ORIGINAL ARTICLE The Exposure Immunogenic Protein of ... - aensi

388 Advances in Environmental Biology, 6(1): 388-396, 2012 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLE

The Exposure Immunogenic Protein of Viral Nervous Necrotic on Humpback Grouper That Influences to Proliferation and Expression of Immune Cells (Interferon γ and NFKb Cell) 1

Uun Yanuhar, 2Ery Gusman, 1Diana Arfiati

1 2

Faculty of Fisheries and Marine Science, Brawijaya University, Malang, Indonesia. Faculty of Fisheries, Borneo University, East Kalimantan, Indonesia. Uun Yanuhar, Ery Gusman, Diana Arfiati; The Exposure Immunogenic Protein of Viral Nervous Necrotic on Humpback Grouper That Influences to Proliferation and Expression of Immune Cells (Interferon γ and NF-Kb Cell)

ABSTRACT The main topic of this study are to find out of the proliferation and expression of IFN- γ and NF-kB cell to Humpback grouper that exposured by immunogenic protein of Viral Nervous Necrosis (VNN). The methods used on this study were exploration by doing isolation to Humpback grouper organs both normal and infected by immunogenic protein of VNN, coat protein isolation, protein fractionation by SDS-PAGE, separation and cutting of protein bands from SDS-PAGE result, coat protein measured by Nano drop spectrophotometer, Hemagglutination (HA) Test of VNN immunogenic protein, and IFN- γ and NF-kB cell proliferation and expression qualitative detection by Immunohistochemistry (IHC).The results of this research confirm that HA test was positive. It showed that 45,9 kDa VNN immunogenic protein had been reacted by agglutination reaction between 45.9 kDa VNN immunogenic protein and erythrocytes of humpback grouper with sensitivity of 0,015625. Positive concentration result was up to 1/64 or its sensitivity of 0.015625. It means antigen determinant (epitope) from immunogenic protein have highly sensitivity from range 0.006 to 0.06. IHC test result show that 45,9 kDa VNN protein immunogenic epitope have ability to induce humpback grouper tissue receptors by creating immune cell qualitatively, there are IFN γ and NF-kB cell immune to humpback grouper specific tissue organ, eye, brain and kidney after labeled by secondary antibody anti IFN γ and NF-kB cell anti mouse . ELISA test using the secondary antibody that same with examination test of IHC were showed that proliferation and expression of IFN γ and NF-kB cell quantitatively had been confirmed value positive around 0.9 mg/ml concentration. The conclusion of this studies showed that immunogenic protein of VNN with weight molecule of 45.9 kDa have ability to induce proliferation and expression of immune cells of humback grouper as like IFN γ and NF-kB cells both qualitative and quantitative on tissue organ especially eye, brain, and kidney organ. Key words: Humpback grouper, immunogenic protein, IFNγ, NF-kB, VNN Introduction One of the constraints in the cultivation of Humpback grouper (Cromileptes altivelis) is a limited supply of seed due to pathogen infection. The Infectious pathogens in farmed fish can cause the deaths of more than 80% even up to 100% [4]. Viral Nervous Necrosis (VNN) is the virus that causes disease and encephalopathy retinopathy which has a wide host range. In Indonesia reported that the VNN has invaded most of the grouper fish cultivation by 100% mortality rate. Symptoms are the fish circling or whirling, there sleeping or dead fish at the bottom like the dead and the presence of symptoms of fish behavior that is not fair [16].

A detailed knowledge about the molecules associated with immune system can also be used for further and deeper subsequent applications in aquaculture, among others, 1) for the development of biomarkers, 2) for the development of vaccines or adjuvants, and 3) development of transgenic fish. VNN is an RNA virus which has genetic material in the form of single-stranded RNA (+). Interferon (IFN) is protein or glycoprotein having ability to prevent viral replication. IFN-γ activates the cells to the immune response including natural killer cells (NK) and macrophages. Nuclear Factor-kappa lightchain-enhancer of activated B cells (NF-kB) is found in almost all types of animal cells and is also related to the cellular response to stimuli such as stress, cytokines, free radicals, ultraviolet radiation, LDL

Coresponding Author: Uun Yanuhar, Faculty of Fisheries and Marine Science, Brawijaya University, Malang, Indonesia E-mail: [email protected], Mob: 081252495924

389 Adv. Environ. Biol., 6(1): 388-396, 2012

oxidation and bacterial or viral antigens. Tues IFN γ is expressed on B cells is the result of differentiation of the B220 isoform population which is a modulation in the immune response produced by B cells and T cells. NF-kB is a protein complex that controls transcription of the DNA. NF-kB is found in almost all types of animal cells and is also related to the cellular response to stimuli such as stress, cytokines, free radicals, ultraviolet radiation, LDL oxidation and bacterial or viral antigens. NF-kB plays an important role in the regulation of immune responses to infection. Conversely, errors on NF-kB regulation is closely related to cell proliferation, inflammation and autoimmune diseases, septic shock, viral infection, and development of the immune system that is not appropriate [2]. NF-kappaB is a transcription factor that is critical for innate and adaptive immunity. Recently, a noncanonical pathway for NF-kappaB activation has emerged. NF-KappaB (Nuclear Factor-KappaB) is a heterodimeric protein composed of different combinations of members of the Rel family of transcription factors. The Rel/ NF-KappaB family of transcription factors are involved mainly in stressinduced, immune, and inflammatory responses. In addition, these molecules play important roles during the development of certain hemopoietic cells, keratinocytes, and lymphoid organ structures. NFKappaB (Nuclear Factor-KappaB) is a heterodimeric protein composed of different combinations of members of the Rel family of transcription factors. The Rel/ NF-KappaB family of transcription factors are involved mainly in stress-induced, immune, and inflammatory responses. In addition, these molecules play important roles during the development of certain hemopoietic cells, keratinocytes, and lymphoid organ structures. More recently, NFKappaB family members have been implicated in neoplastic progression and the formation of neuronal synapses. NF-KappaB is also an important regulator in cell fate decisions, such as programmed cell death and proliferation control, and is critical in tumorigenesis [3]. NF-KappaB can be activated by exposure of cells to LPS (Lipopolysaccharides) or inflammatory cytokines such as TNF (Tumour Necrosis Factor) or IL-1 (Interleukin-1), growth factors, lymphokines, oxidant-free radicals, inhaled particles, viral infection or expression of certain viral or bacterial gene products, UV irradiation, B or T-Cell activation, and by other physiological and non physiological stimuli. The most potent NF-KappaB activators are the proinflammatory cytokines IL-1 and TNF, which cause rapid phosphorylation of KappaBs at two sites within their N-terminal regulatory domain. TNF, which is the best-studied activator, binds to its receptor and recruits a protein called TRADD (TNFAssociated Receptor Death Domain). TRADD binds to the TRAF2 (TNF Receptor-Associated Factor-2)

that recruits NIK (NF-KappaB-Inducible Kinase). Both IKK1 and IKK2 have canonical sequences that can be phosphorylated by the MAP (Mitogen Activated Protein) kinase NIK/MEKK1 and both kinases can independently phosphorylate I-KappaBalpha or I-KappaB-beta. The recognition of bacterial and viral products by Toll-like receptors on cells of the innate immune system also results in NF-KappaB induction, leading to the production of proinflammatory cytokines and the activation of Antigen Presenting Cell for T-Cell costimulation in the adaptive immune response. Viral infection leads to the increased expression and secretion of the cytokine interferon gamma (IFNgamma) from host cells. IFN-gamma activates the double-stranded RNA (dsRNA)-dependent serinethreonine protein kinase R (PKR). dsRNA produced during viral replication induces PKR dimerization, autophosphorylation, and activation of the eIF2alpha kinase activity. When eIF-2alpha is phosphorylated, cellular and viral protein translation cannot efficiently occur. Alternatively, bacterial products or cellular stress can also activate PKR by an endogenous gene product called PACT. The binding of PACT to PKR promotes conformational changes that allow PKR to activate the downstream signaling pathways leading to the activation of NFKappaB. Several survival and stimulatory factors that is important in the development and BAFF (B-Cell activating factor) coordinated response of B and T lymphocytes also employ NF-KappaB to carry out their instructive functions. BAFF induces B-Cell survival and development and requires the specific BAFF receptor, BAFFR (BR3), as well as NFKappaB2, and the NIK kinase. In both immature and mature B cells, BAFF induced processing of p100 to p52, dependent on BAFF-R and NIK, and in addition this process is independent of the canonical IKK complex [5]. The interaction of the Antigen Presenting Cell and T-Cell also causes NF-KappaB activation in both cell types NF-KappaB activation is triggered in TCells by the engagement of the T cell receptor and the CD28 receptor with their ligands, MHC class II, and the costimulatory molecules CD80 and CD86 presented by Antigen Presenting Cells. The T-Cell receptor and CD28 synergize in induction of the NFKappaB-dependent genes required for T-Cell activation and proliferation, such as IL-2, IL-2 receptor (IL-2R), and IFN (Interferons). Activated TCells, in turn, elicit NF-KappaB activation in Antigen Presenting Cells. The activation of NF-KappaB has been linked to inflammatory events associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS. In contrast, complete and persistent inhibition of NFKappaB has been linked directly to apoptosis, inappropriate immune cell development, and delayed cell growth.

390 Adv. Environ. Biol., 6(1): 388-396, 2012

This study aims to determine the molecular expression of IFN-γ cells and NF-kB Humpback grouper (Cromileptes altivelis) as a responsif and activation of process immunity in the cell on tissue organ that appears causes inflammatory and stimulatory reaction in the cell system. The reaction immunity of the cell on tissue organ of Humback grouper were including proliferation and expression of IFN-y and NF-kB after fish induced by VNN immunogenic proteins of 45,9 kDa qualitatifely. Material and Methods Location and Time of Study: The research was conducted at the Central Laboratory of Biological Sciences, Central Laboratory of Biomedical Faculty of Medicine, Laboratory of Aquatic Sciences and Biotechnology Faculty of Fisheries and Marine Sciences Brawijaya University, Malang. Isolation ofa VNN coat protein: Coat proteins isolated from Humpback grouper organs (eyes, brain, and kidney) that were positive infected by VNN. Brain, eyes, and kidney from normal Humpback grouper coat protein isolation were also performed for comparison. Coat protein isolation method is to perform homogenizing organs by mortar under laminar flow then added with extract buffer (1:2w/v). The next homogenates were centrifuged at 50,000 rpm for 1 hour at 40C and followed with 150,000 rpm for 3 hours at 40C by using ultra high centrifuge (Optima TMMAX-XP, Beckman Coulter, USA). Coat protein VNN and normal fish stored in deep freezer -800C until for further test. Separation of coat protein and cutting the protein bands: Coat protein VNN separated using SDS-PAGE (Biorad) according to the method by Widyarti using minislab 12.5% and 4% with gel tracking Voltage 90V, 400 mA, for 120 minutes using coomassie brilliant blue color material and Low molecules standard by PRO-STAINTM range Marker (Fermentas). Molecular weight protein bands were calculated using linear equations from a distance separating gel. Protein bands from VNN selected for use as a candidate adhesion proteins on the basis of the major bands that appear and do not join with other protein bands (non-dimer).The protein bands cutting using gel cutter. Gel bands of protein that has been cut are stored in the running buffer solution for elution performed. Electro elution and Dialysis:

The VNN candidate adhesion proteins on the protein bands were separated by using horizontal electrophoresis electro elution method (Biorad). Pieces of gel from each candidate included in the membrane protein adhesion cellophane then electrophoresed at 120V, 400 mA for 60 minutes. Next Proteins dialyzed in PBS solution pH 7.4 at 40C for 48 hours. The liquid in a cellophane bag then taken and put into micro tube and precipitated by incubating the solution of acetone (1:1v/v) overnight at 40C. Acetone mixture of protein sand then centrifuged at 10,000 rpm for 20 minutes at 40C. Proteins found in the form of pellets, dried and dissolved in 100 mLof 0.5 M tris HCl, pH8.6. Furthermore, the protein concentration was measured using a Nanodrop ND1000 spectrophotometry with an absorbance at 280 nm. Hemagglutination (HA) Test of VNN Protein Adhesion: VNN candidate adhesion proteins performed hem agglutination (HA) test as directed by Hanne and Finkelstein. Blood isolated from normal grouper using 1 ml syringes GX26½" (Therumo) previously wetted with 10% EDTA solution. Fish blood then washed twice using PBS, homogenized and then centrifuged at 3500 rpm for 10 minutes. Erythrocytes obtained from subsequent centrifugation sediment was diluted with PBS (1:200) for use in the HA test. HA test is done by using micro plate V-bottom 96 well. A total of 50m Leach candidate protein VNN sinks and inserted in a dilution series was made using PBS solution at a dilution of up to2-10(1 / 1024). For comparison is used as a negative control wells containing only PBS. Furthermore, all added erythrocytes has been diluted with PBS to all wells, shake it and micro plate hem agglutination reaction was observed at least after the 20 minute. Positive reaction was characterized by the absence of the erythrocyte sedimentation (in the form of dot) in the bottom of wells. Clinical test and the isolation of adhesion antiprotein: The protein shows the highest value of VNN hemagglutination then performed clinical tests on grouper for anti-protein antibodies VNN adhesion. Clinical test conducted in the Central Aquaculture of Situbondo by using the4 Humpback groupers (size 150-200g) for each VNN protein adhesion and 4 fishes are used for the control (normal fish). Before the injection treatment, fish acclimatized for 1 week in advance. Fish maintained in concrete tanks (2x 5x 1.5m3) and each treatment separated by cage net (2x 1x 1.5m3). Adhesion protein from each sample were injected intra-peritoneal (ip) with a concentration of33g/150g of fish with a volume of 0.1 ml/fish.

391 Adv. Environ. Biol., 6(1): 388-396, 2012

Inoculation is done by mixing the protein adhesion with Complete Freund's adjuvant (CFA) (1:1 v/v). Seven days after first injection carried out the second injection (booster) with the volume and concentration of the same protein with the addition of Incomplete Freund's adjuvant (IFA). Fish maintained for 14 days with feeding using trash fish until full (adlibitum) with aeration and water turnover of about 200% /day. Serum from each treatment fishes and the control, were obtained by taking blood at day 14th after the first injection. Blood were centrifuged at 10,000 rpm for 10 minutes at 40C to obtain serum. Serum is stored in a-200C to the next test. Expression Detection of IFN- and NF-kB cells by immunohistochemistry technique: Detection methods of IFN- and NF-kB cell by immunohistochemistry based on Nanda et al. (2008) they are brain, eye, and kidney tissue’s, frozen and placed on a chuck of a previously cooled (-200C) in a CM3050 cryostat (Leica Corp., Deerfield, IL). Tissue sections (6 μm) in thaw-mounted on Super frost Plus glass slides (Fisher Scientific, Itasca, IL). Preparations eye tissue, brain, and kidneys of infected humpback grouper by VNN and normal were fixed in 2% paraformaldehyde (pH 7.3) for 10 minutes, washed, incubated with 2% blocking serum (serum from the horse or goat), and incubated overnight at 40C with primary antibody of IFN- and

Marker

NF-kB antibody. Next, countered with secondary antibodies conjugated with biotin for 30 minutes at room temperature. Biotin was detected with avidinbiotin peroxidase kit (ABC-Elite, Vector Laboratories) using diaminobenzidine tetrachloride as chromogene. The last stage is a piece of tissue dehydrated gradually with alcohol, cleaned with xylene, and coverslip with Permount (Fisher Scientific). Tissue sections counter stained with hematoxylin (1:4) as blue counterstain nuclei. Data Analysis: Analysis of expression of IFN- and NF-kB were conducted by using the program of CorelPaint 11 or Corel Draw Graphics Suite X4 to know the size of the specificity of colored tissue. Results: Profile of VNN Immunogenic Protein Bands: Protein immunogenic derived from infected Humpback grouper by VNN. Results from VNN immunogenic protein electrophoresis using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) at a concentration of 12.5% in profiles get 15 bands of protein. Results Protein electrophoresis with SDS-PAGE for Marker and immunogenic proteins of VNN can be seen in Figure 1.

VNN Immunogenic Protein Gel

Fig. 1: Electrophoresis result of VNN protein bands by SDS-PAGE. Table 1: Hemagglutination Test Result of VNN Coat Protein. Sampel Titer pengenceran 1/2 1/4 1/8 1/16 1/32 1/64 1/128 VNN + + + + + + _ 45.9 kDa

1/256 _

1/512 _

1/1024 _

Kontrol -

3992 Adv. Enviroon. Biol., 6(1): 3888-396, 2012

The resultts of the calcuulation on thee profile view w bands of protein p molecu ular weight marker m proteinss are exposeed to 9 bands with a mollecular weightt starting froom 9 kDa, 18 8.5 kDa, 26 kD Da, 37.8 kDa,, 46.2 kDa, 61.5 kDa, 90..5 kDa, 115 kDa, up to 1988 kDa. Proteeins that play a role in virus attachment too host recepttors is the coaat protein. VNN N coat proteinn molecular weight ranges from 37.18 kD Da and 42 kDaa [12]. Basedd on previous research that the bands off proteins that serve as the canndidate VNN N i a protein band with a immunogeenic protein is molecular weight of 45.99 kDa [15]. Protein dialysis d and Electro E elutionn SDS-PAGE E results and Coat Protein Concentrationn Measurement with a Nan nodrop spectroffotometer: e Proteinn Basedd on the dialysis and Electro elution SDS-PAGE E results, it can c further bee Coat Proteinn Concentrattion Measurem ment VNN with w Nanodropp

spectrophhotometer. S Spectrophotom meter Nanodroop readings to the concenntrations of sp pecific adhesioon 45.9 kDa) withh absorbance of o 0.51 mg/ml. protein (4 Results of o Hemagglutinnation (HA) Teest of VNN Cooat Protein: Baseed on HA testt results of VN NN Coat Proteein to Hum mpback grouuper (Cromileeptes altiveliis) erythrocy ytes obtained thhe highest dilu ution titer at 1/664 (Table 1), which meanns that the coaat protein is abble to aggluttinating VNN red blood cellls (erythrocytees) normal Humpback H groouper up to 64 4 times dilutioon. This is clearly seen in tthe absence off red color on thhe s 6. Positivve reactions alsso occurred daata bottom sinks dilution 1/2, 1/4, 1/8, 11/16 and 1/32, because of Cooat Protein VNN V can aggluutinating erythhrocytes does nnot occur as indicated by the t dot at the bottom b of wellls. NN immunogeenic protein 455.9 HA test results that VN kDa aggainst Humpbback grouperr (Cromilepttes altivelis)) erythrocytes can c be seen in Table T 1.

n Test Result of o VNN Coat P Protein against Humpback groouper erythrocy ytes. Fig. 2: Hemagglutination The concentration c o HA with a dilution 1/644 of showed thhat the agglutin nation reactionn between thee protein imm munogenic VN NN 45 kDa an nd erythrocytess of Humpbaack grouper sh howed levels of sensitivity too concentratiions are 0,0155625. Goldsby y [6] said thatt the agglutiination reactioon which show wed a positivee hemagglutinin directly give the value v of thee sensitivity of 0.3ug/m ml, results inn a positivee hemagglutination admin nisteredat a concentration off 0.006 to 0.06. In this researcch producedd ve(+)on the haemagglutinin h n concentratiions of positiv test up to o a concentraation1 / 64or the level off sensitivity of 0.015625 5 which meeans that thee determinattion of anntigen (epitoope) of ann immunogeenic protein haas a high sen nsitivity in thee range of 0..006 to 0.06. Accorrding to figu ures of haem maglutinin testt results shoowed that the concentration of dilution upp to 1/128 iss formed whichh point indicattes no bindingg between the immunoggenic proteins VNN andd erythrocytees (negative)) grouper to thee dilution1/11024, while in much smaller concentrationss are not forrmed point, thiss means that thhere is bindingg between thhe immunogen nic proteins wiith erythrocytee cell groupper, as said byy previous ressearchers, thiss suggests that the protein p is haemaglutinin. h . Haemagluttinin protein found on VNN viruss glycoproteein is a residue that is harmfull to fish [15].

The resuults of immunohistochemistryy test on speciffic tissue off Humpback grouper g on th he expression of IFN-: results of immunnohistochemisttry The examinattion performed on grouperr that has beeen clinically y tested with a VNN immuunogenic proteein 45.9 kDaa and has beenn tested has an nti-grouper IgM M antibody y response. Im mmunohistochhemistry test is performeed on humpbacck grouper tisssue covering thhe eye, braiin and kidneyss. Results of teesting performeed to detectt expression off IFN- which is an immunne cell that is formed as a response to the presence of feration detectioon antibodiees anti IFN- . IFN - prolife can be done d by lookiing at the bond between thhe antigen (45.9 kDa off VNN immunnogenic proteiin) and IFN N - monooclonal antiboody anti-mouuse secondarry antibody biootin conjugate. The results indicaate that expresssion of antigeen NN determinnants (epitopes) of 45.99 kDa VN immunog genic proteinss specificablee to induce an a immune cell receptorrs through thhe formation of FN - cells of proliferattion and exprression on IF Humpback grouper. IF FN - antiboddy used showeed similaritiies to regionss that are reccognized by thhe mouse, so s to see the reaction of antiggen and antiboddy in induccing an immunne response cellular c can be b done.

393 Adv. Environ. Biol., C(): CC-CC, 2012

Some results of the expression of IFN - cell positively confirmed by reaction of gold brown colour on tissue organ of grouper . That was used to

A

seeing the expression of immune cells in tissues that have been tested grouper with VNN immunogenic proteins 45.9 kDa, as shown in Figure 3.

B

C

D

E

F

Fig. 3: The results of the molecular expression of IFN - cells in tissues grouper with immunohistochemistry examination of the organs of the eye, M100x (A), M1000x (B); brain, M100x (C), M1000x (D), eye, M100x (E), M1000x (F), indicated by brown color. The results demonstrated the expression of IFN  cells by the presence of browncolor (indicated by arrows), brownish color because of the peroxidese enzyme reaction with the DAB and Hematoxilenkromogen eosin. The reaction that forms a golden brown color, indicating a strong bond between the epitopes of the VNN immunogenic protein of 45.9 kDa with anti- IFN - antibody identify specific parts of the protein immunogenic, if at grouper tissue did not test the cells expressing the

IFN - golden brown color as the antigen and antibody IFN - cells will not beformed. The results of immunohistochemistry test on specific tissue of Humpback grouper on the expression of NFkB molecule: The results of immunohistochemistry examination in humpback Grouper that have been tested clinically with VNN immunogenic proteins 45.9 kDa to see the tissue-specific immune responses


through NF-kB cell molecules. Cell immune responses through the expression of NF-kBis showed by the presence of anti-rabbit secondary antibody binding of NF-kB biotin conjugates with antigen (VNN immunogenic protein of 45.9 kDa). The results of this expression shows that the VNN immunogenic protein epitopes of 45.9 kDa able to induce cell molecule NF-kB in humpback grouper, where the antibody NF-kB is also in

common with the area which is also recognized by anti-grouper antibodies (IgM), this by cross-reaction based on the specific antibody with antigenic determinants (epitopes) of specific VNN viral. The results of the expression of immune cells is NF-kB cell molecule on the tissue expression of grouper have been tested with a VNN immunogenic protein of 45.9 kDa as shown in Figure 4.

Fig. 4: Expression of NF-kB cell by immunohistochemistry with anti-rabbit secondary antibody biotin conjugate in normal organ (kidney (A)); treatment on the organs of the eye (B), brain (C), and kidney (D). The results indicated a positive expression by the presence of brown color (arrow), brown color indicates presence of antigen by antibody reaction with peroxidase enzyme and substrate chromogenic labeling. Discussion: VNN infected in fish with the expression of resistance is very weak immune cells, because immune cells are used for system protection against viruses, as well as the fish are not infected by the virus gained IFN - cell molecule expression as IFNγ and NF-kB cell molecules. The number of immune cells induced expression differs depending on the target cells of each organ. To see the expression of immune cells that is IFN-γ molecules and NF-kB in the test grouper fish in this study used a VNN immunogenic protein coating of 45.9 kDa. The results of the expression of IFN - cells and NF-kB study are shown in the highest organ of the brain to NF-kB and the eye for IFN - cells. In a previous study [15] has conducted a test crossreaction of anti-adhesin algino, suggesting that CD4 and CD8 can be recognized by both the brain organ

Humpback grouper (Cromileptes altivelis). CD4 cells are formed by the immune response to VNN viral proteins after proliferation on Th1 cells capable to stimulate formation of further proliferation of immune cells ie cells IL4 and IL5. In macrophage cells were differentiated cell activity to induce IFNgamma plays an important role in chronic inflammatory. This is reinforced by Goldsby et al [6] writes that during chronic inflammationin the host cell is exposed to the antigen material will produce cytokines, which activates the cytokine IFN-gamma formed secreted by Th1 cells, NK cells and Tc cells that play a role the action of the immune system defenses. Furthermore, it is said that Crude Protein adhesion of VNN 45.9 kDa in cells expressing grouper proved CD4 cells and CD8+ cells and also IFN- and NF-kB. IFN - and NF-kB has a higher expression in some organs of the grouper on the


brain and eyes, this is due to the proliferation and expression of immune cells on humback grouper against the VNN immunogenic protein virus response is regulated by nerve cells in the center. This suggests that the organ systems are more likely to have antibody mediated immunity (AMI) that play a role in controlling VNN infection compared to cell mediated immunity (CMI). The cells in the brain organ responsible for the immune system, among others, microglia cells that have a variety of receptors in the immune system such as toll-like receptor (TLR), complement receptors, immunoglobulin and lymphokine receptors. Immune response in Humpback grouper cell adhesion was triggered by VNN protein of 45.9 kDa as a protein that is immunogenic and haemaglutinin. This is indicated by a high value (up to 1/64 dilution) on the hemagglutination (HA) test of normal erythrocytes grouper. The ability of VNN immunogenic proteins of 45.9 kDa was demonstrated by the ability to agglutination red blood cells (erythrocytes) normal humpback grouperat up to 64 times dilutions. Viral infection process always begins with the attachment between the protein adhesion (ligand) on pathogens with receptors on host [15]. Therefore, not all organs or tissues in the host is a good place for a particular pathogen. In this case known as the portal of entry of the host cell or tissue which is a special target of the entry of pathogens into the host's body [10]. Two kinds of immune response that developed host defenses against viral infection is the body that mediated antibody (antibody mediated immunity, AMI) and cell-mediated immune (cellular mediated immunity, CMI). AMI system involves antibodies that are synthesized by plasma cells that are derived from B cells To activate B cells in synthesizing antibodies, antigens enter the body presented by APC (antigen presenting cells) via MHC II molecules to CD4+ T cells (T helper cells). Th cells would then secrete cytokines that provide signals to B cells to conduct proliferation and differentiation into plasma cells and memory B cells while the CMI is intended to antigens contained in the cell. Antigens will be broken down by proteases into molecules smaller then bound by MHC I molecules for presentation to the cell surface to CD8+ T cells (cytotoxic T cells). Tc cell that activated will seek a target cell characterized by the expression of cell surface MHC I in the present foreign antigens. Tc cell would bind to target cells through Fas and Fas ligand and secrete perforin and granzyme that induces apoptosis in target cells. Humpback Grouper test with a size of about 150 g in a clinical trial is expected that the fish at that size has a perfect immune system so that the production of cells and molecules that play a role in the immune system of grouper fish is done optimally. In addition, the use of fish with a large size will facilitate the retrieval of blood and organ tissues.

IFN - and NF-kB cell molecule expression in grouper that have been tested clinically with immunogenic proteins showed that the 45.9 kDa molecular VNN IFN - cells and NF-kB expressed in organs of eyes, brain, and kidneys. It is shown from the results of immunohistochemistry test. IFN  cell molecule expression and NF-kB in the kidney and the brain is also supported from the results of molecular photoelectrons which showed IFN - cells and NF-kB on the surface of the kidney and brain cells. In the study by Graham and Secombes (1988, 1990), suggesting that leukocytes from rainbow trout kidneys secrete IFN-γ molecule in the presence of anti viral and MAF activity when leukocytes are stimulated with mitogen. Also described by Lu et al that the ability of molecules of IFN - cells to induce T cell differentiation (in particular on T cells with low proliferative capacity, and produce IFN-γ, IL-10 and TGF-β) can increase expression of IFN - cells which play a role in the maintenance tolerance or equipment also imposes limits on the reactivity of the immune system cellular. These results confirm a research that immunogenic proteins 45.9 kDa an VNN immunogenic protein capable of triggering an immune response of humpback grouper fish and have demonstrated the molecular expression of IFN γ and NF-kB cell molecules expressed in the organs of the brain, eyes, kidneys, with expression levels that vary depending on the cell target organ of grouper. The existence of a virus infection or antigen (immunogenic proteins) of several viruses in host cells that are nature protection inhibits the function of immune cells. VNN infection by the virus has proved capable of inhibiting immune cell expression of MHC class I [15]. In this study has generated MHC class I that has been measured can be triggered by exposure to VNN immunogenic protein with 45.9 kDa. MHC class I expression in this study turned out after testing cellular expression of immune cell IFN-γ and NF-kB showed measurable positive qualities. Positive reaction indicated by the reaction of antigen with antibody, either by NF-kB and IFN-γ antibodies labeled by biotin labeling. Immune reaction that formed through formation of MHC which further differentiate and proliferate into CD4+ cells due to exposure to an VNN immunogenic protein 45.9 kDa occurs rapidly in cells with the expression levels very quickly after exposure to immunogenic proteins. Similarly, when exposed to the VNN virus, the formation of MHC class I cells and immune cells of IFN-γ and NF-kB cell also occurs very rapidly, but the amount produced is very low, because the immune cells are used to maintain the life of the cell or cell survival system [1]. At the molecular mechanisms that occur indicated further that the virus is able to suppressor reduce the expression of immune cell MHC class I


on the cell surface, it is intended to counter act the inhibition of antigen presented by CD4 + or CD8 + depending on the level of virus infection [6]. The results using VNN immunogenic protein 45.9 kDa has been known to express CD4 + and CD8+ cells. Base on molecular technics has been demonstrated that expression of CD4+ cells have been formed because differentiation and proliferation of MHC class I. Thus in this study has been produced that the expression of IFN-gamma cells and B220 cells can be triggered by treatment with exposure to a VNN immunogenic protein 45.9 kDa. Several viruses have been reported also in addition can reduce the function of MHC class I cells are also able to reduce MHC class II cell function at the cell surface, where the function includes a block function to antigen-specific T helper cells function as an antiviral, aproper T helper cells capable of proliferation induce a formation of MHC class II because of a virus infection, the function to be reduced and even disappear, causing fish to die [6].

7. 8.

9. 10.

11.

Conclusions: From the results of this research can take the conclusion that: VNN immunogenic proteins with 45.9 kDa molecule weight, capable to induce proliferation and expressi0n of IFN-γ cells and NFkBcell molecules on humback grouper as like immune system cellular that confirmed qualitatively uses immunohistochemistry staining. The recommended for further research needs to be done for the measurement of expression and proliferation of immune cell as IFN-y and Nf-kB with flowcytometry.

12.

13. 14.

References 1.

2.

3. 4.

5. 6.

Abbas, A.K. and A.H. Lichtman, 2005. Cellular and Molecular Immunology, fifth edition, updated edition. Elsevier saunders, Pennsylvania. Albensi, B.C., M.P. Mattson, 2000. "Evidence for the involvement of TNF and NF-κB in hippocampal synaptic plasticity". Synapse, 35(2): 151-9. Baldwin, A.S. Jr., 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol., 14: 649-83. Erazo-Pagador, G., 2001. Rapid wound healing in African catfish, Clariasgariepinus, fed diets supplemented with ascorbic acid. Israeli J. Aquacult-Bamidgeh, 53: 69-79. Fulvio D'Acquisto and Sankar Ghosh, 2001. PACT and PKR: Turning on NF- B in the Absence of Virus. Science's STKE, 3 July. Goldsby A. Richard, Kindt J. Thomas, Osborne A. Barbara, 2000. Kuby-Immunology, Fourth Edition. W.H. Freeman and Company, All Right Reserved. United Stated of Amerika.

15.

16.

Graham, S. and C.J. Secombes, 1990. Do Fish Lymphocytes Secrete Interferon γ ?. J. Fish Biol., 36: 563-573. Gunawan, Irawadi, 2009. Identification of Coat Protein Viral Nervous Necrosis (VNN) in Humpback Grouper (Cromileptesaltivelis). Theses, Post graduate Program Aquaculture Program Study, Fisheries and marine Science, University of Brawijaya. Malang, Indonesia. (Unpublished). Joel L. Pomerantz and David Baltimore, 2002. Two pathways to NF-KappaB. Molecular Cell, 10: 693-701, October. Madigan, M.T., J.M. Martinko, dan J. Parker, 2003. Brock Biology of Microorganisms, Tenth edition. Prentice Hall, Pearson education, Inc., New Jersey, pp: 1019. Michael Hinz, Daniel Krappmann, Alexandra Eichten, Andreas Heder, Claus Scheidereit and Michael Strauss, 1999. NF- B Function in Growth Control: Regulation of Cyclin D1 Expression and G0/G1-to-S-Phase Transition. Molecular and Cellular Biology, 19(4): 26902698. Nishizawa, T., K. Mori, M. Furuhashi, T. Nakai, I. Furusawaand, K. Muroga, 1995. Comparison of the Coat Protein Genes of Five Fish Nodaviruses, the Causative Agents of Viral Nervous Necrosis in Marine Fish. Journal of General Virology, 76: 1563-1569. Vishva Dixit and Tak. W. Mak, 2002. NFKappaB signaling: Many Roads Lead To Madrid. Cell, 111: 615-619. Yanuhar, U., 2006. The Molecular Characterization and Identification of Adhesin Protein of Pili Vibrio alginolyticus and Its Receptor in Humpbak Grouper (Cromileptes altivelis) (The Study of Molecular Pathogenese of Vibriosis). Dissertation, Post-Graduate Program, Medical Sciences, University of Brawijaya, Malang, Indonesia. (Unpublished). Yanuhar, U., 2008; 2009; 2010. Development of Vaccine based on Receptor Peptide of Grouper recognizing Antigen as Transgenic Antibody for Preparing Preeminent Seed, Research Report, State Ministry of Research and Technology of Republic Indonesia (Unpublished). Yuasa, K., I. Des Roza, Koesharyani, F. Johnny and K. Mahardika, 2000. General Remarks On Fish Disease Diagnosis. Pp. 5-18. Textbook for the Training Course on Fish Disease Diagnosis.Lolitkanta-JICA Booklet No. 12.