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Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination
Fish & Shellfish Immunology 17 (2004) 367e374 www.elsevier.com/locate/fsi
Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination Tomokazu Takano, Akiko Iwahori, Ikuo Hirono, Takashi Aoki) Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan Received 4 February 2004; accepted 14 April 2004
Abstract Intramuscular injection of Japanese flounder, Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 mg of a plasmid DNA vaccine encoding the hirame rhabdovirus (HIRRV) glycoprotein gene ( pCMV-HRVg) was found to provide strong protection against HIRRV. We also conducted a real-time PCR analysis to quantify immunerelated genes, e.g. MHC class Ia, IIa, IIb, TCR-a, b1, b2 and d, to characterize the immune response at 1 and 7 days after DNA vaccination. In general, the copy numbers were at least 2-fold higher than those of the non-vaccinated fish. Interestingly, the gene expression of TCR b1 and b2 increased 1 day post-DNA vaccination, after which their copy numbers returned to levels similar to those before vaccination. These results suggest that the immune system of Japanese flounder was activated immediately after DNA immunization. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Japanese flounder; Rhabdovirus; DNA vaccine; Glycoprotein gene; MHC; TCR; Immune-related gene
1. Introduction Hirame rhabdovirus (HIRRV) was first isolated in Hyogo prefecture, Japan [1] and has been subsequently found in Japanese flounder farms in Japan and Korea [2]. HIRRV-infected fish show hemorrhage in the muscle, congestion of the gonad and accumulation of ascitic fluid. The pathogen is a negative stranded RNA virus and is classified into the genus Novirhabdovirus of the Rhabdoviridae family [3]. Eou et al. [4] sequenced the whole genome of the HIRRV Korean strain, and it consists of 11 034 nucleotides. The virion of HIRRV consists of six viral proteins, namely, RNA polymerase, envelope glycoprotein, nucleocapsid protein, two matrix proteins and non-viral proteins [5].
) Corresponding author. Tel.: +81-3-5463-0556; fax: +81-3-5463-0690. E-mail address: [email protected] (T. Aoki). 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2004.04.012
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Previous studies reported the antigenic effects of the glycoprotein gene of fish rhabdoviruses. Rainbow trout infected with an attenuated Aeromonas salmonicida strain expressing the glycoprotein epitope of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) showed resistance when challenged with these viruses [6]. Further, the recombinant protein that was synthesized from a fragment of the HIRRV glycoprotein gene conferred protection to Japanese flounder against HIRRV infection [4]. However, the effect of recombinant protein as a vaccine is not yet well understood. In addition, the cost of making a recombinant protein vaccine is expensive, thus making its suitability for commercial use in aquaculture difficult. DNA vaccines are known to be more effective than traditional vaccines in stimulating the host cytotoxic T-lymphocyte activity [7]. Hence, DNA vaccination is effective at preventing intracellular pathogen infection and spread. DNA vaccines also activate the humoral immune system of the host animal [7]. An advantage of DNA vaccines over other types of vaccines is that they do not require development of new antigen purification or pathogen attenuation methods since DNA vaccines can be purified using standard DNA purification techniques. DNA vaccines have been studied not only in mammals but also in fishes. A DNA vaccine based on the IHNV glycoprotein gene protected rainbow trout against an IHNV challenge [8], and similar results have been reported for VHSV [9]. Moreover, a vaccine dose as low as 10 ng of IHNV glycoprotein gene DNA conferred significant protection against IHNV infection in rainbow trout [10]. The authors speculated that the fish rhabdovirus glycoprotein was very immunogenic. However, the DNA vaccines using other IHNV viral protein genes did not induce significant protection against experimental challenge in rainbow trout [11]. In this study, we confirmed the efficacy of the HIRRV glycoprotein gene as a DNA vaccine in Japanese flounder juveniles. Real-time PCR analysis of immune-related genes of the flounder was also conducted to elucidate the mechanisms by which DNA vaccination stimulates the host’s immune response.
2. Materials and methods 2.1. Virus and cell culture The hirame natural embryo (HINAE) cell line [12] was used for the propagation of HIRRV strain 8601H. Cells were cultured in a 25 cm2 culture flask with Leibovitz’s L-15 medium (Gibco BRL, Grand Island, USA) supplemented with 20% fetal bovine serum (JRH Biosciences, Lenexa, USA), 100 units/ml penicillin and 100 mg/ml streptomycin (Gibco BRL). HIRRV strain 8601H was kindly provided by Dr. M. Yoshimizu (Hokkaido University). The virus was used to infect a monolayer of HINAE cells cultured in a 75 cm2 cell culture flask containing 15 ml of medium and incubated at 15 (C for 5 days. When a cytopathic effect (CPE) was observed, the viral culture medium was collected. The virus titer was calculated as 50% tissue-culture infective dose (TCID50) [13]. 2.2. Construction and purification of plasmid DNA Total RNA was isolated from HIRRV strain 8601H culture medium using TRIzol (Invitrogen, Carlsbad, USA). The cDNA was synthesized from total RNA using a cDNA synthesis kit 1st strand with AMV reverse transcriptase (Takara, Otsu, Japan) following the manufacturer’s protocol. PCR primers HRV-Glyf (5#-GAGAATTCGTCATGGACCCTCGAATAAT-3#) and HRV-Glyr (5#-GAGTCGACTTACCCTCGACTGGCG-3#) were designed from the published HIRRV glycoprotein gene nucleotide sequence [14]. Restriction enzyme sites EcoRI and SalI were added at the forward and reverse primer sites, respectively. The PCR primers were used for the amplification of full-length glycoprotein gene of HIRRV
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strain 8601H. PCR was performed with an initial denaturation step of 2 min at 95 (C, and then 30 cycles were run as follows: 30 s of denaturation at 95 (C, 30 s of annealing at 55 (C, and 1 min of extension at 72 (C. The 1546 bp PCR product was digested with EcoRI and SalI, and ligated into the multiple cloning site of pCI-neo vector (Promega, Madison, USA). The plasmid encoding HIRRV glycoprotein gene was designated pCMV-HRVg. The nucleotide sequence was determined using ThermoSequenase (Amersham Bioscience, Little Chalfont, UK) and an automated DNA sequencer LC4200 (Li-Cor, Lincoln, USA). The recombinant plasmid was extracted by ultracentrifugation using a CsCleethidium bromide gradient [15]. In vitro transcriptionetranslation of HIRRV glycoprotein was also determined. TNT Quick coupled transcription/translation system (Promega) was used for the synthesis of the glycoprotein. The pCMVHRVg (0.5 mg) and other reagents were mixed following the protocol of the manufacturer. A similar reaction using pCI-neo vector was also used as a negative control. The reaction mixture was incubated at 30 (C for 90 min. Then, 5.0 ml of the sample was mixed with the same volume of SDS-sample buffer and boiled for 2 min. The samples were applied to a SDSe20% polyacrylamide gel with a 5% stacking gel. The gel was electroblotted to a PVDF membrane, bound with Streptavidin-AP solution and colorimetrically detected using Western blue stabilized substrate for alkaline phosphatase (Promega). A band of approximately 65 kDa was seen, indicating that the HIRRV glycoprotein was translated in vitro (data not shown). 2.3. Vaccination Japanese flounder juveniles with an average weight of 2 g were injected intramuscularly with 1 or 10 mg of pCMV-HRVg in 50 ml saline buffer. As negative control, groups of fish were vaccinated with 10 mg of pCI-neo vector or 50 ml saline buffer. After vaccination, each group of 45 fish was kept in a 60-l tank with a flow-through water system supplying filtered seawater at 13 (C and fed commercial dry pellets corresponding to 5% of total body weight, twice per day. 2.4. Challenge test Each vaccinated group (45 fishes) was transferred to four separate 60-l tanks at 21 days after vaccination. These tanks were provided with UV treated artificial seawater in a recirculating system. At 28 days after vaccination, each fish was injected intramuscularly with 4.0!103 TCID50 of live HIRRV strain 8601H in 50 ml saline buffer. Mortalities were recorded daily for 14 days. 2.5. Real time PCR analysis of gene expression of immune-related genes mRNAs were isolated from the exact site of injection from three individual fish at 1 and 7 days postpCMV-HRVg DNA injection using a micro mRNA purification kit (Amersham Bioscience). The cDNA was synthesized from purified mRNA (1 mg) by cDNA synthesis kit 1st strand with AMV reverse transcriptase. After cDNA synthesis, the sample was diluted to 100 ml with distilled water. To determine the absolute copy number of the target transcript, a cloned plasmid DNA for each gene (MHC class Ia, class IIa and IIb, TCR-a, b1, b2 and d) [16,17] was used to generate a standard curve. The PCR primers used for generating the standard curves are listed in Table 1. PCR was performed as described above. All amplified products were 200 bp in size. The products were purified using Amicon Microcon-PCR Centrifugal Filter Devices (Millipore, Bedford, USA). The copy number of each reacted product was calculated according to its molecular weight and then converted into the copy number based on Avogadro’s number (1 mol=6.022!1023 molecules). The real-time PCR reaction mixture contained 1 ml of diluted sample, PCR primers and the SYBR Green PCR core reagents (ABI, Foster City, USA). Amplification was carried out as follows: 2 min at 50 (C,
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Table 1 PCR primers used in this study Primer name
Oligonucleotides
For construction of standard curve SG200 b-actin F SG200 b-actin R SG200 MRC I a F SG200 MHC I a R SG200 MHC II a F SG200 MHC II a R SG200 MHC II b F SG200 MHC II b R SG200 TCR a F SG200 TCR a R SG200 TCR b F SG200 TCR b R SG200 TCR b2 F SG200 TCR b2 R SG200 TCR d F SG200 TCR d R
5#-TTTCCCTCCATTGTTGGTCG-3# 5#-GCGACTCTCAGCTCGTTGTA-3# 5#-GGACTGGCTGAAGATATTCG-3# 5#-TGGAAGTCCCGTCATGGTTG-3# 5#-GACGTGACTGAAGGAACAAG-3# 5#-ACCCACTCCACAAAACACAG-3# 5#-TCCATGCCTGAGTCAGAAAG-3# 5#-AAAGCAGGTTGAAGCAGCAG-3# 5#-TCAGAAGAGTCTCTGTACAGT-3# 5#-ACGGTCTTGAGGAAGAGGA-3# 5#-CCATATACCTGGAGTCCCTT-3# 5#-CTGTTTCAACACGTGTAGGTG-3# 5#-TTAGCAGCTCACAAACCAGC-3# 5#-TGCTGCTTGTCCTCCAAACT-3# 5#-AGTGAAGAAGAAGCCGTCGT-3# 5#-CAGCGTTCTGATGGTCAGUA-3#
For detection of mRNA SG b-actin F SG b-actin R SG MHC I a F SG MHC I a R SG MHC II a F SG MHC II a R SG MHC II b F SG MHC II b R SG TCR a F SG TCR a R SG TCR b F SG TCR b R SG TCR b2 F SG TCR b2 R SG TCR d F SG TCR d R
5#-TGATGAAGCCCAGAGCAAGA-3# 5#-CTCCATGTCATCCCAGTTGGT-3# 5#-AAGTCTCCCTCCTCTCCAGTCA-3# 5#-TCTCCGTCTTCCTCCAGAACA-3# 5#-GCCAGACTGAAATTCATCGCT-3# 5#-CCAGATCTTGGTCAGTGATTGG-3# 5#-CCTGGGTCTGACCTTATCTCTG-3# 5#-CCAAGTCCAGGACCAGACTCAG-3# 5#-GGTCTGATGCTTCACAGTGTGAG-3# 5#-ACCGCCGGATCTTTCTTCA-3# 5#-GCACCATTCACACACTGTGGTT-3# 5#-AACAGGCTGGTTTGTGAGCTG-3# 5#-CACCTACACATGTTGAAACAGCTC-3# 5#-GTCACTGTTGAATCATTTCTCAG-3# 5#-CGTCAGGTTCAACTCGTACCTG-3# 5#-TGGTAAACGCCACGATCTTG-3#
10 min at 95 (C, 40 cycles of 15 s at 95 (C and 60 s at 58 (C. Thermal cycling and fluorescence detection were conducted using the GeneAmp 5700 Sequence Detection System as described above. All samples were run in triplicate. The threshold cycle (CT) represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected. The ratio between the b-actin content in standard samples (106 copies) and test samples was defined as the normalization factor. Standardized cytokine mRNA quantities (cytokine copies/106 b-actin copies) were determined by dividing the interpolationderived values from the cytokine standard curve by the normalization factor.
2.6. Statistical analysis Statistical analysis was carried using the c2 test. The relative percentage survival (RPS) was determined following the formula RPS=(1ÿ[% loss of immunized fish/% loss of control fish])!100 [18].
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3. Results and discussion 3.1. Protection against HIRRV The nucleotide sequence of the HIRRV strain 8601H (accession no. AB103462) was compared with previously reported viral genes using the BLAST program. The sequence shared 99.8% identity (507/508 amino acids) with the HIRRV glycoprotein gene [14]. Fish vaccinated with the pCMV-HRVg DNA vaccine were better able to survive the HIRRV challenge. Fourteen days after the HIRRV challenge, cumulative mortalities of the fish injected with 1 and 10 mg of pCMV-HRVg were about 28.8 and 8.8%, respectively, while the cumulative mortality of the control groups was 100% (Table 2). The time when mortality was first observed after HIRRV challenge was also different between the vaccinated and control groups. Almost all control fish exhibited acute mortality starting on the 5th day until the 8th day after challenge, following which low mortalities were observed until the termination of the experiment. The vaccinated Japanese flounder, however, had lower mortality rates throughout the challenge period. Recently, DNA vaccines for the prevention of rhabdovirus infections, such as viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in salmonid fish have been demonstrated to be efficacious using the viral glycoprotein gene in a DNA vaccine construct [8,19,20]. In addition, a recent study by Sommerset et al. [21] has shown that a VHSV glycoprotein DNA vaccine was able to provide cross-protection in fish against a heterologous fish virus, the Atlantic halibut nodavirus (AHNV). The HIRRV glycoprotein gene DNA vaccine that we have developed is also efficacious in protecting against HIRRV infection in marine fish such as Japanese flounder, as shown by the higher survival rates of the vaccinated fish in comparison with the control fish upon virus infection. Thus, the glycoprotein gene is a good antigen that protects not only salmonids but also other marine fish, such as Japanese flounder, against rhabdovirus infection. In addition, HIRRV glycoprotein gene DNA vaccine provided protection with no side effects observed after a single intramuscular injection of either 1 or 10 mg of DNA per fish. Our results suggest that this DNA vaccine can provide a high level of protection and may be feasible for use in the aquaculture industry. 3.2. Quantification of expression of immune-related genes In mammals, MHC class I molecule, which consists of a chain and b2-microglobulin, is present on the surface of all kinds of cells [22]. The molecule presents epitopes of intracellular pathogens and activates CD8+ T cells. On the other hand, MHC class II molecule, which consists of a and b chains, exists only on the surface of antigen presenting cells and activates CD4+ T cells [22]. TCRs are heterodimers consisting of Table 2 Cumulative percentage mortality (%) and calculated RPS values following challenge with HIRRV in experimental groups of DNA vaccinated fish by i.m. injection Injected plasmids
pCMV-HRVg (10 mg) pCMV-HRVg (1 mg) pCI-neo (10 mg) PBS a
No. of injected virus: 4.0!103 TCID50/fish Mortalities death/total
RPSa
c2
P value
8.8% (4/45) 28.8% (13/45) 100% (45/45) 100% (45/45)
90.1 70.5 0 e
75.306 49.655 e e
!0.01 !0.01 e e
Relative percentage survival (RPS)={1ÿ[% mortality (vaccinated)/% mortality (control)]}!100.
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either a/b or g/d polypeptide combinations, and recognize MHC-presented epitopes [22]. Therefore these molecules are useful markers for understanding the mechanisms of epitope recognition and tracing the migration of immune-related cells. After 1 and 7 days post-pCMV-HRVg DNA vaccination, the copy numbers of MHC class Ia, class IIa, and IIb, TCR-a, b1, b2 and d mRNAs of the injected fish were at least 2-fold higher than those of the nonvaccinated fish (Table 3). The most remarkable change in the gene expression patterns was observed in TCR-a, b1 and b2. The copy numbers of these transcripts dramatically increased 1 day after vaccination but then after 7 days, the expression levels of these genes, especially TCR-b1 and b2, decreased and returned to nearly the gene expression level of non-vaccinated fish. In case of TCRd gene expression, low increments were observed at day 1 and day 7 post-injection of the pCMV-HRVg DNA vaccine. In mice, the g/d T cells are located in epidermis and in epithelial layers of intestine, in lung and ovary [23], so the g/d T cells may have important roles in inhibiting the virus during the initial stage of infection. However, DNA vaccination in this study was delivered using the intramuscular route. Therefore, the g/d T cells might not have been stimulated and a high level of expression of the TCRd gene was not observed. Expression of MHC class Ia, class IIa and IIb genes were activated at the site of the pCMV-HRVg DNA injection. The increased copy numbers of these genes suggest that antigen-presenting cells and antigen recognition cells such as a/b T cells aggregate at the site of pCMV-HRVg DNA injection due to the possible stimulation of the pCMV-HRVg DNA vaccine, and because some cells neighboring this site might express chemokines and cytokines after recognition of the expressed glycoprotein. The activity and number of cytotoxic T cells might be related to the copy number of TCR mRNAs. Fischer et al. [24] immunized rainbow trout with a plasmid encoding the glycoprotein gene of IHNV. They also infected a MHC class I matched rainbow trout gonad cell line (RTG-2) with IHNV. They found that peripheral blood leukocytes isolated from the immunized fish killed the IHNV-infected cells. Hence, an increase in MHC class Ia gene expression found in the present work implies an increased number of antigen presenting cells and stimulation of specific cytotoxic T cells. In our study, 7 days after pCMV-HRVg DNA injection, antigen recognition cells might gradually leave the injected site, because the expression level of TCR-b1 and b2 returned to nearly the gene expression level of the non-vaccinated fish, even though the expression of MHC class I was higher at 7 days post-injection than at 1 day post-injection. These results suggest that the specific cellular immune functions gradually became established during the 7 days following vaccination. Furthermore, LaPatra et al. [25] examined the immune response of rainbow trout injected with a DNA vaccine against the rhabdovirus IHNV. These fish show an ‘early’ non-specific protection and a ‘late’ specific protection, because rainbow trout immunized with plasmid DNA encoding the IHNV glycoprotein gene showed cross-protection until 14 days post-vaccination against VHSV infection but after this period this cross-protection started to disappear. On the other hand, specific protection against IHNV continued until the end of the experiment. The increase in the copy number of MHC class Ia at 7 days postvaccination in our study might indicate generation of a ‘late’ specific cellular immune response. Table 3 Calculation of copy number of MHC and TCR genes by quantitative real-time PCR Genes MHC class Ia MHC class IIa MHC class IIb TCR a TCR b1 TCR b2 TCR d
Control
DNA vaccine day 1 8
3.6 ! 10 3.5 ! 106 1.7 ! 106 9.0 ! 105 2.9 ! 107 7.1 ! 108 5.4 ! 106
8
7.6 ! 10 3.1 ! 107 6.0 ! 106 5.2 ! 106 2.3 ! 108 5.3 ! 109 6.4 ! 106
(2.1)* (8.7)* (3.3)* (5.8)* (7.9)* (7.5)* (1.2)*
*Increased fold of the copy number of mRNA of DNA injected samples when compared with that of control.
DNA vaccine day 7 1.6 ! 109 1.7 ! 107 9.2 ! 106 3.4 ! 106 4.4 ! 107 1.3 ! 109 1.4 ! 107
(4.4)* (4.8)* (5.5)* (3.8)* (1.5)* (1.8)* (2.6)*
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Our results also suggest that DNA immunization activates both the cellular and humoral defenses of the specific immune system of Japanese flounder. Boudinot et al. [9] observed that DNA vaccination with VHSV glycoprotein genes triggered strong expression of MHC class II and Mx genes at the site of injection. They concluded that DNA immunization was able to activate specialized cells of the immune system as well as nonspecific defense mechanisms. Furthermore, McLauchlan et al. [26] showed strong expression of Mx gene from rainbow trout that was injected with pcDNA3-vhsG intramuscularly but not intraperitoneally, and they suggested that the early non-specific immune response is due to the induction of interferon. Immune-related genes of Japanese flounder have been found from expressed sequence tag (EST) studies [16,17,27]. Further studies on the analysis of their expression as a consequence of DNA vaccination are required.
Acknowledgements This research was supported in part by a Grant-in-Aid for Scientific Research (S) (No. 15108003) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
References [1] Kimura T, Yoshimizu M, Gorie S. A new rhabdovirus isolated in Japan from cultured hirame (Japanese flounder) Paralichthys olivaceus and ayu Plecoglossus altivelis. Dis Aquat Organ 1986;1:209e17. [2] Oh MJ, Choi TJ. A new rhabdovirus (HRV-like) isolated from cultured Japanese flounder Paralichthys olivaceus. J Fish Pathol 1998;11:129e36. [3] Van Regenmortel MHV, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, et al. Virus taxonomy. Seventh report of the International Committee on Taxonomy of Viruses. New York: Academic Press; 2000. [4] Eou JI, Oh MJ, Jung SJ, Song YH, Choi TJ. The protective effect of recombinant glycoprotein vaccine against HIRRV infection. Fish Pathol 2001;36:67e72. [5] Kurath G, Leong JC. Characterization of infectious hematopoietic necrosis virus mRNA species reveals a nonvirion rhabdovirus protein. J Virol 1985;53:462e8. [6] Noonan B, Enzmann PJ, Trust TJ. Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss). Appl Environ Microbiol 1995;61:3586e91. [7] Davis HL, McCluskie MJ. DNA vaccines for viral diseases. Microb Infect 1999;1:7e21. [8] Anderson ED, Mourich DV, Fahrenkrug SC, LaPatra S, Shepherd J, Leong JA. Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Mol Mar Biol Biotechnol 1996;5:114e22. [9] Boudinot P, Blanco M, de Kinkelin P, Benmansour A. Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout. Virology 1998;249:297e306. [10] Corbeil S, LaPatra SE, Anderson ED, Kurath G. Nanogram quantities of a DNA vaccine protect rainbow trout fry against heterologous strains of infectious hematopoietic necrosis virus. Vaccine 2000;18:2817e24. [11] Corbeil S, Lapatra SE, Anderson ED, Jones J, Vincent B, Hsu YL, et al. Evaluation of the protective immunogenicity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss using DNA vaccines. Dis Aquat Organ 1999;39:29e36. [12] Kasai H, Yoshimizu M. Establishment of two Japanese flounder embryo cell lines. Bull Fisheries Sci Hokkaido Univ 2001;52: 67e70. [13] Rovozzo GC, Burke CN. A manual of basic virological techniques. New Jersey: Prentice-Hall; 1973. [14] Bjorklund HV, Higman KH, Kurath G. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: analysis of relationships with other rhabdoviruses. Virus Res 1996;42:65e80. [15] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning, a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989. [16] Aoki T, Nam BH, Hirono II, Yamamoto E. Sequences of 596 cDNA Clones (565,977 bp) of Japanese flounder (Paralichthys olivaceus) leukocytes infected with hirame rhabdovirus. Mar Biotechnol 1999;1:477e88.
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[17] Nam BH, Yamamoto E, Hirono I, Aoki T. A survey of expressed genes in the leukocytes of Japanese flounder, Paralichthys olivaceus, infected with hirame rhabdovirus. Dev Comp Immunol 2000;24:13e24. [18] Amend D. Potency testing of fish vaccines. Dev Biol Stand 1981;49:447e54. [19] Lorenzen N, Lorenzen E, Einer-Jensen K, Heppell J, Wu T, Davis H. Protective immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following DNA vaccination. Fish Shellfish Immunol 1998;8:261e70. [20] Lorenzen N, Lorenzen E, Einer-Jensen K, LaPatra SE. DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout. Fish Shellfish Immunol 2002;12:439e53. [21] Sommerset I, Lorenzen E, Lorenzen N, Bleie H, Nerland AH. A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot. Vaccine 2003;21:4661e7. [22] Roitt I, Brostoff J, Male D. Immunology. 5th ed. New York Mosby International; 1998. [23] Salerno A, Dieli F. Role of gamma delta T lymphocytes in immune response in humans and mice. Crit Rev Immunol 1998;18: 327e57. [24] Fischer U, Dijkstra JM, Aoyagi K, Xia C, Ototake M, Nakanishi T. MHC class I restricted cytotoxicity in rainbow trout, Oncorhynchus mykiss. Dev Comp Immunol 2000;24:S76. [25] LaPatra SE, Corbeil S, Jones GR, Shewmaker WD, Lorenzen N, Anderson ED, et al. Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination. Vaccine 2001;19:4011e9. [26] McLauchlan PE, Collet B, Ingerslev E, Secombes CJ, Lorenzen N, Ellis AE. DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size, dose, route of injection and duration of protection-early protection correlates with Mx expression. Fish Shellfish Immunol 2003;15:39e50. [27] Inoue S, Nam BH, Hirono I, Aoki T. A survey of expressed genes in Japanese flounder (Paralichthys olivaceus) liver and spleen. Mol Mar Biol Biotechnol 1997;6:376e80.
Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination Tomokazu Takano, Akiko Iwahori, Ikuo Hirono, Takashi Aoki) Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan Received 4 February 2004; accepted 14 April 2004
Abstract Intramuscular injection of Japanese flounder, Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 mg of a plasmid DNA vaccine encoding the hirame rhabdovirus (HIRRV) glycoprotein gene ( pCMV-HRVg) was found to provide strong protection against HIRRV. We also conducted a real-time PCR analysis to quantify immunerelated genes, e.g. MHC class Ia, IIa, IIb, TCR-a, b1, b2 and d, to characterize the immune response at 1 and 7 days after DNA vaccination. In general, the copy numbers were at least 2-fold higher than those of the non-vaccinated fish. Interestingly, the gene expression of TCR b1 and b2 increased 1 day post-DNA vaccination, after which their copy numbers returned to levels similar to those before vaccination. These results suggest that the immune system of Japanese flounder was activated immediately after DNA immunization. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Japanese flounder; Rhabdovirus; DNA vaccine; Glycoprotein gene; MHC; TCR; Immune-related gene
1. Introduction Hirame rhabdovirus (HIRRV) was first isolated in Hyogo prefecture, Japan [1] and has been subsequently found in Japanese flounder farms in Japan and Korea [2]. HIRRV-infected fish show hemorrhage in the muscle, congestion of the gonad and accumulation of ascitic fluid. The pathogen is a negative stranded RNA virus and is classified into the genus Novirhabdovirus of the Rhabdoviridae family [3]. Eou et al. [4] sequenced the whole genome of the HIRRV Korean strain, and it consists of 11 034 nucleotides. The virion of HIRRV consists of six viral proteins, namely, RNA polymerase, envelope glycoprotein, nucleocapsid protein, two matrix proteins and non-viral proteins [5].
) Corresponding author. Tel.: +81-3-5463-0556; fax: +81-3-5463-0690. E-mail address: [email protected] (T. Aoki). 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2004.04.012
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Previous studies reported the antigenic effects of the glycoprotein gene of fish rhabdoviruses. Rainbow trout infected with an attenuated Aeromonas salmonicida strain expressing the glycoprotein epitope of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) showed resistance when challenged with these viruses [6]. Further, the recombinant protein that was synthesized from a fragment of the HIRRV glycoprotein gene conferred protection to Japanese flounder against HIRRV infection [4]. However, the effect of recombinant protein as a vaccine is not yet well understood. In addition, the cost of making a recombinant protein vaccine is expensive, thus making its suitability for commercial use in aquaculture difficult. DNA vaccines are known to be more effective than traditional vaccines in stimulating the host cytotoxic T-lymphocyte activity [7]. Hence, DNA vaccination is effective at preventing intracellular pathogen infection and spread. DNA vaccines also activate the humoral immune system of the host animal [7]. An advantage of DNA vaccines over other types of vaccines is that they do not require development of new antigen purification or pathogen attenuation methods since DNA vaccines can be purified using standard DNA purification techniques. DNA vaccines have been studied not only in mammals but also in fishes. A DNA vaccine based on the IHNV glycoprotein gene protected rainbow trout against an IHNV challenge [8], and similar results have been reported for VHSV [9]. Moreover, a vaccine dose as low as 10 ng of IHNV glycoprotein gene DNA conferred significant protection against IHNV infection in rainbow trout [10]. The authors speculated that the fish rhabdovirus glycoprotein was very immunogenic. However, the DNA vaccines using other IHNV viral protein genes did not induce significant protection against experimental challenge in rainbow trout [11]. In this study, we confirmed the efficacy of the HIRRV glycoprotein gene as a DNA vaccine in Japanese flounder juveniles. Real-time PCR analysis of immune-related genes of the flounder was also conducted to elucidate the mechanisms by which DNA vaccination stimulates the host’s immune response.
2. Materials and methods 2.1. Virus and cell culture The hirame natural embryo (HINAE) cell line [12] was used for the propagation of HIRRV strain 8601H. Cells were cultured in a 25 cm2 culture flask with Leibovitz’s L-15 medium (Gibco BRL, Grand Island, USA) supplemented with 20% fetal bovine serum (JRH Biosciences, Lenexa, USA), 100 units/ml penicillin and 100 mg/ml streptomycin (Gibco BRL). HIRRV strain 8601H was kindly provided by Dr. M. Yoshimizu (Hokkaido University). The virus was used to infect a monolayer of HINAE cells cultured in a 75 cm2 cell culture flask containing 15 ml of medium and incubated at 15 (C for 5 days. When a cytopathic effect (CPE) was observed, the viral culture medium was collected. The virus titer was calculated as 50% tissue-culture infective dose (TCID50) [13]. 2.2. Construction and purification of plasmid DNA Total RNA was isolated from HIRRV strain 8601H culture medium using TRIzol (Invitrogen, Carlsbad, USA). The cDNA was synthesized from total RNA using a cDNA synthesis kit 1st strand with AMV reverse transcriptase (Takara, Otsu, Japan) following the manufacturer’s protocol. PCR primers HRV-Glyf (5#-GAGAATTCGTCATGGACCCTCGAATAAT-3#) and HRV-Glyr (5#-GAGTCGACTTACCCTCGACTGGCG-3#) were designed from the published HIRRV glycoprotein gene nucleotide sequence [14]. Restriction enzyme sites EcoRI and SalI were added at the forward and reverse primer sites, respectively. The PCR primers were used for the amplification of full-length glycoprotein gene of HIRRV
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strain 8601H. PCR was performed with an initial denaturation step of 2 min at 95 (C, and then 30 cycles were run as follows: 30 s of denaturation at 95 (C, 30 s of annealing at 55 (C, and 1 min of extension at 72 (C. The 1546 bp PCR product was digested with EcoRI and SalI, and ligated into the multiple cloning site of pCI-neo vector (Promega, Madison, USA). The plasmid encoding HIRRV glycoprotein gene was designated pCMV-HRVg. The nucleotide sequence was determined using ThermoSequenase (Amersham Bioscience, Little Chalfont, UK) and an automated DNA sequencer LC4200 (Li-Cor, Lincoln, USA). The recombinant plasmid was extracted by ultracentrifugation using a CsCleethidium bromide gradient [15]. In vitro transcriptionetranslation of HIRRV glycoprotein was also determined. TNT Quick coupled transcription/translation system (Promega) was used for the synthesis of the glycoprotein. The pCMVHRVg (0.5 mg) and other reagents were mixed following the protocol of the manufacturer. A similar reaction using pCI-neo vector was also used as a negative control. The reaction mixture was incubated at 30 (C for 90 min. Then, 5.0 ml of the sample was mixed with the same volume of SDS-sample buffer and boiled for 2 min. The samples were applied to a SDSe20% polyacrylamide gel with a 5% stacking gel. The gel was electroblotted to a PVDF membrane, bound with Streptavidin-AP solution and colorimetrically detected using Western blue stabilized substrate for alkaline phosphatase (Promega). A band of approximately 65 kDa was seen, indicating that the HIRRV glycoprotein was translated in vitro (data not shown). 2.3. Vaccination Japanese flounder juveniles with an average weight of 2 g were injected intramuscularly with 1 or 10 mg of pCMV-HRVg in 50 ml saline buffer. As negative control, groups of fish were vaccinated with 10 mg of pCI-neo vector or 50 ml saline buffer. After vaccination, each group of 45 fish was kept in a 60-l tank with a flow-through water system supplying filtered seawater at 13 (C and fed commercial dry pellets corresponding to 5% of total body weight, twice per day. 2.4. Challenge test Each vaccinated group (45 fishes) was transferred to four separate 60-l tanks at 21 days after vaccination. These tanks were provided with UV treated artificial seawater in a recirculating system. At 28 days after vaccination, each fish was injected intramuscularly with 4.0!103 TCID50 of live HIRRV strain 8601H in 50 ml saline buffer. Mortalities were recorded daily for 14 days. 2.5. Real time PCR analysis of gene expression of immune-related genes mRNAs were isolated from the exact site of injection from three individual fish at 1 and 7 days postpCMV-HRVg DNA injection using a micro mRNA purification kit (Amersham Bioscience). The cDNA was synthesized from purified mRNA (1 mg) by cDNA synthesis kit 1st strand with AMV reverse transcriptase. After cDNA synthesis, the sample was diluted to 100 ml with distilled water. To determine the absolute copy number of the target transcript, a cloned plasmid DNA for each gene (MHC class Ia, class IIa and IIb, TCR-a, b1, b2 and d) [16,17] was used to generate a standard curve. The PCR primers used for generating the standard curves are listed in Table 1. PCR was performed as described above. All amplified products were 200 bp in size. The products were purified using Amicon Microcon-PCR Centrifugal Filter Devices (Millipore, Bedford, USA). The copy number of each reacted product was calculated according to its molecular weight and then converted into the copy number based on Avogadro’s number (1 mol=6.022!1023 molecules). The real-time PCR reaction mixture contained 1 ml of diluted sample, PCR primers and the SYBR Green PCR core reagents (ABI, Foster City, USA). Amplification was carried out as follows: 2 min at 50 (C,
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Table 1 PCR primers used in this study Primer name
Oligonucleotides
For construction of standard curve SG200 b-actin F SG200 b-actin R SG200 MRC I a F SG200 MHC I a R SG200 MHC II a F SG200 MHC II a R SG200 MHC II b F SG200 MHC II b R SG200 TCR a F SG200 TCR a R SG200 TCR b F SG200 TCR b R SG200 TCR b2 F SG200 TCR b2 R SG200 TCR d F SG200 TCR d R
5#-TTTCCCTCCATTGTTGGTCG-3# 5#-GCGACTCTCAGCTCGTTGTA-3# 5#-GGACTGGCTGAAGATATTCG-3# 5#-TGGAAGTCCCGTCATGGTTG-3# 5#-GACGTGACTGAAGGAACAAG-3# 5#-ACCCACTCCACAAAACACAG-3# 5#-TCCATGCCTGAGTCAGAAAG-3# 5#-AAAGCAGGTTGAAGCAGCAG-3# 5#-TCAGAAGAGTCTCTGTACAGT-3# 5#-ACGGTCTTGAGGAAGAGGA-3# 5#-CCATATACCTGGAGTCCCTT-3# 5#-CTGTTTCAACACGTGTAGGTG-3# 5#-TTAGCAGCTCACAAACCAGC-3# 5#-TGCTGCTTGTCCTCCAAACT-3# 5#-AGTGAAGAAGAAGCCGTCGT-3# 5#-CAGCGTTCTGATGGTCAGUA-3#
For detection of mRNA SG b-actin F SG b-actin R SG MHC I a F SG MHC I a R SG MHC II a F SG MHC II a R SG MHC II b F SG MHC II b R SG TCR a F SG TCR a R SG TCR b F SG TCR b R SG TCR b2 F SG TCR b2 R SG TCR d F SG TCR d R
5#-TGATGAAGCCCAGAGCAAGA-3# 5#-CTCCATGTCATCCCAGTTGGT-3# 5#-AAGTCTCCCTCCTCTCCAGTCA-3# 5#-TCTCCGTCTTCCTCCAGAACA-3# 5#-GCCAGACTGAAATTCATCGCT-3# 5#-CCAGATCTTGGTCAGTGATTGG-3# 5#-CCTGGGTCTGACCTTATCTCTG-3# 5#-CCAAGTCCAGGACCAGACTCAG-3# 5#-GGTCTGATGCTTCACAGTGTGAG-3# 5#-ACCGCCGGATCTTTCTTCA-3# 5#-GCACCATTCACACACTGTGGTT-3# 5#-AACAGGCTGGTTTGTGAGCTG-3# 5#-CACCTACACATGTTGAAACAGCTC-3# 5#-GTCACTGTTGAATCATTTCTCAG-3# 5#-CGTCAGGTTCAACTCGTACCTG-3# 5#-TGGTAAACGCCACGATCTTG-3#
10 min at 95 (C, 40 cycles of 15 s at 95 (C and 60 s at 58 (C. Thermal cycling and fluorescence detection were conducted using the GeneAmp 5700 Sequence Detection System as described above. All samples were run in triplicate. The threshold cycle (CT) represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected. The ratio between the b-actin content in standard samples (106 copies) and test samples was defined as the normalization factor. Standardized cytokine mRNA quantities (cytokine copies/106 b-actin copies) were determined by dividing the interpolationderived values from the cytokine standard curve by the normalization factor.
2.6. Statistical analysis Statistical analysis was carried using the c2 test. The relative percentage survival (RPS) was determined following the formula RPS=(1ÿ[% loss of immunized fish/% loss of control fish])!100 [18].
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3. Results and discussion 3.1. Protection against HIRRV The nucleotide sequence of the HIRRV strain 8601H (accession no. AB103462) was compared with previously reported viral genes using the BLAST program. The sequence shared 99.8% identity (507/508 amino acids) with the HIRRV glycoprotein gene [14]. Fish vaccinated with the pCMV-HRVg DNA vaccine were better able to survive the HIRRV challenge. Fourteen days after the HIRRV challenge, cumulative mortalities of the fish injected with 1 and 10 mg of pCMV-HRVg were about 28.8 and 8.8%, respectively, while the cumulative mortality of the control groups was 100% (Table 2). The time when mortality was first observed after HIRRV challenge was also different between the vaccinated and control groups. Almost all control fish exhibited acute mortality starting on the 5th day until the 8th day after challenge, following which low mortalities were observed until the termination of the experiment. The vaccinated Japanese flounder, however, had lower mortality rates throughout the challenge period. Recently, DNA vaccines for the prevention of rhabdovirus infections, such as viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in salmonid fish have been demonstrated to be efficacious using the viral glycoprotein gene in a DNA vaccine construct [8,19,20]. In addition, a recent study by Sommerset et al. [21] has shown that a VHSV glycoprotein DNA vaccine was able to provide cross-protection in fish against a heterologous fish virus, the Atlantic halibut nodavirus (AHNV). The HIRRV glycoprotein gene DNA vaccine that we have developed is also efficacious in protecting against HIRRV infection in marine fish such as Japanese flounder, as shown by the higher survival rates of the vaccinated fish in comparison with the control fish upon virus infection. Thus, the glycoprotein gene is a good antigen that protects not only salmonids but also other marine fish, such as Japanese flounder, against rhabdovirus infection. In addition, HIRRV glycoprotein gene DNA vaccine provided protection with no side effects observed after a single intramuscular injection of either 1 or 10 mg of DNA per fish. Our results suggest that this DNA vaccine can provide a high level of protection and may be feasible for use in the aquaculture industry. 3.2. Quantification of expression of immune-related genes In mammals, MHC class I molecule, which consists of a chain and b2-microglobulin, is present on the surface of all kinds of cells [22]. The molecule presents epitopes of intracellular pathogens and activates CD8+ T cells. On the other hand, MHC class II molecule, which consists of a and b chains, exists only on the surface of antigen presenting cells and activates CD4+ T cells [22]. TCRs are heterodimers consisting of Table 2 Cumulative percentage mortality (%) and calculated RPS values following challenge with HIRRV in experimental groups of DNA vaccinated fish by i.m. injection Injected plasmids
pCMV-HRVg (10 mg) pCMV-HRVg (1 mg) pCI-neo (10 mg) PBS a
No. of injected virus: 4.0!103 TCID50/fish Mortalities death/total
RPSa
c2
P value
8.8% (4/45) 28.8% (13/45) 100% (45/45) 100% (45/45)
90.1 70.5 0 e
75.306 49.655 e e
!0.01 !0.01 e e
Relative percentage survival (RPS)={1ÿ[% mortality (vaccinated)/% mortality (control)]}!100.
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either a/b or g/d polypeptide combinations, and recognize MHC-presented epitopes [22]. Therefore these molecules are useful markers for understanding the mechanisms of epitope recognition and tracing the migration of immune-related cells. After 1 and 7 days post-pCMV-HRVg DNA vaccination, the copy numbers of MHC class Ia, class IIa, and IIb, TCR-a, b1, b2 and d mRNAs of the injected fish were at least 2-fold higher than those of the nonvaccinated fish (Table 3). The most remarkable change in the gene expression patterns was observed in TCR-a, b1 and b2. The copy numbers of these transcripts dramatically increased 1 day after vaccination but then after 7 days, the expression levels of these genes, especially TCR-b1 and b2, decreased and returned to nearly the gene expression level of non-vaccinated fish. In case of TCRd gene expression, low increments were observed at day 1 and day 7 post-injection of the pCMV-HRVg DNA vaccine. In mice, the g/d T cells are located in epidermis and in epithelial layers of intestine, in lung and ovary [23], so the g/d T cells may have important roles in inhibiting the virus during the initial stage of infection. However, DNA vaccination in this study was delivered using the intramuscular route. Therefore, the g/d T cells might not have been stimulated and a high level of expression of the TCRd gene was not observed. Expression of MHC class Ia, class IIa and IIb genes were activated at the site of the pCMV-HRVg DNA injection. The increased copy numbers of these genes suggest that antigen-presenting cells and antigen recognition cells such as a/b T cells aggregate at the site of pCMV-HRVg DNA injection due to the possible stimulation of the pCMV-HRVg DNA vaccine, and because some cells neighboring this site might express chemokines and cytokines after recognition of the expressed glycoprotein. The activity and number of cytotoxic T cells might be related to the copy number of TCR mRNAs. Fischer et al. [24] immunized rainbow trout with a plasmid encoding the glycoprotein gene of IHNV. They also infected a MHC class I matched rainbow trout gonad cell line (RTG-2) with IHNV. They found that peripheral blood leukocytes isolated from the immunized fish killed the IHNV-infected cells. Hence, an increase in MHC class Ia gene expression found in the present work implies an increased number of antigen presenting cells and stimulation of specific cytotoxic T cells. In our study, 7 days after pCMV-HRVg DNA injection, antigen recognition cells might gradually leave the injected site, because the expression level of TCR-b1 and b2 returned to nearly the gene expression level of the non-vaccinated fish, even though the expression of MHC class I was higher at 7 days post-injection than at 1 day post-injection. These results suggest that the specific cellular immune functions gradually became established during the 7 days following vaccination. Furthermore, LaPatra et al. [25] examined the immune response of rainbow trout injected with a DNA vaccine against the rhabdovirus IHNV. These fish show an ‘early’ non-specific protection and a ‘late’ specific protection, because rainbow trout immunized with plasmid DNA encoding the IHNV glycoprotein gene showed cross-protection until 14 days post-vaccination against VHSV infection but after this period this cross-protection started to disappear. On the other hand, specific protection against IHNV continued until the end of the experiment. The increase in the copy number of MHC class Ia at 7 days postvaccination in our study might indicate generation of a ‘late’ specific cellular immune response. Table 3 Calculation of copy number of MHC and TCR genes by quantitative real-time PCR Genes MHC class Ia MHC class IIa MHC class IIb TCR a TCR b1 TCR b2 TCR d
Control
DNA vaccine day 1 8
3.6 ! 10 3.5 ! 106 1.7 ! 106 9.0 ! 105 2.9 ! 107 7.1 ! 108 5.4 ! 106
8
7.6 ! 10 3.1 ! 107 6.0 ! 106 5.2 ! 106 2.3 ! 108 5.3 ! 109 6.4 ! 106
(2.1)* (8.7)* (3.3)* (5.8)* (7.9)* (7.5)* (1.2)*
*Increased fold of the copy number of mRNA of DNA injected samples when compared with that of control.
DNA vaccine day 7 1.6 ! 109 1.7 ! 107 9.2 ! 106 3.4 ! 106 4.4 ! 107 1.3 ! 109 1.4 ! 107
(4.4)* (4.8)* (5.5)* (3.8)* (1.5)* (1.8)* (2.6)*
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Our results also suggest that DNA immunization activates both the cellular and humoral defenses of the specific immune system of Japanese flounder. Boudinot et al. [9] observed that DNA vaccination with VHSV glycoprotein genes triggered strong expression of MHC class II and Mx genes at the site of injection. They concluded that DNA immunization was able to activate specialized cells of the immune system as well as nonspecific defense mechanisms. Furthermore, McLauchlan et al. [26] showed strong expression of Mx gene from rainbow trout that was injected with pcDNA3-vhsG intramuscularly but not intraperitoneally, and they suggested that the early non-specific immune response is due to the induction of interferon. Immune-related genes of Japanese flounder have been found from expressed sequence tag (EST) studies [16,17,27]. Further studies on the analysis of their expression as a consequence of DNA vaccination are required.
Acknowledgements This research was supported in part by a Grant-in-Aid for Scientific Research (S) (No. 15108003) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
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