Immunogenicity, retention and protective effects of the protein derivatives of formalin-inactivated red seabream iridovirus (RSIV) vaccine in red seabream, Pagrus major

Abstract

A formalin-inactivated virus was previously found to be efficient in protecting fish against challenge with red seabream iridovirus (RSIV), a DNA virus belonging to the Iridoviridae family. In the present study, we determined the amount of the virus in the vaccine in terms of the number of copies of the gene for the major capsid protein (MCP) gene by quantitative real-time PCR and examined the longevity and types of immune response generated after intramuscular vaccination. We also tested whether the protein components of the vaccine are able to mount a protective immune response in fish. The vaccine contained 10⁷ MCP copies per microliter of vaccine, and was detected in blood, kidney and spleen of vaccinated fish up to 15 days post-vaccination. Fish vaccinated with either the intact formalin-inactivated vaccine or its protein derivatives had increased serum neutralization antibodies and enhanced expression of MHC class I, although the kinetics of expression varied among groups. However, only those vaccinated with the intact vaccine survived the virus challenge, and this indicates that serum neutralization antibodies have scarce role in protecting the fish against RSIV. We hypothesize that the cell-mediated immunity, particularly the MHC class I pathway is responsible for such protection.

Introduction

Iridoviruses are large icosahedral cytoplasmic DNA viruses that have been isolated from a diverse number of invertebrate and vertebrate hosts. The Iridoviridae family is classified into four genera: Iridovirus, Chloriridovirus, Ranavirus and Lymphocystivirus [1]. Systemic iridovirus infections in fish raise serious economic concerns in modern aquaculture because these affect the various stages of the life cycle [2], [3], [4]. Piscine iridoviruses have been observed in more than 100 different species of fish worldwide [5] in both freshwater and marine environments [6].

The red seabream iridovirus (RSIV) is the causative agent of a serious marine viral disease in Japan. The virus was first isolated from diseased red seabream, Pagrus major, in the western part of the country in 1992 [7] and has since infected 31 cultured marine species spanning three taxonomic orders [8]. Its biological and physico-chemical properties include an optimal temperature of 20 or 25 °C for growth, a sensitivity to acid at pH 3, chloroform and ether, and heat lability but not a sensitivity to ultrasonic treatment or repeated freezing and thawing [9]. Infected fish are anemic, exhibit petechiae of the gills, congestion of the liver, and hypertrophy of the spleen and kidney. This viral pathogen has a wide geographical range since epizootics of this disease have been reported in other Asian countries as well [10], [11], [12], [13], [14], [15].

Due to the devastating effects of RSIV to marine aquaculture, a formalin-inactivated virus was used to vaccinate juvenile fish against this viral disease. Laboratory [16] and field studies [17] have shown the effectiveness of the inactivated vaccine in protecting fish against subsequent viral infection. However, the mechanisms behind the protective immunity conferred by the formalin-inactivated RSIV vaccine in fish are not yet fully characterized although the specific immune response was speculated to provide such protection [16]. Identification of such mechanisms would provide an important basis for developing new, more efficient and cheaper methods of control by vaccination.

Previous studies found that RSIV produced antigenic proteins [18], [19]. To investigate the contribution of formalin-inactivated RSIV vaccine and its protein components to immunity against RSIV, we extracted the heat-denatured form of the protein component as well as its structurally altered form from a commercially prepared formalin-inactivated vaccine and used these to vaccinate juvenile red seabream. The amount of the virus in the formalin-inactivated vaccine was determined by quantifying the number of copies of the gene for the major capsid protein (MCP) by quantitative real-time PCR. The longevity and distribution of the vaccine in fish were also determined by PCR amplification of the MCP gene. Lastly, the immune responses generated by the formalin-inactivated vaccine and its protein components were determined by a serum neutralization test and RT-PCR of some immune-related genes post-vaccination.