Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination

https://doi.org/10.1016/j.fsi.2004.04.012Get rights and content

Abstract

Intramuscular injection of Japanese flounder, Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 μg 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 immune-related genes, e.g. MHC class Iα, IIα, IIβ, TCR-α, β1, β2 and δ, 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 β1 and β2 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.

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].

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.

Section snippets

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 μg/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

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 μg of pCMV-HRVg were about 28.8 and 8.8%, respectively,

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 (27)

  • T. Kimura et al.

    A new rhabdovirus isolated in Japan from cultured hirame (Japanese flounder) Paralichthys olivaceus and ayu Plecoglossus altivelis

    Dis Aquat Organ

    (1986)
  • M.J. Oh et al.

    A new rhabdovirus (HRV-like) isolated from cultured Japanese flounder Paralichthys olivaceus

    J Fish Pathol

    (1998)
  • M.H.V. Van Regenmortel et al.

    Virus taxonomy. Seventh report of the International Committee on Taxonomy of Viruses

    (2000)
  • Cited by (0)

    View full text