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Host responses of Japanese flounder Paralichthys olivaceus with lymphocystis cell formation

https://doi.org/10.1016/j.fsi.2014.03.028Get rights and content

Highlights

  • We investigated the mechanisms of lymphocystis cell formation in fin cells.

  • Lots of apoptosis related genes on the KEGG pathway were down-regulated in the cell.

  • The cell might be inhibited apoptotic death before cell hypertrophy.

Abstract

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease (LCD). In this study, we investigated the mechanisms of lymphocystis cell (LCC) formation from the viewpoint of gene expression changes in the infected fish. LCC occurrence and virus titers in the experimentally infected Japanese flounder, Paralichthys olivaceus were monitored by visual confirmation and real-time PCR, respectively. The gene expression changes in the fish fin were investigated by microarray experiments. LCCs firstly appeared in the fish at 21 days post infection (dpi). LCD incidence increased with time and reached 92.9% at 62 dpi. LCDV genome was firstly detected from dorsal fins at 14 dpi, and the relative amount of the genome gradually-increased until 56 dpi. Since the occurrence of LCC was approximately synchronized with increasing of the virus genome, virus replication might play important roles for LCC formation. The microarray detected a few gene expression changes until 28 dpi. However, the number of expression changed genes dramatically increased between 28 and 42 dpi in which LCCs formation was active. From the microarray data analyses, apoptosis and cell division related genes were down-regulated, whereas cell fusion and collagen related genes were up-regulated at 42 dpi. Together with the observation of morphological changes of LCCs in previous reports, it is suggested that the following steps are involved in LCC formation: the virus infected cells were (1) inhibited apoptotic death and (2) cell division before enlargement, (3) hypertrophied by cell fusion, and (4) surrounded by a hyaline capsule associated with the alteration of collagen fibers.

Introduction

Lymphocystis disease (LCD) has been reported in over 100 different marine and freshwater teleost fish species worldwide [1]. In Japan and Korea, the disease is often observed in cultured fish, especially Japanese flounder, Paralichthys olivaceus, which is the most economically important fish species in the aquaculture industry [2]. LCD usually occurs in spring and autumn in both countries when water temperatures reach approximately 20 °C [2]. Generally, LCD-affected fish naturally recover with increasing or decreasing water temperature. However, rapidly changing water temperature is a stressor that enhances LCD outbreaks [3].

LCD is characterized by enlarged cells called lymphocystis cells (LCCs) on the skin and fins of affected fish [1]. Massive hypertrophy and encapsidation of host cells by an extracellular hyaline matrix are induced by viral infection [4]. The formation of LCCs is not lethal; however the diseased fish often die due to feeding disturbance by the formation of nodular lesions on the jaws. The structures of LCCs are different among growth stages [5]. Immature LCCs have cytoplasm containing high basophilic reticulations of inclusion bodies and a hypertrophied nucleus. The cells are encapsulated with a thick hyaline wall. In the cytoplasm, there are viral assembly sites, rough endoplasmic reticula, ribosomes, smooth endoplasmic reticula and mitochondria. In contrast, mature LCCs have an eosinophilic cytoplasm containing low inclusion bodies. The cytoplasm has enormously large numbers of virions showing crystalline array and degenerated rough endoplasmic reticula and mitochondria.

Lymphocystis disease virus (LCDV), which belongs to the genus Lymphocystivirus in the family Iridoviridae, is the causative agent of LCD [6]. The virus forms a double-layer icosahedral capsid approximately 200 ± 50 nm in diameter with a fringe of fibril-like external filaments [1], [7], [8]. The LCDV genome is a single linear dsDNA molecule, in which the structure is circularly permuted and terminally redundant [9], [10]. The complete genome sequences of two LCDV strains, LCDV-1 isolated from European flounder Platichthys flesus [11] and LCDV-C isolated from Japanese flounder [12], were determined. The former showed 102,653 bp encoding with 195 potential ORFs, while the latter showed 186,250 bp with 240 potential ORFs. Comparing the LCDV-1 and LCDV-C genomes, the highest nucleotide sequence identity was observed in a major capsid protein (MCP) gene (78.9%) encoding a single polypeptide with a molecular mass of approximately 50 kDa [12], [13]. The MCP gene is one of the most important targets for analysis of genetic relationships among iridoviruses because its nucleotide sequence is relatively conserved within the family Iridoviridae [14], [15]. Based on the MCP gene sequence and pathogenicity of lymphocystiviruses, the viruses were divided into nine genotypes [16], [17], [18].

The morphologic, genomic and taxonomic characterizations of the virus have been well investigated so far. However, the molecular mechanisms of LCC formation have not been clarified due to the lack of efficient cell lines for the propagation of LCDV. The aim of this study is to elucidate the mechanisms of LCC formation from the viewpoint of gene expression changes in the infected fish. LCC occurrence and virus titers in the experimentally infected fish were monitored by visual confirmation and real-time PCR, respectively. Additionally, gene expression changes in the fish were investigated by microarray experiments.

Section snippets

LCDV Inoculum

LCCs were collected from the skin of Japanese flounder naturally affected with LCD at a fish farm in Ehime prefecture, Japan in 2009. The collected LCCs were homogenized with a 10-fold volume of minimum essential medium (MEM, Nissui Pharmaceutical Co., Ltd.) and stored at −80 °C. In this study, LCDV titer for experimental infection was estimated by PCR [16]. The virus titer in the inoculum was 109 PCR titer/ml.

Experimental fish

Japanese flounder (average body weight 1.0 g) were obtained from a fish farm in

Occurrence of LCD and detection of viral genome

LCCs firstly appeared on the skin, fins and/or jaw at 21 dpi. LCD incidence increased with time and reached 92.9% at the end of the experimental period (Fig. 1). The cumulative mortalities in the virus infected and control groups were 8.3% and 3.3%, respectively. From these results, we confirm that LCD is a chronic disease with low mortality. The ATPase gene of LCDV was firstly detected from dorsal fins at 14 dpi in the virus infected fish group, and the relative amount of the genome

Conclusion

From the results of microarray experiments, apoptosis inhibition, cell cycle arrest and alterations of collagen fibers were suspected in fin cells infected with LCDV at 42 dpi. Together with the observation of morphological changes of LCCs [1], [5], [28], we concluded that the following steps are involved in LCC formation (Fig. 4): the virus infected cells were (1) inhibited apoptotic death and (2) cell division before enlargement, (3) hypertrophied by cell fusion, and (4) surrounded by a

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (no. 23780195) of the Japan Society for the Promotion of Science.

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