♀,Zhujiang River ♂ and Their F_1 Hybrids of Juvenile Grass Carp

[ Objective] The aim of this study was to investigate the growth and morphological characteristics of juvenile hybrids of grass carp. [Method] Three inbred generations of YR （Yangtze River stock ♀×Yangtze River stock ♂ ）, ZR （Zhujiang River stock ♀ ×Zhujiang River stock♂ ） and hybrids F1 （ Yangtze River stock ♀ × Zhujiang River stock ♂ ） were established, and their growth or morphological data were also measured. [ Result] The orders of body weight, standard length and absolute growth rate were F1 〉 ZR 〉 YR. No significant difference was found in body weight or standard length of 50 day-old generations （ P〉0. 05）, while there was an extremely significantly difference in those of 170 day-old generations （ P 〈0.01 ）. However, the absolute growth rate of F1 was 20.00% and 50.00% higher than that of ZR and YR respectively, and no significant difference was found between F1 and ZR （ P 〉 0.05）, but significant difference between F1 and YR （ P 〈 0.05）. F1 showed a significant hybrid vigor with rate of 20.09%. There was no significant difference among three inbred generations in standard length/total length （ P 〉 0.05）, while significant difference in head length/total length, body height/standard length and body width/standard length （ P 〈 0.05）, which indicated that F1 had the characteristics of shorter head, higher and wider body. [ Conclusion]F1 has advantages in growth performance and morphological characteristics.

Study on the Toxicity Effect of Abamectin and Chlorpyrifos on Grass Carp(Ctenopharyngodon idllus)

[ Objective] The study aimed to discuss the toxicity effect of abamectin and ehlorpyrifos on grass carp （ Ctenopharyngdon idllus ） and their interrela- tions. [ Method] Taking healthy and active grass carp as the object, the acute toxicity test of single pesticide and the combined toxicity test of two pesticides on grass carp were carried out. [ Result] The LC50 of abamectin and chlorpyrifes and the mixture of two pesticides against grass carp at 24, 48, 72 and 96 h were as follows： abamectin： 0.54, 0.49, 0. 17 and 0.10 rag/L; chlorpyrifes ： 0.29, 0.21, 0.12 and 0.05 rag/L; the mixture of two pesticides ： 1.22, 1.08, 0.99 and 0. 86 mg/L. The safe concentration （SC） of ahamectin, chlorpyrifos and the mixture of two pesticides were 0.010, 0.005 and 0.086 rag/L, respectively. The tox- icity of the pesticides in sequence was ablorpyrifos 〉 ahamectin 〉 the mixture of two pesticides. [ Conclusion ] Different concentrations of abamectin, chlorpyrifos and the mixture of two pesticides had remarkable effect on the growth of grass carp, the higher the concentration was, the greater the toxicity effect was. Ahamectin at low toxicity intensity had certain relief function on the toxicity of ehlorpyrifos.

The Therapeutic Effect of Onion on the Pathogenic Bacteria of Gill Rot Disease in Grass Carp

[Objective] The curative effect of onion on bacterial rotted gill disease in grass carp was researched [Method] The combination method of taking medicine through oral and spraying was used to cure sick grass carp for 1 period of treatment in room under artificial conditions. [Result] Different concentrations of onion generated different cure rates. When the combination was adding 1.0% - 2.0% medicine into feed and spraying 2.0 g,/m3 - 5.0 g,/m3, the curative result was the best with cure rate was 70% -90% [ Conclusion] The onion was effective on curing bacterial rotted gill disease in grass carp and could be taken as curative medicine.

Optimization of Antibacterial Conditions in Freshcut Grass Carp Belly and Comparison of Shelf Life

With fresh-cut grass carp belly as the test material, the antibacterial con- ditions of a disinfection agent sodium hypochlorite were optimized. In addition, the shelf lives of various grass carp products were compared after disinfection, so as to provide certain basis for the processing of fresh-cut grass carp products. The re- sults showed that the optimal disinfection conditions of sodium hypochlorite were as follows： concentration 300 mg/L, soak time 5 min and solid-liquid ratio 1 g ： 5 ml. Under the optimal disinfection conditions, the inhibition rates of total bacteria and Pseudomonas reached 83% and 81%, respectively. The shelf life of refrigerated fresh-cut grass carp belly disinfected by sodium hypochlorite and packed in bag filled with gases could be extended as long as 11 d, which was increased by 5 d （83%） compared with that in the control group. The disinfection by sodium hypochlorite could significantly reduce the initial bacterial colony number and improve the sensory quality of fresh-cut grass carp belly, as well as extend the shelf life of refrigerated and modified gas-packed grass carp belly.

Growth Performance of Hybrid Progenies of Grass Carp (Ctenopharyngodon idella) Populations from Different Geographical Origins

[Objective] This study aimed to evaluate growth performance of hybrid progenies of 10 grass carp （Ctenopharyngodon idella） populations from different ge- ographical origins （Foshan population, Zhaoqing population, Jingzhou population 1, Jingzhou population 2, Jingzhou population 3, Ezhou population, Yiyang population, Changsha population 1, Changsha population 2 and Jiangsu population）. [Method] Fifteen hybrid combinations were established with these ten grass carp populations and cultured in the same pond. The body mass of hybrid progenies was measured at the age of 4, 7, 8, 10, 12, 15 and 18 months, respectively. Using body mass at the age of 4 months as a concomitant variable, the growth performance of proge- nies from different hybrid combinations was analyzed by covariance analysis. [Re- suit] The average body mass of hybrid progenies of Jingzhou population 2 c2 x Foshan population $ reached the maximum （1 892.90 g） at the age of 18 months, which was 3.51%-32.36% higher than that of other hybrid progenies. Multiple com- parison analysis showed that average body mass of hybrid progenies of Jingzhou population 2 2 x Foshan population ♂ and Jingzhou population 1 ♀× Foshan population ♂ was significantly higher than that of other hybrid progenies （P〈0.05）. [Conclusion] In the production process, Jingzhou population 2 ♀ × Foshan popula- tion 5 and Jingzhou population 1 ♀×Foshan population ♂ can be used to pro- duce high-quality fries, thereby greatly improving economic benefits. This study laid the foundation for establishment of rapid-growing grass carp core populations.

Comparison of intestinal bacterial communities in grass carp,Ctenopharyngodon idellus,from two different habitats

The intestinal bacteria of vertebrates form a close relationship with their host.External and internal conditions of the host,including its habitat,affect the intestinal bacterial community.Similarly,the intestinal bacterial community can,in turn,influence the host,particularly with respect to disease resistance.We compared the intestinal bacterial communities of grass carp that were collected from farm-ponds or a lake.We conducted denaturing gradient gel electrophoresis of amplified 16S rRNA genes,from which 66 different operational taxonomic units were identified.Using both the unweighted pair-group method with arithmetic means clustering and principal component analysis ordination,we found that the intestinal bacterial communities from the two groups of pond fish were clustered together and inset into the clusters of wild fish,except for DF-7,and there was no significant correlation between genetic diversity of grass carp and their intestinal bacterial communities(Mantel one-tailed test,R=0.157,P=0.175).Cetobacterium appeared more frequently in the intestine of grass carp collected from pond.A more thorough understanding of the role played by intestinal microbiota on fish health would be of considerable benefit to the aquaculture industry.

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

Growth,digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp Ctenopharyngodonidella fed graded levels of dietary threonine

Background： This study was carried out to investigate effects of threonine levels on growth, digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp （ Ctenopharyngodonidella）. Results： Weight gain, specific growth rate, feed intake and feed efficiency were significantly improved by dietary threonine （P 〈 0.05）. Intestinal activities of trypsin, chymotrypsin, alpha-amylase, lipase, alkaline phosphatase, y-glutamyl transpeptidase and creatine kinase took the similar trends. Contents of malondialdehyde and protein carbonyl in intestine and hepatopancreas were significantly decreased by dietary optimal threonine supplementation （P 〈 0.05）. Anti-superoxide anion capacity, anti-hydroxyl radical capacity, glutathione content and activities of superoxide dismutase, catalase and glutathione-S-transferase in intestine and hepatopancreas were enhanced by dietary threonine （P 〈 0.05）. Conclusions： Dietary threonine could improve growth, enhance digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp. The dietary threonine requirement of sub-adult grass carp （441.9-1,013.4 g） based on weight gain was 11.6 g/kg diet or 41.5 g/kg of dietary protein by quadratic regression analysis.

Molecular cloning and mRNA expression analysis of myosin heavy chain(MyHC)from fast skeletal muscle of grass carp,Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

Detection of Grass Carp Hemorrhage Virus (GCHV) from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as "Red Spot Disease", is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and comparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplification. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain induces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the 6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines, including CAB, FHM, CIK, EPC, CCO and GCO, infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHV strains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and the two strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-V viruses have been replicated and amplified despite there being no cytopathogenic effects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published recently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp fragment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is identical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously, the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product, and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.

Molecular Characterization of Nonstructural Protein NS38 of Grass Carp Reovirus

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.

The VP2 protein of grass carp reovirus(GCRV) expressed in a baculovirus exhibits RNA polymerase activity

The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA-dependent RNA polymerase （RdRp）.In previous work,we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity （denoted as rVP2390-900） in E.coil and have prepared a polyclonal antibody against VP2.To characterize the GCRV RNA polymerase,a recombinant full-length VP2 （rVP2） was first constructed and expressed in a baculovirus system,as a fusion protein with an attached His-tag.Immunofluorescence （IF） assays,together with immunoblot （IB） analyses from both expressed cell extracts and purified Histagged rVP2,showed that rVP2 was successfully expressed in Sf9 cells.Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly（C）-dependent poly（G） polymerase activity.The RNA enzymatic activity required the divalent cation Mg2＋,and was optimal at 28 ℃.The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.